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Destruction and injury of escherichia coli under vacuum microwave : death kinetics and transcriptional… Yaghmaee, Parastoo 2004

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DESTRUCTION AND INJURY OF ESCHERICHIA COLI UNDER V A C U U M M I C R O W A V E : D E A T H KINETICS AND TRANSCRIPTIONAL RESPONSES by  PARASTOO Y A G H M A E E  B.Sc. Shahid Beheshti University, Tehran, Iran, 1991 A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Food Science) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A May 2004 ©Parastoo Yaghmaee 2004  ABSTRACT Rapid development in microwave applications in the home and industry along with the increase in the possibility o f exposure to microwave radiation have raised concerns about the effect of microwaves on living cells.  Although numerous studies have been conducted,  microwave effects on living cells still are not fully understood. Some scientists believe that the effect is solely attributable to microwave heating while others suggested that additional effects, other than thermal, are required to explain various types of molecular formations and alterations in a target organism.  The present work was designed to study the effect of 2450 M H z  microwave radiation under vacuum (vacuum microwave or V M ) on kinetic parameters and transcriptional response of mid-stationary Escherichia coli (ATCC 11775) cells and to search for possible non- thermal effects associated with V M . In addition, the E. coli transcriptome in latelog and mid-stationary phase of growth was studied. In a preliminary study, the lethal effect of microwave radiation on the microorganisms naturally occurring on parsley during dehydration under vacuum was investigated. Fresh parsley leaves were dried with air-drying (AD) and vacuum microwave drying (VMD) at the same final temperature.  This study showed that parsley leaves treated with V M D had lower microbial  populations than A D samples at comparable water activity.  In addition V M D was more  effective against yeast and mould than against total aerobic populations. Since higher reduction in microbial population of fresh parsley leaves occurred not only in a shorter time but also at a lower final temperature as a result of V M D compared to A D , it can be concluded that V M drying was an effective method of reducing the number of naturally occurring microorganisms in parsley.  Death kinetics of E. coli in peptone water were determined in a continuous-flow vacuum system with a water bath or microwave as the heating source. Vacuum was used to control the boiling point of water and to maintain the bacterial suspensions at specified temperatures (49°C to 64°C). The z value in the water bath under vacuum was 9.0 °C whereas for V M treatments at 510W and 711W it was 6.0 °C and 5.9 °C respectively, suggesting that E. coli is more sensitive to temperature changes during microwave heating than conventional heat treatments.  Based  upon the Arrhenius calculation of the activation energy it is proposed that the mechanism of E. coli inactivation in V M treatment is different from the inactivation that occurs during conventional heat treatment.  Thus the impact of temperature on E. coli destruction under  vacuum was not the same when microwaves were the medium of heat transfer. Further, a molecular biology approach, D N A microarray technology, was used to investigate E. coli transcriptional response to sub-lethal V M and water bath treatment at 50°C for 3 minutes. The results showed that the number of E. coli genes that their expression altered through water bath treatment was higher than during V M treatment. V M treatment had a larger effect on genes related to membrane structure and membrane transport systems suggesting that microwave destruction may follow the dielectric cell-membrane rupture theory. In addition V M affected t he e xpression o f g enes e ncode f or e nzymes r elated t o r n etabolism o f c arbohydrates, lipids and amino acids to a greater extent than the water bath treatment. Conversely the effect of conventional water bath treatment on ribosomal subunits was higher. Although both treatments were employed under vacuum and signs of anaerobic respiration would be expected, there was more evidence at the transcriptional level for the start of anaerobic respiration in water bath treated cells than in V M treated cells.  iii  In the present work, the focus of the kinetic and gene expression studies was on stationary phase cells, while other gene expression studies have mostly worked with cells at the exponential phase of growth. To close the loop, another study was conducted to investigate the changes a 11 he t ranscription 1 evel i n E. c oli c ells b etween 1 ate-exponential a nd m id-stationary phase of growth. In mid-stationary phase, genes encoding for energy metabolism as well as amino acids and carbohydrate metabolism were down regulated. In addition csg genes, required for curli synthesis, were induced and 70.5% of genes involved in cell motility were down regulated or were not detected in mid-stationary cells indicating that in this stage cells may have been less mobile and had more tendency to clump or stick to surfaces.  The transcription of  hupA, hupB, hlpA, himA and himD genes previously reported to show up-regulation upon entry into stationary phase were down regulated in mid-stationary cells suggesting that the mechanisms involved in cell function are not only different between lag, log and stationary phase of growth but also may differ in early, mid and late stationary phases.  iv  T A B L E OF CONTENTS page  ABSTRACT  "  T A B L E OF CONTENTS.  v  LIST OF T A B L E S  xiii  LIST OF FIGURES  xv  LIST OF S Y M B O L S & ABBREVIATIONS  xvii  ACKNOWLEDGEMENTS  xx  A WISH  xxi  CHAPTER ONE: INTRODUCTION  1  1.1 General introduction  2  1.2 Hypotheses  3  1.3 Overview of work plan  4  CHAPTER TWO: REVIEW OF T H E RELATED LITERATURE 2.1 Electromagnetic radiation and electromagnetic spectrum 2.1.1  6 7  Microwaves  7  2.2 Microwave heating  9  2.2.1  Factors involved in microwave heating  2.2.1.1 Dielectric properties  10 10  2.3 Microwave applications  11  2.3.1  Microwave dehydration  12  2.3.2  Vacuum microwave  13  2.3.3  Microwave pasteurization and sterilization  15  2.3.3.1 Microwave pasteurization and sterilization systems  V  18  2.4 Effect of microwaves on microorganisms  20  2.4.1  Microwave kinetic parameters  25  2.4.2  Mechanism of thermal destruction  26  2.4.3  Mechanism of microwave destruction  27  2.4.4  Injured microorganisms  30  2.5 Biological effects of microwaves  32  2.6 Escherichia coli  34  2.7 Stress response and stress proteins  36  2.7.1  Function of stress proteins  37  2.7.2  Heat shock response in Escherichia coli  38  2.7.2.1 Regulation of heat shock response in E. coli 2.7.3  Microwaves and stress response  40 42  2.8 D N A microarray technology  43  2.8.1  D N A microarray applications  44  2.8.2  D N A microarray limitations  45  CHAPTER THREE: E F F E C T OF V A C U U M MICROWAVE DRYING ON NATURALLY OCCURING MICROORGANISMS OF PARSLEY  46  3.1 Introduction  47  3.2 Materials & methods  49  3.2.1  Plant source  49  3.2.2  Drying  49  3.2.2.1 Air drying (AD)  49  3.2.2.2 Vacuum microwave drying (VMD)  50  vi  3.2.3  Temperature measurement  of parsley during vacuum microwave  drying  50  3.2.4  Determination of moisture content  50  3.2.5  Water activity measurement  51  3.2.6  Microbiological analysis  51  3.2.6.1 Microbiological sampling  51  3.2.6.2 Total microbial count  51  3.2.6.3 Yeast and mould counts  51  3.2.7  Statistical analysis  52  3.3 Results  52  3.4 Discussion  53  3.5 Conclusion  56  CHAPTER ESCHERICHIA  FOUR:  EFFECT  OF  VACUUM  MICROWAVE  ON  COLI : A STUDY OF D E A T H KINETIC PARAMETERS  AND DIELECTRIC PROPERTIES  61  4.1 Introduction  62  4.2 Materials & methods  65  4.2.1  Bacterial strain  65  4.2.2  Stock culture and inoculum preparation  65  4.2.3  Growth index  66  4.2.4  Sample preparation  66  4.2.5  Microwave power determination  66  4.2.6  Continuous flow vacuum system  67  vii  4.2.7  Determination of temperature consistency inside the glass vacuum chamber  68  4.2.8  Determination of residence time distribution  68  4.2.9  Sanitizing  69  4.2.10 Vacuum microwave (VM) and water bath under vacuum treatments  69  4.2.11 Enumeration of surviving and injured E. coli  70  4.2.12 Correction for loss of heating medium during experiments  70  4.2.13 Check for microorganism loss and possible bio-film formation in the chamber  71  4.2.14 Calculation of kinetic parameters  72  4.2.15 Dielectric measurement of pure culture  72  4.2.16 Statistical analysis  73  4.3 Results  73  4.3.1  Monitoring E. coli growth  74  4.3.2  D value  74  4.3.3  z value  74  4.3.4  Activation energy  75  4.3.5  Injured microorganisms  76  4.3.6  Dielectric properties  77  4.4 Discussion  77  4.5 Conclusion  80  CHAPTER FIVE: C HANGES IN ESCHERICHIA COLIT RANSCRIPTOME DUE TO SUB-LETHAL V A C U U M MICROWAVE T R E A T M E N T  viii  96  5.1 Introduction  97  5.2 Materials & methods  99  5.2.1  Bacterial strain  99  5.2.2  Sample preparation  100  5.2.3  Vacuum microwave (VM) and water bath under vacuum treatments....  100  5.2.4  Untreated sample  101  5.2.5  Total R N A extraction  101  5.2.6  m R N A enrichment  102  5.2.6.1 c D N A synthesis  102  5.2.6.2 rRNA digestion  103  5.2.6.3 c D N A digestion  103  5.2.7  Labeling and fragmentation  104  5.2.8  Hybridization, washing and staining  104  5.2.9  Scanning  104  5.2.10 Data analysis  105  5.2.10.1  Data normalization  105  5.2.10.2  Statistical analysis  105  5.2.10.3  Calculation of fold change  106  5.2.10.4  Data filtering  106  5.2.10.5  Gene annotation  107  5.3 Results  107  5.3.1  Correlation among replicates  107  5.3.2  Present, absent and marginal calls in single arrays  108  ix  5.3.3  Number of up and down-regulated genes  108  5.3.4  Overview of E. coli response  108  5.3.4.1 Heat shock genes  109  5.3.4.2 Genes changed > two fold in both treatments compared to untreated cells  109  5.3.4.3 Genes changed > two fold by water bath or V M treatments compared to untreated cells  HO  5.3.4.4 Genes significantly changed in V M compared to water bath treatment 5.4 Discussion  110 Ill  5.4.1  Heat shock response  Ill  5.4.2  Membrane structure and membrane transport system  112  5.4.3  Enzyme activity  113  5.4.4  Ribosomal R N A  114  5.4.5  Transfer R N A (tRNA)  116  5.4.6  Cell respiration  117  5.5 Conclusion  119  CHAPTER SIX: ESCHERICHIA COLI TRANSCRIPTOME IN L A T E - L O G AND MID-STATIONARY PHASE OF GROWTH  145  6.1 Introduction  146  6.2 Materials & methods  148  6.2.1 Bacterial strain  148  6.2.2 Growth curve determination  148  6.2.3 Batch cultures  148  6.2.4 D N A microarray analysis  149  6.2.5 Statistical analysis  149  6.3 Results  149  6.3.1 Genes up-regulated (>2 fold) in mid-stationary phase cells  150  6.3.2 Genes down-regulated (>2 fold) in mid-stationary phase cells  151  6.3.2.1 Translation and transcription  151  6.3.2.2 Energy metabolism  152  6.3.2.3 Cell motility  152  6.3.2.4 Carbohydrate metabolism  152  6.3.2.5 Fatty acid biosynthesis  153  6.3.2.6 Membrane and transport system  153  6.4 Discussion  153  6.4.1 Curli synthesis  153  6.4.2 Cell motility  154  6.4.3 Transcription and translation  155  6.4.4 Regulatory systems  155  6.4.5 Early stationary phase genes  157  6.5 Conclusion CHAPTER SEVEN:  157 GENERAL DISCUSSION, GENERAL CONCLUSION  AND RECOMMENDATIONS FOR FUTURE STUDIES 7.1 General discussion  177 178  7.1.1 Effect of V M on E. coli cells  178  xi  7.1.2 Effect of growth phase on E. coli transcriptome  183  7.2 General conclusion  186  7.3 Proposed theories  187  7.4 Recommendations for future studies  188  CHAPTER EIGHT: REFERENCES  190  CHAPTER NINE: APPENDICES  212  9.1 A P P E N D I X I: Checking the purity of culture  213  9.2 A P P E N D I X II: Continuous Vacuum System: Schematics and suppliers  214  9.3 A P P E N D I X III: Microwave power determinations  220  9.4 A P P E N D I X IV: Micro pump flow rate determinations  221  9.5 A P P E N D I X V : Thermocouple calibration  222  9.6 A P P E N D I X VI: Survival curves for E. coli plated on P C A and P C A - B S  223  9.7 A P P E N D I X VII: Genes altered less than two fold between late-log and midstationary cells (p<0.05)  229  xii  LIST OF TABLES  Table 3.1. Total microbial and yeast & mould counts for fresh, air dried and vacuum microwave dried parsley  page 60  Table 4.1. Regression equations and D-values of E. coli exposed to vacuum microwave at 711 W treatments  90  Table 4.2. Regression equations and D-values of E. coli exposed to vacuum microwave at 510 W treatments  91  Table 4.3. Regression equations and D-values of E. coli exposed to water-bath under vacuum treatments (control)  92  Table 4.4. Regression equations of temperature sensitivity of E. coli for water-bath under vacuum treatment and vacuum microwave treatments at 510 W and 711W  93  Table 4.5. Regression equations of activation energy (E ) for E. coli in water-bath under vacuum treatment and vacuum microwave treatments at 510 W and 711 W  94  Table 4.6. Dielectric constant and loss factor of sterile peptone water, and centrifuged pellet of pure culture of E. coli at room temperature  95  Table 5.1. vacuum  121  a  Treatment conditions for vacuum microwave (VM) and water bath under  Table 5.2. Sequence of primers for 16S and 23S r R N A used in this study (Affymetrix manual 2000)  122  Table 5.3. Correlation among replicates for treated and untreated E. coli  123  Table 5.4. E. coli gene probe set signals from E. coli exposed to water bath under vacuum and vacuum microwave treatments as well as from untreated stationary phase E. coli cells  124  Table 5.5. Number of significant up- regulated, down-regulated or unchanged genes (p< 0.05) between treatments  125  Table 5.6. List of previously known heat shock genes and their calls in untreated, water bath and vacuum microwave treated E. coli  126  Table 5.7. Genes displaying up-regulation in vacuum microwave and water bath under vacuum treated cells compared to untreated stationary phase E. coli cells  132  xiii  Table 5.8. Genes displaying down-regulation in vacuum microwave and water bath under vacuum treated cells compared to untreated E. coli cells  134  Table 5.9. Genes down-regulated (>2 fold) in water bath under vacuum treated E. coli compared to untreated stationary phase E. coli cells (p<0.05)  135  Table 5.10. Genes up-regulated (>2 fold) in water bath under vacuum treated E. coli compared to untreated stationary phase E. coli cells (p<0.05)  136  Table 5.11. G enes down regulated (>2 fold) in V M treated cells compared to untreated stationary phase E. coli cells (p<0.05)  137  Table 5.12. Genes up-regulated (>2 folds) in V M treated cells compared to untreated stationary phase E. coli cells (p<0.05)  138  Table 5.13. E. coli genes down regulated in V M treatment compared to water bath under vacuum treatment (p<0.05)  139  Table 5.14. E. coli genes up-regulated in V M treatment compared to water bath under vacuum treatment (p<0.05)  142  Table 6.1. E. coli probe sets signals in mid-stationary phase and late-log phase cells  160  Table 6.2. Distribution of gene transcription in mid-stationary phase E. coli, compared to late-log phase E. coli  161  Table 6.3. Genes up-regulated (>2 fold) in mid-stationary phase cells compared to late-log phase E. coli cells (p<0.05)  162  Table 6.4. Genes down regulated (>2 fold) in mid-stationary phase cells compared to latelog phase E. coli cells (p<0.05)  163  Table 9.1. Tests and outcomes for checking the purity of the E. coli culture  213  Table 9.2. Microwave power determined using IMPI2-Liter test (Buffler 1993)  220  Table 9.3. The flow rate of micro pump was d etermined under normal atmosphere and vacuum (22, 24 and 26 in Hg)  221  Table 9.4. Genes up-regulated less than 2 fold in mid-stationary phase cells compared to late-log phase E. coli cells (p<0.05)  229  Table 9.5. Genes down-regulated less than two fold in mid-stationary phase cells compared to late-log phase E. coli cells (p<0.05)  231  xiv  LIST OF FIGURES page  Figure 2.1. The electromagnetic spectrum  8  Figure 2.2. The E. coli heat shock regulon  41  Figure 3.1. Vacuum microwave drier-door closed  57  Figure 3.2. Vacuum microwave drier- door open  58  Figure 3.3. Time-temperature profile of fresh parsley leaves during vacuum microwave drying process: 2450 M H z , 1.5 K W , 26-28 in Hg vacuum with basket rotating at 3 rpm  59  Figure 4.1. Continuous-flow vacuum system  81  Figure 4.2. Continuous vacuum system with microwave as heating source-front view  82  Figure 4.3. Continuous vacuum system with microwave as heating source-outside view..  83  Figure 4.4. Time-temperature profile of 1000 ml distilled water in microwave (2450 MHz) under vacuum (22.5 mmHg) with fiber optic probe  84  Figure 4.5. Sampled population of E. coli cells in the continuous vacuum system with no heating source as a function of time indicated homogeneous mixing of injected bacteria within 30 seconds  85  Figure 4.6.a Temperature sensitivity curves for E. coli treated under vacuum microwave at 510 W  86  Figure 4.6.b Temperature sensitivity curves for E. coli treated under vacuum microwave at 711 W  87  Figure 4.6.C Temperature sensitivity curves for E. coli treated in water-bath under vacuum (control)  88  Figure 4.6.d Temperature sensitivity curves ofE. coli treated by vacuum microwave 711 W, vacuum microwave 510 W and water bath under vacuum treatment  89  Figure 5.1. Simplified flow diagram of role of glnS and glnA in glutamine synthesis  120  Figure 6.1. Growth of 10 CFU/ml stationary phase E. coli (ATCC 11775) transferred to 50 ml Nutrient Broth at 37°C over 22 hours Figure 9.1. Overview of Continuous Vacuum System  159 215  7  XV  Figure 9.2. Vacuum Chamber body; Side-view  216  Figure 9.3. Vacuum Chamber body; Inside-view  216  Figure 9.4. Vacuum Chamber body; Top-view  217  Figure 9.5. Vacuum Chamber lid; Top-view  218  Figure 9.6. Vacuum Chamber lid; Side-view  219  Figure 9.7. Regression equation for temperatures from the data logger versus recorded temperatures from the A S T M thermometer, as a correction factor for T type thermocouple  222  Figure 9.8. Differential counts of E. coli on P C A and P C A - B S during vacuum microwave (711W) at 58.43°C  223  Figure 9.9. Differential counts of E. coli on P C A and P C A - B S during vacuum microwave (510W) at 58.19°C  224  Figure 9.10. Differential counts of E. coli on P C A and P C A - B S during water bath treatment under vacuum at 58.62°C  225  Figure 9.11. Differential counts of E. coli on P C A and PCA-BS during vacuum microwave (711W) at 51.84°C  226  Figure 9.12. Differential counts of E. coli on P C A and P C A - B S during vacuum microwave (51 OW) at 50.21°C  227  Figure 9.13. Differential counts of E. coli on P C A and P C A - B S during water bath treatment under vacuum at 5 0.5 ° C  xvi  228  LIST OF SYMBOLS & ABBREVIATIONS %  °c °F Hg uL uM u.m g 16S r R N A A AD ANOVA ASTM ATCC ATP aw b no. c cDNA CFU cm DNA Dnase I dNTP DTT D-values E. coli e' e" E EDTA ELF Eq EVOH f F Fig g GHz HPLC hsp HTST Hz x  a  0  Percent Degree(s) Celsius Degree(s) Fahrenheit Microgram(s) Microliter(s) Micro Molar Micrometer(s) Multiples of the Earth's gravitational field 16 Svedberg unit ribosomal R N A Absent, not detected Air drying Analysis of variance American Society for Testing and Materials American Type Culture Collection Adenosine triphosphate Water activity Blattner number Velocity of electromagnetic wave complimentary D N A Colony forming unit Centimeter(s) Deoxyribonucleic acid Deoxyribonuclease I Deoxyribonucleoside triphosphate DL-Dithiothreitol Decimal reduction time (min) Escherichia coli Dielectric constant Dielectric loss factor Activation energy Ethylenediamine tetra-acetic acid Extremely low frequency Equation Ethylene vinyl alcohol copolymer Frequency (s" ) Sterilization value or accumulated lethality (min) Figure gram(s) Gigahertz (s" x 10 ) High Performance Liquid Chromatography Heat shock protein High temperature short time Hertz (one cycle per second) or (s" ) 1  1  9  1  xvii  ID inHg ISM J/mole K kD kJ/mole kW log LSD LTLT M m m /s mbar MCWC MHz min mL mM mm MM mmHg M M L V RT MMLV mRNA mT MUG mW/cm NA NB nm OD orf P PCA PCA-BS PM pM PVDC r RNA Rnase H rpm rRNA RT-PCR s 3  2  2  Inside diameter Inches of mercury Industrial, Scientific and Medical Joules per mole per degree Kelvin Kilo Dalton Kilo joules per mole Kilowatt(s) Logarithm Least significant difference Low temperature, long-time Marginal Meter(s) Cubic meter per second Millibar Microwave Circulated Water Combination system Megahertz (s" * 10 ) Minute(s) Milliliter(s) milli Molar Millimeter(s) MisMatch Millimeters of mercury Moloney Murine Leukemia Virus Reverse Transcriptase enzyme Moloney Murine Leukemia Virus Messenger R N A M i l l i Tesla 4-Methylumbelliferyl-P-D-glucuronide Milliwatts per square centimeter Nutrient Agar Nutrient Broth Nanometer(s) (mx 10" ) Outside diameter Open reading frame Present, detected Plate Count Agar Plate Count Agar + 1.5 g/L bile salts #3 Perfect Match pico Molar Polyvinylidene chloride Coefficient of determination Ribonucleic acid Ribonuclease H Rotations per minute Ribosomal Ribonucleic Acid Reverse transcription-polymerase chain reaction Second 1  6  9  xviii  stdv T tan 8 TDT TE buffer fRNA U USDA v/v VM VMD VM-U VM-W W w/v W-U z value X a 32  Standard deviation Tesla (unit of magnetic flux density) Loss tangent Thermal death time (min) Tris/EDTA buffer Transfer R N A Unit United States Department of Agriculture Volume per volume Vacuum microwave Vacuum microwave drying Vacuum microwave at 711W compared to stationary phase sample Vacuum microwave at711W compared to water bath treatment Watt(s) (J/s) Weight per volume Water bath treatment compared to stationary phase sample Temperature sensitivity value (C or K degrees) Wavelength (m) Sigma 32  xix  ACKNOWLEDGMENTS  I would like to offer my warmest thanks to my Ph.D. supervisor, Professor Timothy Douglas Durance, who was both a friend and an adviser to me. I shall always appreciate his patience in putting up with me all the way along. Now he can sit back and relax. A big trouble has gone out of his working life.  I also wish to express m y special gratitude to other members of my advisory committee, Dr. Barbara Dill, Dr. Christine Seaman and Dr. Brent J. Skura for their wise suggestions and kind and caring criticism that lit my way through difficulties of research.  I also wish to thank Mr. Sherman Yee and Mrs. Valerie Skura for their endless technical support, no matter when or where, they were always ready for help. I wish all the students had the opportunity to work with them.  Thanks to Dr. George van der Merve and Mr. Brad Greatrix in genomic lab at U B C wine research centre, for their technical help.  Thanks to Mr. Jochen Brum for his guidance in  statistical analysis without whose advice I would be lost.  M y special salute goes to all of my friends with whom my path crossed during the school years. Those who made my days colorful and my thoughts full of happiness, those that made me smile when I was sad and shook me when I was smug.  And my last but not the least thanks goes to my little precious, Darya, whose presence along with all the trouble and responsibilities, made me stronger and more persistent on my way and kept me honest on my difficult days.  XX  A WISH  I don't think that I like to dedicate my thesis to anyone in specific. It already belongs to science. Hope it's worthy.  In the future, if this smoothes the bumpy road of research, I would be content. I would feel happy if I am around. M y soul would lighten up when I am gone.  xxi  CHAPTER ONE  INTRODUCTION  i  1.1  General introduction  From 1945, the time that Dr. Percy LaBaron Spencer "father of the microwave oven" (Schiffmann 1997) discovered the specific heating properties of microwaves, till today, many microwave applications have b een developed. Nowadays, microwave o vens c an b e found i n almost every household and are used on a regular basis (Regier & Schubert 2001; Housova et al. 1996). Some industrial microwave applications, such as microwave tempering units, microwave pasteurization plants and microwave driers (Housova et al. 1996), have been adopted across the world. The chance of exposure to microwave radiation (Saffer & Profenno 1989) has increased. Thus a long w ith i ts d evelopment, c oncern a bout t he e ffect o f m icrowaves o n 1 iving c ells h as been raised. Many studies have been conducted on microorganisms (Woo et al. 2000; Kakita et al. 1995), nematodes (Adams et al. 1999; Daniells et al. 1998), rats (Trosic et al. 1999) and human cells (Liu et al. 2002) to determine the effect of a wide range of frequency of electromagnetic radiation. There is evidence that microwaves cause different biological effects depending upon field strength, frequencies and duration of exposure (Banik et al. 2003). It is clear that microwave heating is not identical to conventional heating, at the molecular 1 evel, b ut w hether t his d ifference c ould c ause e ffects o ther t nan h eat i s n ot c learly understood.  There are conflicting reports in the literature regarding the mechanism of  microwaves effects.  Some scientists believe that the effect is exclusively attributable to  microwave heating and could be considered as a pure thermal effect (Yeo et al. 1999; Fujikawa et al. 1992) whilst others show evidence of changes in physicochemical characteristics of bacterial cells (Dreyfuss & Chipley, 1980) and suggested that additional effects other than  2  thermal are required to cause various types of molecular formations and alterations in target organisms (Banik et al. 2003; Woo et al. 2000; Papadopoulou et al. 1995). The present research was initiated to investigate the effects of microwaves under vacuum (vacuum microwave) on a simple microorganism.  A molecular biology approach, D N A  microarray technology, was used to learn about microwave interactions with the bacteria. This technique provides a format for whole-genome expression profiling which enables global approaches to biological function in living cells (Blattner et al. 1997) give a better understanding of bacterial response to any factor, in this case microwave radiation. A simple bacterial model, Escherichia coli was chosen, as its genome map and physiology have been well studied (Blattner et al. 1997; Adams & Moss 2000) and it has been successfully used as a model for electromagnetic related studies (Nakasono & Saiki 2000; Saffer & Profenno 1989). In addition, if electromagnetic fields are found to affect the E. coli cells, a wide range of biochemical and molecular biology techniques can be applied, leading to deeper levels of understanding (Saffer & Profenno 1989).  1.2  Hypotheses •  The destructive effect of vacuum microwave on microorganisms is not purely due to the thermal effect.  Other factor or factors are involved in the  destruction process. •  Kinetic parameters of E. coli death under vacuum microwave heating condition are different from kinetic parameters of E. coli death during convective heating under vacuum.  3  •  The transcriptional response of E. coli cells subjected to sub-lethal vacuum microwave heating is different from transcriptional response during sub-lethal convective heating for the same time and temperature.  •  E. coli transcriptional response in late-exponential and mid-stationary phase of growth is different.  1.3  Overview of work plan The aim of the present work was to study the effect of microwave radiation 2450 M H z on  mid-stationary E. coli cells under vacuum. The first step was to determine the destruction effect of vacuum microwave on E. coli, followed by an investigation to search for non-thermal effect(s) through determination of kinetic parameters.  The work continued with an examination of  changes in transcriptional response of mid-stationary E. coli cells following vacuum microwave treatment. Since reported kinetic studies in the literature have been mostly carried out with stationary phase cells, and gene expression studies have focused on cells in exponential growth, another study was conducted to investigate the changes in E. coli cells at the transcription level between late-exponential and mid-stationary phase of growth. The complete plan consisted of four phases: Phase I involved a preliminary study of the destructive effect of vacuum microwave drying on naturally occurring microorganisms in parsley. Phase II focused on the effects of 2450 M H z microwave radiation on survival and injury of E. coli under vacuum, along with determination of kinetic parameters of death as well as dielectric properties of the pellet of pure E. coli culture and heating medium.  Phase III was an investigation into changes in transcriptional response of mid-stationary phase E. coli cells subjected to sub-lethal treatment with vacuum microwave 2450 M H z and conventional heating. Phase IV was a study of differences in E. coli gene expression at the transcription level in cells at late-exponential and mid-stationary phases of growth, to verify the changes in E. coli gene expression between these two stages of growth.  5  CHAPTER  TWO  LITERATURE REVIEW  2.1  Electromagnetic radiation and the electromagnetic spectrum The electromagnetic radiation is characterized by variations of electric and magnetic  fields (Khalil 1987). In microwave and infrared frequencies, the electric field applies a force on charged particles. As a result they are impelled to migrate or rotate. Due to the movement of charged particles further polarization of polar particles may take place (Galema 1997). The electromagnetic spectrum includes several regions, that in order of increasing frequency and photon energy, consist of: radio waves, microwaves, infrared, visible light, ultraviolet, x-ray and gamma rays (Knutson et al. 1987; Copson 1975) (Figure 2.1).  2.1.1  Microwaves Microwaves are located between  the 300 M H z and 300 GHz bands in the  electromagnetic spectrum. They travel at the speed of light (186,282 miles per second or 3><10  8  meters per second in vacuum) and their wavelength varies between 1 mm and 1 m (Equation 2.1). A.=c/f  Eq(2.1)  where X = wavelength (m) c = velocity of electromagnetic energy (m/s) f = frequency (s ) _1  For example the wavelength at 2450 M H z is 12.24 cm (Meredith 1998). Only restricted microwave frequencies are allowed for heating purposes. Currently, in North America assigned frequencies for industrial, scientific and medical (ISM) applications are specified by the Federal Communications Commission (Knutson et al. 1987; Buffler 1993).  7  0.76 um  0.38 um  Visible light  Gamma rays  Wavelength  X-rays  0.01 n m  Infra-red rays  Ultra violet rays  1 nm  1 mm  1 um J  3x10  20  3x10  17  3x10  Frequency (Hz)  Figure 2.1. The electromagnetic spectrum  8  lm  100 m  _J  L_ 3x10  1 4  Radio waves  Microwaves  1 1  3xl0  l 8  L_ 3xl0  6  Those frequencies are: 915 ± 25 M H z , 2450 ± 50 M H z , 5800 ± 75 M H z , 22125 ± 125 M H z (Regier & Schubert 2001; Knutson et al. 1987; Khalil 1987). Other countries permit the use of these and/or additional frequencies. Household microwave ovens operate at 2450 MHz. In industrial food processing 2450 M H z is commonly used in Europe, while 915 M H z dominates in North America and 896 M H z is used in the U K (Ryynanen 2002). In addition 433.92 M H z in Austria, Liechtenstein, Portugal, Switzerland and Yugoslavia (Metaxas & Meredith 1983) and 2375 M H z in some other countries (Datta & Davidson 2000) are used for heating purposes.  2.2  M i c r o w a v e heating  Microwave heating is defined as the heating of a substance by electromagnetic energy (Buffler 1993). Heat is a secondary effect of an electromagnetic field interacting with matter. There are two mechanisms by which the microwave electric field is converted to heat within a material. The first, the ionic mechanism, comes from a linear acceleration of ions, usually from salts, within a non-metallic material. As the dissolved charged particles (ions) in a food or material, oscillate back and forth under the influence of the microwave field, they collide with their neighbour atoms or molecules. defined as heat (Buffler 1993).  These collisions impart agitation or motion, which is  The second mechanism is the molecular rotation of polar  molecules, primarily water as the major constituent of nearly all food products, as well as weaker interactions with carbohydrates, proteins and fats. Polar molecules try to align themselves to the rapidly changing direction of the electric field (Buffler 1993). This alignment requires energy that is taken from the electric field. When the field changes direction, the molecules relax, and  9  the energy previously absorbed is dissipated to the surroundings directly inside the food (Ohlsson 2000).  2.2.1 Factors involved in microwave heating Microwave heating is a complex phenomenon, which depends on several factors (Gunasekaran 1999; Prakash et al. 1997).  The combination of these factors, influence the  temperature development of material during microwave heating (Housova et al. 1996). Factors include: volume, density, shape and dimension of material (Ohlsson 2000; Dorantes-Alvarez et al. 2000; Housova et al. 1996; Ryynanen & Ohlssson 1996; Buffler 1993), packaging composition and geometry (Ohlsson 2000), specific heat capacity, thermal conductivity of heated material (Dorantes-Alvarez et al. 2000; Gunasekaran 1999; Buffler 1993), sample composition and dielectric properties (Ohlsson 2000; Dorantes-Alvarez et al. 2000; Gunasekaran 1999; Housova et al. 1996; Ryynanen & Ohlssson 1996; Buffler 1993), initial temperature of the material (Dorantes-Alvarez et al. 2000; Gunasekaran 1999; Housova et al. 1996; Buffler 1993) as well as process parameters, such as type of magnetron (Dorantes-Alvarez et al. 2000; Buffler 1993), microwave frequency, power supply (Ohlsson 2000; Dorantes-Alvarez et al. 2000; Housova et al. 1996; Buffler 1993), field intensity (Gunasekaran 1999), load in the oven (Dorantes-Alvarez et al. 2000; Buffler 1993), and process time (Dorantes-Alvarez et al. 2000; Housova et al. 1996; Buffler 1993).  2.2.1.1 Dielectric properties Dielectric properties of a material are a measure of the dielectric charge movement inside that material in response to an external electric field (Kuang & Nelson 1998). When a sample is  10  placed in the path of microwaves, it will absorb energy from the waves, depending upon its dielectric properties (Engelder & Buffler 1991). T hus, the dielectric properties describe how non-magnetic  materials  interact  with  electromagnetic  radiation.  Dielectric constant  (permittivity) is the ability of any material to absorb, transmit, and reflect energy from the electric portion of microwave fields (Engelder & Buffler 1991). In other word, the dielectric constant (e') shows the amount of energy absorbed by a specific material in a specific electric field while the loss factor (e") shows how much of this energy can be converted into heat. In addition, the loss tangent (tan 8= e"/e') defines the ability of a medium to convert electromagnetic energy into heat energy at a given frequency and temperature (Engelder & Buffler 1991; Galema 1997). Factors that affect the dielectric properties are the frequency of the electromagnetic waves, temperature, density, water content, salt content, percentage of solutes, and state (liquid, solid, or gas) of the material under examination (Yaghmaee & Durance 2001; Galema 1997). Magnetic properties of microwaves must be accounted for when magnetic materials such as ferrites or metals are under study (Buffler 1993). In food science, only electrical interaction is considered, for no foods magnetically interact with microwaves (Buffler 1993).  2.3  Microwave applications Microwaves were originally used for communication and radar (Coronel et al. 2003), but  nowadays m any o f m icrowave a pplications i nvolve t he u tilization o f h igher e nergy for d irect interior heating (Ohlsson 2000). Researchers in many fields have conducted studies on various applications of microwaves.  For example the use of microwave energy in chemistry for  heterogeneous esterification (Chemat et al. 1998), analytical chemistry, synthesis of radio-  11  pharmaceuticals, inorganic complexes, intercalation reactions, polymer curing (Galema 1997), and decomposition reactions (Michael et al. 1991) have been reported.  Other applications  include use of microwaves in formation of an aerated cheese product (Jeffry 2003), microwave assisted digestion of seafood for analytical purposes (Li et al. 2003), use of microwaves to shorten P C R total reaction time (Fermer et al. 2003), microwave lysis (Menon & Nagendra 2001), microwave hydrolysis (Marconi et al. 2000), use of microwaves for eukaryotic D N A isolation (Goodwin & Lee 1993), ceramic processing (Michael et al. 1991), moisture determination ( A O A C 16.239), removal of the feathers from poultry (Rosenberg & Bogl 1987b) and microwave rendering of fats (Decareau 1985). The use of microwave energy in food processing can be classified into six main groups: heating and re-heating (Coronel et al. 2003; Heddleson et al. 1996), baking, cooking and precooking (Regier & Schubert 2001; Knutson et al. 1987; Khalil 1987; Rosenberg & Bogl 1987a; Schiffmann 1986), tempering and thawing (Regier & Schubert 2001; Knutson et al. 1987; Edgar 1986) blanching (Dorantes-Alvarez et al. 2000; Knutson et al. 1987), dehydration (Kaensup et al. 2002; Ohlsson 2000; Mudgett 1989; Decareau 1985), pasteurization and sterilization (Regier & Schubert 2001; Knutson et al. 1987). Although microwave applications have a wide range of objectives, they are all established based on microwave heating properties and increase in temperature (Regier & Schubert 2001).  2.3.1  Microwave dehydration The main rational for application of microwaves to dehydration is the shortened process  time. I n t raditional a ir-drying m ethods p rocess time i s 1 imited b y 1 ow t hernial c onductivities (Regier & Schubert 2001; Fellows 2000; Garcia et al. 1988). Microwaves excite water and fat  12  molecules for some depth into the food. Moisture from the interior of the food can be expelled due to the increase in vapor pressure (Kaensup et al. 2002). Oxidation by atmospheric oxygen is minimized during microwave heating, since it is not necessary to heat large volumes of air (Fellows 2000). This can lead to rapid drying without overheating the atmosphere or creating surface d amage (Kaensup e t a 1. 2 002). In a ddition m ore h omogeneous drying, w ithout 1 arge moisture gradients, improves moisture transfer during the later stages of drying and eliminates case hardening (Regier & Schubert 2001; Fellows 2000; Knutson et al. 1987). At the same time, unwanted changes in sensory attributes and nutrient loss due to long drying times or high surface temperatures, can be prevented (Regier & Schubert 2001).  However the higher cost of  microwaves and smaller scale of operation, compared with traditional methods of dehydration, has restricted microwave drying as a sole source of energy in dehydration (Fellows 2000). In most cases, microwaves are used in combination with conventional hot air drying for dehydration in pilot and industry levels (Ohlsson 2000). One of the earliest examples was drying of pasta (Decareau 1985) and the production of dried onions (Regier & Schubert 2001; Metaxas & Meredith 1983). Later, drying of vegetables and cereal products (Ohlsson 2000), agar gel and Gelidium (Garcia & Bueno 1998), spices, tomato paste, wild rice, snack foods and bacon pieces (Kaensup et al. 2002; Mudgett 1989) and final drying of potato chips (Kaensup et al. 2002; Knutson et al. 1987; Decareau 1985) were also reported.  2.3.2  Vacuum microwave Microwave energy, by overcoming the low heat transfer rates of conduction, has led to  higher drying rates and less shrinkage in the final product. Unfortunately the higher drying rates cause the loss of aromas (Regier & Schubert 2001; Decareau 1985). On the other hand vacuum  13  drying systems are normally used for sensitive materials that would be damaged or decomposed at high temperatures. During vacuum drying, high-energy water molecules rapidly diffuse to the surface and evaporate into the vacuum atmosphere (Gunasekaran 1999). Since vacuum drying takes place by evaporation at reduced boiling points in a low-pressure chamber, the product may be dried at a lower temperature at reduced pressure than at atmospheric pressure. Moreover, removal of air due to vacuum, during dehydration, diminishes oxidation reactions (Gunasekaran 1999). Conversely conduction or radiant heating that are normally used for vacuum drying maintain low drying rates because the moisture front is retracted and thermal conduction is slower (Kaensup et al. 2002; Gunasekaran 1999). The use of vacuum along with microwaves has proved a good combination in production of high quality materials. While microwaves provide the fastest means available of transferring energy into the interior of biological solids (Durance & Wang, 2002), the reduced pressure keeps the product temperatures low, as long as a certain amount of free water is present. Therefore temperature sensitive substances like vitamins, colours, volatiles and flavours will be retained (Regier & Schubert 2001; Decareau 1 985). K im and colleagues (2000) reported retention o f chicoric acid and caftaric acid i n Echinacea purpurea flowers dried with vacuum microwave dryer. K i m and colleagues (1997) dried concentrated yogurt in a laboratory scale microwave vacuum dryer (10 mm Hg, 250 W, 2450 MHz) at 35°C and reported a substantial retention of lactic acid bacteria (S. thermophilus and L. bulgaricus). Decareau (1985) reported that retention of vitamin C and volatile compounds in orange juice powder was higher after vacuum microwave drying compared to other drying processes (Knutson et al. 1987). Sobiech (1980) also reported that microwave vacuum drying enhanced the flavour of dried sliced parsley root and retained the properties of fresh raw material.  14  Vacuum microwave drying has been used in dehydration of a wide range of products, starting from fruits and vegetables such as: banana slices (Mousa & Farid 2002, Mui et al. 2002); tomato slices (Durance & Wang 2002); chilli (Kaensup et al. 2002), parsley (Bohm et al. 2002), sweet basil (Yousif et a l . 1 999), carrot slices ( L i n e t a l . 1 998), potato chips (Durance & L i u 1996), cranberry (Yongsawatdiguul & Gunasekaran, 1996), and sliced parsley root (Sobiech 1980), to fruit juice (Regier & Schubert 2 001) and tea powder (Schiffmann 1 995) as well as grains (Decareau 1985), enzymes (Schiffmann 1995), pectin gel (Drouzas et al. 1999) and shrimp (Lin etal. 1999).  2.3.3 Microwave pasteurization & sterilization The fast and effective heating with microwaves, short process times and the relatively low thermal exposure of the food material r esulting in less changes in physical and chemical properties of the product, along with destruction effect of microwave on microorganisms, has made microwave radiation a promising candidate for pasteurization and sterilization purposes. Researchers have intensively studied the possibility of using microwaves in pasteurization and sterilization (Regier & Schubert 2001). Some of the studies have focused on prolonging product shelf life. Cunningham (1980) studied the effect of microwave radiation (915 M H z ) on total microbial counts of fresh cut chicken and suggested that minimal microwave radiation might be used to extend the refrigerated shelf life of fresh poultry. Wu and Gao (1996) reported that moon cake, breads and spring rolls treated at 850W of 2450 M H z microwaves had a 30 day shelf life, that was significantly longer than the 3-4 days of untreated samples. Herve and colleagues (1998) studied the effect of microwave treatment (2450 and 915 M H z ) on inactivating surface spoilage  15  microorganisms in cottage cheese and suggested that microwave treatment decreased the number of psychrotrophs and would increase the shelf life of the cheese. Ohlsson (2000) pasteurized ready-made foods by microwave heating to 75°C to 80°C and prolonged their shelf life to approximately three to four weeks Other scientists and researchers have focused on the calculation of process times or have checked the sterility of the contaminated product. Yang and co-workers (1947) reported that after pasteurizing wine at 140°F for 4 seconds with 26 to 34 M H z microwaves, no detectable microorganisms were found. Douglas and colleagues (1990) studied the effect of microwaves on sterilization of urinary catheters at home. Catheters were incubated for sixty minutes at 37°C in a phosphate buffer suspension containing sixteen species of microorganisms (10 -10 cells/ml), 4  6  isolated from patients with urinary tract infections including two strains of E. coli, Klebsiella sp., Pseudomonas sp., Proteus sp., Enterobacter sp., Streptococcus sp., Staphylococcus sp. and Candida sp. Catheters were removed from suspension and placed in a sterile plastic bag. They reported no live bacteria or yeast after 12 minutes exposure to microwaves (2450 M H Z , 65 0W). They concluded that microwave sterilization is a practical, efficient and cost-effective method of home catheter sterilization.  Kudra and co-workers described a simple laboratory scale  microwave s ystem f or t he c ontinuous h eating o f m ilk, u sing 7 00W, 2 450 M Hz ( Kudra e t a l . 1991). Diaz-Cino & Martinelli (1991) studied the effect of microwave (700W, 2450 MHz) on Aspergillus nidulans, Escherichia coli, Bacillus subtilis and Bacteriophage T4. They reported complete sterilization at 85°C, 30 min for all tested microorganisms except for B. subtilis. They concluded that the microwave method was faster, but while it can be used for pasteurization, is not suitable for sterilization process, since viable spores remained (Diaz-Cino & Martinelli 1991).  Odani and colleagues (1995) performed a study on microwave pasteurization and  16  reported that bacteria in frozen shrimp (4.1xl0 CFU/g), refrigerated thick custard (1.2><10 3  2  CFU/g) and frozen pilaf (1.2x10/ CFU/g) were killed by microwave radiation after 40 seconds, 60 seconds and 4 minutes, respectively.  They also exposed cultures of E. coli (4.2xl0  3  CFU/ml), S. aureus (7.0xl0 CFU/ml) and B. cereus (1.6xl0 CFU/ml) diluted in saline to 3  5  microwave and reported a pasteurization time of 30 seconds at 50°C for E. coli and S. aureus cultures and 90 seconds at 100°C for B. cereus. They observed that spores of B. cereus survived even after 30 minutes at 100°C. Lau and Tang (2002) pasteurized pickled asparagus using 915 M H z microwave and reported better heating uniformity, shorter process time and marked reduction in thermal degradation of asparagus compared to the conventional method. Hiti and colleagues (2001) used microwave (2450 M H z , 600W) to sterilize contact lens cases inoculated with Acanthamoeba {A. comandoni, A. castellanii, A. hatchetti) and their cysts and stated that Trophozoites as well as cysts, were effectively killed by microwave treatment in 3 minutes, regardless of the type of lens case used. Guan and colleagues (2003) conducted a study on macaroni and cheese inoculated with Clostridium sporogenes (PA 3679) spores packed in trays flushed with nitrogen, heat sealed and sterilized in a 915 M H z Microwave-Circulated Water Combination (MCWC) system. They reported that microbial destruction by M C W C system matched with calculated F values of sterilization. 0  Microwave pasteurization and sterilization of many other products have been described. For e xample m icrowave p asteurization o f r eady m ade f ood a nd p acked f ood (Ohlsson 2 000), shell eggs (Sullivan & Padua 1999), fruit juices (Copson 1975), apple juice, apple cider, pineapple juice (Kozempel et al. 1998), raw cow's milk and goat's milk (Mann 1997; Villamiel et al. 1996), yogurt and pouch-packed meals (Regier & Schubert 2001; Decareau 1985), pasta meals, soft bakery goods and peeled potatoes, fruits in syrup (Fellows 2000), baby foods,  17  puddings, custard, sauces, soups, pharmaceuticals and gelatines (Armfield 2001) have been reported.  In addition microwave sterilization of reusable pharmaceutical glass vials, tissue  culture plates,  culture media, contact  lenses,  dental  instruments,  baby bottles,  and  decontamination of clinical specimens containing bacterial pathogens has also been studied (Douglas et al. 1990).  2.3.3.1 Microwave pasteurization and sterilization systems Although using microwaves in pasteurization and sterilization has been investigated for many years, introduction on a commercial level has only happened in the past few years (Ohlsson 2000). For both processes it is very important to be able to properly control heating uniformity within the product to ensure microbial destruction and the microbiological safety of the processed foods (Guan et al. 2003; Regier & Schubert 2001; Ohlsson 2000; Prakash et al. 1997). The existence of hot and cold spots in microwave ovens due to uneven microwave distribution (Tassinari & Landgraf 1997; Sieber et al. 1996; Sigman-Grant et al. 1992; Knutson et al. 1987), is the main reason that up to now microwave pasteurization and sterilization has been mostly utilized for batch sterilization operations (Regier & Schubert 2001). In general, techniques that have been used to improve heat uniformity include rotating, oscillating and moving of samples, using cooling medium immediately after or simultaneous with microwave exposure, surrounding samples with a medium of higher dielectric constant, applying microwave in cycles (Datta & Davidson 2000), or applying microwaves along with high pressure. Researchers and scientists have used either one or a mixture of these techniques to design their pasteurizer or sterilizer systems.  18  Some of the most common approaches to achieve uniform heating are: microwave pasteurizers with conveyor belt systems (Knutson et al. 1987); microwave sterilizers with conveyor belt and heating chamber with sliding door (Kumeta 1997); inserting a stainless steel cooling tube with cold water flow inside the plastic process tube within the microwave to maintain the temperature of the liquid (Kozempel et al. 1998); conveyor tunnel with a combination of microwaves and hot air (Fellows 2000); continuous hydrostatic microwave sterilizer for laminated microwave transparent pouches made from polypropylene / E V O H or PVDC/polypropylene while they are submerged in a medium with higher dielectric constant than the product (Fellows 2000); pressurized HTST sterilization system with water immersion technique (Tang et al. 2001; Ohlsson 1991); continuous fluid pasteurization and sterilization systems with tubes intersecting waveguides or small resonators, in such a way that heating is accomplished across the tube cross section (Regier & Schubert 2001; Ohlsson 2000; Decareau 1985); microwave heaters with sliding doors and special compression and decompression systems (Regier & Schubert 2001); UHT/HTST microwave pasteurizer with water cooling (Armfield Limited, Ringwood, England 2001) and Microwave-Circulated Water Combination (MCWC) system, consisting of microwave generator, pressurized microwave heating vessel and a water circulation heating and cooling system (Guan et al. 2003). Continuous microwave pasteurization systems are commercially used in Belgium (Tops Foods, Belgium), Japan (Otsuka Chemical Co., Osaka, Japan), and England (Armfield Limited, Ringwood, England) but to the knowledge of the author do not exist in North America (Tang et al. 2001; Ohlsson 2000).  19  2.4  Effect of microwaves on microorganisms There have been conflicting reports in the literature regarding the effects of microwaves  on microorganisms (Vasavada 1986) along with an ongoing debate for over fifty years on the existence of effects other than heat associated with electromagnetic energy (Kozempel et al. 2000).  Since the mid 1920s, numerous studies have been carried out at various microwave  frequencies in an attempt to solve the debate (Mertens & Knorr 1992).  Some researchers  attribute the destruction of microorganisms subjected to microwave energy solely to thermal effects, whereas others have indicated injury to cells regardless of temperature (Datta & Davidson 2000; Vasavada 1986; Dreyfuss & Chipley 1980). Those researchers believing in a microwave effect either have reported greater lethality with microwave treatment than conventional heating (Tajchakavit & Ramaswamy 1995; Khalil & Villota 1988) or have found smaller D-values for the microwave-treated cells compared to conventional destruction method (Tajchavit et al. 1998) or showed evidence of characteristic biological changes such as changes in metabolic function of the microorganism under study (Woo et al. 2000; Dreyfuss & Chipley 1980). Olsen (1965) treated loaves of bread, inoculated with cultures of Aspergillus niger, Penicillium sp. and Rhizopus nigricans, with microwave radiation (5 kW, 2450 M H z ) and reported that the numbers of viable spores were greatly reduced. He concluded that since the destruction happened  at a temperature lower than the thermal death point of these  microorganisms, the results of microwave treatment were probably not due to conventional thermal kill. Culkin & Fung (1975) studied the pattern of E. coli and Salmonella typhimurium destruction with microwave (915 M H z ) in cooked soups while measuring temperatures with temperature sensitive strips.  They found that although the top portion of the soup was the  20  coolest region, it showed the greater decrease in microbial survival for any given exposure time. They stated that if the lethal action of microwaves on microorganisms were solely due to the heat generated by the w aves, then samples from the w armest region of the soups would show the lowest survival values. Dreyfuss & Chip ley (1980) studied the effect of sublethal microwave radiation (2450 MHz) on enzymatic activity of Staphylococcus aureus compared to conventional heat treatment for 10, 20, 30 and 40 seconds, while internal temperatures of microwave treated flasks did not exceed 46°C. They reported some changes in physicochemical characteristics in microwave-treated cells, such as higher activity of malate dehydrogenase, dehydrogenase, cytochrome oxidase, and cytoplasmic ATPase.  a-ketoglutarate  They also observed that the  activity of glucose-6-phosphate dehydrogenase was decreased by microwave radiation but increased by conventional heat treatment.  In general they concluded that the effect of  microwave radiation on the metabolic activity of S. aureus can not be explained by thermal effects alone. Khalil & Villota (1988) also studied the effect of microwave radiation on S. aureus metabolic function and reported that microwave-treated cells regained their ability to produce enterotoxin A at a slower rate, and did not reach the amount produced by untreated cells after 72 hours of recovery, while conventionally treated cells regained production levels almost identical to the unheated cells.  They concluded that microwaves have intrinsic injurious effects on  biological s ystems, o ther t han t hose b rought o n b y heat (Khalil & V illota, 1988). R eznik & Knipper (1994) studied the microwave pasteurization of liquid egg and found a higher degree of microbial kill with microwaves compared to a conventional pasteurizer. They also reported less re-growth of bacteria even when the egg was maintained at room temperature (Kozempel et al. 2000). Woo and colleagues (2000) performed a study on the effect of microwave radiation on  21  E. c oli a nd B. s ubtilis c ells a nd r eported a h igher a mount o f n ucleic a cid 1 eakage, r ough a nd swollen cell surfaces, along with the presence of dark spots in the cytoplasm of microwave treated cells. Odani and colleagues (1995) investigated the presence of protein in the cell-free supernatant of E. coli cells exposed to microwave radiation for 0-12 seconds at 15°C. The result of acrylamide gel electrophoresis with silver staining showed release of proteins from microwave treated cells compared to untreated samples. They suggested that the mechanisms of killing of bacteria depends not only on temperature, but also on other effects of microwave irradiation. Other authors reporting results which appeared to indicate nonthermal effects are Papadopoulou et al. (1995), Rosaspina et al. (1994), Galuska et al. (1988) and Webb & Dodds (1968). At the same time there are several scientists who reported that there is no non-thermal effect associated with microwaves (Fujikawa et al. 1992). This group have found no difference between microwave and conventional heat destruction or reported no destruction with microwaves at lower temperature. They argued that reported nonthermal effects are due to the lack of precise measurements of the time-temperature history (Datta & Davidson 2000). Goldblith & Wang (1967) heated suspensions of E. coli and Bacillus subtilis with microwave (2450MHz) and conventional heating.  They reported an identical degree of  deactivation for both treatments. Hamrick & Butler (1973) exposed E. coli and Pseudomonas aeruginosa cultures to microwave radiation (2450 M H z , 60mW/cm2) and maintained the temperature at 37°C for 12 hours.  They plotted the growth curve of microorganisms and  detected no deviation of cell replication rate. Vela & Wu (1979) studied the effect of 2450 M H z microwaves on various bacteria, Actinomycetes, fungi, and bacteriophages in the presence and  22  absence of water. They reported that dry or lyophilized organisms were not affected even by extended exposure time. The authors stated that microorganisms were killed by a thermal effect (Dreyfuss & Chipley 1980; Vela & Wu 1979). Welt and colleagues (1994) found no difference between conventional and microwave inactivation of Clostridium sporogenes PA3679 spores at 90, 100 and 110°C.  In their study they continuously cooled suspensions and reported no  detectable inactivation (Datta & Davidson 2000). Yeo and colleagues (1999) studied the effect of microwave radiation (2450MHz, 800W) on Staphylococcus aureus on stainless steel discs arid reported that destruction of microorganisms was mainly due to heat transfer from the stainless steel substrate and very little direct energy was absorbed from the microwaves. A third group of researchers has suggested that electromagnetic energy acts in a way to magnify the thermal effect (Kozempel et al. 2000; Brunkhorst et al. 2000; Reznik & Knipper 1994; Ramaswamy & Tajchakavit 1993).  Mittenzwey and co-workers (1996) reported that  extremely low-frequency magnetic fields (2-50 Hz, 1-10 mT) act on Photobacterium phosphoreum only as a co-stressing factor which activates processes or reactions already initiated by other stresses. Tajchakavit and co-workers (1998) reported rapid destruction of S. cervisiae and L. plantarum at temperatures of 60-65°C, which were 10-15°C lower than those used for thermal destruction. Their data for S. cervisiae showed a D-value of 25.1 s for thermal destruction and 2.08 s for microwave destruction at an intermediate temperature of 55°C. For L. plantarum, at an intermediate temperature of 60°C, the D-values were 21.9 and 3.83 s, under thermal and microwave h eating modes respectively. They c oncluded that microwave heating was more efficient than conventional heating, and indicated the possibility of some non-thermal or enhanced thermal effects associated with microwave heating.  Kozempel and co-workers  (1998) studied microwave destruction of Pediococcus sp. in water, 10% glucose solution, apple  23  juice, tomato juice and apple cider and beer using a continuous microwave system with water flow as a cooling medium to keep the temperature of the hot spot at 40°C. Bacterial counts were more readily reduced in water, glucose solution and apple juice than in apple cider, tomato or pineapple juice, and none were killed in skim milk.  They suggested that microorganisms  become more susceptible to stresses like acidic pH when the temperature goes up. Kozempel and co-workers (2000) described a continuous steady state microwave process (7kW, 2450 MHz) while simultaneously removing the thermal energy to maintain low temperature. They testedE. coli, Pediococcus sp., Listeria innocua, Enterobacter aerogenes and yeast i n water, beer, whole egg, egg white, apple juice and tomato juice in the temperature range of 26-45°C for 2-9.7 minutes. Microwave energy in the absence of other stresses such as heat, pH or antimicrobial did not destroy microorganisms at low temperature.  The authors added that it is  possible that microwave energy may complement or magnify thermal effects. Khalil & Villota (1988) indicated that added lethality of microwaves lies in their ability to distribute the thermal energy instantaneously to the heat sensitive subcelluar components.  Accordingly, higher  amounts of thermal energy would be generated within the cellular suspension, thus more heatinduced injury to the cellular components would occur. Mittenzwey and colleagues (1996) studied the effect of extremely low-frequency electromagnetic fields (magnetic strength 1 to 8 mT and frequency of 2-50 Hz) on different Escherichia coli, Proteus vulgaris, Photobacterium phosphoreum, Photobacterium fisheri at temperatures ranging from 25 to 37°C. They reported that extremely low-frequency electromagnetic fields might act on bacteria as a co-stressing factor by activating a process or reaction already initiated by other stresses like heat.  24  2.4.1  Microwave kinetic parameters The lethal effect of heat on bacteria is a function of time, temperature, bacterial  population and bacterial thermal resistance. The D-value, the decimal reduction time, is a means of characterizing the death rate or lethal effect of a temperature at different times. It is the length of time necessary to produce a "one-log" reduction in microbial population at a specific temperature.  The z is a measure of the sensitivity of an organism to changing lethal  temperatures.  Calculating microbial destruction for a microwave heating process is more  complicated than for a conventional thermal process, because of the difficulty in keeping precise constant temperatures inside the microwave oven (Heddleson & Doores 1994). Fujikawa and colleagues (1992) exposed E. coli culture to microwaves in a container placed on a rotary plate inside a microwave oven. They found that the profile of destruction of bacteria by microwave radiation was approximated by a set of three linear relationships and was difficult to understand. Fujikawa & Ohta (1994) also reported that the survival curves of E. coli and Staphylococcus aureus exposed to microwave radiation at 200, 300 and 500 W approximated a set of three linear phases. The destruction profile of Bacillus cereus spores in saline showed two linear phases at 200 W and was approximated by a single linear function at 300 or 500 W. When Kakita and colleagues (1995) plotted the relative survival populations of bacteriophage P L-1 as a f unction o f t ime o n a s emi-logarithmic g raph, t hey r eported t hat t he bacteriophage were inactivated by microwave irradiation according to almost first-order kinetics with some lag period at the beginning. Odani and colleagues (1995) reported two linear survival curve for E. coli, S. aureus and B. cereus in saline after microwave irradiation. Tajchakavit and co-workers (1998) studied the destruction kinetics of Saccharomyces cervisiae and Lactobacillus plantarum in apple juice and reported that the inactivation profiles in continuous microwave  25  system followed a first-order kinetic model. They also reported D52.5 of 4.8 s, D 5 5 of 2.1 s and D57.5  of 1.1 s for S. cervisiae and D . of 14 s, D 6 0 of 3.8 s, and 57  5  D 2.5 6  of 0.79 s for L. plantarum  with corresponding z-values of 7 and 4.5°C. D-values under conventional heating were 58, 25 and 10 s at 50, 55 and 60°C for S cervisiae and 52, 22 and 8,4 s at 55, 60 and 70°C for L. plantarum with corresponding z-values of 13.4 and 15.9°C. Rosenberg & Sinell (1990) studied the effect of microwave (2450 MHz), on Staphylococcus aureus, Salmonella typhimurium and E.  coli and reported D55 of 11.6, 2.3 and 2.9 min respectively while in water bath treated cells D55 was 17.8, 2.4 and 3.0 min. They also reported corresponding z-values of 11.6, 4.7 and 24.4 °C for Staphylococcus aureus, Salmonella typhimurium and E. coli treated with microwave while  reported z for water bath treated cells was 6.5, 4.6 and 13.6 °C respectively.  2.4.2  Mechanism of thermal destruction Many factors affect the heat resistance of an organism, including type of organism,  inherent resistance (the differences among species and strains within the same species, spores and vegetative cells), number of cells, age of cells, stage of growth, growth condition (growth temperature, growth medium), and environmental condition during the time of heating (pH, water activity, type of medium, salts and other organic and inorganic compounds). The preservative effect of heat processing is due to the irreversible heat denaturation of proteins, nucleic acids, enzymes or other vital components of microorganisms (Datta & Davidson 2000; Fellows 2000; Heddleson & Doores 1994). Denaturation stops enzyme activity and as a result metabolic functions related to that specific enzyme will be stopped and cell death will occur (Fellows 2000). Some of the enzymatic activities reported to be affected by heat are:  26  glucose phosphate isomerase, fructose diphosphate aldolase and lactose dehydrogenase (Khalil & Villota, 1988; Bluhm & Ordal 1969). Another reported cellular effect of heat is perturbation of the integrity of D N A including D N A damage and loss of negative superhelicity (Champomier-Verges et al. 2002; Delaney 1989). In addition, membrane damage or disruption of membranes has been observed as a result of thermal treatment (Champomier-Verges et al. 2002; Datta & Davidson 2000; Heddleson & Doores 1994; Khalil & Villota, 1988). Metabolites and cofactors crucial to cellular function may leak through damaged membrane and cause cellular death (Heddleson & Doores 1994). The presence of intra cellular compounds such as ninhydrin positive material, purines, pyrimidines and ribonucleotides in the medium indicated damage to the cell at the membrane level (Khalil & Villota, 1988).  2.4.3  Mechanism of microwave destruction Several theories  have been advanced to explain how microwave energy  kills  microorganisms (Brunkhorst et al. 2000) such as: breakage of hydrogen bonds and secondary linkages (Kalant 1959), release of bound water (Ballario et al. 1975), electron tunneling (Cope 1976), pearl chain formation (Khalil & Villota, 1988; Lambert 1980), particle orientation and molecular resonance (Lambert 1980), change in the charged nucleus surface (Shckorbatov et al. 1998), interference with cell signaling pathways (de Pomerai et al. 2000), and changes in secondary and tertiary structure of proteins (Banik et al. 2003).  In general there are four  predominant theories with supporting evidence that have been focused on in literature: electroporation, dielectric cell membrane rupture, magnetic field coupling, and selective heating (Kozempel etal. 1998).  27  The electroporation theory states that the electrical potential across the c ell membrane causes pore formation in the weakened membrane of the microorganisms, resulting in leakage of cellular material and cell lysis (Datta & Davidson 2000; Kozempel et al. 1998). Rosaspina and co-workers (1994) examined microwave treated Mycobacterium bovis and reported a series of progressive changes in bacterial morphology. These changes consisted of formation of pits, which penetrated deeply into the bacterial cell until they passed through the entire width. Liquefaction appeared to occur, so that individual cells could no longer be distinguished. They added that these phenomena increased progressively with increasing exposure time until nearly total disintegration of the cells was achieved and the remaining fragments appeared to be a shadow of the destroyed cellular body. With the application of dry or moist heat, the changes were less extensive and complete cellular disintegration was never observed (Rosaspina et al. 1994). Woo and colleagues (2000) studied destruction of E. coli and B. subtilis exposed to microwave radiation (2450 MHz). They reported that most of the microwave treated cells were ghost cells from which intracellular materials were released into the cell suspension.  At the  same time they did not find any decrease in cell optical density at 600 nm in spite of a significant reduction in the viable count. Therefore they suggested that this might be due to the fact that microwave-treated cells were not completely lysed.  They also found that the surface of  microwave treated E. coli cells were damaged and had become rough and swollen, while no damage to surface structure was observed for B. subtilis. Considering that both microorganisms were inactivated by microwave irradiation, they suggested that the damage to the surface structure of microorganisms might not be the main reason for inactivation by microwave heating. In the dielectric cell-membrane rupture theory, an external electric field is thought to induce an additional trans-membrane electric potential, which is larger than the normal potential  28  of the cell. This drop of voltage across the cell membrane may be sufficient for membrane rupture (Datta & Davidson 2000; Kozempel et al. 1998; Zimmermann et al. 1974). It also may result in pore formation, increased permeability, and lost of cell integrity (Brunkhorst et al. 2000, Kozempel et al. 2000). Ke and co-workers (1978) reported positive correlation of peroxide value in fresh mackerel fillets with the length of exposure to microwave energy at 2450 M H z and suggested that the energy from microwaves might disrupt the membrane and/or subcellular structure, thus releasing the lipids. In the magnetic field coupling theory, cell lysis was explained by a coupling of the electromagnetic energy with critical molecules within the cells, such as protein or D N A . Disrupting the internal components of the cells may cause them to die (Kozempel et al. 1998). Mertens and Knorr (1992) suggested that the oscillating magnetic field couples energy into the magneto-active parts of large biological molecules with several oscillations. When a large number of magnetic dipoles are present in one molecule, enough energy can be transferred to the molecule to break a covalent bond. Therefore certain critical molecules in a microorganism, like D N A , or proteins, could be broken, hence death of microorganisms or at least reproductive inactivation will occur (Brunkhorst et al. 2000; Kozempel et al. 2000).  On the other hand  Heddleson & Doores (1994) reported that the quantum energy of microwave is 1.2xlO* eV, 5  whereas the energy needed to break hydrogen bonds is 5.2 eV, thus microwaves are unable to break the hydrogen bonds.  Woo and colleagues (2000) observed several dark spots in the  cytoplasm of microwave treated cells, examined by scanning electron microscopy, while no dark spots were observed in the untreated cells.  They suggested that the dark spots could be  aggregated proteins caused by microwave heating. Kakita and co-workers (1995) conducted a study on the effect of microwave radiation on the survival of bacteriophage PL-1. When phage  29  particles were exposed to microwave radiation (2450 M H z , 500W) for 60 s with the maximum temperature of 75°C, D N A molecules within the phage particles were randomly broken into small fragments, whereas D N A treated with conventional heating (70°C or 80°C for 75 s) or untreated phages remained intact. In the fourth proposed theory, the selective heating theory, the microorganisms are thought to selectively absorb the electromagnetic energy. The solid microorganisms are thought to heat faster than the surrounding fluid and reach lethal temperatures while the surrounding fluid remains below lethal temperatures (Kozempel et al. 1998). Nelson and Charity (1972) conducted a study on energy absorption of winter wheat Triticum aestivum and adults of the rice weevil Sitophilus oryzae and found the degree o f selective heating depends upon the relative values of the dielectric properties and the loss factor between insects and grain. They observed a better selective heating of insect at the frequency of 40 M H z than at 2450 M H z (Kozempel et al. 2000; Nelson & Charity 1972).  Wang and colleagues (2003) also studied the effect of  microwave radiation (27 and 915 M H z ) on in-shell walnuts and gellan gel as a model for coldling moth larvae and reported 1.4 to 1.7 times greater heating of insects than walnuts at 27 M H z while no detectable preferential heating was observed at 915 M H z .  2.4.4  Injured microorganisms Microorganisms may be injured by sublethal environmental stresses such as heat,  freezing, ionizing or non-ionizing radiation (Kang & Siragusa 1 999; Aktas & Ozilgen 1 992). The injured bacteria may escape detection by common food microbiology techniques as used by the food industry and regulatory agencies (Vasavada 1986). Injured or stressed microorganisms are characterized by their inability to form colonies and multiply in a medium that contains a  30  selective agent, which has no inhibitory effect on unstressed cells (Kang & Siragusa 1999). The differential in counts between selective and nonselective media is a means to determine the sublethally injured population (Kang & Siragusa 1999). The occurrence of injured or stressed organisms in thermally processed food is a matter of concern in the food industry (Vasavada 1986). Following heat treatment, sub-lethally injured food-borne pathogens could be assumed to be dead while they are alive and potentially as dangerous as their uninjured counterparts (Kang & Siragusa 1999). The stressed organisms can undergo repair and produce toxins thus cause public health hazard (Vasavada 1986). Although the existence and the extent of injury in bacteria resulting from microwave irradiation have been stressed by some researchers, very little information is available on injured organisms with respect to microwave application (Vasavada 1986). Khalil & Villota (1988) studied effects of microwave radiation at 50°C for 6 hours on destruction and injury of S. aureus in phosphate buffer compared to conventional heat treatment and reported greater injury in microwave-treated cells. They also observed that the stationary lag phase, which often indicates a repair and adaptation period, was approximately twice as long for the microwave-injured cells compared to conventionally injured cells (Khalil & Villota 1988).  S. aureus injured by  microwave treatment often displays minimal metabolic capacity and an inhibition of enterotoxin synthesis (Khalil & Villota 1988; Bluhm & Ordal 1969). Aktas & Ozilgen (1992) studied injury and death of E. coli by microwaves in a tubular pasteurization flow reactor and reported that generally 15 to 25% of the surviving microorganisms were injured, but this ratio increased drastically near total sterilization conditions.  Shin & Pyun (1997) exposed suspensions of  Lactobacillus plantarum cells to conventional heating, continuous microwave or pulsed microwave irradiation at 50°C for 30 minutes. They reported a higher injury in cells treated with  31  pulsed microwave irradiation followed by continuous microwave and conventional heating. They also observed that lactic acid production in injured cells was restored during recovery and acid production at a detectable level for conventionally and both microwave treatment was started after 10 and 20 hours respectively.  2.5  Biological effects of microwaves Although the concern about the biological effects of non-ionizing radiation on humans  and other eukaryotes began years ago, very little information about the effect of microwaves at frequencies of 2450 and 915 M H z is available. Most of the studies have focused either on very low electromagnetic frequencies (de Pomerai et al. 2000; Mittenzwey et al. 1996) or very high frequency range (Banik et al. 2003; Pakhomov et al. 1998; Lambert 1980). Thus these results can not be applied to the whole electromagnetic spectrum. Evidence indicates that alternating electromagnetic fields interfere with the functioning of D N A and R N A , stimulate the activity in certain biochemical systems linked with cancer growth, affect molecules that are essential for the functioning of the nervous system and may disturb the normal function of the cell membrane (Mertens & Knorr 1992). The human body begins to significantly absorb electromagnetic radiation when the frequency exceeds about 15 M H z and the absorption varies for different body parts (Banik et al. 2003). A number of studies indicated that microwaves could affect the fine chromosome structure and function of cells, cell tolerance to standard mutagens, and lesion repairs (Banik et al. 2003). In the 1960s and 70s researchers showed that protein, R N A and D N A could absorb microwaves at the frequency o f 6 5-75 G H z , and that microwaves were able to interfere with  32  repair mechanisms or even to induce gene mutation in bacteria (Banik et al. 2003; Pakhomov et al. 1998). Lambert (1980) comprehensively reviewed the studies on the biological effects of microwaves for the period of 1940-1980 (Knutson et al. 1987). In his review, he stated that the result of various studies, on blood cells, macromolecules, organs and organ systems, bone marrow, cell membrane, testes and blood forming systems, showed that microwave radiation at higher frequencies and higher power densities, caused biological responses that were adverse to living organisms. For example at frequency of 10000 M H z , skin heats with sensation of warmth. Lens of the eye and testicles are susceptible at frequencies between 3300 to 10000 M H z . Frequencies of 150 to 1200 M H z could damage the internal organs by overheating. The body is transparent for frequencies less than 150 MHz, which have a wavelength over 200cm (Lambert 1980). Smialowicz and co-workers (1980) showed that exposure of male albino rats, injected with bacterial endotoxin, to continuous-wave microwave radiation (2450 MHz) was associated with significant elevation of body temperature directly related to the power density (10 mW/cm > 5mW/cm > 1 mW/cm ). 2  2  2  Some researchers studied the effects of microwave exposure over time on people who are in daily contact with microwave radiation such as welders, television and radio transmitter technicians, particle accelerator workers and steel factory workers engaged in tempering steel. They reported a predominance of fatigue in some of the exposed groups as well as a reduction in alertness (Baranski & Czerski 1976). But to the knowledge of the author there have been no confirmed cases of people being seriously injured from exposure to microwaves. Yao (1978) reported that the exposure of corneal epithelium of Chinese hamsters to microwaves (2450 M H z , 100 mW/cm ) produced an abnormal configuration in the animal's 2  33  chromosomes.  Liburdy and his group (1985) found that exposure of rabbit erythrocytes to  microwaves (2450 M H z , 100 mW/g) increased sodium passive transport only at membrane phase transition. In animals and humans, local microwave exposure stimulated tissue repair and regeneration, alleviated stress reactions and facilitated recovery in a wide range of diseases such as gastric, duodenal ulcers, tuberculosis, cardiovascular and skin diseases (Banik et al. 2003; Pakhomov et al. 1998). Ortner and colleagues (1983) reported that continuous exposure to 2450 M H z microwave radiation had no effect on microtubular polymerization or depolymerization, or on the secondary structure of purified tubulin in vitro. Galvin and colleagues (1984) exposed the whole body of pregnant mice to 2450 M H z microwave radiation at a power density of 30 mW/cm for two, four hour periods per day in total 2  for 6 or 15 days.  They found no effect on peripheral blood morphology (no change in  lymphocytes, neutrophils or monocytes number). Trosic and co-workers (1999) exposed male Wistar rats (13 week old) to 2450 M H z microwave at 5-15 mW/cm , 2 hours per day, maximum 5 days a week for the period of 1,8,16 and 30 days. The result of peripheral blood cell response showed a decreasing tendency in total leukocyte count as well as lymphocyte percentage in the treated rats. They also reported an increase in the percentage of granulocytes while the absolute erythrocyte count was increased over the first eight days, and kept falling afterwards, yet still remained within the physiological range.  2.6  Escherichia  coli  E. coli is the abbreviated name for the bacterium Escherichia (Genus) coli (Species) (Adams & Moss 2000) a member of Enterobacteriaceae family. The name Escherichia comes from the name of Theodor Escherich, who in 1885 isolated and characterized this bacterium for  34  the first time.  This enteric bacterium is gram negative, non-spore forming, rod shaped and  facultative anaerobe, which is an almost universal inhabitant of the lower intestinal tract of humans, warm blooded animals, and birds (Adams & Moss 2000; Neidhardt 1987). E. coli is a typical mesophile and its optimum growth temperature is around 37-39°C, with the maximum limit of 48-50°C and the minimum border of 7-10°C (Madigan et al. 2003; Adams & Moss 2000). A near-neutral pH is optimal for their growth but they also can grow at pH level as low as 4.4 (Adams & Moss 2000). This bacterium can grow in media with glucose as a sole source of energy, and carbon and ammonium salt as sole source of nitrogen (Magasanik 2000) and metabolically can transform glucose into all the necessary macromolecular  components  (Madigan et al. 2003). E. coli is a catalase-positive, oxidase negative, fermentative bacterium (Xdams & Moss 2000). Physiologically, E. coli is flexible and can adapt to the characteristic of its environment (Bell & Kyriakides 1998). It inhabits in the lower gut of animals and survives when released to the natural environment, allowing widespread distribution to new hosts (Bell & Kyriakides 1998; Blattner et al. 1997).  It is well known that pathogenic E. coli strains are responsible for  infections of the enteric, urinary, pulmonary, and nervous systems (Madigan et al. 2003; Blattner et al. 1997). Initially E. coli were used as indicators of direct or indirect fecal contamination and possible presence of enteric pathogens in food. Their presence in heat-processed food causes great concern and is a sign of incomplete processing or post-process contamination. E. coli can respond to environmental signals such as chemicals, pH, osmolality (Ramaswamy et al. 2003), heat (Ramaswamy et al. 2003; Arsene et al. 2000; Delaney 1989), acetate and propionate (Polen et al. 2002), peroxides and superoxides (Lindquist 1992), ethanol, ultraviolet light, nalidixic acid, coumermycin (Delaney 1989: Neidhardt & VanBogelen 1987),  35  and hydrogen peroxide (Zheng et al. 2001). The mechanisms of Escherichia coli stress response is well studied (Nakasono & Saiki 2000). In addition, Escherichia coli has been used as a model system in many studies. Nakasono & Saiki (2000) used this bacterium to determine whether e xtremely low frequency magnetic fields (5-100 Hz) can be considered as a general stress factor. E. coli containing the plasmid pUC8 also has been used as model to detect athermal effects of non-ionizing electromagnetic radiation through assessment of P-galactosidase activity (Saffer & Profenno 1989). Because of its unique position as a preferred model in biochemical genetics, molecular biology, and biotechnology, E. coli K-12 was the earliest organism to be suggested as a candidate for whole genome sequencing (Blattner et al. 1997).  Today Escherichia coli is  probably one of the best understood living organisms in terms of genome map and physiology (Nakasono & Saiki 2000; Adams & Moss 2000).  2.7  Stress response and stress proteins Living organisms respond at the cellular level to stressful conditions by a rapid and  temporary acceleration in the expression rate of stress genes (Morimoto et al. 1990). It is well established that a general stress response is universal among prokaryotes and eukaryotes (Champomier-Verges et al. 2002; Nakasono & Saiki 2000; Goodman et al. 1994; Delaney 1989). Overall, the stress response represents a general mechanism for coping with increased protein damage while cells or organisms are under stressful conditions. Protein damage appears to be the common signal that elicits the activation of most stress-inducible genes (Daniells et al. 1998). In addition other stress conditions such as oxidative and acid stress can affect the gene  36  expression (Teixeira-Gomes et al. 2000). The product of these genes is commonly referred to as stress proteins or heat shock proteins (Morimoto et al. 1990). In the literature, the abbreviation "hsp" is used for the whole stress protein family. Stress proteins are induced by a large variety of stress conditions such as heat (Zhang & Griffiths 2003), cold (Gualerzi et al. 2003), toxic chemicals, reactive oxygen species (Shallom et al. 2002), ethanol, anoxia, electron transport inhibitors, amino acid analogs, virus infections (Weigl et al. 1999), arsenite and cadmium, starvation (Lindquist 1992), complex metabolic processes (Arsene et al. 2000) and low frequency magnetic field (Mittenzwey et al. 1996). Their induction is often accompanied by tolerance to these stresses (Lindquist 1992). Stress proteins can be clustered in two main groups: general stress proteins and specific stress proteins. General stress proteins are the most studied in all kinds of stress and probably all kinds of bacteria. They are induced non-specifically by several stimuli and are involved in D N A or protein repair including chaperons DnaK, GroEL, GroES, or proteases such as Clp proteases. The specific stress proteins are induced as a result of a given specific stress such as cold shock or acid shock. In addition there is another group that some researchers consider as stress proteins. Proteins in this group normally belong to general metabolism but they can be affected by some specific stresses, for example, the proteins of the glycolytic pathway (Champomier-Verges et al. 2002).  2.7.1  Function of stress proteins The primary function of stress proteins is to protect cells or organisms from  environmental conditions, allowing them to recover and continue their normal metabolic processes (Morimoto et al. 1990; Delaney 1989).  37  Some are required for growth at high  temperatures w hile o thers a re r equired f or 1 ong-term s urvi val a 11 emperatures j ust b eyond t he normal growth range, and yet others are specialized to provide protection against extremes (Lindquist 1992). Several hsps are also present at normal conditions and play vital roles in cell growth as well as in stress tolerance (Lindquist 1992; Morimoto et al. 1990). Hsps are generally directly or indirectly involved in protein degradation (Heitzer et al. 1992). They promote the folding and unfolding of other proteins, the assembly and disassembly of proteins in oligomeric structures and the degradation of proteins that are improperly assembled or denatured (Lindquist 1992). Hsps, as molecular chaperones, help other proteins to assemble with their proper partners (Weigl et al. 1999; Lindquist 1992). They also bind to unfolded polypeptides during their movement in the cell, enabling the transport of these polypeptides through membranes or their integration into cell organelles (Weigl et al. 1999). Hsp involvement in synthesis of various macromolecule such as  bacteriophage  development, chromosomal and plasmid D N A replication, R N A synthesis and protein synthesis have been reported (Heitzer et al. 1992). Their function in the immune response of organisms have also been studied (Weigl et al. 1999).  2.7.2  Heat shock response in Escherichia coli The cellular response of an organism to heat shock was first described when a brief pulse  of heat induced puffs in specific locations on the polytene chromosomes in the salivary glands of Drosophila buskii (Delaney 1989). Since then heat shock response has been studied in a wide range o f organisms: for example, Mycobacterium tuberculosis (Stewart et al. 2 002), Brucella melitensis (Teixeira-Gomes et al. 2000), Haloferax volcanii (Kuo et al. 1997), Archaea (de Macario & Macario 1994), maize seedlings (Greyson et al. 1996), soybean seedlings (Krishnan  38  & Pueppke 1987), mammalian cells (Landry et al. 1982), and Chinese hamster fibroblasts (Laszlo 1988). In E. coli, temperature increase from 30 to 42°C causes a rapid increase up to 15fold induction of more than 20 heat shock proteins, followed by an adaptation period where the rate of hsp synthesis decreases to reach a new steady-state level (Arsene et al. 2000). The conditions that induce expression of heat shock proteins in E. coli and the effect of heat shock protein on E. coli resistibility to stress factors has been studied extensively. Yamamori & Yura (1980) stated that up-shift of temperature by 3°C above 34°C in batch cultures of log phase E. coli result in induction of heat shock proteins. Seyer and co-workers (2003) studied the production of DnaK in exponentially growing E. coli (ATCC 25922) culture immersed in a shaking water bath at 50 or 55°C for 3 and 5 minutes. The heated E. coli were cooled to 37°C in an ice-water bath. They observed higher DnaK in heat-treated cells at 50 and 55°C compared to cells grown at 37°C. Heitzer and colleagues (1992) heated E. coli cultures grown at 37°C to 42°C for 2 or over 60 minutes and reported that htpG was induced faster in the 2 minute treatment. They also observed a strong correlation between temperature increase and expression pattern of htpG gene. Pagan & Mackey (2000) studied the effect of heat shock on the resistance of E. coli H1071 to pressure. E. coli cells were harvested at 4°C and pellets were re-suspended in a 45°C pre-heated phosphate buffered saline (pH 7.0) for 45 minutes. Pagan & Mackey (2000) observed an increase in E. coli resistant to pressures between 200 and 400 M P a due to induction of heat shock proteins. Yamamori & Yura (1982) reported that E. coli cells grown at 30°C, then shifted directly to a lethal temperature (50°C) were rapidly killed. However, i f the cells were first preheated by growth at 42°C for 30 minutes, the rate of killing upon shift to lethal temperature was dramatically decreased. Chow & Tung (1998) showed that recovery rate of early log phase E.  39  coli after 24 hours frozen storage at -80°C was ten times higher in cells that were exposed to heat shock o f 42°C for 30 min, prior to freeze treatment compared to unhealed control cells. They also added that this higher recovery was related to the accumulation of heat shock proteins induced before frozen storage.  2.7.2.1 Regulation of heat shock response in E. coli The regulation of heat shock response for different organisms and even different cell types within an organism varies. For instance, heat shock response in yeast is controlled at the transcriptional level while in Drosophila both transcriptional and translational regulations are involved (Delaney 1989). The E. coli heat shock response is positively controlled at the transcriptional level by the product o f t he rpoH gene. A t first, t he t ranscription o f rpoH g ene i s i ncreased t hrough four different promoters. Three promoters are a -dependent and one promoter requires the a 70  E  factor  (Arsene et al. 2000). Each of these promoters is responsible for expression of some genes at various metabolic and environmental conditions (Kallipolitis & Valentin-Hansen 1998). The second step is an increase in translation of rpoH m R N A and results in an increase in rj  32  level (Arsene et al. 2000). E. coli strains lacking the heat shock transcription factor (a ) 32  cannot grow above 20°C and they are unable to induce hsps at higher temperatures (Lindquist 1992). The a  3 2  along with RNA-polymerase induce the expression of heat shock genes and heat  40  PI  P3  P4  P5  rpoH  ~~~ m R N A  32  degradation  R N A polymerase  .(H). hs  heat shock genes  DnaK, DnaJ, G r p E  misfolded proteins  Figure 2.2. The E. coli heat shock regulon (Arsene et al. 2000).  41  shock proteins. The heat shock proteins start the task of refolding or degrading the misfolded, proteins. This induces a signal to shut off the heat shock response. During the shut off phase, the DnaK, DnaJ and GrpE heat shock proteins act as negative modulators by repressing the translation of rpoH mRNA, causing efficient degradation of rj , and repression of a 32  3 2  activity  (Arsene et al. 2000) (Figure 2.2).  2.7.3  Microwaves and stress response Extremely low frequency electromagnetic fields at frequencies of 50 and 60 Hz have  been shown to induce a classic stress response in cells, enhanced induction of stress proteins and altering cellular metabolism in a number of models including cell cultures, Caenorhabditis elegans, Drosophila melanogaster and Escherichia coli (Shallom et al. 2002; Daniells et al. 1998). Goodman and co-workers (1994) exposed human HL-60 cells to 60 Hz electromagnetic field for 20 minutes and reported that the transcript level of hsp70 was increased. Nakasono & Saiki (2000) also reported that yeast and H L 6 0 human leukemia cells responded to magnetic fields by the synthesis of hsp 70 or the transcription factor a , which is similar to the general 32  environmental stress response,  de Pomerai and co-workers (2000) exposed soil nematode  Caenorhabditis elegans to microwave radiation at 750 M H z , 0.5 W for 18 hours and reported the induction of hsps. Shallom and co-workers (2002) investigated the effect of microwaves (915 M H z , 3.5 or 5 W) on chick embryo and showed that microwave exposure increased the induction of hsp 70 by 20 to 60% while the temperature rise was not enough for activation of heat shock pathway. They also reported that after 30 minutes, chicks exposed to microwaves had significantly higher survival rates than controls.  42  Induction of stress protein in E. coli cells after exposure to electromagnetic fields (50 Hz) was reported by Chow & Tung (2000). Nakasono & Saiki (2000) found no reproducible change in the level of protein synthesis after exposing E. coli to frequencies 5-100 Hz. Cleary and colleagues (1997) also saw no significant induction in hsps levels following exposure of HeLa cells to microwaves (2450 M H z , 25 W/kg, 2 hours) compared to mild heat stress (40°C, 2 hours).  2.8  D N A microarray technology In the past, to define stress-related, global regulatory responses researchers have often  relied upon either separation of protein fractions from stressed cultures or the use of transgenic test organisms carrying a stress gene whose product could be easily detected (such as betagalactosidase from the Escherichia coli lac Z gene) (Daniells et al. 1998). However, traditional methods usually work with one gene in one experiment, which is time consuming and unable to show an overview of the organism's response. In the last several years, a new technology, called D N A microarray has become available and received a great deal of attention (Schulze & Downward 2001).  This technology has  increased the speed of the investigation of gene regulation and has provided a system for the simultaneous measurement  of the expression level of thousands of genes in a single  hybridization assay with the possibility for further understanding of the total organism response to a specific condition. The most commonly used microarray systems, classified according to the arrayed material are: complementary D N A (cDNA) and oligonucleotide microarrays. Probes for c D N A arrays are usually products of the polymerase chain reaction generated from cDNA libraries or  43  clone collections. In oligonucleotide arrays, short 20-25mers are synthesized in situ (Schulze & Downward 2001) or by conventional synthesis. In general, a microarray consists of a series of DNAs target sequences (primarily P C R products or oligonucleotides) spotted on to a carrier (glass slide, nylon filter, silica "chip", or membrane), in an orderly manner.  Subsequently, labeled R N A or c D N A probes synthesized  from m R N A isolated from a sample are hybridized on to the array (Snijders et al. 2000). Hybridization intensities for each D N A sequence are determined using an automated process and converted to a quantitative read-out of relative gene expression levels (Harrington et al. 2000), which provides a measure of the expression of thousands of genes in a single experiment (Snijders et al. 2000). The data can be further analyzed to identify expression patterns and variation correlated with cellular development, physiology and function (Harrington et al. 2000).  2.8.1 DNA microarray applications One of the most significant applications of this technique was the gene expression profiling of the whole genome.  Genomic-wide expression levels of Saccharomyces cervisiae  (Wodicka et al. 1997) and Escherichia coli (Richmond et al. 1999) have been monitored with microarray technology (Oh & Liao 2000). A n attempt to monitor the genomic-wide expression of Caenorhabditis elegans (Kim et al. 2001) has been reported. Gene expression patterns can be used to assign functions to unknown g enes, improve understanding of cellular function, identify potential drug targets, generate genome-wide snapshots of transcriptional activity in response to any stimulus (Harrington et al. 2000), resolve the changes in gene expression that accompany adjustments to cellular physiology, identify genes differentially expressed in response to changes in environmental parameters, define  44  developmental programs or to evaluate mutations in regulatory and metabolic pathways (Conway & Schoolnik 2003). In addition, gene expression patterns of tumors can often tell the oncologist in advance whether a patient will respond to certain chemotherapeutical or hormonal agents (Snijders etal. 2000). At the same time for manyresearchers, the ultimate goal is to investigate the transcriptome of bacteria growing within infected tissues thus to study the hostadapted transcriptional responses (Conway & Schoolnik 2003).  2.8.2  DNA microarray limitations Like any other technique, some limitations are associated with D N A microarray  technology including, the high costs, the need for specialized technical expertise, the need for collaboration between different disciplines, difficulty in coping with very large amounts of data, and uncertainty about the biological meaning and clinical relevance of the results (Snijders et al. 2000). In addition this technique shows the expression of genes in transcription, which does not necessarily matche the translational changes.  The expression of a specific gene is not an  indicator for the presence of related protein or enzyme. Thus any r egulatory c hange at posttranscriptional level can not be detected. Nevertheless, arrays have proved to be quite successful in describing trends in gene expression patterns that reflect operon, regulon and stimulon organization (Conway & Schoolnik 2003).  45  CHAPTER THREE PRELIMINARY STUDY: EFFECT OF VACUUM MICROWAVE DRYING ON NATURALLY OCCURING MICROORGANISMS OF PARSLEY  46  3.1  Introduction Parsley, a member of the Umbelliferae family, has been cultivated since the days of the  Romans (Sobiech 1980). The fresh leaves are employed for garnishing, seasoning and flavoring. They also were used as medicine by the ancient Greeks and Romans (Small 1997). Fresh parsley is a good source of vitamin C (133 mg/lOOg fresh), (3-carotene (5054 ug/lOOg fresh) and vitamin K (phylloquinone 1640 ug/lOOg fresh) (USDA database). It is the most popular of all garnishing herbs in the West and many other parts of the world (Small 1997). Microbial contamination of vegetables and herbs may occur in the field through irrigation, harvesting and handling. Generally, harvested fresh parsley is chilled with cold water and carried on ice to the market. Fresh parsley can be stored for a month at 0°C and relative humidity o f 9 0-95% ( Small 1 997). D ue t o h igh w ater c ontent and h igh n umber o f n aturally occurring bacteria and fungi, parsley was the source of several food poisoning outbreaks in 1998 (Crowe et al. 1999). Drying, as the most commonly applied method of increasing shelf life, inhibits the growth of microorganisms and delays the onset of some biochemical reactions (Bohm et al. 2002). The common method for drying parsley is hot air blast drying. Hot air causes heat and oxidative damage to the plant tissue and changes the physical and chemical characteristics of the product (Bohm et al. 2002).  In the market, quality of dried herbs mainly depends on their  colour, aroma, and absence of off-flavour defects (Bohm et al. 2002). The food industry is always searching for improvements in dehydration process to preserve product quality while using less heat, mechanical shear, and additives. The use of vacuum along with microwave has proved a good combination in production of high quality materials. While microwaves provide the fastest means available of transferring energy into the  47  interior of foods (Durance & Wang 2002), the reduced pressure keeps the product temperatures low, as long as a certain amount of free water is present. Thus temperature sensitive substances like vitamins, colours and flavours will be retained (Regier & Schubert 2001; Decareau 1985). Vacuum m icrowave d rying h as b een e mployed t o d ehydrate a w ide r ange o f p roducts including banana slices (Mousa & F a r i d 2002, M u i et al. 2002), chilli (Kaensup et al. 2002), pectin gel (Drouzas et al. 1999), shrimp (Lin et al. 1999), sweet basil (Yousif et al. 1999), carrot slices (Lin et al. 1998), potato chips (Durance & Liu 1996) and cranberry (Yongsawatdiguul & Gunasekaran, 1996). Bohm and co-workers (2002) reported that the retained colour and odour of parsley dried under vacuum microwave (642 W, pulsed microwave, 40 mbar vacuum) was better than with the air drying method (75°C, 50-70 min). Sobiech (1980) also reported that microwave vacuum drying (2450 M H z , 2.0 kW) enhanced the flavour of the dried sliced parsley root and retained the properties of fresh raw material. Although destruction of microorganisms under microwave radiation has been studied for years (Dreyfuss & Chipley 1980; Welt et al. 1994; Kozempel et al. 2000), very little information on the effect of microwave drying or vacuum microwave on microbial reduction can be found. Daglioglu and colleagues (2002) dried tarhana dough (fermented product of yogurt-cereal mixture) inoculated with Staphylococcus aureus (10 CFU/g) with a hot air oven at 55 +/-2°C 4  for 36 hours or in microwave oven (1500 W, 2450 M H z , 30% power level) for 10 minutes. They reported that S. aureus was completely eliminated after microwave drying.  They  recommended microwave drying as a more efficient way to decrease the microbial population. K i m and colleagues (1997) dried concentrated yogurt in a laboratory scale microwave vacuum dryer (10 mmHg, 250 W, 2450 MHz) at 35°C and reported a great survival of lactic acid bacteria (S. thermophilus and L. bulgaricus).  48  The main objective of this study was to gain knowledge of the lethal effect of microwave radiation on the naturally occurring microorganisms of parsley during dehydration under vacuum as part of a long-term objective to investigate the response of microorganisms to vacuum microwave. In addition we wished to determine the impact of the drying method on microbial reduction.  3.2  Materials & Methods  3.2.1  Plant source Parsley, Petroselinum crispum, was purchased from a local herb cultivator in Vancouver,  B C , then washed with tap water and stems were removed.  Only the leaves of the freshly  harvested plant were used for all experiments  3.2.2. Drying Parsley leaves were dried with two different methods.  3.2.2.1 Air drying (AD) Five hundred g fresh parsley leaves were air-dried in duplicate using a commercial airdrier (Vers-A-Belt, Wal-Dor Industries Ltd. New Hamburg, Ontario, Canada) for 35, 60, 70 and 105 minutes.  A i r flow rate was 0.9 m /s with initial relative humidity of 10%. The dryer 3  temperature was set at 60-73°C and samples were placed on conveyor belt exposed to hot air.  49  3.2.2.2 Vacuum microwave drying (VMD) Three hundred to five hundred grams of fresh parsley leaves from the same batch were placed in a perforated cylindrical high density polyethylene drying basket (0.26 m radius and 0.23 m length) (Figure 3.2-d).  The basket was rotated on its horizontal axis in a vacuum  microwave drier (2450 M H z , 4KW, E N W A V E Corp, Vancouver, B C , Canada) at a rate of 3 rotations per minute. Samples were dried in duplicate at 1.5 K W power, 26-28 in Hg vacuum for 9, 17, 19 and 20 minutes (Figure 3.1, 3.2). The final temperature of samples was measured using an infrared thermometer (Model 39650-04, Cole-Parmer Instruments, Co. USA). A l l the dried samples were stored in polyethylene vegetable bags (Reggie Veggie, Richmond, BC), heat sealed and stored at 4°C for 48 hours before further analysis.  3.2.3  Temperature measurement of parsley during vacuum microwave drying Three hundred to five hundred grams fresh parsley at room temperature (20-25 °C) were  placed in the V M drier under the same drying condition (1.5 k W power, 26-28 in Hg vacuum, 3 rpm, 17-20 min). The drying process was stopped at timed intervals and the temperature of the sample was measured using the infrared thermometer immediately after opening the drier door. Temperature measurements were performed in triplicate.  3.2.4  Determination of moisture content To determine the moisture content, duplicate samples (2-4 g) were placed in aluminum  dishes a nd d ried i n a n air-drying o ven (Blue M , B lue M -electric C ompany, Illinois, U S A) a t 103°C for 4-6 hours (AOAC method 6.004) or until constant weight.  50  3.2.5 Water activity measurement Water activity of the samples was measured in duplicate using a water activity meter (Rotronic-Hygroskop DT, Rotronic Instrument corp. Huntington, N Y ) .  The instrument was  calibrated before measurements using humidity standards (Rotronic, Rotronic Instrument Corp. Huntington, N Y ) .  3.2.6 Microbiological Analysis 3.2.6.1 Microbiological sampling Twenty five grams of dried sample or 50g fresh parsley leaves were transferred aseptically into a stomacher bag, then 225 ml or 450 ml sterile peptone water (0.1% w/v) was added to dried and fresh samples respectively.  Samples were soaked 15 minutes at room  temperature before homogenizing in stomacher unit (Seward - Stomacher 400- Lab System Seward Stomacher England) for 4 minutes at medium speed. Serial dilutions of 10" to 10" were 1  4  prepared.  3.2.6.2 Total microbial count One ml aliquot from each dilution was pour plated in duplicate using Plate Count Agar (Difco) for total microbial count and incubated at 35 ± 1°C for 48 hours.  3.2.6.3 Yeast & mould counts A 1 ml aliquot of each dilution was pour-plated in Potato Dextrose Agar (Difco) +12 ml/L sterile tartaric acid (1:10 w/v), final pH of 3.5 (Beuchat & Nail 1985).  Plates were  completely wrapped in aluminum foil and incubated at room temperature 20-25°C for 5 days.  51  Plates with 25-250 colonies were selected for calculation. The average number of colonies per plate was multiplied by the corresponding dilution factor. Final values for dry and fresh samples were reported as colony forming units (CFU) per gram of sample (dry weight) and calculated as following: w= d  100-m 100  Eq(3.1)  where w = percent dry matter d  m= moisture content (wb) CFU/g sample w  = C F U / g sample (dry weight)  Eq (3.2)  d  where: CFU/g sample= colony forming unit per gram sample (dry weight)  3.2.7 Statistical Analysis Analysis of Variance: Estimate Model (SYSTAT 8.0, 1998) was used to determine the significant among treatments. L SD ( S Y S T A T 8.0, 1 998) was used to compare the treatment means (p< 0.05).  3.3  Results The initial moisture content of the fresh parsley leaves was 83.9 +/- 0.9 % on a wet basis.  The average initial microbial population of fresh parsley was 9.3 x 10 and 1.8 6  x  10 C F U per 5  gram dry sample for total microbial counts, and yeast and mould counts, respectively. Unfortunately, due to basket rotation which would damage the fiber optic probe, it was not possible to measure the temperature of the product during the V M D process. Thus the final  52  temperature of product was considered as a temperature indicator instead of the actual temperature of the sample. The results of batch temperature measurements of parsley during V M D showed good reproducibility and the average coefficient of variation among replicates was 4.17% (Figure 3.3). To check the effect of drying level on microbial population, samples were divided into three groups according to their post-drying water activity values: i) equal or greater than 0.9 ii) from 0.5 to 0.7, and iii) from 0.2 to 0.5 (Table 3.1). Total microbial counts, for air dried samples with water activity 0.964 were 3.2 * 10 CFU/g dry sample while for V M dried parsley leaves 5  with water activity 0.952 were 4.9 * 10 CFU/g dry sample. 4  Although V M dried samples  showed one log more reduction in microbial population compared to A D , no significant difference in microbial population between drying method and fresh sample was observed. This could be explained by variation among replicates. In the second group, dried samples with water activity values from 0.5 to 0.7, a significant difference in both total aerobic and yeast and mould counts between V M D and A D samples was observed (p<0.05). For samples with water activity values between 0.2 to 0.5, there was a significant difference for total microbial counts and yeast and moulds between V M D and A D and fresh samples. There was no significant difference in total microbial counts and yeast and mould population of dried parsley between treatments in the 0.2 to 0.5 water activity range (Table 3.1).  3.4  Discussion Actively growing microorganisms may contain more than 80% water. The process of  dehydration removes water from the bacterial environment and cells, thus multiplication stops. Partial drying is less effective than total drying, although for some microorganisms, partial  53  drying as in concentration may be sufficient to arrest bacterial growth. Bacteria and yeasts generally require more moisture for growth than molds (Potter & Hotchkiss 1995). Other factors influencing microbial survival in dried samples are drying temperature, drying time and water activity of final product. Nutrient transportation into microbial cells is affected by the reduction in water activity. Cells can only adapt to environmental conditions within a limited individual range (Rodel 2001), beyond which they are no longer capable of reproduction. Almost all microbial activities are inhibited below a water activity of 0.6 (Fellows 2000). The minimum water activity for multiplication of bacteria is 0.75; for yeast and moulds it is 0.62 and 0.61 respectively (Rodel 2001). In the present study the total microbial count for A D samples was not significantly different from fresh parsley leaves for samples in a ranging from 0.2 to 0.5. w  One possible explanation for higher population on A D samples in water activity of 0.2-0.5 is that the b atch o f fresh s ample u sed h ad a h igher m icrobial p opulation c ompared t o t he o ther t wo batches and it might have contained spores and microorganisms with higher heat resistance. Thus exposure to drying condition of 65°C was not enough for the reduction of total aerobic population. While in V M D the presence of vacuum along with higher final temperature 75°C may have increased the reduction in microbial population. One possible explanation for higher microbial population in samples with a > 0.9 is that w  these samples were exposed to heat and/or microwave for a shorter period of time compared to drier samples, 9 minutes compared to 20 min for V M dried and 34-35 min compared to 105 min for A D dried samples.  Therefore, although there was a reduction in the number of  microorganisms compared to fresh parsley, it was not enough to show a significant difference. The results of this experiment indicated that V M D was more effective than A D for reduction in total microbial population and yeast and mould population in the water activity  54  range of 0.5 -0.7. V M D could be an efficient method for yeast and mould reduction considering that fungal spoilage of foods occurs more often than bacterial spoilage at a 0.61-0.85 (Beuchat w  1983). Hamid and colleagues (2001) exposed inoculated air to microwave 2450 M H z for a total of 35 min exposure time (with a 2.5 min on and 5 min off cycle). They detected no fungi after 10 min microwave radiation and recommended using microwave in the cheese packaging section of a dairy plant to eliminate fungi. Legnani and co-workers (2001), in a study on the effect of microwave treatment (100°C for 15 minutes) on black pepper, red chili, oregano, rosemary and sage, reported that microwave heating had little effect on spore forming bacteria but was effective on the moulds and bacteria that were indicators of fecal contamination. V M dried parsley samples in the present study showed 1.04 to 3.04 log reduction in total microbial count and a 1.85 to 2.97 log reduction in yeast and mould counts while only 0.22 to 1.20 log reduction in total microbial count and a 0.23 to 1.3 log decrease in yeast and moulds population was detected for air dried samples. These results are in agreement with the data presented by Daglioglu and colleagues (2002) who reported total mesophile aerobic bacteria counts decreased approximately 2 log in conventionally dried tarhana dough (fermented product of yogurt-cereal mixture) in an air oven at 55 +/-2°C for 36 hours and 4 log in microwave dried samples in microwave oven (1500 W, 2450 M H z , 30% power level) for 10 minutes. They observed 3 log reduction after conventional drying and about 5 log reduction after microwave drying for yeast and moulds. The batch measurement of the temperature of parsley throughout V M D showed that temperature gradually rose in the process and that parsley samples were not at the maximal final temperature for longer than 2 to 3 minutes compared to a total drying time of 17-20 minutes (Figure 3.3).  Thus times at a specific temperature is shorter with V M D , 17-20 minutes  55  compared to 60-105 minutes in A D .  In addition, the average temperature needed to reduce  microbial population in V M D was less than in A D .  3.5  Conclusion  This study showed that parsley leaves treated with V M D had microbial populations less than A D samples at comparable water activity. V M dried parsley samples in the present study showed 1.04 to 3.04 log reduction in total microbial count and a 1.85 to 2.97 log reduction in yeast and mould counts while only 0.22 to 1.20 log reduction in total microbial count and a 0.23 to 1.3 log decrease in yeast and moulds population was detected for air dried samples.  In  addition V M D was more effective against yeast and mould than total aerobic population. Since higher reduction in microbial population of fresh parsley leaves occurred not only in a shorter time but also at a lower final temperature in V M D compared to A D , it can be concluded that V M drying was an effective method of reducing the number of naturally occurring microorganisms in parsley. These data support the hypothesis of existence of lethal factor(s) other than heat associated with V M . However, there were limitations in the preliminary data. No attempt was made to identify the microorganisms in the microbial population. In addition batch temperature measurements during V M D were not as accurate as measuring temperature continuously over the drying process.  Therefore to define differences between V M heating and conventional  heating, more precise and meticulous measurements of time and temperature on specific microbial population were needed.  56  Figure 3.1. Vacuum microwave drier- door closed: a) microwave generator, b) vacuum chamber, c) waveguide.  57  Figure  3.2. Vacuum microwave drier- door open: a) microwave generator, b) vacuum  chamber door, c) waveguide, d) drying basket  58  0  -I  1  1  1  1  0  5  10  15  20  Time (min)  Figure 3.3. Time-temperature profile of fresh parsley leaves during vacuum microwave drying process: 2450 M H z , 1.5 kW, 26-28 in Hg vacuum with basket rotating at the speed of 3 rpm. Each value is average of 3 readings ± standard deviation.  59  Table 3.1. Total microbial and yeast & mould counts for fresh, air dried and vacuum microwave dried parsley. Total microbial count CFU/g dry weight  log reduction  Yeast & Moulds count CFU/g dry weight  log reduction  Final temperature of parsley  0.49 1.85  73.0°C 42.1°C  1.30 2.51  65.0°C (47.9-62.7)°C  a > 0.9 w  0.97 ± 0 . 0 2 Fresh parsley Air dried 0.964 V M dried 0.952 a w = 0.5 - 0.7  Fresh 0.923 parsley Air dried 0.67 ± 0 . 0 1 V M dried 0.66 ± 0.03 a = 0.2 - 0.5  5.4 x 105 a  3.2 x 10 ,4 a 4.9 x 10 5  ,4 a 5.6 x 10  1.8 x 104a 7.9 x 102 a  0.22 1.04  6a 3.7 x 10 5b  2.4x10 4c 5.0 x 10  2.2 x 105 a :  1.18 1.86  ,4 b  1.1 x 10 2c 6.7 x 10 ;  w  5a  Fresh 0.929 3.0 x 107a 1.7 x 10 parsley 3b 7a 9.9 x 10 0.27 0.23 (64.0-65.0)°C Air dried 0.496 1.6 x 10 2b ,4 b 2.97 3.04 1.8 x 10 (66.5 - 75.0)°C V M dried 0.323 ± 0 . 1 3 2.7 x 10 Each reported value is the average of two samples. ' ' Values within the same column for a given water activity which are not sharing the same superscript letter are significantly different (p< 0.05) from each other. a  b  c  60  CHAPTER FOUR EFFECT OF VACUUM MICROWAVE ON E  S C H E R I C H I A  A STUDY OF DEATH KINETIC PARAMETERS AND DIELECTRIC PROPERTIES  61  COLI  4.1  Introduction Special attributes such as faster heating rate and greater penetration depth have made  microwaves a unique tool for many industrial applications such as tempering, thawing, blanching, cooking, dehydration, sterilization, and pasteurization (Knutson et al. 1987; Rosenberg & Bogl 1987a, b). Attempts to use microwaves to destroy microorganisms had begun before the microwave oven was built (Fleming 1944).  One of the earliest studies applied  microwave energy to extend the shelf life of bread (Olsen 1965). That study was successful in reducing the number of viable spores of Aspergillus niger, Penicillium sp. and Rhizopus nigricans by exposure to microwave energy (5 kW, 2450 MHz) at a temperature lower than their thermal death point.  Goldblith and Wang (1967) exposed E. coli cultures suspended in a  phosphate buffer/ice mixture to 2450 M H z microwaves. They observed no change in microbial population in the bacterial suspensions after 100 s of microwave radiation with the final temperature of 51.5 °C, and concluded that inactivation of E. coli was due solely to the thermal effect. Kakita and colleagues (1999) showed that the complete sterilization of a piece of cloth, experimentally contaminated with bacteria, could be achieved quite rapidly by microwave irradiation before the cloth was dried to the water content of clothes usually worn (about 2.4 %). Papadopoulou and co-workers (1995) studied the bactericidal effect of microwaves on certain pathogenic enterobacteria and first reported the possibility of differences between thermal and electromagnetic lethal effects. The mechanism of destruction of microorganisms by microwaves is controversial. Some have stated that inactivation of microorganisms by microwaves is entirely by heat, through the same mechanisms as other biophysical processes induced by heat, such as denaturation of proteins, nucleic acids, or other vital components, as well as disruption of membranes (Datta &  62  Davidson 2000; Heddleson & Doores 1994).  Others have linked destruction to nonthermal  effects, since a lower final temperature may be needed to kill microorganisms.  Woo and  colleagues (2000) studied the effect of microwave radiation on E. coli and B. subtilis and reported protein and D N A leakage, severe damage on the surface of cells and cell walls, and appearance of dark spots in bacterial cells, as a result of microwave treatment.  They also  indicated that most of the microwave- treated cells were "ghost cells" from which intracellular materials had been released into the cell suspension. Kakita and co-workers (1995) studied the effect of microwave radiation on the survival of bacteriophage PL-1 and observed that most of the particles turned out to be the ghost particles (with empty heads). They reported microwave radiation broke the D N A located deep in the phages core, whereas heating the phage particles from the outside did not. Although there is a controversy about the mechanisms of microwave-induced death of microorganisms, there is no doubt about the destructive effect of microwaves.  Microwave  destruction of many bacteria {Bacillus cereus, Campylobacter jejuni, Clostridium perfringens, Escherichia coli, Enterococcus, Listeria monocytogenes, Staphylococcus aureus, Salmonella enteritidis, Salmonella sofia, Proteus mirabilis, and Pseudomonas aeruginosa) has been reported (Chipley 1980; Datta & Davidson 2000; Heddleson & Doores 1994; Knutson et al. 1987; Papadopoulou et al. 1995; Rosenberg & Bogl 1987b). Uneven temperature distribution is one of the biggest limitations of microwave applications.  Non-uniform heat distribution in products can result in survival of unwanted  microorganisms and incomplete pasteurization. Knutson et al (1988) attempted to simulate hightemperature, short time (HTST) and low temperature, long-time (LTLT) milk pasteurization processes using microwave heating, and found that the non-uniform heat distribution in the milk  63  resulted in recovery of viable Salmonella typhimurium, initially inoculated a population of 10 3  10 CFU/ml. 4  With respect to studies of microwave destruction of bacteria, four main experimental difficulties are evident in the literature: i) lack of convenient technology for on-line temperature measurement in a microwave field, ii) uneven heating due to inconsistent microwave field distributions, a nd t he p hysical and electrical n ature o f t he s ample, i ii) i nability t o e ontrol t he temperature of microwave-heated samples at a specific level, iv) uncontrolled concentration of solutes due to evaporative losses from the sample during heating (Welt et al. 1994). Since microwave absorption continuously generates heat, temperatures tend to rise throughout the microwave process. To keep the temperature consistent, either the microwave power needs to be turned on and off during the process or some cooling medium needs to be applied. In this section, vacuum was used to control the boiling point of water and thus maintain temperatures in microwave-treated bacterial suspensions at specific levels while the microwave power remained constant. In such a system, once the boiling point of water at a specific pressure is achieved, the temperature of the medium remains constant as long a s the pressure remains unchanged. Thermal destruction of organisms follows classical first order destruction kinetics of time versus absolute temperature, known as the Arrhenius relationship (Fellows 2000; Stumbo 1965). If microwave lethality is entirely thermal, the Arrhenius activation energies should be equivalent whether the heat is supplied by thermal conduction or generated by microwave. The aim of this study was to investigate the lethal effect of microwave irradiation under vacuum on pure cultures of E. coli c ompared t o conventional h eating i n a w ater b ath u nder v acuum a nd t o s earch f or possible non-thermal effects of the vacuum microwave treatment. In addition we wished to  64  determine i f the level of microwave power had an impact on E. coli destruction independent of temperature.  4.2  Materials and methods  4.2.1  Bacterial Strain Escherichia coli (ATCC 11775) isolated from human urine was purchased as a freeze  dried sample from American Type Culture Collection, Rockville, U S A .  4.2.2  Stock culture and inoculum preparation Lyophilized Escherichia coli culture was transferred to 50 ml of Nutrient Broth (Difco)  and i ncubated at 3 7°C. S ome m icrobiological t ests w ere i nitially c arried o ut t o a scertain t he purity of the culture (Appendix I, Table 8.1). Static culture was maintained by daily transfer of incubated culture to fresh Nutrient Broth. After propagating the microorganism, 1 ml of 18-hour culture was transferred into a 1.5 ml flat top sterilized micro-centrifuge tube (Siliconized Flat Top Microtubes, Fisher Scientific, Pittsburgh, PA, USA) containing 1ml sterilized glycerol and mixed. A l l the micro-centrifuge tubes were kept at -80 °C as stock culture. To prepare inocula, one micro-centrifuge tube was removed from -80°C every two weeks and kept at 4°C for two to three hours to completely thaw. Next it was transferred to room temperature (20-25 °C) for 2 hours to avoid any sudden temperature change. Finally the whole liquid was aseptically poured into 50 ml of room temperature Nutrient Broth and incubated at 37 °C for 24 hours. A sub-culture was then prepared by transferring 1ml of bacteria suspension into 50 ml Nutrient Broth after 16-18 hours. Each sample was sub-cultured three times before being used in an experiment.  65  4.2.3  Growth index The turbidity of Nutrient Broth containing E. coli was measured at 580 nm with a  spectrophotometer ( U N I C A M UV/VIS Spectrometer U V 2 A T I U N I C A M , Cambridge, U K ) and simultaneously t he s ame b roth w as s pread p lated i n d uplicate o n P late Count A gar ( PC A) t o count the population of microorganisms. A o nm was taken as an index of the population of the 58  microorganism in the liquid medium.  4.2.4  Sample preparation Stationary phase E. coli (10 to 10 CFU/ml) were separated from Nutrient Broth by 7  8  centrifuging at 1310xg for 4 minutes at 4°C using a Micro Centrifuge (Micro Centrifuge 5415C, Brinkmann Instruments, Inc. N . Y . , U S A ) .  The supernatant was discarded, 1 ml room  temperature 0.1 % peptone water (Bacto peptone, Difco) was mixed with the culture and it was centrifuged again at 1310 x g for 4 minutes at 4°C to wash the microorganisms and remove the nutrient broth residues. After discarding the supernatant, cells were suspended in 1 ml 0.1 % (w/v) peptone water for water bath treatment and vacuum microwave treatments respectively. The microorganisms and peptone water were mixed using a sterile syringe by passing them through the syringe several times just before the experiment.  4.2.5  Microwave power determination Microwave power was d etermined using IMPI2-Liter test (Buffler 1993). Two 1-liter  Pyrex beakers of distilled water (20 ± 2°C) were placed in the center of the microwave chamber and heated for 122 s, then stirred and temperature was measured immediately using mercury in  66  glass thermometer (Fisherbrand, Fisher Scientific co. USA). The power output was calculated by multiplying 70 b y the average of temperature rise of two beakers. M easurement of power output was repeated three times for e ach power and the average a nd standard deviation were reported (Section 9, Appendix III).  4.2.6 Continuousflowvacuum system Microwave and water bath treatments were c onducted in a continuous-vacuum system (Figure 4.1). More detailed information about the continuous-vacuum system can ba found in Appendix II (Figure 9.1-9.5). A glass container (glass vacuum chamber) with three long sidearms was built and placed in the heating environment. For microwave treatments, three holes were made in the back of a microwave oven (General Electric- JE435, Mississauga, Canada), and a copper tube (OD = 45 mm. ID = 41 mm, 89mm long) placed in each hole. The glass vacuum chamber was placed in a microwave oven with the side arms projecting through apertures in the oven wall.  Apertures were sized so as not to propagate microwaves and  microwave leakage was monitored with a microwave radiation detector (HI-3520, Holaday Industries, Inc., Eden Prairie, M N , USA). The two lower side arms were connected to stainless steel tubing and a pump (pressure-loaded compact low-flow pump head without canister, Micropump, Inc. Vancouver, W A , U S A ) for liquid circulation at a flow rate of 410 - 645 ml/min. The upper side arm was connected to the water-ring vacuum pump (SIHI pumps Ltd. Guelph, Canada) to reduce pressure in the chamber (Figure 4.2, 4.3). The temperature was monitored with a data logger (DATA T A K E R , F I E L D L O G G E R , D T 100F, Data electronics (Aust.) Pty, Ltd. Australia) through a union Tee-connector outside the microwave oven with a 1 mm diameter needle thermocouple tip connected to an 11 mm diameter copper-constantan  67  thermocouple.  Thermocouples were previously calibrated with an A S T M mercury-in-glass  thermometer ( A S T M  l c , -20/150 CP, VWRbrand, V W R ) .  The data logger collected  temperatures every 20 s. Samples were taken with a syringe through another union Tee with septum (SEPTA 11.5 CS, SGE Analytical Science, Austin, T X , USA) (Fig. 4.1., F). In water bath treatments under vacuum, the glass vacuum chamber was placed in a water bath (VersaBath, Fisher Scientific Co. USA) and the bath level was higher than liquid level in the container.  4.2.7  Determination of temperature consistency inside the glass vacuum chamber To ensure the recorded temperature using thermocouple during the treatment is the true  representative of temperature inside the glass vacuum chamber, a fiber optic probe (Luxtron Fluoroptic temperature probe M S A , Luxtron corporation, Santa Clara, C A , USA) was placed inside a glass beaker containing 1000 ml distilled water. Time temperature profile of water at 22.5 in Hg vacuum was monitored (Figure 4.4). Although start temperature varied by 5°C, final temperature was constant (±0.3 °C) once equilibrium was established.  4.2.8  Determination of residence time distribution To determine the time needed for E. coli cells to distribute evenly in the system, a simple  residence Time Distribution Study was performed. E. coli cells were used as the tracer material. The circulation system with no heating source was run at 410 and 580 ml/min under the highest 7  8  vacuum (26 inHg). Stationary phase E. coli cells (10 to 10 CFU/ml) were introduced at the inlet and samples were taken every 5 s. After serial dilution they were spread plated on P C A in duplicate and incubated at 37 °C for 24 hours before enumeration.  68  4.2.9 Sanitizing To prevent any contamination before and after each experiment the micro pump and tubing were autoclaved (121 °C, 20 minutes) and the whole system was sanitized with 500 ml 70 % (v/v) ethanol for 10 - 15 minutes at room temperature (20-25 °C), then rinsed with 500 ml sterile distilled water for 10 - 15 minutes. To check asepsis, before injecting the culture for each treatment, 0.5 - 1 ml peptone water was taken from the equilibrated liquid system and spread plated directly on P C A .  4.2.10 Vacuum microwave (VM) and water bath under vacuum (control) treatments The glass chamber was filled with 700 - 900 ml 0.1 % (w/v) peptone water. The lid was sealed with vacuum grease (Dow Corning Corporation, Midland, Mich, USA) and the vacuum pump and microwave were turned on. Vacuum pressure was controlled with an adjustable aperture (as a screw-clamp on a tygon tube) connected to the vacuum trap to vary the boiling point of water inside the glass chamber to set points between 49 °C and 65 °C. After the desired 7  8  temperature equilibrium was achieved, 1ml of suspended E. coli (10 to 10 CFU/ml) was injected into the liquid system. At time intervals of 35 - 40 seconds, 0.5 ml samples were taken with a sterile syringe (Latex free syringe, Becton, Dickinson, USA) and needle (Precision Glide needle 2 0 G 1 A" Becton Dickinson, U S A ) and injected into 9.5 m l room temperature 0.1 % l  (w/v) peptone water. Microwave experiments were conducted at two microwave powers; 711 ± 20 W and 510 ± 5 W. Control treatment followed the same protocol except the glass vacuum chamber was immersed in a water bath with the side-arms and pump projecting out of the water.  69  For temperature less than 53 °C, treatment continued until a 2 log reduction in bacterial population was achieved (except for 49°C at 510 W) and for temperature greater than 53°C treatment was continued until no microorganisms survived ( 5 - 6 log reduction).  4.2.11 Enumeration of surviving and injured E. coli Immediately after each experiment, dilutions of 10" to 10" were prepared by adding 1ml 1  4  samples to 9 ml 0.1 % (w/v) peptone water. Serial dilutions were spread plated in duplicate on P C A , and P C A to which Difco bile salts #3 had been added at a final concentration of 1.5 g/L (PCA-BS) (Facon & Skura 1996). A l l plates were incubated at 37 °C for 24 hours. Total number of surviving microorganisms was determined on P C A and injured microorganisms were calculated by subtracting the number of bacteria growing on P C A - B S from those that growing on P C A .  4.2.12 Correction for loss of heating medium during experiments As the liquid in the continuous-flow vacuum system was boiling, the volume of liquid bacterial suspension continuously decreased. Therefore the volume of peptone water in the glass vacuum chamber at the time of sample injection and after taking the last sample was recorded. The evaporation rate was calculated:  evaporation rate = (v, - v)/t  Eq (4.1)  where Vj = initial volume v = final volume t = process time (min)  70  Then the survival population at each sampling time was corrected using the following equations:  Vj - (Evaporation rate x At) = v  Eq (4.2)  c  where Vj = initial volume At = sampling time v = corrected volume c  (dilution factor x C F U x v )/ v; = C F U c  C  Eq(4.3)  where Vj = initial volume v = corrected volume c  C F U = bacterial population enumerated by plating C F U = corrected C F U C  4.2.13 Check for microorganism loss and possible bio-film formation in the chamber Two swab samples using sterile calcium alginate swabs (Puritan, Calgiswab type 3, Hardwood Products Co., Guilford, Maine, USA) were taken from the internal surface of the lid (307 cm ) and inside wall (764 cm ) of glass chamber after each treatment. The swabs were 2  2  soaked in 9 ml 0.1 % (w/v) peptone water for 15 minutes.  Then 1 ml sodium  hexamethaphosphate (10 %) (Fisher Scientific) w as added to completely d issolve the calcium alginate swabs. Then 0.1 ml of each suspension was spread plated in duplicate on P C A followed by incubation at 37°C for 24 hours.  71  4.2.14 Calculation of kinetic parameters The survival curves were prepared by plotting the log surviving population versus time for each experiment. The decimal reduction time (D-value) was computed for each temperature as the negative reciprocal regression slope of these curves. Two methods were used, Arrhenius and Thermal Death Time (TDT), to define temperature sensitivity (Lund 1975).  In TDT  technique the D-values on logarithmic scale were plotted against the temperature and z, as the negative reciprocal regression slope of the log D versus temperature, was calculated (using Microsoft Excel 1998). The reaction constant (k) in the Arrhenius technique, was calculated with equation (4.4): k = 2.303/D  Eq (4.4)  where D = the D-value for that specific temperature Then ln k was plotted against 1 / T (T is the treatment temperature in degree Kelvin) and the slope of the regression line was calculated. Activation energy (E ) was calculated using a  equation (4.5):  Slope = -E /R  Eq (4.5)  a  where R is the gas constant = 8.3144 (J/mole K ) E = activation energy (J/mole). a  4.2.15 Dielectric measurement of pure culture Four litres of Nutrient Broth containing a 16 - 18 hours Escherichia coli culture (stationary phase) were centrifuged at 10 - 15 °C (GSA rotor, S O R V A L L R C 5B, Dupont,  72  Newtown, CT, USA) at 27300 x g for 30 minutes. The supernatant was discarded; the pellets were pooled and resuspended in nutrient broth and centrifuged again at the same condition. This was repeated four times till the pellet was hardened and a grayish paste was formed. Dielectric properties, apparent dielectric constant s' and apparent dielectric loss factor s" at 2450 M H z , of paste and sterile peptone water were measured in triplicate with an Open-ended coaxial Probe and network analyzer (HP 8752C, HP 85070B, Hewlett-Packard Company, Fullerton, C A , USA). The probe was calibrated with air, a metal fitting that provided an electrical short, and deionized distilled water before each set of experiments. The probe was washed with water, 75% (v/v) ethanol and rinsed with deionized distilled water and dried after each measurement.  4.2.16 Statistical analysis The differences between slopes of temperature sensitivity curves and activation energies were evaluated by homogeneity of regression test (Steel & Torrie 1980). Regression test- Linear regression (SYSTAT 1998) was used to determine correlation between temperature and population of injured microorganisms in different treatments (p < 0.05).  4.3  Results Liquid inside the chamber under vacuum was in turbulent flow and well mixed in all  treatments. The temperature variation during this study did not exceed ±1.5 °C, and in most cases was within ± 1 °C. The result of Residence Time distribution showed that E. coli cells were distributed evenly in the liquid system after 25 - 30 seconds (Fig. 4.4), and had reached the  73  expected population density of 2 x 10 CFU/ml, given the volume of the system and the number 6  of bacteria introduced.  4.3.1  Monitoring E. coli growth The growth of E. coli after transferring into 50 ml Nutrient broth was monitored to  determine the time required to reach to stationary phase of growth. The population density of 1.9 x 10 CFU/ml was achieved after 18 hours incubation at 37°C (Figure 6.1). 8  4.3.2  D-value The regression equations for survival curves of E. coli in V M at 711 W, 510 W and  control treatment are shown in Tables 4.1, 4.2 and 4.3 respectively. Log C F U versus time data from all the experiments formed straight lines on semi-log plots, indicating that regardless of the type of treatment, the inactivation profiles were approximated by first-order kinetic models in this range of temperatures. The destruction rate increased with increasing temperature for all the treatments. These results are in agreement with the data reported by Tajchakavit and co-workers (1998).  4.3.3  z value The temperature sensitivity curves of E. coli (z value) for V M 711 W and 510 W and  control treatments are shown in Fig. 4.5 a, b and c. The regression equations of the temperature sensitivity curves for all treatments are shown in Table 4.4. Since the relationship between log D and temperature is linear, z values can be used to predict the impact of changing temperature on D-values. As can be observed in Fig. 4.5-d, for temperatures less than 53 °C in V M at 711 W  74  and temperature less than 56 °C in V M at 510 W, microbial destruction in the control treatment was faster than in V M treatments. For example D  5 2  °c calculated from the regression equation  for the water bath under vacuum treatment was 3.65 minutes while for V M 711 W and V M at 510 W it was 4.2 and 6.0 minutes respectively. At the same time for temperatures higher than 53°C for V M at 711 W and higher than 56°C in V M at 510 W, the D-values obtained for V M treatments were considerably shorter than those obtained for water bath under vacuum treatments. For instance D  5 7  °c for control treatment was 1.0 minute while for V M at 711 W and  510 W it was 0.6 and 0.9 minutes. At the same time, as can be seen in Table 4.4, the value for z in the control treatment was 9.0 °C while for V M at 510 W and 711 W it was 6.0 °C and 5.9 °C, respectively. This indicates that E. coli was more sensitive to temperature changes during V M treatments than water bath under vacuum treatments. Statistical analysis showed that there was no significant difference in z values for E. coli exposed to 510 W and 711 W microwave power levels. In other words, microwave power level did not affect temperature sensitivity of E. coli in this temperature range. Similar results have been reported for E. coli exposed to microwaves (2450 MHz) in apple juice at 720W and 900W (Canumir et al. 2002).  4.3.4  Activation energy Activation energy for E. coli destruction in water bath under vacuum treatment was 232  kJ/mole while for V M at 510 W and 711 W it was 338 and 372 kJ/mole respectively (Table 4.5). Statistical analysis showed that between V M treatments and the control there was a significant difference in activation energy, while there was no significant difference between the values for V M 510 W and V M 711 W. Therefore, V M treatments needed higher levels of energy to initiate  75  destruction, relative to the control. Activation energies have not often been reported for E. coli but can be calculated from reported z values according to the following equation  z = 2.303RTT / E 0  Eq (4.6)  a  where z = temperature sensitivity (z value) R= gas constant (8.3144 J/mole K ) T & To = minimum and maximum temperature for calculation of z value (°K) For example Huang & Juneja (2003) reported a z value of 7.6 °C, equivalent to 279.3 KJ/mole fox E. c oli O 157:H7 i n 9 3% 1 ean b eef d uring thernial p rocessing i n t he t emperature range of 55-65 °C. Dock and colleagues (2000) reported a z value of 6 °C equivalent to 332.9 KJ/mole for E. coli 0157:H7 in apple cider while reported value for another strain of E. coli in apple cider by Splittstoesser and colleagues (1996) was 435.5 KJ/mole which is equivalent to z value of4.8°C.  4.3.5  Injured microorganisms Differential plate counts on P C A and PCA-BS for all the treatments showed the number  of injured microorganisms. Survival curves of E. coli plated on P C A and PCA-BS for V M 711W, 510W and water bath treatment at 50 and 58°C are shown in Appendix VI, Figures 9.8 to 9.13. Correlation coefficient tests indicated that there was no correlation between temperature and injured microorganisms population indicating that temperature variation did not have a significant effect on microbial injury in this experiment. Statistical analysis of the number of injured microorganisms among different treatments showed no significant difference. Likewise the type of heat treatment, whether microwave or water bath, had no significant effect on the  76  population of injured microorganisms. The present data is in contrast with the study of Shin & Pyun (1997) on Lactobacillus plantarum cells in M R S broth exposed to microwave and conventional heat at 50°C for 30 min. Shin & Pyun (1997) reported a greater injury in cells treated with microwave radiation. Khalil & Villota (1988) also exposed S. aureus in phosphate buffer to microwave at 50°C for 30 min under aerobic and anaerobic conditions. Although they reported a greater injury in microwave treated cells compared to water bath treated cells for both conditions, the difference in the number of recovered S. aureus was less in anaerobic conditions. They observed that while the percent of recovery for water bath treated  S. aureus was not  affected by the absence of oxygen, anaerobic conditions enhanced the recovery of microwave treated S. aureus.  4.3.6  Dielectric properties The final weight of the E. coli pellet for dielectric measurements was (5.01 ± 1.62 g).  Dielectric constant, loss factor and loss tangent of sterile peptone water and of the pellet of E. coli culture at room temperature are shown i n Table 4.6. D ielectric properties o f the E. coli pellet and peptone water were significantly different.  4.4  Discussion The range of temperatures employed in this experiment was restricted due to very high  rate of E. coli destruction at temperatures above 64 °C. Furthermore, our vacuum microwave equipment was not capable of maintaining constant temperatures below 49 °C. Goldblith & Wang (1967) also reported that because of the sensitivity of E. coli to heat, they could not obtain accurate inactivation studies at temperatures greater than 60 °C.  77  The present study showed that vacuum microwave treatment inactivated E. coli cells. The larger D-values for V M treatments at lower temperatures and small D-values at higher temperatures compared to conventional heat treatments provides evidence that there is a factor(s) in V M , different from water bath treatment, which delays E. coli destruction at lower temperatures and increases destruction rates at higher temperatures. One possible explanation for lower destruction rates at lower temperatures is that direct heating of microorganisms with microwave enhances the production of heat shock proteins, thereby increasing their resistance compared to the control.  Heat resistance of some bacteria increases upon exposure to  temperatures slightly higher than their optimum (Foster & Spector 1995; Kaur et al. 1998). Another possible explanation is the difference between heating rates of bacteria in the microwave treatment compared to water bath under vacuum treatments. Kaur and colleagues (1998) also studied the effect of heating rate on the survival of E. coli at 60 °C for 40s. They reported that for heating rate of 1 °C/min the mean number of survivors was 1.4 log CFU/ml while for heating rate 10 °C /min it was 2.6 log CFU/ml. They concluded that this might be due to exposure to potentially lethal temperatures for longer during heating period. Heitzer and colleagues (1992) also studied the effect of temperature elevation from 37 to 42°C in 2 and 60 minutes on expression of heat shock gene (htpG) in E. coli. They reported that the expression pattern was strongly dependent on heating rate. Activation energies show that V M treatments needed higher levels of energy for E. coli destruction, compared to the control. Since activation energy represents the minimum kinetic energy that must be possessed by a molecule in order to react, it can be concluded that destruction of E. coli under V M treatment occurs by a different mechanism than under the control treatment. The higher level of activation energy for microbial destruction associated  78  with microwave is in agreement with the study of Khalil (1987). He reported activation energy of 353.13 and 369.44 KJ/mol for Bacillus stearothermophilus spores and Bacillus subtilis spores for m icrowave t reatment a 11 emperature r ange o f 9 5 t o 100 °C. While for conventional heat treatment at the same temperature range was 316.73 and 308.36 KJ/mol for the spores of B. stearothermophilus and B. subtilis. The dielectric constant represents the amount of microwave energy absorbed by the sample and the loss factor indicates how much of that energy will be converted to heat. Loss tangent defines the ability of the medium to convert electromagnetic energy into heat at a given frequency and temperature.  Therefore, when the mixture of culture and peptone water was  exposed to microwave radiation, E. coli with higher loss factor, produced more heat than the surrounding liquid environment (peptone water). This higher capacity of E. coli to generate heat may cause a slight local temperature increase inside the cell. This lends credence to the selective heating theory, one of the four predominant theories of microwave inactivation (Kozempel et al. 1998).  The selective heating theory hypothesizes that microorganisms are heated more  effectively by microwaves than their surrounding medium, they can be killed more rapidly (Datta & Davidson 2000).  On the other hand, Sastry & Palaniappan (1991) studied the  temperature difference between a microorganism and its surrounding medium using simple mathematical relationships based on heat transfer principles. Results of their analysis showed a rapid heat loss to the surrounding environment due to the high ratio of the surface area to the volume of bacteria.  In the future i f actual direct temperature measurements of bacteria are  possible, the correlation between values predicted by Sastry & Palaniappan (1991) model and actual bacterial temperature could be used to prove or reject the selective heating theory.  79  4.5  Conclusion  In this study, although temperatures within experiments and between treatments were kept constant, slower inactivation at temperatures less than 53°C and higher reduction in microbial population at temperatures above 53°C for V M treated E. coli was observed, z value in the water bath treatment was 9 °C while for V M at 510W and 711W it was 6.0°C and 5.9 °C, suggesting that E. coli is more sensitive to temperature changes under microwave heating. Arrhenius calculations of the activation energies of the destruction reactions suggest that the mechanism of destruction in V M is different from that of conventional heat. Thus the presence of factor(s) other than heat involve in microwave under vacuum was established.  80  Figure 4.1. Continuous-flow vacuum system. A) glass vacuum chamber, B) heating source (microwave oven or water bath), C) micropump, D) data logger, E) thermocouple connector, F) sampling port, G) computer, H) vacuum pump.  81  82  Figure 4.3. Continuous vacuum system with microwave as heating source-outside view: a) micro-pump, b) circulation tube, c) connection to the vacuum pump, d) microwave oven.  83  0  1  1  1  2  1  3  1  4  1  5  1  6  1  7  1  8  1  9  I  I  I  10  11  12  Time (min)  Figure 4.4. Time-temperature profile of 1000 ml distilled water in microwave (2450 MHz) under vacuum (22.5 mmHg) with fiber optic probe. Each line is the representative of one measurement.  84  U)  o  4 3 2  -I  0  ,  ,  ,  ,  25  50  75  100  ,  125  1  150  ,  175  1  1  1  225  250  275  ,  200  1  300  time (second)  Figure 4.5. Sampled population of E. coli cells in the continuous-vacuum system with no heating source as a function of time indicated homogeneous mixing of injected bacteria within 30 seconds (each value is an average of two measurements).  85  Figure 4.6.a Temperature sensitivity curves for E. coli treated under vacuum microwave at 510 W.  86  temperature (C) Figure 4.6.b Temperature sensitivity curves for E. coli treated under vacuum microwave at 711 W.  87  1.5 -, 1-  -1 -1.5 -  temperature C  Figure 4.6.c Temperature sensitivity curves for E. coli treated in water-bath under vacuum.  88  temperature (C)  Figure 4.6.d Temperature sensitivity curves of E. coli treated by vacuum microwave 711 W — ™ "™ " , vacuum microwave 510 W  and water bath under vacuum  treatment  89  Table 4.1. Regression equations and D-values of E. coli exposed to vacuum microwave 711 W treatments. Temperature ( ° C )  D-value  Regression Equation  r*  (min) 49.2 +/- 0.6  13  y= -0.0013 x + 6.25  0.84  51.7+/- 0.7  6.9  y = -0.0024 x + 6.45  0.74  51.8+/- 0.6  4.1  y =-0.0041 x +7.10  0.88  55.2+/- 1.2  0.7  y = -0.025 x + 6.99  0.89  56.5 +/- 0.4  0.6  y =-0.03 x +6.68  0.97  57.1 +/- 1.1  0.8  y =-0.021 x +5.83  0.78  57.3 +/- 0.6  0.5  y =-0.031 x +6.69  0.99  58.4 +/- 0.5  0.5  y =-0.034 x +6.01  0.94  61.1 +/- 0.5  0.1  y =-0.13 x +7.28  0.91  90  Table 4.2. Regression equations and D-values of E. coli exposed to vacuum microwave 510 W treatments. Temperature (°C )  D-value  Regression Equation  r  2  (min) 48.9 +/- 0.7  28  y =-0.036 x +5.41  0.94  50.2 +/- 0.5  15  y =-0.0651 x+4.83  0.54  52.8 +/- 0.5  9.8  y =-0.0017 x +6.35  0.70  53.3 +/- 0.5  3. 8  y = -0.0044 x + 6.27  0.94  54.2 +/- 1.5  2.2  y =-0.0076 x +6.90  0.64  55.6 +/- 0.6  0.5  y =-0.031 x +6.82  0.98  56.3 +/- 0.4  1.0  y = -0.017 x + 6.03  0.98  56.3 +/- 0.5  0.4  y =-0.044 x +6.18  0.91  58.1 +/- 0.8  0.4  y =-0.039 x +6.52  0.99  58.2 +/- 0.8  0.7  y =-0.019 x +5.72  0.87  60.3+/- 1.0  0.5  y = -0.037 x + 6.44  0.91  60.5 +/- 0.7  0.3  y = -0.058 x + 5.64  0.76  63.6+/- 0.7  0.1  y =-0.15 x +6.28  0.81  91  Table 4.3. Regression equations and D-values of E. coli exposed to water-bath under treatments. Temperature (°C)  D-value  Regression Equation  r  2  (min) 50.4 +/- 0.5  5.1  y=-0.0033 x +6.16  0.82  52.9+/- 1.5  3.6  y =-0.0046 x +6.09  0.38  53.1 +/- 1.5  4.1  y =-0.0041 x +6.06  0.75  54.7 +/- 0.9  2.6  y =-0.0065 x +5.89  0.81  55.0+/- 1.3  1.3  y =-0.013 x +6.45  0.97  57.6 +/- 0.5  0.6  y - -0.029 x + 6.96  0.94  58.6 +/- 0.3  0.7  y =-0.024 x +6.41  0.94  60.0 +/- 0.5  0.5  y = -0.036 x + 5.65  0.76  60.1 +/- 0.3  0.24  y = -0.070 x + 6.52  0.87  63.5 +/- 0.1  0.19  y =-0.087 x +5.88  0.76  65.7 +/- 0.6  0.18  y =-0.14 x +6.52  1.00  92  Table 4.4 Regression equations of temperature sensitivity of E. coli for water-bath under vacuum treatment and vacuum microwave treatments at 510 W and 711 W. Treatment  z value  Regression Equation  r" 2  (°C) Water bath under vacuum (control)  9.0  Vacuum microwave (510 W)  6.0  Vacuum microwave (711 W)  5.9  a  y =-0.11 x + 6.3241  092  b  y = -0.17 x + 9.3883  0.88  b  y =-0.17 x + 9.4155  0.96  a  ' In each column values not sharing the common superscript are significantly different b  (p < 0.05) from each other.  93  Table 4.5. Regression equations of activation energy (E ) for E. coli in water-bath under vacuum a  treatment and vacuum microwave treatments at 510 W and 711 W. Treatment  E  a  Regression equation  r  (kJ/mole) Water bath under vacuum (control)  232  s  y = -28023 x + 85.71  092  Vacuum microwave (510 W)  338  b  y = -40659 x + 124.13  0.89  Vacuum microwave (711 W)  372  b  y = -44769 x + 136.93  0.95  a  '  In each column values not sharing the common superscript are significantly different  (p < 0.05) from each other.  94  Table 4.6. Dielectric constant and loss factor of sterile peptone water, and centrifuged pellet of pure culture of E. coli at room temperature. Dielectric constant  Loss factor  Sterile peptone water  77.3 ± 0.60  9.86±0.55  E. coli pellet  55.8±2.94  Sample  I  a  - I —  a  b  Loss tangent a  11.22 ± 1.14  b  0.128 ± 0 . 0 0 7  a  0.179 ± 0 . 0 2 6  b  .........  ' In each column values not sharing the common superscript are significantly different  (p < 0.05) from each other.  95  CHAPTER FIVE CHANGES IN  E S C H E R I C H I A  COLI  TRANSCRIPTOME  DUE TO SUB-LETHAL VACUUM MICROWAVE TREATMENT  96  5.1  Introduction A l l cells have complex signalling pathways, which help them to survive or adapt to a  destructive force in their environment at some certain level. These pathways are essential to cell viability and determine resistance to individual stimuli. Therefore cellular stress responses play an important role in the sensitivity of microorganisms to any external factor (Downes et al. 1999). Several attempts have been made to investigate the effect of microwaves on various cell function and response systems since the discovery of microwave heating properties (Mayne et al. 1999; Trosic et al. 1999). A number of studies showed evidence indicating that alternating electromagnetic fields interfere with D N A and R N A , affect molecules essential for the nervous system function, disturb the normal function of cell membrane (Mertens & Knorr 1992) and affect chromosome structure (Banik et al. 2003). In addition, changes in growth rate of Spirulina platensis (Pakhomov et al. 1998), Methanoscarcina barkeri (Banik et al. 2003), and sprouting of barley seeds (Pakhomov et al. 1998), and sensitivity of S. aureus to antibiotics (Bulgakova et al. 1996; Pakhomov et al. 1998) have been reported. Galvin and colleagues (1984) exposed the whole body of pregnant mice to 2450 M H z microwave radiation at a power density of 30 mW/cm for two, four hour periods per day in total 2  for 6 and 15 days. They found no change in lymphocyte, neutrophil or monocyte numbers. Trosic and co-workers (1999), who exposed male Wistar rats (13 week old) to 2450 M H z microwave at 5-15 mW/cm , 2 hours per day, maximum 5 days a week for the period of 1,8,16 2  and 30 days, however, showed a decrease in total leukocyte count as well as lymphocyte percentage in the treated rats.  97  Liu and co-workers (2002) studied the effects of 2450 M H z microwave radiation at a power density of 10 mW/cm on gene expression transcription of cultured human retina pigment 2  epithelial cells. They found at temperatures that did not exceed the heat shock temperature (32°C), seven of 97 genes were up-regulated about 2.07-3.68 fold compared to cells exposed to water bath while no significant down-regulation was observed. Harada and co-workers (2001) studied the effect of magnetic field (60 Hz, 0.25-0.5 T) to Klenow enzyme-catalyzed D N A synthesis and R N A polymerase driven R N A synthesis in vitro. They indicated that neither the polymerase activity nor proof reading was affected by magnetic fields in the condition employed. So far, published studies have focused on a specific function, cell structure or tissue of the organism under examination. To get an overview of cell response, study of the expression of the whole genome is required.  D N A microarray technology has proven to be a powerful  technique for investigating gene regulation by providing a system for simultaneous measurement of gene expression of the whole genome in a single hybridization assay.  Genome-wide  expression of Saccharomyces cervisiae (Wodicka et al. 1997) and Escherichia coli (Richmond et al. 1999) have been monitored with microarray technology. In addition, E. coli gene expression under a number of different conditions including minimal and rich media (Tao et al. 1999), responses to protein overproduction (Oh & Liao 2000), response to hydrogen peroxide (Zheng et al. 2001), and changes due to transition from exponential-phase to stationary phase growth in minimal medium (Wei et al. 2001) have been successfully studied. The results of the previous section on the injury and inactivation of stationary phase E. coli cells during microwave heating under vacuum showed that E. coli cells were significantly more sensitive to temperature changes when microwaves were the medium of heat transfer. In  98  addition, the activation energy of E. coli exposed to microwaves under vacuum was significantly higher t han t he cells e xposed t o h eat i n w ater b ath u nder v acuum w hile t he t emperature w as constant. This suggests that the destruction mechanism of vacuum microwave heating could be different from water bath heating. The aim of this section was to study the changes in gene expression of E. coli cells under vacuum microwave and water bath to achieve a better understanding of the mechanism of the microwave effects on microorganisms through global gene expression response.  The lowest  temperature that showed a difference in destruction rate was 49°C and the calculated D49.5 from regression equations for water bath treatment and vacuum microwave at 711 W were 6.9 and 11.2 minutes respectively (Table 4.4). Thus in the present work, the sub-lethal condition of 3 minutes t reatment at 4 9.5°C w as c hosen t o i nvestigate E. c oli s hort-term r esponse t o v acuum microwave ( V M ) and water bath treatments.  D N A microarray technology was used to  characterize the changes in gene expressions at the transcription level as a result of short-term exposure to microwaves under vacuum compared to water bath treatment and to identify the activated and inactivated pathways involved in E. coli response to vacuum microwave treatment.  5.2  Materials & Methods  5.2.1  Bacterial Strain Escherichia coli (ATCC 11775) isolated from human urine was purchased as a freeze  dried sample from the American Type Culture Collection, Rockville, U S A . For stock culture and inoculum preparation, refer to chapter 4, section 4.2.2.  99  5.2.2  Sample preparation A stationary phase E. coli (10 to 10 CFU/ml) culture (75-90 ml) was aseptically 7  8  transferred into six sterile, 15 ml centrifuge tubes (Fisher brand disposable sterile centrifuge tubes, seal cap, modified polystyrene, Fisher Scientific, Pittsburgh, PA, USA) and centrifuged at 2060xg at 4°C for 8 minutes ( B E C K M A N GS-6 centrifuge, Beckman Instrument, USA). The supernatant was discarded and cells were re-suspended in 4 ml room temperature 0.1 % (w/v) peptone water (Bacto peptone, Difco). They were mixed using a sterile syringe by passing them through a syringe several times just before the experiment.  5.2.3  Vacuum microwave (VM) and water bath under vacuum treatments Cells were exposed to microwaves (2450 M H z , 711W) under vacuum or heated in water  bath under vacuum in a continuous-flow vacuum system. For details about the system and sanitizing, refer to chapter 4, sections 4.2.6 and 4.2.8. Peptone water (825 ± 31.62 ml; 0.1 % (w/v)) was poured into the glass chamber. The lid was sealed with vacuum grease and the vacuum pump and microwave were turned on or the chamber was immersed in the water bath. Vacuum pressure was adjusted to 24.56 ± 0.31 in Hg with an adjustable aperture connected to the vacuum trap. 7  After the desired temperature 8  equilibrium was achieved, 4ml of suspended E. coli (10 to 10 CFU/ml) was injected into the liquid system. The length of treatment for all the experiments was 3 minutes. Next the heating source was eliminated (microwave turned off or the g lass g lass vacuum c hamber taken out of water bath) and the sample was cooled under full vacuum (26 in Hg). The liquid medium cooled to 44°C after 40-60 s, and cooling process continued for 4-5 minutes until the sample reached  100  <30°C.  The final temperature of the liquid medium was measured using an infrared  thermometer (Model 39650-04, Cole-Parmer Instruments, Co. Vernon Hills, USA). The detailed condition for each treatment is shown in Table 5.1.  Immediately following treatment, the  medium was aseptically transferred into sterile centrifuge tubes and cells were harvested at 25400xg, 4°C, 20 minutes ( S O R V A L L R C 5B, G S A rotor, Mandel Scientific, Norwalk, USA). Each treatment was repeated three times.  5.2.4  Untreated sample Forty eight ml stationary phase E. coli pure culture (10 CFU/ml) were aseptically 7  transferred into six sterile 15 ml centrifuge tubes (Fisher Scientific, Pittsburgh, P A , USA) and centrifuged at 2060xg at 4°C for 8 minutes.  5.2.5  Total RNA extraction After harvesting the cells, the supernatant was removed and 133 ul of the lysis mixture,  (15 p. 1 Ready-Lyse Lysozyme solution (Epicentre Technologies, Madison, WI, U S A ) + 785 yx\ Tris/EDTA (TE) buffer) were added to each tube, vortexed and incubated at room temperature (21-23°C) for 20 minutes. L ysed cell suspension was 1 oaded onto a QiagenRNeasy column (100 ul each column) from a Qiagen RNeasy Total R N A Isolation Mini kit (Valencia, C A , USA). Total R N A isolation was completed using the protocol supplied with the kit. Isolated R N A w as d iluted i n T E b uffer a nd q uantified b ased o n t he a bsorption a t 2 6 0 n m ( UNIC A M UV/VIS Spectrometer U V 2 A T I U N I C A M ) . The quality of R N A was checked by running the sample on an agarose/formaldehyde gel (Ausubel et al.1999) as well as calculating the ratio of absorbance at 260 / 280 nm.  101  The extracted total R N A was divided into 25 ug aliquots and frozen at -20°C over night then vacuum-dried using a Speed-vac (Sci 10, Savant Instrument Inc. Holbrook, N Y , USA), at low drying rate then stored at -20°C until further use.  5.2.6  m R N A enrichment To enrich mRNA, Affymetrix protocols (Gene expression analysis, Affymetrix technical  manual 2000 & 2001) were used with some modification as described below:  5.2.6.1 cDNA synthesis Five tubes of 25 ug total R N A were used for each sample.  To each tube, 1 ul Spike  control (500 pM) (Affymetrix Technical Manual 2001), 15 ul r R N A removal stock (5 uM) (16S r R N A Primers and 23S rRNA Primers, desalted, H P L C filtered, Sigma Genosys, Oakville, ON) (Table 5.2), and 24 ul of nuclease free water (Ambion, Austin, T X , U S A ) (total volume 40 ul) was added, mixed and incubated at: 70°C (5 min), 4°C (5min), 1 cycle, using a thermal cycler (PERKTN_ELMER, D N A Thermal cycler 480, Norwalk, CT, USA). Then 10 (al of l O x M M L V Reverse Transcriptase buffer (Moloney Murine Leukemia Virus ( M M L V ) RT, Epicentre Technologies, Madison, WI, U S A ) , 5 ul of 100 m M DTT (Epicentre Technologies, Madison, WI, USA), 2 ul of dNTP mix (25mM) (dATP, dCTP, dGTP, dTTP, Amersham Pharmacia Biotech), 2.5 ul of RNAguard (34.29 U/ul) (RNAguard RNase INHIBITOR Porcine, Amersham Biosciences corp, Piscataway, N J , USA), 30.5 ul of nuclease free water, 10 ul of M M L V Reverse Transcriptase enzyme (50U/ul) (Epicentre Technologies, Madison, WI, USA) (total volume 60 ul) were added, mixed briefly and incubated at: 42°C (25  102  min), 45°C (20 min), 4°C (3 min), 1 cycle, followed by: 65°C (5 min), 4°C (5 min), 1 cycle to inactivate M M L V RT enzyme, using a thermal cycler.  5.2.6.2 r R N A digestion rRNA digestion followed immediately after c D N A synthesis. Four ul of Rnase H (10 U/pl) (Ribonuclease Ff, E. coli, Epicentre Technologies, Madison, WI, U S A ) , 1.7 pi of R N A guard (34.29 U/pl) and 1.3 pi of nuclease free water (total volume 7 pi) were added and each tube was incubated at: 37 °C (25 min), 4 °C (4 min), 1 cycle, using a thermal cycler.  5.2.6.3 c D N A digestion Immediately after r R N A digestion, 1.5 pi Dnase I (10 U/pl) (Deoxyribonuclease I, Amersham Pharmacia Biotech) was mixed with 2.1 pi nuclease free water, followed by adding 1.4 pi R N A guard (34.29 U/pl) (total volume of mixture for each tube was 5pl). Tubes were incubated at 37 °C for 18 minutes. Enzyme was inactivated by adding 3 pi E D T A (500 mM) (0.5M E D T A , pH=8.0, Gibco B R L , Life Technologies, Maryland, USA). Samples were cleaned up using Q I A G E N E Rneasy mini column (Valencia, C A , USA) and enriched mRNA was eluted in 45pi of nuclease-free water, quantified at  A260  nm and frozen at -20 °C before drying.  Enriched mRNA samples were dried in a Speed Vac, at low drying rate, and stored at -20 °C till required for use.  103  5.2.7  Labeling and fragmentation Enriched m R N A (25-38 ug) was used for fragmentation and direct labeling (Affymetrix  Protocol for Prokaryotic Sample, Technical Manual, Gene Chip Expression Analysis, July 2001). F irst, m R N A was fragmented b y heat and ion-mediated hydrolysis. T hen the 5'-end R N A termini were modified by T4 polynucleotide kinase and y-S-ATP, and a biotin group. After clean-up the quantity of enriched, fragmented, and labeled m R N A samples was checked at A260  rim. The efficiency of the labeling procedure was assessed using the gel-shift assay  (Affymetrix technical manual 2001). Fragmented and labeled m R N A samples were stored at 20°C until use.  5.2.8  Hybridization, washing and staining Fragmented-biotin labelled m R N A (1.5 \ig) was added to a GeneChip sense E. coli  Genome array (Affymetrix Inc., Santa Clara, C A , USA) containing 7312 probe sets, and was hybridized in an Affymetrix GeneChip Hybridization Oven 640 (16 hours, 45°C, 60 rpm). Chips were then washed and stained using a GeneChip fluidics station 400, ProkGE-WS2 protocol (Affymetrix, Santa Clara, C A USA) in three steps: (1) binding of streptavidin to biotin, (2) binding of biotin-conjugated anti-streptavidin antibody to streptavidin, and (3) binding of phycoerythrin-conjugated streptavidin to the antibody biotin.  5.2.9  Scanning Each probe array was scanned twice in a GeneArray scanner (Agilent Technologies, Palo  Alto, C A ) . The computer workstation automatically overlaid the two scanned-images and averaged the intensities of each probe cell.  104  5.2.10 Data analysis The data for each array were collected and initially analyzed using Affymetrix Microarray Suite 5.0 software. calculated.  First, the average intensity value for all probe cells was  The degree of variation within the same probe cells were used to calculate the  background noise.  Other matrices compared the intensities of the sequence-specific Perfect  Match (PM) probe cells with their control Mismatch (MM) probe cells for each probe set, and then were used in a decision matrix to determine i f a transcript was Present (P), Marginal (M), or Absent (A, undetected) (Affymetrix Technical Manual 2001).  5.2.10.1 Data normalization Log base 2 of probe set intensities and the median of the gene intensity for each array was calculated for normalization. Then the median of each set was subtracted from each probe set intensity value. This normalization method was based on the geometric midpoint (average of the logarithmic measures of the ratios) rather than the arithmetic midpoint of ratios, as the geometric midpoint accounts for down- as well as up-regulation.  5.2.10.2 Statistical analysis The correlation among present calls of replicates was calculated using Excel (Microsoft Excel 1998). One way A N O V A was used to find genes in which expression was significantly different among treatments (p<0.05, n=6).  105  5.2.10.3 C a l c u l a t i o n o f f o l d c h a n g e  The log fold change was calculated as the difference between the average log intensity of three microarrays for each treatment compared with average log intensity of three microarrays of the base (untreated samples or water bath treatment). The log fold change for the transcript is a positive value when the expression level in treatment has increased compared to base line and is a negative number when the relative expression level in the treatment has decreased. Then an antilog was performed using equations 1 and 2 for up-regulated and down-regulated genes respectively, to calculate the fold change. For up-regulated genes: Fold change = 2  Eq (5.1)  ( l o g f o l d c h a n g e )  For down-regulated genes: Fold change = 1/ (2  5.2.10.4 D a t a  ( l o g f o l d change)  )  Eq (5.2)  filtering  Significant genes (p<0.05) had to be sorted before further analysis. Uninformative genes, genes that were expressed less than two fold and genes that were not present in any of the experiments were filtered out. Only those genes that either were present in all three replicates or evaluated twice as present and once as marginal in a data set were accepted. The probe sets related to intergenic regions were not considered. In this study the term "probe sets" refer to data on single array before data filtering which include all the 7312 probe sets and the term "gene" refers to those probe sets with a specific gene name from the E. coli genome.  106  5.2.10.5 Gene annotation To search for activated or deactivated metabolic pathways gene, data were divided into functional groups followed by individual analysis. Affymetrix  gene  chip data  base,  Colibri  The following web sites were used:  (genolist.pasteur.fr/Colibri), K E G G  (Kyoto  Encyclopedia of Genes and Genomes, http:Wwww.genome.ad.jp/kegg/), GenProtEC (Genes and Proteins of E.coli, http:Wwww.mbl.edu/html/ecoli.html.) (Riley 1998).  5.3  Results The  A260/A280  ratio of total R N A for all the samples was 1.97-2.1 and they showed two  clear bands for 16S and 23S R N A on an agarose/formaldehyde gel. The average yield for untreated E. coli samples was 5.47 ± 0.27 pg total RNA/ml culture (10 -10 CFU/ml) and for 7  8  treated samples with V M and water bath was 2.39 ± 1 . 1 and 1.79 ± 0.23 pg total RNA/ml culture respectively. From each 100 pg of total R N A , 49.78 ± 7.54 pg enriched m R N A was extracted. The yield for fragmentation and labeling was 15.98 ± 7.13 %.  5.3.1  Correlation among replicates The average correlation among present calls of replicates for each sample was 0.9 (Table  5.3). A fresh E. coli inoculum from the stock culture was used for each treatment. Therefore samples are biological replicates not technical replicates and the maximum calculated coefficient of variation of 5.1% and standard deviations less than 0.05 indicates that there was a good reproducibility among replicates.  107  5.3.2  Present, absent, and marginal probe sets in single arrays The number of probe sets detected as present or absent or marginal after exposure to  microwave and water bath and for untreated cells is shown in Table 5.4. In single arrays 57-58% of probe sets were identified as present after both treatments and 37-38% were not detected and considered as absent. In untreated cells 49% and 46% of probe sets were present and absent respectively. For all the samples 3.8-4.5% of probe sets were in the marginal area (Table 5.4). Those probe sets related to intergenic regions were not considered in this study.  5.3.3  Number of up and down-regulated genes After water bath treatment 123 genes (1.67%) were up-regulated and 135 genes (1.85%)  were down-regulated compared to untreated samples. V M caused 109 genes (1.49%) and 91 genes (1.24%) to be up and down-regulated respectively, when compared to untreated samples. In both cases > 96% of genes remained unchanged. Comparison between treatments showed 55 (0.75%) up-regulated and 49 (0.67%) down-regulated genes in V M compared to water bath treatment while 98.5% of genes did not show any significant change (Table 5.5).  5.3.4  Overview of E. coli response The analysis was divided into three overlapping sets of genes to obtain an overview of E.  coli genome response. Set 1 included all previously known heat-inducible genes, set 2 included all common genes showing significant change in both treatments to identify the similar response between treatments, and set 3 includes genes that showed significant change (> 2 fold) in each treatment compared to untreated samples and between treatments.  108  5.3.4.1 Heat shock genes A list of previously known heat shock genes along with their present/absent call and fold change for each treatment is shown in Table 5.6. O f the 76 known genes related to heat shock stress, 22 genes were not detected in any of the samples. The expression level of some heat shock related genes were up-regulated by both V M and water bath treatment.  However, the  degree of transcriptional response varied among the treatments as well as among heat shock genes. Only two genes significantly altered expression due to V M treatment, while the water bath treatment resulted in 6 genes that were significantly changed compared to untreated samples. T wo genes showed significant change (p<0.05) as a result o f each treatment. The bl600 gene increased in expression by 1.45 and 1.71 fold, and the secB gene decreased 1.72 and 1.79 fold as a result of water bath and V M compared to untreated samples respectively. The htgA (+1.56 fold), msbB (+1.90 fold), uspA (-1.70 fold) and yfiA (-3.57 fold) genes were significantly changed (p<0.05) by the water bath treatment compared to untreated samples, while these genes showed no significant change in V M treated E. coli. Any changes in the rest of the genes were not significant. Although a higher number of heat shock genes were altered due to water bath treatment, the change was less than two fold except in yfiA. None of the heat shock genes showed a significant difference between the two treatments.  5.3.4.2 Genes changed > two fold in both treatments compared to untreated cells Genes significantly altered in both treatments were studied to determine similarity in E. coli response to treatments (Tables 5.7 & 5.8). O f the 39 up-regulated genes, cysW, rrlD, trpT, ybaR were only induced by the treatments, but were not detected in the untreated sample. Of the  109  16 down-regulated genes, rrfG, rrfD, rrfE, rrfF, and ompF were the only genes that changed > 2 fold in both treatments.  5.3.4.3 Genes changed > two fold by water bath or V M treatments compared to untreated cells Genes that altered > 2 fold (p<0.05) due to water bath treatment in comparison to the untreated samples are shown in Tables 5.9 and 5.10.  The name and description of genes  changed > 2 fold (p<0.05) due to V M treatment compared to untreated samples are shown in Tables 5.11 and 5.12. Both treatments seem to affect genes involved in amino acid metabolism, membrane transport and translation as well as genes that encode for putative proteins with unknown functions.  5.3.4.4 Genes significantly changed in V M compare to water bath treatment A l l of the genes that were significantly different between the two treatments have been expressed less than two fold: 1.10-1.78 fold for down-regulated genes and 1.11-1.62 fold for upregulated genes (Tables 5.13 & 5.14).  The fliG, fecA, b2496, b3694 and ycgL genes were  induced by V M treatment, but were not expressed in the water bath treated E. coli. Conversely, the rscA, ydgB,fliiA,  b0538, b0878, b2660 and b2999 genes were not detected in V M treated E.  coli, but were present in the E. coli subjected to water bath treatment.  110  5.4.  Discussion  5.4.1. Heat shock response The heat shock response in E. coli is controlled at the transcriptional level by the sigma factor rpoH (sigma 32) and rpoE (sigma E). Although the expression level of rpoE and rpoH showed slight increases in both treatments, the change was not significant. At the same time htgA, a positive regulator for sigma 32, was expressed more in water bath treatment than in untreated cells, while uspA global regulatory gene for stress response was down-regulated in water bath treated E. coli and remained unchanged in V M treated cells compared to untreated cells. This suggests that the conditions employed in this study were not sufficient to stimulate the heat shock response. Thus no significant difference in expression level of E. coli heat shock genes was detected. The present data are in contrast with Chow & Tung (2000) who reported that heat shock proteins DnaK/J (Hsp70/40) are overproduced when E. coli cultures are exposed to a low frequency magnetic field (50Hz, 1 hour), while Nakasono and Saiki (2000) found no detectable change in protein synthesis of cells exposed to extremely low frequency (ELF) magnetic field (7.8-14 mT, 5-100 Hz) for (0.5-16 hours). One possible explanation is that the temperature less than or equal to 50°C for 3 minutes is not high enough to induce a heat shock response. In addition stimulated genes may have gone back to their normal condition during the 3-4 minute cooling period. The third explanation could be related to the presence of heat shock genes in cells at stationary phase of growth. Entering stationary phase of growth is considered a mild stress condition and accompanied by production of some heat shock proteins.  Stationary-phase cells of E. coli have the ability to survive  prolonged periods of starvation and have a strong multiple-stress resistance (Hengge-Aronis  ill  1996). Therefore, having a mild treatment along with enough time to recover from the stress may have decreased the relative expression level of stimulated genes.  5.4.2  Membrane structure and membrane transport system The cysW and ybaR genes, related to copper and sulfate transport system respectively,  were up-regulated in both treatments.  ompF, which encodes porins and is responsible for  dipeptide permease, was significantly down-regulated by both treatments.  Porins, which are  transmembrane proteins, associate to form small membrane holes about 1 nm in diameter for the diffusion of organic molecules through the outer membrane and into the periplasm (Madigan et al. 2003). The relative abundance of porins is regulated by the media osmotic activity and temperature (Nikaido & Vaara 1987). Chang and colleagues (2002) also reported that the outer membrane proteins encoded by ompT and ompF were down-regulated during growth arrest (Pratt & Silhavy 1996; Chang et al. 2002). In addition yejE, btuC, exuT, ycjO, ydiQ, yfcC and b0878 involved in the membrane transport systems for peptides, vitamin B12, putative S-transferase, sugar and A B C transporter system were down-regulated while fee A, which encodes for ferric dicitrate outer membrane receptor protein, was up-regulated in V M treated E. coli compared to E. coli exposed to water bath treatment. This suggested that transcription for genes involved in ion transfer was increased while transcription of genes involved in transfer of larger molecules including peptide, sugar and vitamin transport were decreased as a result of V M treatment. Nascimento and co-workers (2003), who reported a higher level of glucose transported into E. coli cells exposed to electromagnetic field (60 Hz, 8 hours, 28°C), suggested that electromagnetic field stimulated the periplasm-binding protein-dependent transport system. Liburdy and his group (1985) also found  112  that exposure of rabbit erythrocytes to microwaves (2450 M H z ) increased sodium passive transport only at membrane phase transition. The fimC, fimD and fimG genes, related to outer membrane protein, periplasmic chaperone and morphology of fimbriae, and fliG, which encodes for the flagellar motor switch, were expressed more in V M treated E. coli compared to water bath treated E. coli. The murG gene, that encodes for an enzyme involved in peptidoglycan biosynthesis, was expressed less in V M treated E. coli compared to water bath treated E. coli. Peptidoglycan present in the cell wall is responsible for mechanical strength and maintaining the shape of the cell (Singleton & Sainsbury 2000). This suggests that while genes related to membrane structure and transport system were affected by both treatments, the effect was greater as a result of microwave than the conventional heat treatment. This may lend credence to the dielectric cell-membrane rupture theory.  This theory hypothesizes that an external electric field is induced and causes an  additional trans-membrane electric potential to the normal potential of the cell, which could result in a voltage drop across the cell membrane and may be sufficient for pore formation, increased permeability, loss of cell integrity (Brunkhorst et al. 2000, Kozempel et al. 2000) or membrane rupture (Datta & Davidson 2000; Kozempel et al. 1998; Zimmermann et al. 1974).  5.4.3  Enzymatic activity The t ranscription 1 evel o f s ome g enes e ncoding f or e nzymes i nvolved i n c arbohydrate  metabolism including gloA (lactoylglutathione lyase), murD (glutamate ligase), glgC (glucose-1phosphate adenylyltransferase) and yrfE (putative A D P compounds hydrolase) was increased in V M treated E. coli compared to the water bath treated E. coli. The amyA (cytoplasmic alphaamylase) and murG (N-acetylglucosamine transferase) genes from the same group were  113  expressed more in water bath treated cells. In addition other genes encoding other enzymes including gpsA (glycerol-3-phosphate dehydrogenase), spoT (guanosine bis-pyrophosphate pyrophosphohydrolase) and ppiA (peptidyl-propyl isomerase A ) were expressed more in V M treatment than water bath treatment. On the other hand menD (2-oxoglutarate decarboxylase), sfsA (probable regulator for maltose metabolism) were down-regulated in V M treated E. coli compared to the water bath treated E. coli. Dreyfuss and Chipley (1980) also reported higher malate dehydrogenase, a-ketoglutarate dehydrogenase, cytochrome oxidase, and cytoplasmic ATPase activities and lower glucose-6-phosphate dehydrogenase  activity in sub-lethal  microwave (2450 MHz) irradiated S. aureus cells compared to water bath conventional heated cells at 46°C. Saffer and Profenno (1992) reported higher production of a chromophore  (A402.5)  as a result of higher beta-galactosidase activity in E. coli cells radiated with low-level microwave radiation (10 kW/kg).  Rebrova (1992) also indicated increased stimulation of fibrinolytic  enzymes in Bacillus firmus irradiated cells.  5.4.4  Ribosomal RNA The r ibosome i s a c omplex r ibonucleoprotein r esponsible f or t ranslation 0 f m essenger  RNAs into proteins. The E. coli ribosome is composed of 23S, 16S and 5S ribosomal R N A and about 53 proteins. Twenty-one of these proteins assemble with the 16S rRNA to form the 30S ribosomal subunit, while the other 31 proteins assemble with the 23S and 5S rRNA to form the 50S ribosomal subunit. Ribosomal proteins and rRNAs cooperate both in the assembly and activity o f the ribosome and ribosomal functions are dependent o n the presence o f the major R N A species (Madigan et al. 2003).  114  In this study the rrlD gene related to 23 S ribosomal R N A in rrnD operon was upregulated about 250 and 300 fold in water bath and V M treated E. coli respectively. At the same time, the expression level of genes related to 5S rRNA of rrnD, rrnE and rrnG operones including rrfD, rrfE, rrfF, rrfG in E. coli subjected to both treatments and rrfH and rrfA in water bath treated E. coli was decreased. rrsH gene related to 16S ribosomal R N A in rrnH operon also showed down-regulation in water bath treated E. coli. Hansen and colleagues (2001) investigated the level of r R N A before and after a heat shock from 3 0 to 43°C on exponential cells of wild-type Lactococous lactis subsp. cremoris. They reported that the amount of 23S rRNA and 16S r R N A decreased by the same rate through the heat shock. Rosenthal and Iandolo (1970) described a heat-induced dissociation of the 30S particle and degradation of 16S rRNA in Staphylococcus aureus at 55°C while, the 50S ribosomal subunit and 23 S rRNA appeared to be stable. Similar degradation patterns have been found in Salmonella enterica serovar Typhimurium due to heat treatment (Tolker-Nielsen et al. 1997). Khalil & Villota (1989) reported a selective destruction of the 16S R N A subunits after exposure of S. aureus cells to conventional heat whereas the destruction of the 16S R N A as well as 23S R N A subunits was reported with microwave sub-lethal heating (50°C, 30min). The present study showed down-regulation i n one gene related to 1 6S r R N A due to conventional water bath treatment of E. coli while that gene remained unchanged in V M treated E. coli compared to untreated cells. In addition, the expression of genes related to 5S rRNA as part of 50S ribosomal subunit was down-regulated in both V M treated and water bath treated E. coli. But the number of down-regulated genes (6 in water bath treated E. coli and 4 in V M treated E. coli) as well as the average fold change (42.5 in water bath treated E. coli compared to 17.5 in  115  V M treated E. coli) was higher in water bath treated cells. The effect of sub-lethal V M treatment on ribosomal R N A may be less than conventional water bath treatment. Simultaneously we observed a very high expression level of one gene related to 23S r R N A in V M treated and water bath treated E. coli while other related genes remained unchanged. This is in agreement with Lopez and colleagues (2002) who reported an increase in the occurrence of 20S R N A and 23S R N A in wild and industrial Saccharomyces cerevisiae after exposure to nutritional stress conditions. Those authors concluded that these R N A species could be used as indicators of yeast stress condition in industrial processing. Further studies are needed to be able to state that rrlD gene could be used as a stress related gene or stress indicator i n i i . coli. More down-regulation in 5S and 16S rRNA due to water bath treatment means that ribosomal subunits in V M treated cells were affected less, and thus are more stable. This could be a reason for less destruction at 50°C of V M treated E. coli compared to water bath treated E. coli.  5.4.5  Transfer RNA (tRNA) Transfer RNAs serve as adapter molecules matching amino acids to their codons on  mRNA (Singleton & Sainsbury 2000). The tRNA and its related amino acids are brought together by amino-acyl-tRNA synthetases, which ensure a particular tRNA receives its correct amino acid (Madigan et al. 2003). In this study, genes related to tRNAs specific to glutamine, tryptophan and leucine including glnV, trpT in V M and glnX, trpT and leuX in water bath treated E. coli were upregulated compared to untreated samples. T he glnA, which encode for glutamine synthetase,  116  was not detected in water bath treated cells while remaining unchanged in V M treated E. coli. In addition the comparison between treatments showed that the expression of glnS coding for glutaminyl-tRNA synthetase was significantly higher (1.34 fold) in V M treated E. coli compared to water bath treated E. coli. In V M treated cells induction of glnA gene could activate L glutamine synthesis by assimilation of ammonia and converting L-glutamate to L-glutamine. In addition, in the amino acyl-tRNA biosynthesis pathway, higher expression of glnS could increase the connection of the L-glutamine to tRNA (glutamine) and as a result increase production of Lglutaminyl-tRNA (glutamine).  Thus higher production of glutamine in V M treated E. coli  compared to water bath treated E. coli would be expected (Figure 5.1). Almost all the nitrogenous compounds in an enteric bacterium derive their nitrogen atoms from either glutamate or glutamine. About 88% of the cellular nitrogen in E. coli is derived from glutamate and the remaining 12% is derived from glutamine (Reitzer 1996). Thus, glutamine is one of the key intermediates in cellular nitrogen metabolism.  5.4.6  C e l l respiration  The E. coli respiratory chain contains a number of dehydrogenase and oxidase complexes (Poole & Ingledew 1987). Quinones are non-protein electron carriers which can diffuse freely through the membrane and mediate electron transfer between protein components of the respiratory chain, generally by transferring electrons from iron-sulfate proteins to cytochromes (Madigan et al. 2003; Gennis & Stewart 1996). E. coli can synthesize three types of quinones including ubiquinone and menaquinone and demethylmenaquinone (Gennis & Stewart 1996). The amount of quinone and menaquinone in the cell depends on growth conditions (Singleton & Sainsbury 2000) especially the presence of oxygen in the growth environment (Poole &  117  Ingledew 1987). Studies have shown that quinone is used for oxygen respiration, while both quinone  and  menaquinone  are  used  for  nitrate  respiration but  menaquinone  and  demethylmenaquinone are used for anaerobic respiration with acceptors other than nitrate (Gennis & Stewart 1996). In general, under aerobic conditions, ubiquinone is predominant while menaquinone is dominant at reduced oxygen levels (Wallace & Young 1977; Hollander 1976). The ubiB gene is one of the genes responsible for ubiquinone biosynthesis and was expressed 1.28 fold more in V M treated E. coli compared to water bath treated E. coli. On the other hand, menD is one among five genes (menA,B,C,D and E) necessary for menaquinone synthesis that were expressed 1.32 fold less in V M treated E. coli compared to water bath treated E. coli. fliG expression level showed 1.2 fold decrease in water bath treated E. coli as well, meaning flagellar motility was r educed. This is in agreement with Poole & Ingledew (1987) who reported mutation in quinone biosynthesis gives rise to immobility and lack of flagella. At the same time, in water bath treated E. coli, genes involved in energy metabolism through oxidative phosphorylation (ppa) and nitrogen metabolism (aspA) were not detected or were expressed less compared to untreated E. coli while these genes remained unchanged after V M treatment. Simultaneously, transcription levels for genes related to copper, sulfate and ferric, ions functioning in electron acceptors in anaerobic respiration were shown to be up-regulated for copper and sulfate in both V M treated and water bath treated E. coli, and ferric just in V M treated E. coli. These data suggest that although in both treatments E. coli responded to vacuum by higher expression in transcription level of genes related to anaerobic respiration, the evidence for the start of anaerobic respiration is higher for water bath treated E. coli than for V M treated E. coli.  118  5.5  Conclusion  This study was designed to investigate the effect of microwaves on cell stress response in a sub-lethal condition. The very first step was to check for heat shock genes as an indicator of heat stress and g eneral stress response. Although some of the heat shock genes were altered significantly in treated E. coli, in general the result of this experiment did not show any major change in heat shock gene expression levels. V M treatment had larger effects on genes related to membrane structure and membrane transport systems as well as the activity of enzymes related to metabolism of carbohydrates, lipids and amino acids. Meanwhile, the effect of conventional water bath treatment on ribosomal subunits was higher. Interestingly, although both treatments employed vacuum and signs of anaerobic r espiration w ould b e e xpected, w ater b ath t reated E. c oli s howed m ore e vidence a t transcriptional level for the start of anaerobic respiration.  119  Glutamate Metabolism L-glutamate" j  Aminoacyl-tRNA Biosynthesis glnA tRNA (Gin)  Nitrogen metabolism L-glutamine  vlnA  Ammonia  glnS  L-Glutaminyl-tRNA (Gin)  Figure 5.1. Simplified flow diagram of role of glnS and glnA in glutamine synthesis  120  Table  5.1. Treatment conditions for vacuum microwave (VM) and water bath under vacuum. Treatment  Final temperature  temperature ( ° C )  after c o o l i n g ( ° C )  1 2 3  49.6±0.22 49.910.32 49.910.99  30.0 28.3 30.0  1 2 3  49.712.89 49.911.58 50.410.25  27.6 30.1 30.0  Treatment  VM711W  Water bath under vacuum  121  Table 5.2. Sequence of primers for 16S and 23S rRNA used in this study (Affymetrix manual 2000). Name 16S r R N A Primers 16S1514 16S889 16S541 23S r R N A Primers 23S2878 23SEco2064 23SEcol595 23S539  Sequence 5' - C C T A C G G T T A C C T T G TT-3' 5' - T T A A C CTTGC G G C C G T A C T C - 3 ' 5' - T C C G A T T A A C G C T T G C A C C C - 3 ' 5' 5' 5' 5'  -CCTCA -CTATA -CCTGT -CCATT  CGGTT C A T T A GT-3' G T A A A GGTTC A C G G G - 3 ' G T C G G T T T G G GGT-3' A T A C A A A A G G TAC-3'  122  T a b l e 5.3.  Correlation among replicates for treated and untreated E. coli. r  Treatment  Untreated E. coli  Vacuum microwave treated E. coli  Water bath treated E. coli  Average  2  0 92 0 86 0 95  0.91+/-0.04  0 90 0 97 0 88  0.92+/-0.04  0 93 0 95 0 88  0.92+/-0.03  123  Table 5.4. E. coli array probe set signals from E. coli exposed to water bath under vacuum, vacuum microwave treatments, untreated stationary phase E. coli cells. Samples  Present call (%)  Absent call (%)  Marginal call (%)  Untreated E. coli  49.20+/-15.02  46.30+/-14.72  4.50+/-0.32  Water bath under vacuum  57.26+/-12.43  38.47+/-11.73  4.27+/-0.72  Vacuum microwave 711W  58.61+/-16.00  37.5+/-14.77  3.89+/-1.23  124  Table 5.5. Number of significantly up-regulated, down-regulated or unchanged genes (p< 0.05) between treatments. Up-regulated genes Total  Down-regulated genes  > 2 fold  Total  > 2 fold  Water bath compare to untreated  123  10  135  6  V M compared to untreated  109  12  91  15  V M compared to water bath  55  0  49  0  125  Table 5.6. List of previously known heat shock genes and their calls in untreated, water bath and vacuum microwave treated E. coli. Gene b No. Fold change Call Description name W-U V M - U VM-W untreated water V M bath o A Diadenosine tetraphosphatase ' ; stress response; complex P -1.34 -1.29 1.04 b0049 apaH operon P Possible chaperone P P -1.05 1.34 1.28 bl599 P Possible chaperone P P 1.18 M600 1.45f 1.71t P Recognizes a curved D N A sequence similarity to DnaJ ; P A 1.02 -1.09 blOOO cbpA 1.11 curved DNA-binding protein; functions closely related to DnaJ 1 23 P P -1.04 1.05 b2592 clpB 1.10 P ClpB protease, ATP dependent ; heat shock protein ' clpB protein (heat shock protein f84.1) P P -1.04 -1.22 1.18 b0437 clpP P ClpP ATP-dependent protease proteolytic subunit ; heat shock protein F21.5 ; ATP-dependent proteolytic subunit of clpAclpP serine protease P P 1.09 1.10 1.00 clpX b0438 P ClpX protease, which activates ClpP ; ATP-dependent specificity component of clpP serine protease ' , chaperone P P -1.52 -1.26 -1.21 b0598 cstA 7  p  1 2  1  2  2  1  2  5  1  2  3  1  2  ddg dksA  b2378 b0145  1.41 -1.32  1.67 -1.38  1.18 -1.05  P P  P P  dnaJ  b0015  -1.04  -1.14  -1.09  P  P  2  12  P P P  Starvation induced stress response protein ' Acetyltransferase ; putative heat shock protein ' High copy suppresses muK and TS growth and filamentation of dnaK mutant ; dnaK suppressor protein Chaperone with DnaK ' ' ; D N A chain elongation ; stressrelated D N A biosynthesis , responsive to heat shock ; heat shock protein ' HSP-70-type molecular chaperone ' ' , with DnaJ ; D N A biosynthesis ; stress-related heat-shock D N A biosynthesis ' ; ATP-regulated binding and release of polypeptide substrates'; auto-regulated heat shock protein ; dnaK protein (heat shock protein 70) (HSP70) Stress response DNA-binding protein; starvation induced resistance to H2O2 phase ; global regulator, starvation condition 1  2 5  1  P  2  1 2 3  1  1  dnaK  b0014  1.10  -1.02  -1.12  P  P  1  2 3  P  1 2 3  3  1 2  2  dps  b0812  -1.02  -1.29  -1.27  P  P  5  P  1  2  126  1  Table 5.6. Continued. Call Fold change bNo. Gene name W-U" V M - l T VM-W"* untreated water bath A A b0140 ecpD  Description VM A  Possible pilin chaperone ; probable pilin chaperone similar to PapD Biosynthesis of fimbriae; periplasmic chaperone for type 1 fimbriae ' Anti-sigma F factor (FliA) '' ; regulator of FlhD; also known as RflB protein Transcription sigma factor for class 3a and 3b operons; regulation of late gene expression ; flagellar biosynthesis; alternative sigma factor 28; regulation of flagellar operons Cell division and growth; heat inducible , cell division protein ' Heat shock protein ' ; mutant survives induction of prophage lambda; stimulates DnaK ATPase; nucleotide exchange function ; phage lambda replication; host D N A synthesis; heat shock protein ' ; protein repair heat shock protein grpE (heat shock protein b25.3) (HSP24) Subunit of protease specific for phage A, CII repressor ; HflX GTpase, putative ; GTP - binding subunit of protease specific for phage lambda ell repressor 1  2  fimC  b4316  -1.08  1.45  1.12  P  P  P  1 2  flgM  A  bl071  A  A  2  2  fliA  A  bl922  A  A  1  2  ftsJ  b3179  1.47  1.46  -1.01  P  P  P  1  2 3  grpE  b2614  -1.00  1.02  1.02  P  P  P  1 2  1  2 3  2  5  hflX  b4173  -1.32  1.02  1.35  P  A  P  3  1  2  hfq hscA  b4172 b2526  -0.97 1.21  1.28 1.28  1.24 1.02  P A  P P  P P  Host factor I for bacteriophase Q (3 replication ' Stress response gene ; Hsp70 family; heat shock protein ' , chaperone ; heat shock protein hscA (HSC66) Heat-inducible; regulatory gene , heat shock protein hslJ ' Heat-inducible ATP-dependent protease HslVU; heat shock protein D48.5 :heat shock protein h s l V U ' ' , ATPase subunit ' , homologous to chaperones Heat shock regulon ; heat shock protein h s l V U ' ' , proteasome-related peptidase subunit ' 1 3  1  1 2  2  hslJ hslU  bl379 b3931  1.01 -1.48  1.10 -1.26  1.09 1.29  P P  P P  P P  5  1  2 4  1  2  2 3  hslV  b3932  1.80  1.22  1.04  P  P  P  1  5  2  2 3  127  3  2  3  5  Table 5.6. Continued. Gene b No. Fold change name W-U V M - U V M - W htgA  b00T2  1.56|  L21  -1.29  htpG  b0473  -1.41  -1.06  1.40  Call Description untreated water V M bath P P P Positive regulator for sigma 32 heat shock promoters ; heat shock protein htgA (heat shock protein htpy) P A P Heat shock protein C 6 2 . 5 ; chaperone'; chaperone Hsp90 ' ;heat shock protein htpG (high temperature protein G) (heat shock protein c62.5) P P P Protein expressed as heat shock regulon member ; heat shock protein, integral membrane protein ; probable protease htpX (heat shock protein htpX) A A A Periplasmic serine protease Do; heat shock protein HtrA P P P Not under heat shock regulation; membrane protein affecting cell division, growth, and high-temperature survival ; heat shock protein A A A Essential for growth at high temperature, under sigma 32 (heat shock) regulation ; heat shock protein h t r C ; heat shock protein C A A A Outer membrane usher protein htrE precursor (heat shock protein E) ; probable outer membrane porin protein involved in fimbrial assembly ; Sequence homology with pilin protein PapC A A A Formate-sensing regulator for hyf operon ; putative 2component regulator, interaction with sigma 54 P P P Chaperone, heat-inducible protein of HSP20 family ; heat shock protein Inclusion body protein A ; 16 kD heat shock protein A P P P Chaperone, heat-inducible protein of HSP20 family ; heat shock protein Inclusion body protein B ; 16 kD heat shock protein B P P P Protein induced by acid, independent of SoxRS regulation ; pH-inducible protein involved in stress response 2  5  1,2,5  2 3  5  htpX  bl829  1.04  1.08  1.04  1  2,3  5  htrA htrB  b0161 bl054  2  1.27  1.22  -1.04  1  htrC  b3989  1  2  5  htrE  bl39  5  2  1  hyfR  b2491  1  2  ibpA  b3687  -1.31  1.03  1.34  1  2  3  2  3  5  ibpB  b3686  -1.46  -1.12  1.30  1  5  inaA  b2237  -1.16  1.18  1.36  1  2  128  Table 5.6. Continued. Description Call Fold change bNo. Gene name V M W-U* V M - l T VM-W*" untreated water bath 1.14 P P P DNA-binding, ATP-dependent protease L A ' ' ; Ion mutants 1.21 1.38 Ion b0439 1  1  2  2  3  3  *  "  form long cells ; heat shock K-protein ' ; nucleic acid binding heat shock protein Lysyl tPvNA synthetase, inducible ' ; heat shock protein ;nucleic acid-binding heat shock protein A(2)- Isopentenyl pyrophosphate tRNA-adenosine transferase ' ; 2-methylthio-N6-isopentyladenosine tRNA hypermodification GroEL, chaperone Hsp60 ' ; peptide-dependent ATPase; heat shock protein GroES, 10 KDalton chaperone binds to Hsp60 ' in pres. M g ATP, suppressing its ATPase activity Role in outer membrane structure or function ; suppressor of htrB, heat shock protein Nitrate reductase delta-subunit ' ; chaperone ; nitrate reductase 1, delta subunit, assembly function Na+/H+ antiporter ' ; stress response to high salinity and p H ; pH dependent Phosphoprotein phosphatase involved in signalling protein misfolding ' ; heat shock regulon 'protein phosphatase 1 Phosphoprotein phosphatase involved in signalling protein misfolding; heat shock regulon ; protein phosphatase 2 R N A polymerase, sigma 70 ' ; sigma suc-unit, initiates most exponential phase transcription R N A polymerase, sigma E-subunit ' ' , high-temperature transcription ; heat shock and oxidative stress ' R N A polymerase,sigma 32-subunit ' , heat-shock transcription ; regulation of proteins induced at high temperatures 4  lysU  b4129  1.04  1.13  1.08  A  P  A  1 2  2  miaA  b4171  -1.02  1.07  1.09  P  P  P  2  4  3  1  mopA  b4143  -1.31  -1.36  -1.04  P  P  P  2 3  2  mopB  b4142  1.04  -1.19  -1.24  P  P  P  2 3  2  msbB  bl855  1.90|  1.65  -1.15  A  P  P  1  2  narJ  A  bl226  A  A  1 2  1  2  nhaA  b0019  1.62  1.22  -1.33  A  A  P  1 2  1  2  pphA  A  bl838  A  A  1 2  pphB  b2734  1.06  1.25  1.19  P  P  P  1  2  1  rpoD  b3067  -1.13  1.06  1.20  P  P  P  2  3 1  1  rpoE  b2573  1.90  1.41  -1.34  P  P  P  1 2 3  1  rpoH  b3461  2.12  1.68  -1.27  P  P  P  1  2  129  2 3  1 2  Table 5.6. Continued. Description Fold change Call bNo. Gene name W TJ* V M - U " VM-W*" untreated water V M bath P P P R N A polymerase ' sigma S-subunit , sigma S (sigma38) 1.48 -1.16 b2741 rpoS 1.71 factor stationary phase ; synthesis of many growth phase related proteins P Membrane protein ' , negative regulator of sigma E ' ' ; -1.29 P P b2572 1.29 -1.00 rseA P P Binds rseA, negative regulation of sigma E ; regulates activity 1.04 P rseB b2571 -1.00 1.03 of sigma-E factor A A A Deletion does not affect sigma E activity ; sigma-E factor, b2570 rseC negative regulatory protein P P Protein export ' ; chaperone SecB^molecular chaperone; may -1.04 P secB b3609 -1.72| -1.79f bind to signal sequence A Salmonella fimbriae gene homolog ; putative chaperone A A b0531 sfmC P P A Stress response protein ; regulator of transcription; stringent 1.08 -1.17 sspA b3229 1.26 starvation protein A 1 2 P P 1.37 1.10 P sspB b3228 1.25 Stress response protein ; stnngent starvation protein B A A A stpA b2669 Hns-like protein ' , suppresses T4 tf mutant ; DNA-binding protein; chaperone activity; R N A splicing? -1.15 A P A Suppresses groL mutation and mimics effects of gro 1.02 sugE b4148 1.18 1 2  1  2  1  2  1 2  1  2  3  1  2  1  2  1 2  2  1  2  1  2  1 2  1  2  1  suhB  b2533  -1.32  -1.57  -1.19  P  A  A  tig  b0436  -1.49  -1.37  1.08  P  P  P  2  overexpression ; suppresses groEL, may be chaperone Inositol monophosphate ; enhances synthesis of sigma32 in mutant; extragenic suppressor, may modulate RNAse III lethal action Trigger factor; chaperone ' ; a molecular chaperone involved in cell division D N A topoisomerase type I, Q protein ; Topoisomerase I, Omega protein 1 Global regulatory gene for stress response ; broad regulatory function? Function unknown ;putative dnaK protein Function unknown ; putative chaperone 1  2  1 2  2  topA  bl274  -1.10  1.06  1.17  P  P  P  3  1  uspA  b3495  ybeW ybgP  b0650 b0717  -1.70t  -1.36  1.25  P  P  P  A A  A A  A A 130  1  1  1  2  2  Table 5.6. Continued. Call Fold change bNo. |Gene name W-U* VM-U** V M - W * " untreated water V M bath A A A ycaL b0909 A A A ycbF b0944 A A A b0939 ycbR P P A -1.31 -1.47 yciM M280 1.13 A A A yegD b2069 A A A b2110 yehC A A A b2336 yfcs P P P 1.23 b2597 -3.57| -2.91 yM A A A b3215 yhcA A A A yral b3143 P P P -1.13 -1.06 1.07 yrfli b3400  Description  Function unknown ; putative heat shock protein Function unknown ; putative chaperone Function unknown ; putative chaperone Function unknown ; putative heat shock protein Function unknown ; putative heat shock protein Function unknown ; putative chaperone Function unknown ; putative chaperone Function unknown ;putative yhbH sigma 54 modulator Function unknown ; putative chaperone Function unknown ; putative chaperone Binding nucleic acid-heat shock protein ; orf, hypothetical 1  2  1  2  1  2  1  2  1  2  1  2  1  2  1  2  1  2  1  2  4  2  1  protein ; Function unknown  http://genolist.pasteur.fr/Colibri/ Affymetrix gene chip data base Richmond et al. 1999 Korberetal. 1999 http://www.genome.ad.jp/kegg/ f significant genes (p<0.05) W-U= water bath treatment compare to untreated E. coli V M - U = VM711W compare to untreated E. coli *** V M - W = VM711W compare to water bath treatment P= present, detected A = absent, not detected  2  3  4  5  6  7  131  Table 5.7. Genes displaying up-regulation in vacuum microwave and water bath under vacuum treated cells compared to untreated stationary phase E. coli cells. Gene name  b no  aceE  b0114  Fold change VM-lT W-U* 1.71  1.83  untreated P  Call VM P  J  Description water bath P  pyruvate dehydrogenase (decarboxylase component) N-acetyl-gammaglutamylphosphate reductase putative transposase putative DEOR-type transcriptional regulator orf, hypothetical protein possible chaperone orf, hypothetical protein orf, hypothetical protein putative oxidoreductase sodium-calcium/proton antiporter sulfate transport system permease W protein [2FE-2S] ferredoxin, electron carrier protein hydroxamate-dependent iron uptake, cytoplasmic membrane component probable hydroxyacylglutathione hydrolase NAD-dependent 7alphahydroxysteroid dehydrogenase, dehydroxylation of bile acids multiple antibiotic resistance protein; repressor of mar operon suppressor of htrB, heat shock protein 1  argC  b3958  1.36  1.69  A  P  4  P  1  b0257 b0845  1.67 1.7  1.83 1.92  P A  P P  P P  1  1  chaA  bl342 bl600 M680 bl754 b2899 bl216  1.39 1.71 1.54 1.55 1.52 1.36  1.31 1.45 1.44 1.59 1.35 1.51  A A P A P A  P P P P P P  P P P P P P  1  1  1 1  1  1  cysW  b2423  2.48  2.33  A  P  P  1  fdx  b2525  1.24  1.22  A  P  P  1  jhuB  b0153  1.1  1.22  A  P  P  1  gloB  b0212  1.77  1.82  P  P  P  1  hdhA  bl619  1.68  1.75  A  P  P  1  marR  bl530  1.46  1.91  A  P  P  1  msbB  bl855  1.65  1.9  A  P  P  1  132  Table 5.7. Continued. Gene name  b no  murG  b0090  Fold change VM-U  **  1.31  W-U  *  1.44  untreated  Call VM P  A  Description water bath P  UDP-Nacetyl glucosamine :Nacetylmuramyl(pentapeptide) pyrophosphorylundecaprenol N acetylgluco samine transferase 4-amino-4-deoxychorismate lyase phosphatidylglycerophospha tase protease II 23 S r R N A of rrnD operon Tryptophan tRNA inner membrane transport protein orf, hypothetical protein putative ATPase orf, hypothetical protein orf, hypothetical protein orf, hypothetical protein orf, hypothetical protein putative transport protein orf, hypothetical protein orf, hypothetical protein putative transport putative membrane protein IS2 hypothetical protein orf, hypothetical protein orf, hypothetical protein 1  pabC  M096  1.76  1.87  P  A  P  1  PgpA  b0418  1.34  1.48  P  P  P  1  ptrB rrlD trpT yaaJ  bl845 b3275 b3761 b0007  1.51 299.90 3.95 1.64  1.44 250.41 4.11 1.71  P P P P  A A A P  P P P P  1  1  1  1  P P 1.31 b0232 1.39 yaJN P 4.14 A b0484 3.19 ybaR P P 1.46 ybdF b0579 1.41 P P 1.29 ybgA b0707 1.47 P P 1.30 b0792 1.41 ybhR A P 1.70 ybiM b0806 1.28 P P 1.30 b0808 1.40 ybiO A P 1.84 b l l l 2 2.27 ycfR P 1.54 P b l l 7 7 1.61 ycgJ A P 1.60 b2170 1.33 yeiO P P 1.49 1.35 yhil b3487 A P b4272 1.52 1.55 yi21J P P 1.95 yjcB b4060 1.86 P P 1.30 yqjA b3095 1.19 Affymetrix web site W-U= water bath treatment compare to untreated E. coli ** V M - U = VM711W compare to untreated E. coli P= present, detected A = absent, not detected 3  4  133  P P P P P P P P P P P P P P  1  1  1  1  1  1  1  1  1  1  1  1  1  1  Table 5.8.  Genes displaying down-regulation in vacuum microwave and water bath under  vacuum treated cells compared to untreated E. coli cells. Gene name  b no  ft>P folE gW  b4232 b2153 b2240  Fold change VM-U 1.65 1.51 1.64  **  W-U  *  1.85 1.56 1.65  Call untreated V M P P A  P P P  Description water bath P P P  fructose-bisphosphatase GTP cyclohydrolase I sn-glycerol-3-phosphate permease ATP-binding component of histidine transport outer membrane protein l a (IafrF) phosphoribosylaminoimida zole-succinocarboxamide synthetase 5S r R N A of rrnD operon 5S r R N A of rrnE operon 5S r R N A of rrnD operon 5S r R N A of rrnG operon protein export; molecular chaperone; may bind to signel sequence outer membrane protein induced after carbon starvation orf, fragment 2 methyl-accepting chemotaxis protein I, serine sensor receptor putative filament protein orf, hypothetical protein 1  1  1  hisP  b2306  1.54  1.31  P  P  P  1  ompF  b0929  2.37  2.04  P  P  P  1  purC  b2476  1.75  1.93  P  P  P  1  rrfD rrfE rrfF rrfG secB  b3274 b4010 b3272 b2588 b3609  29.90 9.85 19.42 11.22 1.788  75.75 24.12 71.88 74.23 1.72  P P P P P  P P P P P  P P P P P  1  1  1  1  1  sip  b3506  1.627  1.41  P  P  P  1  smf_2 b3285 tsr b4355  1.43 1.38  1.40 1.35  P P  P P  P P  1  1  ynaF  bl376 b4216  1.84 1.53  2.10 1.64  P P  P P  y}P Affymetrix web site W-U= water bath treatment compare to untreated E. coli ** V M - U = VM711W compare to untreated E. coli P= present, detected A = absent, not detected I  3  4  134  P P  1  1  Table 5.9. Genes down-regulated (>2 fold) in water bath under vacuum treated E. coli compared to untreated stationary phase E. coli cells (p<0.05). Gene name aspA glnA  Fold Call number change untreated water bath P b4139 2.38 A 3.52 P b3870 4  Description  aspartate ammonia-lyase (aspartase) ' glutamine synthetase ' (glutamate— ammonia ligase) glycerol uptake facilitator protein , facilitated diffusion of glycerol glycerophosphoryl diester phosphodiesterase periplasmic precursor (glycerophosphodiester phosphodiesterase) ' outer membrane protein F precursor (outer membrane protein la, ia, or B ) ' , outer membrane protein l a (Ia;b;F) hatase ' 5S ribosomal R N A 5S rRNA of rrnA operon 5S ribosomal R N A 5S rRNA of rrnD operon 5S ribosomal R N A , 5S rRNA of rrnE operon , 5S rRNA of rrnD 1 2  1  glpF  b3927  2.16  P  P  1  2  gipQ  b2239  2.58  P  P  1  l 2  ompF  b0929  2.04  P  P  ppa rrfA  b4226 b3855  1.96 3.70  P P  A P  2  1 2  1  2  rrfD  b3274  75.75  P  P  1  2  rrfE  b4010  24.12  P  P  1  2  rrfF  b3272  71.88  P  P  rrfG  b2588  74.23  P  P  operon 5S ribosomal R N A , 5S rRNA of rrnG operon 5S ribosomal R N A ,, 5S rRNA of rrnH operon 16S ribosomal R N A , 16S R N A of rrnH operon 12.7 kD protein in sfhB-pheL intergenic region (URF1) (ORFS54) , putative yhbH sigma 54 modulator 1  2  rrfH  b0205  5.79  P  P  1  2  rrsH  b0201  2.64  P  P  1  2  yM  b2597  3.57  P  P  1  2  P ynaF bl376 2.09 http: //www. genome. ad.j p/kegg/ Affymetrix gene chip data base P= present, detected A = absent, not detected  P  • 1 2  putative filament protein '  2  3  4  135  Table 5.10. Genes up-regulated (>2 fold) in water bath under vacuum treated E. coli compared to untreated stationary phase E. coli cells (p<0.05).  Gene name cysW  b no. bl657 b2423  Fold change 1.99 2.33  Call untreated water bath P A P A 4  J  Description putative transport protein ' sulfate transport system permease protein , sulfate transport system permease W protein glutamine tRNA 2 leucine tRNA 5 , Leucine tRNA5 (amber [UAG] suppressor) pyruvate dehydrogenase complex repressor , transcriptional regulator for pyruvate dehydrogenase complex colanic acid capsular biosynthesis activation protein A , positive regulator for ctr capsule biosynthesis, positive transcription factor 23S ribosomal R N A , 23S rRNA of rrnD operon tryptophan t R N A ' probable copper-transporting ATPase , putative ATPase hypothetical 22.9 kD protein in purB-icdA intergenic region (ORF- 23) , orf, hypothetical protein hypothetical 13.0 kD protein in ssb-soxS intergenic region (Fl 16) \ orf, hypothetical protein hypothetical 44.4 kD protein in argl-valS intergenic region , orf, hypothetical protein 1 2  1  2  glnX leuX  b0664 b4270  5.67 2.29  A A  P P  pdhR  b0113  2.59  A  P  1,2  1  1  2  rcsA  bl951  2.05  A  P  1  2  rrlD  b3275  250.41  A  P  1  2  trpT ybaR  b3761 b0484  4.11 4.14  A A  P P  1  2  1  2  ycfC  bll32  1.98  A  P  l  2  yjcB  b4060  1.95  P  P  2  b4257  2.10  A  P  1  2  2 3 4  http://www.genome.ad.jp/kegg/ Affymetrix gene chip data base P= present, detected A = absent, not detected  136  Table 5.11.  Genes down-regulated (>2 fold) in V M treated cells compared to untreated  stationary phase E. coli cells (p<0.05). Gene name ompF  b no. bl746 b0929  Fold change  Call untreated V M  2.27 2.37  P P  A P  Description putative aldehyde dehydrogenase ' outer membrane protein F precursor (outer membrane protein la, ia, or B) outer membrane protein l a (Ia;b;F) 5S ribosomal R N A , 5S rRNA of rrnD operon 5S ribosomal R N A , 5S rRNA of rrnE operon 5 S ribosomal R N A , 5 S rRNA of rrnD operon 5S ribosomal R N A , 5 S rRNA of rrnG operon 2  rrfD  b3274  29.90  P  P  1  2  rrfE  b4010  9.85  P  P  1  2  rrfF  b3272  19.42  P  P  1  2  rrfG  b2588  11.22  P  P  1  2  2  3  4  http://www.genome.ad.jp/kegg/ Affymetrix gene chip data base P= present, detected A = absent, not detected  137  Table 5.12. Genes up-regulated (>2 fold) in V M treated cells compared to untreated stationary phase E. coli cells (p<0.05). Gene name cysW  b no.  b2423  Fold change  Description  Call untreated V M  2.48  A  4  P  J  sulfate transport system permease protein , sulfate transport system permease W protein glutamine tRNA 2 hydrogenase isoenzymes formation protein hypE , plays structural role in maturation of all 3 hydrogenases 23S ribosomal R N A , 23S rRNA of rrnD operon soxR protein , redox-sensing activator of soxS tryptophan t R N A probable copper-transporting ATPase , putative ATPase hypothetical 8 .8 k D protein i n ndh-mfd intergenic region , orf, hypothetical protein 1  2  glnV hype  b0665 b2730  3.16 2.53  A A  P P  1,2  1  2  rrlD  b3275  299.90  A  P  1  2  soxR  b4063  1.98  A  P  1  2  trpT ybaR  b3761 b0484  3.95 3.19  A A  P P  1,2  1  2  ycfR  blll2  2.27  A  P  1  2  ycgB yhbU  bll88 b3158  2.04 3.68  A A  P P  •12  putative sporulation protein ' putative protease in sohA-mtr intergenic region precursor , putative collagenase 1  2  3  4  http://www.genome.ad.jp/kegg/ Affymetrix gene chip data base P= present, detected A = absent, not detected  138  2  Table 5.13.  E. coli genes down-regulated in V M treatment compared to water bath under  vacuum treatment (p<0.05). Gene name  b no.  b0878 bl844  Fold change 1.78 1.40  Call water bath P P J  Description VM A P  4  putative membrane protein ' exodeoxyribonuclease X , orf, hypothetical protein putative sensory transduction regulator ' putative oxidoreductase, major subunit, 1  2  b0538 M501  amyA  M837 b2999 bl927  1.40 1.34 1.37 1.26 1.22  P P P P P  A P P A P  1 2  •12  putative oxidoreductase, major subunit ' orf , hypothetical protein ' orf , hypothetical protein ' cytoplasmic alpha-amylase ' , (1,4alpha-D-glucan glucanohydrolase) vitamin B i transport permease protein ' deoxyribose operon repressor , transcriptional repressor for deo operon, tsx, nupG hexuronate transporter '' flagellar biosynthesis , fhiA protein multiple antibiotic resistance protein ' ; repressor of mar operon , marR 2-oxoglutarate decarboxylase; SHCHC synthase , 2-oxoglutarate decarboxylase / 2-succinyl-6-hydroxy-2,4cyclohexadiene-1 -carboxylate synthase UDP-N-acetylglucosamine:Nacetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N acetylglucosamine transferase ' colanic acid capsular biosynthesis activation protein A , positive regulator for ctr capsule biosynthesis, positive transcription factor probable regulator for maltose metabolism , sugar fermentation stimulation protein , hypothetical 12.8 kD protein in speE-gcd intergenic region precursor , orf, hypothetical protein hypothetical 34.6 kD protein in panD-panC intergenic region , orf, hypothetical protein 2  1 2  2  1 2  1 2  1  btuC deoR  bl711 b0840  1.24 1.46  P P  P P  1 2  2  1  2  exuT fhiA marR  b3093 b0229 bl530  1.21 1.15 1.31  P P P  P A P  2  2  1  l  2  menD  b2264  1.32  P  P  2  1  2  1  murG  b0090  1.10  P  P  1 2  rcsA  bl951  1.41  P  A  1  2  sfsA  b0146  1.40  P  P  1  yacC  b0122  1.24  P  P  1  2  yadD  b0132  1.37  P  P  2  139  Table 5.13. Continued. Gene name ybjD  b no.  b0876  Fold change 1.48  Call water bath P  Description VM P  orf, hypothetical protein , hypothetical 63.6 kD protein in aqpZ-cspD intergenic region orf, hypothetical protein , hypothetical 51.8 kD protein in phoH-csgG intergenic region multiple sugar transport system permease protein , putative bindingprotein dependent transport protein hypothetical oxidoreductase in pntArstA intergenic region , putative oxidoreductase putative transport protein , putative electron transfer flavoprotein subunit ydiq orf, hypothetical protein , hypothetical 63.2 kD protein in gapA-rnd intergenic region hypothetical 8.7 kD protein in gapA-rnd intergenic region , orf, hypothetical protein 2  1  ycdT  M025  1.63  P  P  2  1  ycjO  bl311  1.57  P  P  1  2  ydgB  M606  1.60  P  A  1  2  ydiQ  bl697  1.26  P  P  2  1  yeaJ  bl786  1.21  P  P  1  yeaQ  bl795  1.30  P  P  1  2  yeiG yejE  b2154 b2179  1.55 1.19  P P  P P  12  1  putative esterase ' (EC 3.1.1.-) peptide transport system permease protein , putative transport system permease protein putative S-transferase ' hypothetical 48.6 kD protein in alpAgabP intergenic region , orf, hypothetical protein hypothetical 36.3 kD protein in dsbApolA intergenic region , putative endonuclease hypothetical 28.7 kD protein in GJJPfdrA intergenic region , orf, hypothetical protein orf, hypothetical protein , hypothetical 17.8 kD protein in cobb-potB intergenic region 1  2  yfcC ygaF  b2298 b2660  1.16 1.21  P P  P A  1 2  1  2  yihG  b3862  1.32  P  P  1  2  ylbA  b0515  1.47  P  P  1  2  ymfA  bll22  1.39  P  P  2  1  140  Table 5.13. Continued. Gene name ymfN  b no.  bll49  Fold change 1.21  Call water VM bath P P  Description  orf, hypothetical protein, hypothetical 50.9 kD protein in inte-pin intergenic region hypothetical 16.0 kD protein in gntR-ggt intergenic region ( 0 1 3 8 ) o r f , hypothetical protein 1  yrhA  b3443  1.60  P  A  2  2 3 4  http://www.genome.ad.jp/kegg/ Affymetrix gene chip data base P= present, detected A = absent, not detected  141  Table 5.14. E. coli genes up-regulated in V M treatment compared to water bath under vacuum treatment (p<0.05). Gene name  b no.  Fold change  b2496 b2174 b2511 b2595 b2689 b3051 b3694  1.29 1.16 1.29 1.20 1.32 1.22 1.48  Call water VM bath A P P P P P A P P P P P P A 4  J  Description  putative D N A replication factor '  1 2  r2  12  orr, hypothetical protein ' putative GTP-binding factor ' orf , hypothetical protein '  1 2  2  1 2  r2  12  orr, hypothetical protein ' putative membrane protein ' putative F A D A-type transcriptional regulator ' repressor of arg regulon; cer-mediated site specific recombination , arginine repressor ferric citrate outer membrane receptor protein , outer membrane receptor; citratedependent iron transport, outer membrane receptor periplasmic chaperone, required for type 1 fimbriae , chaperone protein fimC precursor outer membrane protein; export and assembly of type 1 fimbriae, interrupted , outer membrane usher protein fimD precursor fimbrial morphology , fimG protein precursor flagellar motor switch protein fliG , flagellar biosynthesis, component of motor switching and energizing, enabling rotation and determining its direction glucose-1 -phosphate adenylyltransferase ' glutamine tRNA synthetase , glutaminyltRNA synthetase 1 2  1 2  argR  b3237  1.16  P  P  2  1  fecA  b4291  1.33  A  P  1  2  fimC  b4316  1.58  P  P  2  1  fimD  b4317  1.54  P  P  2  1  JimG  b4319  1.62  P  P  2  1  fliG  bl939  1.27  A  P  1  2  glgC  b3430  1.33  P  P  1  glnS  b0680  1.34  P  P  2  2  1  gloA gpsA  bl651 b3608  1.22 1.47  P P  P P  12  lactoylglutathione lyase ' glycerol-3 -phosphate dehydrogenase (NAD+) ' regulator of ftsl, penicillin binding protein 3, septation function , rod shapedetermining protein mreB 1 2  mreB  b3251  1.33  P  P  1  142  Table 5.14. Continued. Gene name  b no.  murD b0088  Fold change 1.45  Call water VM bath P P  Description  , TJDP-N-acetylmuramoylalanine-Dglutamate ligase ' (UDP-Nacetylmuranoyl-L-alanyl-D-glutamate synthetase) transcriptional regulatory protein ' phoP peptidyl-prolyl cis-trans isomerase A ' precursor (ppiase A ) (rotamase A) ' ' (cyclophilin A ) guanosine-3',5'-bis(diphosphate) 3'pyrophosphohydrolase , (p)ppGpp synthetase II; also guanosine-3 ,5 -bis pyrophosphate 3 -pyrophosphohydrolase NAD(P)H-flavin reductase (ferrisiderophore reductase C) ,(TN[ADPH:flavin oxidoreductase) orf, hypothetical protein , hypothetical 12.4 kD protein in minC-shea intergenic region orf, hypothetical protein , peptide methionine sulfoxide reductase orf, hypothetical protein , hypothetical 24.1 kD protein in metC-sufl intergenic region putative transcriptional regulator L Y S R type , hypothetical transcriptional regulator in argR-cafA intergenic region orf, hypothetical protein , hypothetical 14.5 kD protein in prkB-CRP intergenic region (F134) putative methylase , orf, hypothetical protein orf, hypothetical protein , hypothetical 10.3 kD protein in ftsY-nikA intergenic region orf, hypothetical protein , hypothetical 62.3 kD protein in gltS-selC intergenic region putative glycoprotein/receptor , hypothetical 17.5 kD protein in mdoBdnaC intergenic region precursor (protein P-18)(F165) 1 2  1  phoP ppiA  M130 b3363  1.39 1.24  P P  P P  1 2  1  1  1  2  2  1  spoT  b3650  1.41  P  P  1  2  ubiB  b3844  1.28  P  P  1,2  ycgL  bll79  1.35  A  P  2  2  1  yeaA  bl778  1.15  P  P  2  1  yghB  b3009  1.11  P  P  2  1  yhcS  b3243  1.41  P  P  2  1  yhfA  b3356  1.27  P  P  2  1  yhhF  b3465  1.31  P  P  1  2  yhhL  b3466  1.22  P  P  2  1  yicH  b3655  1.17  P  P  2  1  yjM  b4360  1.25  P  P  2  1  143  Table 5.14. Continued. Gene name  b no.  Fold change  ytfB  b4206  1.35  Call VM water bath P P  Description  orf, hypothetical protein , hypothetical 24.9 kD protein in rplI-cpdB intergenic region (F224) orf, hypothetical protein , A D P compounds hydrolase 2  1  yrfE  b3397  1.53  P  P  2  1  1,_..._.// 2 3 4  http: //www. genome .ad.jp/kegg/ Affymetrix gene chip data base P= present, detected A = absent, not detected  144  CHAPTER SIX ESCHERICHIA  COLI  T R A N S C R I P T O M E IN L A T E - L O G A N D MID-  S T A T I O N A R Y P H A S E OF G R O W T H  145  6.1  Introduction Bacteria in natural environments are constantly challenged by the need to adapt to  changes in nutrient availability and stress conditions. Bacterial cells growing in an optimal condition are not an exception. They are also exposed to a continuous change of environment due to constant consumption of nutrients and accumulation of waste products (Singleton & Sainsbury 2000). Thus referring to a bacterial cell without mentioning their growth condition is as meaningless as talking about them without specifying their strains or their stage of growth (Neidhardt & Umbarger 1996). Most researchers use the mid-logarithmic phase as their experimental control for studying cell physiology or response either at the transcription or translation level, because cells in this stage are in a steady state and well-defined physiological phase, thus the experimental variability can be minimized and identified easily (Conway & Schoolnik 2003). On the other hand, in nature, bacterial cells spend most of their life under conditions in which the amount of available nutrients is limited and they rarely encounter an environment that allows exponential growth. In addition, in the food industry the focus is on stationary phase bacteria that are able to survive stress conditions better than cells in the logarithmic phase.  The ability of stationary  phase cells to survive prolonged periods of starvation as well as their higher resistance to variable stress conditions compared to cells in exponential phase of growth has been demonstrated (Hengge-Aronis 1996). The stationary-phase influences the entire cell physiology (Hengge-Aronis 1996). In the log phase cells the primary metabolisms such as energy metabolism and synthesis of cell components are the most active metabolism while in stationary phase cells the secondary metabolisms are dominant (Singleton & Sainsbury 2000). Thompson and colleagues (2003)  146  have studied growth-phase-dependent density D N A microarray.  gene expression in Helicobacter pylori using a high-  They conducted their experiment on cells during the late log-to-  stationary phase and reported changes between two stages of growth in genes involved in iron homeostasis and iron-storage protein, neutrophil activating protein and the major flagellin subunit iflaA). de Saizieu and co-workers (1998) compared R N A samples from exponentially growing Streptococcus pneumoniae cells to early stationary phase cells and reported that genes related to the polysaccharide capsule biosynthesis, long-chain fatty acid biosynthesis and cell division were transcribed three to eight times less in stationary phase cells. The primary response of E. coli cells to the limitation of a specific nutrient is activation of certain groups of genes for higher uptake of other nutrients that are present in low concentration, or for the utilization of other substances (Hengge-Aronis 1996). In contrast to the specific response, the stationary phase response is not dependent on the type of the limiting nutrient (Hengge-Aronis 1996). A number of morphological and physiological changes have been identified in stationary phase E. coli cells, including thickened cell wall, condensed cytoplasm (Makinoshima et al. 2002), accumulation of polyphosphates (Kornberg 1995), variation in the compositions and proportions of R N A polymerase a subunit, modulation of nucleoid (Ishihama 1999), decrease in D N A superhelicity (Jaworski et al. 1991; Kusano et al. 1996), smaller cells with a spherical rather t han a r od-shaped m orphology, i ncreased tendency t o f orm a ggregates (Hengge-Aronis 1996), differential protein degradation (Lange & Hengge-Aronis 1994), and alterations in ribosome assembly (Wada et al. 1990). In this section, D N A microarray technology was used to investigate the d ifferences in gene expression of E. coli at mid-stationary phase compared to late-log phase. The goal was to  147  investigate the effect of growth phase on E. coli genome at the transcription level and to identify E. coli metabolism and functions involved in late-log and mid-stationary stage of growth through activation or inactivation of biochemical pathways.  6.2  Materials & Methods  6.2.1  Bacterial Strain Escherichia coli (ATCC 11775) isolated from urine was purchased as freeze dried  sample from American Type Culture Collection, Rockville, U S A . For stock culture and inoculum preparation refer to chapter 4, section 4.2.2.  6.2.2  Growth determination To determine the pattern of E. coli growth under conditions employed for cell growth and  maintenance in chapters 4 and 5, one ml stationary phase culture was transferred into 50 ml Nutrient Broth (Difco) and incubated at 37°C. A 1 ml sample was taken hourly for 21 hours and serial dilutions were prepared with peptone water 0.1% (w/v). The duplicate dilutions were spread plated on Plate Count Agar and incubated at 37°C for 21 hours before enumeration. The average number of colonies for each dilution was calculated and the results were plotted as time versus log of colony forming unit per ml. The experiment was repeated twice.  6.2.3  Batch cultures To prepare samples, one ml stationary phase culture was transferred into 50 ml of  Nutrient Broth and incubated for 5 or 16 hours for late-log and mid-stationary phase samples, respectively. Then 48 ml E. coli pure culture containing 10 or 10 CFU/ml for late-log and mid7  148  8  stationary phase samples respectively was aseptically transferred into six sterile 15 ml centrifuge tubes (Fisher b rand d isposable s terile c entrifuge t ubes, s eal c ap, m odified p olystyrene, F isher Scientific, Pittsburgh, P A , USA) and centrifuged at 2060xg at 4°C for 8 minutes ( B E C K M A N GS-6 centrifuge, Beckman Instrument, USA). Total R N A samples were extracted immediately from the pellet using a Qiagen RNeasy total R N A Isolation Mini kit (Valencia, C A , USA) (Chapter 5, section 5.2.5).  6.2.4  DNA microarray analysis Reverse transcriptase and primers specific to 16S and 23S r R N A were used to synthesize  complementary cDNAs.  Then rRNA was removed enzymatically by Rnase H , which  exclusively digested R N A within an R N A : D N A hybrid. The c D N A molecules are removed with Dnase I digestion and the enriched mRNA was purified on Qiagen RNEasy columns. The procedure  for total R N A extraction, mRNA  enrichment,  fragmentation,  labelling and  hybridization were described previously (Chapter 5, sections 5.2.6, 5.2.7, 5.2.8, 5.2.9).  6.2.5  Statistical analysis Linear regression (SYSTAT 1998) was used to determine the linear section of growth.  One way A N O V A was used to find genes which expression was significantly different among treatments (p<0.05, n=6). (Chapter 5, section 5.2.10)  6.3  Results To v erify t he s tage o f growth, t he n umber o f c ells w as p lotted o n a 1 ogarithmic s cale  against time on an arithmetic scale (Figure 6.1). The result of linear regression test of the data  149  points for the first 6 hours of growth showed a straight line with r of 0.966 and p<0.01. The 2  straight line in a semilogarithmic graph of bacteria growth is an immediate indicator of cells that are growing e xponentially (Madigan e t al. 2 003). In a ddition, c alculation o f generation t ime showed that by 6 hours E. coli population have gone through four generation times, which means that despite the high initial "population, cells were in log phase of growth and should be homogenous. Therefore the first 6 hours of growth were considered as exponential phase and a sample taken at 5 hours was considered as late-exponential phase. The correlation among present calls of replicates for each sample showed an average of 0.90 ± 0.08. In single arrays, 49% and 46% of probe sets were identified as present and absent respectively in mid-stationary phase cells, while the values for late-exponential phase samples were 46% and 49% respectively. For all the samples, 4.3-4.5% of probe sets were in the marginal category (Table 6.1). A comparison of the mid-stationary phase expression data with that obtained from the late-exponential growth identified 494 down-regulated (11.22%) and 8 4 up-regulated (1.91%) genes. O f these, 304 genes were down-regulated and 12 genes were up-regulated more than two fold in mid-stationary phase E. coli cells. Most of genes (86.88%) remained unchanged between the two growth phases. A list of genes altered less than two fold among samples is presented in appendix VII (Tables 9.4, 9.5). The probe sets related to intergenic regions were not considered in this study.  6.3.1  Genes up-regulated (>2 fold) in mid-stationary phase cells Seven of the 12 genes that displayed >2 fold up-regulation in stationary phase cells are  related to hypothetical proteins, which are not assigned to any known pathways. The csgA, csgB  150  and csgD genes involved in curli synthesis were induced in stationary phase cells by 14 and 12 fold for csgA and csgB respectively, and 2.25 fold for csgD. In addition guaB which encodes the enzyme inosine-monophosphate dehydrogenase and is related to purine metabolism, was expressed 3.54 fold more in stationary phase cells. (Table 6.3).  6.3.2 Genes down-regulated (>2 fold) in mid-stationary phase cells The name and description of genes down-regulated more than 2 fold in mid-stationary phase along with their fold change is shown in Table 6.4. The majority of down-regulated genes were assigned to six functional groups, namely translation, amino acid metabolism, carbohydrate metabolism, energy metabolism, cell motility and membrane transport (Table 6.2). About 30% of the expressed genes do not have functional annotation and are not assigned to any specific pathway.  6.3.2.1 Translation and transcription The expression of genes that encode for ribosomal proteins was affected most in midstationary versus late-exponential growth phase. These genes showed the highest reduction in mid-stationary phase cells. Of the 85 genes related to translation, 55 genes were significantly down-regulated in mid-stationary samples (2.01-19.95 fold change). Those genes were mostly related to 30S and 50S ribosomal proteins. In addition, 6 other genes, from 13 genes that encode for translation factors, were also down-regulated in mid-stationary phase cells. Genes that encode for R N A polymerase enzymes including rpoA, rpoB and rpoC, which are involved in transcriptional functions, were expressed more in cells at late-exponential phase of growth.  151  6.3.2.2 Energy metabolism From 9 genes involved in A T P synthesis, 8 genes showed lower expression in midstationary cells.  Twenty of the 41 genes related to oxidative phosphorylation were down-  regulated more than two fold and 5 of the others were expressed 1.6-1.92 fold less in midstationary phase.  6.3.2.3 Cell motility Bacterial cell genes involved i n chemotaxis and flagellar assembly are responsible for cell motility (Madigan et al. 2003). O f the 41 genes known to be involved in flagellar assembly, 32 genes were down-regulated more than two fold in mid-stationary phase E. coli. Six other motility genes were not detected in any of the samples while 3 remained unchanged. Eleven of 20 genes involved in bacterial chemotaxis were down-regulated more than two fold in midstationary E. coli cells, while 3 were not detected in any of the samples, and the change in the rest was not significant.  6.3.2.4 Carbohydrate metabolism The aceE, aceF, IpdA, eno,fba and tdcD genes which are involved in glycolysis, caiB, sucC and sucD which are involved in propanoate metabolism were expressed less in midstationary phase of growth. In addition, the IpdA, acnB, gltA, sdhA, sucA, sucB, sucC and sucD which are involved in citrate cycle were down-regulated in mid-stationary E. coli cells.  152  6.3.2.5 Fatty acid biosynthesis Six genes from the fatty acid biosynthesis pathway were expressed more in lateexponential phase of E. coli growth, while 5 genes were not detected in any samples and one gene (fabH) remained unchanged, de Saizieu and co-workers (1998) showed that the accC gene, which is involved in long-chain fatty acid biosynthesis, was transcribed three to eight fold lower in stationary phase Streptococcus pneumoniae cells than in exponential phase cells.  6.3.2.6 Membrane and transport systems Genes involved in fimbriae proteins were transcribed less in mid-stationary phase E. coli cells. Also opmA, ompC, ompF, tolC and acrB genes involved in membrane transport through pore ion channels were down-regulated in mid-stationary phase E. coli cells.  6.4  Discussion  6.4.1  Curli synthesis  Curli, thin fibres tending to coil up into a fuzzy mass on the surface of bacteria, are one of t he a dhesive o rganelles i n E. coli{ Hultgren et a 1. 1 996). C urli a Iso p romote c lumping o f bacterial cells in culture and binding to abiotic surfaces such as glass and polystyrene, making them important for biofilm formation (Vidal et al. 1998; Austin et al. 1998; Prigent-Combaret et al. 2000). The csg genes are required for curli synthesis (Chirwa & Herrington 2003). Studies have shown that the expression of csg genes is related to temperature, osmolality, and the availability of the nutrients, oxygen and iron (Olsen et al. 1998; Gerstel & Romling 2001). Polymerization  153  of the curlin subunit to insoluble curli is dependent on the presence of a specific protein encoded by the csgB gene (CsgB) (Harnmar et al. 1996). The csgA and csgB genes are co-transcribed (Arnqvist et al. 1994) and csgD encodes for a lipoprotein involved in secretion of curlin and CsgB (Harnmar et al. 1995, 1996; Loferer et al. 1997).  Others have reported that some  regulatory proteins including RpoS, OmpR, and Cpx are responsible for CsgD expression (Arnqvist et al. 1994; Prigent-Combaret et al., 2001; Chirwa & Herrington 2003). In this study the csgA, csgB and csgD genes were induced in mid-stationary phase E. coli cells.  The  expression of rpoS, ompR genes displayed no significant change between mid-stationary and exponential E. coli samples and cpx was not detected in either phase of E. coli growth. This shows that curli synthesis was started in stationary phase of growth and may suggest that other regulatory systems could be involved in the activation of these genes. Hultgren and colleagues (1996) also reported cells develop curli in their stationary phase of growth. Since c urlin c ontains h igh amounts o f glycine, Chirwa & H errington (2003) p roposed that up-regulation of glyA is an essential response for curli formation. In this study, although glyA was detected in all samples, its transcription level did not show any significant change between the two stages of growth. It is possible that glyA was expressed in the late-log phase to produce amino acid necessary for rapid cell growth and its expression continued in the stationary phase to provide necessary glycine for curli synthesis, whereas other genes involved in amino acid biosynthesis were down-regulated.  6.4.2  Cell motility About 61 genes in E. coli are required for flagellar synthesis, chemotaxis and subsequent  motility.  These genes have several functions, including encoding structural proteins of the  154  flagellar apparatus, export of flagellar components through the membrane to the outside of the cell, and regulation of the many biochemical events surrounding the synthesis of new flagella (Madigan et al. 2003). In E. coli cells in the mid-stationary phase of growth, 70.5% of these genes were down-regulated or were not expressed at all. This indicates that E. coli cells in midstationary phase of growth may have less tendency to be mobile.  6.4.3  Transcription and translation  ^  The expression profiles of genes involved in transcription and translation, including the major subunits of R N A polymerase, ribosomal proteins, and translation factors showed downregulation i n m id-stationary p hase E. c oli. T he decrease i n t he o verall t ranslation a ctivity o r protein synthesis, has been reported to occur along with the transition from the exponential growth to the stationary phase in E. coli cells (Wada et al. 1990). Selinger and colleagues (2000) reported a decreased of expression for genes involved in protein synthesis (rRNA, tRNA and ribosomal protein) in stationary E. coli MG1655 cells compared to exponential cells.  These  genes are also reported to be down-regulated due to growth arrest (Chang et al. 2002).  6.4.4  Regulatory systems Fis and Rpos are two regulatory proteins which co-ordinately control the expression of  some of the genes during late-log and stationary phase of growth. While Fis expression is at its maximum in early-to-mid log, the expression of Rpos is turned on in late exponential and stationary phase. Fis reduces the expression of specific genes required for growth under suboptimal nutrient conditions (Xu & Johnson 1995) and the expression of fis has also been reported to decrease during growth arrest (Chang et al. 2002). Rpos is required for expression of genes  155  important under starvation or stationary phase conditions (Xu & Johnson 1995).  In this  experiment the expression of fis gene was 2.25 fold higher in late-exponential phase, but interestingly, although the rpoS gene was detected as present in late-log and mid-stationary of E. coli, its expression did not show any significant change between two stages of growth. Selinger and colleagues (2000) also did not find any increase in rpoS in E. coli K-12 stationary cell compared to exponenetial cells. In addition, X u & Johnson (1995) reported that the products of xylF and mglA are required for E. coli growth under nutrient-poor conditions in which fis levels are low. The result of this experiment supports X u & Johnson as expression of the xylF gene was 1.57 fold higher in mid-stationary phase. However the mglA gene had a higher expression in late-exponential phase of growth where fis level was not reduced. X u & Johnson (1995) also reported the presence of rpoS reduced the expression of xylF, mglA and sdhA. The present study showed that while the expression of rpoS did not change, the expression of other three genes were altered between the two stages of growth.  This could suggest that other regulatory genes are involved in the  induction or repression of these genes in different stages of growth. Schellhorn and co-workers (1998) also found that the expression of some of the growth dependent genes can be changed without t he p resence o f rpoS a nd t hey c oncluded t hat p robably m any growth-phase-regulated functions in E. coli do not require RpoS for expression.  Another explanation is that most  changes reported in literature were detected at the protein level and the correlation between gene transcript and protein activity is not expected to be perfect (Selinger et al. 2000).  156  6.4.5  Early stationary phase genes Previous studies have shown that the expression of genes which encode basic proteins  that non-specifically bind D N A (hupA, hupB and hlpA) (Dersch et al. 1993; Weglenska et al. 1996), and genes that encode for the integration host factor (himA and himD) (Claret & Rouviere-Yaniv 1997) increases upon entry into stationary phase.  The study of Chang and  colleagues (2002) also showed up-regulation for the above genes as a result of growth arrest. In the present study, transcription of hupA (2.42 fold), hupB (3.33 fold), and hlpA (3.26 fold) showed down-regulation in mid-stationary phase E. coli cells. In addition the transcription of himA and himD decreased 2.68 and 1.81 fold in mid-stationary phase E. coli. The transition of E. coli cells to stationary phase of growth has been described as a general stress response (Hengge-Aronis 1996, 1999). Changes in gene expression at early stationary phase is a shortterm response to external stress factors while mid and late stationary phase are long-term stress responses. Differences in short-term and long term stress response, in this case early and mid stationary phase, would be expected. Azam and co-workers (1999) also reported some changes in gene expression of E. coli cells in early and late stationary phase.  6.5  Conclusion In the mid-stationary phase of growth, genes encoding for energy metabolism as well as  amino acid and carbohydrate metabolism were down-regulated. In addition, transcription of genes involved in fimbriae synthesis was reduced while genes encoding for curli synthesis were induced in stationary phase cells. Thus, E. coli cells may be less mobile and may have more tendency to clump or stick to surfaces. Interestingly some genes reported to up-regulate upon entry into stationary phase were shown to be down-regulated in mid-stationary phase cells,  157  indicating that the active metabolic pathways involved in early, mid and late stationary ph; varied.  158  8.5  I  8  D  7 *5 O 5  7 16.5 i  r  -1  8  0  1  1  1  1  1  I  I  10  12  14  16  18  20  22  Time (hour) Figure 6.1. Growth of 10 CFU/ml stationary phase E. coli (ATCC 11775) transferred to 50ml 7  Nutrient Broth at 37°C over 22 hours.  159  Table 6.1. E. coli probe sets signals in mid-stationary phase and late-log phase cells. Samples  Present call (%)  Absent call (%)  Marginal call (%)  Mid-stationary phase E. coli  49.20+/-15.02  46.30+/-14.72  4.50+/-0.32  Late-log phase E. coli  46.25+/-3.27  49.38+/-3.39  4.36+/-0.13  160  Table 6.2. Distribution of gene transcription in mid-stationary phase E. coli, compared to latelog phase E. coli. Gene function  Translation Amino acid metabolism Carbohydrate metabolism Energy metabolism Cell Motility Membrane transport Nucleotide metabolism Metabolism of cofactors and vitamins Lipid metabolism Sorting and degradation Signal transduction Transcription Biodegradation of xenobiotics Biosysnthesis of secondary metabolite Unassigned  Down-regulated > 2 fold number of genes 71 48 46 45 42 16 12 11 9 9 5 4 2 1 93  161  (23.36 %) (15.79%) (15.13 %) (14.80%) (13.82 %) (5.26 %) (3.95 %) (3.62 %) (2.96 %) (2.96 %) (1.64%) (1.32%) (0.66 %) (0.33 %) (30.59 %)  Up-regulated > 2 fold number of genes 0 0 0 0 0 0 1 0 0 0 0 0 0 0 12  0 0 0 0 0 0 (7.69 %) 0 0 0 0 0 0 0 (92.31 %)  Table 6.3. Genes up-regulated (>2 fold) in mid-stationary phase cells compared to late-log phase E. coli cells (p<0.05). Gene name  bfr  b no.  b2670 b3336  Fold change  1.96 2.19  Call Log Stationary phase phase  p  P A  Description  orf , hypothetical protein ' bacterioferritin ' (BFR) (cytochrome B 1) (cytochrome B-557) , an iron storage homoprotein major curlin subunit precursor ' ,coiled surface structures; cryptic minor curlin subunit precursor ' , similar ro CsgA probable csgab operon transcriptional regulatory protein , putative 2-component transcriptional regulator for 2nd curli operon inosine-5'-monophosphate dehydrogenase '(IMP dehydrogenase) ' ,(IMPDH) (IMPD) hypothetical 37.1 kD protein in modCbioA intergenic region , orf, hypothetical protein hypothetical 5.9 kD protein in waam-solA intergenic region , orf, hypothetical protein hypothetical transcriptional regulator in bacA-ttdA intergenic region , putative transcriptional regulator LYSR-type hypothetical 11.2 kD protein in argR-cafA intergenic region , orf, hypothetical protein hypothetical 10.3 kD protein in argR-cafA intergenic region (F90) , orf, hypothetical protein P14 protein , orf, hypothetical protein 2  4  1 2  1 2  1  1  1  csgA  M042  14.06  p  A  1 2  2  csgB  bl041  11.79  p  A  1 2  2  csgD  bl040  2.25  p  A  1  2  guaB  b2508  3.54  p  P  1 2  1  ybhH  b0769  2.15  p  A  1  2  yceO  M058  2.25  p  A  1  2  ygiP  b3060  2.30  p  A  1  yhcN  b3238  2.46  p  P  1  2  yhcR  b3242  2.32  p  P  l  2  p b4363 1.99 yjjB http://www.genome.ad.jp/kegg/ Affymetrix gene chip data base P= Present (gene was detected) A = Absent (gene was not detected)  P  2  3  4  162  1  2  Table 6.4. Genes down-regulated (>2 fold) in mid-stationary phase cells compared to late-log phase E. coli cells (p<0.05). Gene name  b no.  Fold change  accC  M199 M200 bl604 bl722 M839 b2255 b2512 b2529 b2595 b2736 b2737 b2878 b2881 b3256  2.33 2.77 2.18 2.43 2.33 3.24 4.75 2.37 2.45 2.84 2.25 2.37 3.11 2.37  aceE  b0114  4.27  Call stationary log phase phase P A P P P P A P P P P P A P P P P A A P P P P A A P P P 4  P  j  P  aceF  b0115  2.76  A  P  acnB  b0118  2.83  P  P  Description  putative dihydroxyacetone kinase putative dihydroxyacetone kinase hypothetical protein hypothetical protein hypothetical protein putative transformylase '  1  1 1  1  1 2  1 2  putative dehydrogenase ' orf , hypothetical protein ' orf , hypothetical protein ' putative dehydrogenase ' orf , hypothetical protein ' putative oxidoreductase, Fe-S subunit putative dehydrogenase ' biotin carboxylase , acetyl Co A carboxylase, biotin carboxylase subunit pyruvate dehydrogenase ' E l component , (decarboxylase component) dihydrolipoamide acetyltransferase component (E2) of pyruvate dehydrogenase complex ' aconitate hydratase 2 , aconitate hydrase B acyl carrier protein ' acriflavin resistance protein A precursor acriflavin resistance protein B aerotaxis receptor alkyl hydroperoxide reductase c22 protein (scrp-23) (sulfate starvation-induced protein 8) (SSI8) alkyl hydroperoxide reductase f52a protein alanyl-fRNA synthetase shikimate kinase I (SKI) L-arginine transport system substratebinding protein aspartate-semialdehyde dehydrogenase (AsA dehydrogenase) aspartate—ammonia ligase (asparagine synthetase A ) 2  1 2  2  1 2  1 2  2  1 2  1  1 2  1  2  1 2  1  2  1 2  1  2  acpP acrA acrB aer ahpC  M094 b0463 b0462 b3072 b0605  P P  P P  2.72 2.05 2.50 2.31 2.88  A P  P P  P  1 2  1  1  1  1  ahpF  b0606  2.31  A  alaS aroK artl  b2697 b3390 b0863  2.79 2.73 2.86  P P P  P P P  asd  b3433  2.49  P  P  1  1  1  1  asnA  b3744  2.76  P  P  1  1 , 2  163  Table 6.4. Continued. Gene name asnB  b no.  b0674  Fold change 3.33  Call stationary log phase phase P P  Description  asparagine synthetase B ' (glutaminehydrolyzing) aspartate aminotransferase A T P synthase alpha chain , membranebound A T P synthase, F l sector, alphasubunit ATP synthase A chain (protein 6) membrane-bound ATP synthase, F0 sector, subunit a ATP synthase epsilon chain , membranebound A T P synthase, F l sector, epsilonsubunit A T P synthase beta chain , membranebound A T P synthase, F l sector, betasubunit ATP synthase C chain (lipid-binding protein) (dicyclohexylcarbodiimidebinding protein) , membrane-bound ATP synthase, F0 sector, subunit c ATP synthase B chain , membrane-bound ATP synthase, F0 sector, subunit b ATP synthase gamma chain , membranebound A T P synthase, F l sector, gammasubunit A T P synthase delta chain , membranebound A T P synthase, F l sector, deltasubunit probable carnitine operon oxidoreductase ' ; caiA crotonobetainyl-CoAxarnitine CoAtransferase ; 1-carnitine dehydratase carnitine operon protein caiE chemotaxis protein cheA chemotaxis protein methyltransferase purine-binding chemotaxis protein chemotaxis protein cheY chemotaxis protein cheZ cold shock protein cspA (7.4 kD cold shock protein) (CS7.4) , transcriptional activator of hns cold shock-like protein cspg 1  2  1  aspC atpA  b0928 b3734  3.45 3.44  P P  P P  1  1  2  atpB  b3738  2.40  P  P  atpC  b3731  5.73  P  P  1  2  atpD  b3732  5.82  P  P  1  2  atpE  b3737  2.59  P  P  l  2  atpF  b3736  2.48  P  P  atpG  b3733  3.63  P  P  1  2  1  2  atpH  b3735  3.57  P  P  caiA  b0039  2.77  A  P  1  f  caiB  b0038  2.72  P  P  2  1  1  caiE cheA cheR cheW cheY cheZ cspA  b0035 bl888 bl884 bl887 bl882 bl881 b3556  3.39 2.80 1.97 2.73 2.92 4.59 3.97  P A P A A A P  P P P P P P P  2  1  1  1  1  1  1  1>2  2  cspG  b0990  4.43  P  P  164  1  Table 6.4. Continued. Gene name cydA  b no.  b0733  Fold change  Call stationary log phase phase  Description  cytochrome d ubiquinol oxidase ' subunit I,(cytochrome bd-I oxidase subunit I) \ polypeptide subunit 1 cytochrome d ubiquinol oxidase ' subunit II (cytochrome bd-I oxidase  2.28  2  cydB  b0734  2.69  1 2  1  cyoC  b0430  2.80  P  P  cyoD  b0429  3.32  A  P  cyoE  b0428  2.87  A  P  2  subunit II) , polypeptide subunit II cytochrome o ubiquinol oxidase subunit III ' cytochrome o ubiquinol oxidase operon protein cyoD , subunit IV 1 2  1  cysK  b2414  6.21  P  P  2  1 2  protoheme IX farnesyltransferase ' , (haeme O biosynthesis) cysteine synthase A (O-acetylserine sulfhydrylase A ) ' (O- acetylserine (THIOL)-lyase A ) (csase A ) (sulfate starvation-induced protein 5) (SSI5) sulfate transport system thiosulfatebinding protein enolase ' malonyl CoA-acyl carrier protein transacylase ( M C T ) ' 3-oxoacyl-[acyl-carrier-protein synthase II] ' 2  1  cysP  b2425  3.79  A  P  eno fabD  b2779 M092  2.72 3.58  P A  P P  fabF  M095  3.15  P  P  fba fdhF  b2925 b4079  3.25 2.49  P A  P P  fdoG  b3894  2.19  P  P  2  1  1  1 2  x  2  1 2  1 2  fructose-bisphosphate aldolase, class I I ' formate dehydrogenase, major subunit (formate dehydrogenase alpha subunit) \ selenopolypeptide subunit of formate dehydrogenase H 2  1 2  fdoH  b3893  2.36  P  P  formate dehydrogenase, major subunit ' (formate dehydrogenase alpha subunit) ,formate dehydrogenase-0 formate dehydrogenase, iron-sulfur subunit (formate dehydrogenase beta subunit) ' , formate dehydrogenase-O chaperone protein fimC precursor , periplasmic chaperone, required for type 1 fimbriae outer membrane usher protein fimD precursor , outer membrane protein; export and assembly of type 1 fimbriae, interrupted 1  fimC  b4316  3.40  P  P  2  l 2  2  1  fimD  b4317  3.16  P  P  2  1  2  165  Table 6.4. Continued. Gene name fimG  b no.  b4319  Fold change 2.11  Call stationary P  Description log P  fimG protein precursor , fimbrial morphology fimbrin-like protein fiml , fimbrial protein 2  fiml  b4315  3.21  P  P  1  2  fis  b3261  2.25  P  P  factor-for-inversion stimulation protein , site-specific D N A inversion stimulation factor; DNA-binding protein; a trans 1  *  fixA fixB fixX flgA  b0041 b0042 b0044 M072  3.63 3.66 3.02 2.99  A A A A  P P P P  *  2  activator for transcription fixA protein fixB protein ferredoxin like protein flagella basal body P-ring formation protein flgA precursor , flagellar biosynthesis; assembly of basal-body periplasmic P ring protein flgB , flagellar basal-body rod cell-proximal flagellar biosynthesis, portion of basal-body rod flagellar basal-body rod protein flgC , cell-proximal flagellar biosynthesis, portion of basal-body rod basal-body rod modification protein flgD , flagellar biosynthesis, initiation of hook assembly flagellar hook protein flgE , flagellar biosynthesis, hook protein flagellar basal-body rod protein flgF , flagellar biosynthesis, cell-proximal portion of basal-body rod flagellar basal-body rod protein flgG , flagellar biosynthesis, cell-distal portion of basal-body rod flagellar L-ring protein precursor , flagellar biosynthesis, basal-body outermembrane L (lipopolysaccharide layer) ring protein flagellar protein flgJ , flagellar biosynthesis flagellar hook-associated protein 1 (HAP1) ' , flagellar biosynthesis" 1  1  1  1  2  flgB  M073  5.04  A  P  2  flgc  bl074  6.06  A  P  2  flgD  M075  6.96  A  P  1  2  flgE  bl076  7.43  A  P  1  2  flgF  bl077  4.96  A  P  2  flgG  bl078  4.92  A  P  1  2  flgH  bl079  2.09  A  P  1  2  flgJ  bl081  flgK  bl082  2.00  P  P  1  2  5.13  P  P  1 2  166  Table  Gene  6.4. Continued. b no.  flgL  bl083  Description  Call  Fold change  name  stationary  log  phase  phase  flagellar hook-associated protein 3 (HAP3) (hook- filament junction protein) ' , flagellar biosynthesis negative regulator of flagellin synthesis (anti-sigma factor) , also known as RilB protein  3.21  1  l 2  flgM  M071  3.08  A  2  l  flgN flhC  bl070 bl891  3.71 2.14  P P  P P  flhD  bl892  2.24  P  P  fliA  bl922  4.86  A  P  1 2  flagella synthesis protein flgN ' flagellar transcriptional activator flhC , regulator of flagellar biosynthesis acting on class 2 operons; transcription initiation factor flagellar transcriptional activator flhD , regulator of flagellar biosynthesis, acting on class 2 operons; transcriptional initiation factor R N A polymerase sigma factor for flagellar operon , flagellar biosynthesis; alternative sigma factor 28; regulation o f flagellar operons flagellin ' , flagellar biosynthesis; filament structural protein flagellar hook-associated protein 2 (HAP2) (filament C A P protein)', flagellar biosynthesis; filament capping protein; enables filament assembly flagellar hook-basal body complex protein fliE , flagellar biosynthesis; basal-body component, possibly at (MS-ring)-rod junction flagellar motor switch protein fliG flagellar assembly protein fliH , flagellar biosynthesis; export of flagellar proteins? flagellar hook-length control protein ' flagellar fliL protein , flagellar biosynthesis flagellar motor switch protein f l i M , flagellar biosynthesis, component of motor switch and energizing, enabling rotation and determining its direction 1  2  1  1  fliC  bl923  5.02  A  P  fliD  bl924  3.53  A  P  fliE  M937  2.58  P  P  2  1 2  2  2  1  2  fliG fliH  bl939 bl940  4.10 3.17  A A  P P  fliK fliL  bl943 bl944  2.24 3.27  A A  P P  fliM  M945  3.46  P  P  1  1  2  1 2  1  2  1  2  167  Table 6.4. Continued. Gene name  b no.  Fold change  fliN  bl946  8.45  fliO  bl947  3.05  Call stationary log phase phase P A  A  P  Description  flagellar motor switch protein fliN , flagellar biosynthesis, component of motor switch and energizing, enabling rotation and determining its direction flagellar protein fliO , flagellar biosynthesis flagellar biosynthetic protein fliP , flagellar biosynthesis flagellar protein fliS , flagellar biosynthesis; repressor of class 3a and 3b operons (RflA activity) putative polar amino acid transport system substrate-binding protein , putative periplasmic binding transport protein fliZ protein , orf, hypothetical protein elongation factor E F - G galactokinase ' glycine dehydrogenase glutamine synthetase ' (glutamate— ammonia ligase) lactoylglutathione lyase ' glycerol kinase glycerophosphoryl diester phosphodiesterase periplasmic precursor (glycerophosphodiester phosphodiesterase) glycerol-3-phosphate transporter (G-3-P transporter) (G-3-P permease) citrate synthase ' glutamate/aspartate transport system ATPbinding protein 6-phosphogluconate dehydrogenase, decarboxylating ' glycerol-3 -phosphate dehydrogenase (NAD+) D N A gyrase subunit A hflK protein , protease specific for phage lambda e l l repressor integration host factor alpha-subunit histidyl-tRNA synthetase 1  1  2  fliP  M948  2.35  A  P  1  2  fliS  bl925  4.39  A  P  1  2  fliY  M920  3.01  P  P  1  2  fliZ fusA galK gcvP glnA  bl921 b3340 b0757 b2903 b3870  2.15 4.31 1.97 2.25 2.73  A P A P P  P P P P P  1  2  1  1 2  1  1 2  1  gloA glpK gipQ  bl651 b3926 b2239  1.96 2.03 1.96  P P P  P P P  1 2  1  1  glpT  b2240  2.04  P  P  1  gltA gltL  b0720 b0652  1.96 2.01  P P  P P  1 2  1  gnd  b2029  2.725  P  P  1  gpsA  b3608  2.29  P  P  2  1  gyrA hflK  b2231 b4174  2.33 2.32  P P  P P  1  1  2  himA hisS  bl712 b2514  2.68 2.04  P P  P P  168  1  1  Table 6.4. Continued. Gene name hlpA  b no.  b0178  Fold change 3.26  Call stationary P  Description log P  histone-like protein hlp-1 precursor (DNA-binding 17 kD protein) heat shock protein hscA (HSC66) heat shock protein, chaperone, member of Hsp70 protein family DNA-binding protein hu-alpha (HU2) ' (NS2) DNA-binding protein hu-beta (NS1) (HU1  hscA  b2526  1.97  A  P  2  hupA  b4000  2.42  P  P  1 2  hupB  b0440  3.33  P  P  hybE  b2992  1.97  P  P  ileS inaA infB leuL HpA Ion IpdA  b0026 b2237 b3168 b0075 b0628 b0439 b0116  3.65 2.37 4.06 2.19 1.96 2.22 3.02  A P P P P P P  P P P P P P P  1  hydrogenase-2 operon protein hybE , member of hyb operon isoleucyl-fRNA synthetase inaA protein translation initiation factor IF-2 Leu operon leader peptide lipoic acid synthetase ATP-dependent protease L a dihydrolipoamide dehydrogenase (e3 component of pyruvate and 2-oxoglutarate dehydrogenases complexes) (glycine cleavage system L protein) UDP-3-0-[3-hydroxymyristoyl glucosamine N - acyltransferase (firA protein) (rifampicin resistance protein) leucine-responsive regulatory protein lysyl-tRNA synthetase methionyl-tRNA synthetase methionine tRNA-m ' ; duplicate gene D-galactose transport system ATPbinding protein molybdate transport system substratebinding protein chemotaxis motA protein (motility protein 1  1  1  1  1  1  1  IpxD  b0179  2.88  P  P  1  Irp lysS metG metT mglA  b0889 b2890 b2114 b0673 b2149  3.14 2.66 2.03 2.43 2.00  A P A P P  P P P P P  1  1  1  1  2  2  1  modA  b0763  2.00  A  P  motA  M890  2.27  P  P  mukE murD  b0923 b0088  2.63 2.95  A A  P P  mukE protein (kica protein) UDP-N-acetylmuramoylalanine—Dglutamate ligase (UDP-Nacetylmuranoyl-L-alanyl-D-glutamate synthetase) UDP-N-acetylmuramoylalanyl-Dglutamate—2,6-diaminopimelate ligase 1  murE  b0085  2.18  A  P  169  1  Table 6.4. Continued.  Gene name  b no.  Fold change  Call stationary log phase phase  murG  b0090  2.22  A  P  nlpB ntpA nuoC  b2477 M865 b2286  2.45 2.36 2.48  P P P  P P P  Description  UDP-N-acetylglucosamine~Nacetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N acetylglucosamine transferase lipoprotein-34 precursor dATP pyrophosphohydrolase N A D H dehydrogenase I chain C, chainD N A D H dehydrogenase I chain F N A D H dehydrogenase I chain I N A D H dehydrogenase I chain J N A D H dehydrogenase I chain L (NADHubiquinone oxidoreductase chain 12) (NU012) N A D H dehydrogenase I chain M ' (NADH-ubiquinone oxidoreductase chain 13) ( N U 0 1 3 ) N utilization substance protein A N utilization substance protein B (nusB protein) outer membrane protein A precursor (outer membrane protein II*) outer membrane protein C precursor (outer membrane protein l b ) outer membrane protein F precursor (outer membrane protein la, ia, or B ) oligopeptide transport system substratebinding protein peptidoglycan-associated lipoprotein precursor 4-hydroxythreonine-4-phosphate dehydrogenase formate acetyltransferase l ' (pyruvate formate-lyase l ) phenylalanyl-tRNA synthetase beta chain fatty acid/phospholipid synthesis protein polyribonucleotide nucleotidyltransferase (polynucleotide phosphorylase ' ) (PNPase)cytidylate kinase activity NAD(P) transhydrogenase subunit alpha 1  1,2  1  1  1,2  nuoF nuol nuoJ nuoL  b2284 b2281 b2280 b2278  2.64 2.62 2.622.81  P A P A  P P P P  1  1  1 , 2  1  nuoM  b2277  2.33  A  P  1  2  1  nusA nusB  b3169 b0416  3.30 2.59  P A  P P  1  1  ompA  b0957  2.66  P  P  1  ompC  b2215  4.70  P  P  1  ompF  b0929  4.57  P  P  1  oppA  bl243  2.32  P  P  1  pal  b0741  4.82  P  P  1  pdxA  b0052  2.04  A  P  1  pflB  b0903  3.30  P  P  1  2  1  pheT plsX pnp  bl713 bl090 b3164  2.56 2.05 3.17  P P P  P P P  1  1  1 2  2  pntA  M603  2.23  P  P  170  1  Table 6.4. Continued.  Gene name potD  b no.  bll23  Fold change 2.26  Call stationary log phase phase A  P  Description  spermidine/putrescine transport system substrate-binding protein primosomal replication protein N ribose-phosphate pyrophosphokinase , phosphoribosylpyrophosphate synthetase CTP synthase (TJTP--ammonia ligase) (CTP synthetase) uridylate kinase ribosome-binding factor A relF protein riboflavin synthase beta chain 16s r R N A processing protein rimm 50S ribosomal protein L I 5 OS ribosomal protein L 2 50S ribosomal protein L 3 50S ribosomal protein L 4 5 OS ribosomal protein L 5 50S ribosomal protein L 6 50S ribosomal protein L 9 50S ribosomal protein L10 5OS ribosomal protein L I 1 5 OS ribosomal protein L7/L12 5 OS ribosomal protein L13 50S ribosomal protein L14 50S ribosomal protein L15 50S ribosomal protein L16 50S ribosomal protein L17 50S ribosomal protein L18 50S ribosomal protein L19 50S ribosomal protein L20 5 OS ribosomal protein L22 5 OS ribosomal protein L23 50S ribosomal protein L24 50S ribosomal protein L25 5 OS ribosomal protein L27 50S ribosomal protein L28 5 OS ribosomal protein L29 50S ribosomal protein L30 50S ribosomal protein L31 50S ribosomal protein L32 5 OS ribosomal protein L33 1  priB prsA  b4201 M207  6.90 3.74  P P  P P  1  1  2  pyrG  b2780  2.26  P  P  pyrH rbfA relF ribH rimM rplA rplB rplC rplD rplE rplF rpll rplJ rplK rplL rplM rplN rplO rplP rplQ rplR rplS rplT rplV rplW rplX rplY rpmA rpmB rpmC rpmD rpmE rpmF rpmG  b0171 b3167 bl562 b0415 b2608 b3984 b3317 b3320 b3319 b3308 b3305 b4203 b3985 b3983 b3986 b3231 b3310 b3301 b3313 b3294 b3304 b2606 bl716 b3315 b3318 b3309 b2185 b3185 b3637 b3312 b3302 b3936 bl089 b3636  2.14 2.66 3.46 2.33 6.19 6.02 5.96 5.57 6.27 4.37 6.63 6.63 7.23 5.48 4.83 4.45 3.92 6.21 7.76 6.16 5.71 3.84 5.38 7.17 6.38 3.93 3.87 3.31 2.75 19.95 5.96 2.98 4.04 2.42  P A P P P P P A P P P P P P P P P P P P P P P P P P P P P A P P P P  P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P  1  171  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  Table 6.4. Continued.  Gene name rpmH rpml rpmJ rpoA  b no.  b3703 bl717 b3299 b3295  Fold change 2.01 7.144 4.02 6.64  Call stationary log phase phase P P P P  P P P P  Description  50S ribosomal protein L34 5 OS ribosomal protein L35 50S ribosomal protein L36 DNA-directed R N A polymerase alpha chain , R N A polymerase, alpha subunit DNA-directed R N A polymerase beta chain , R N A polymerase, beta subunit DNA-directed R N A polymerase beta' chain , R N A polymerase, beta subunit 30S ribosomal protein S l 3 OS ribosomal protein S2 30S ribosomal protein S3 30S ribosomal protein S4 30S ribosomal protein S5 30S ribosomal protein S6 30S ribosomal protein S7 3OS ribosomal protein S8 30S ribosomal protein S9 30S ribosomal protein S10 3OS ribosomal protein S l 1 3 OS ribosomal protein S12 3 OS ribosomal protein S13 3 OS ribosomal protein S14 3 OS ribosomal protein S15 30S ribosomal protein S16 30S ribosomal protein S17 30S ribosomal protein S18 30S ribosomal protein S19 30S ribosomal protein S20 sulfate transport system sulfate-binding protein succinate dehydrogenase flavoprotein subunit ' preprotein translocase sec A subunit , protein-export membrane protein secD protein-export membrane protein secF protein-export membrane protein secG (preprotein translocase band 1 subunit) (P12) preprotein translocase SecY subunit phosphoserine aminotransferase 1  1  1  1  rpoB  b3987  5.51  P  P  1  rpoC  b3988  5.51  A  P  2  1  rpsA rpsB rpsC rpsD rpsE rpsF rpsG rpsH rpsl rpsJ rpsK rpsL rpsM rpsN rpsO rpsP rpsQ rpsR rpsS rpsT sbp  b0911 b0169 b3314 b3296 b3303 b4200 b3341 b3306 b3230 b3321 b3297 b3342 b3298 b3307 b3165 b2609 b3311 b4202 b3316 b0023 b3917  5.21 6.24 9.77 5.20 5.88 4.35 4.61 6.19 5.42 5.50 5.18 3.66 4.82 4.62 3.64 5.22 8.97 3.58 7.84 5.30 2.01  P P P P P P P P P P P P P P P P P P P P P  P P P P P P P P P P P P P P P P P P P P P  2  1  1  1 1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  sdhA  b0723  2.61  P  P  1 2  secA secD secF secG  b3609 b0408 b0409 b3175  2.00 2.33 2.20 2.98  P P A P  P P P P  1  1  1  1  secY serC  b3300 b0907  4.36 3.14  P A  P P  172  1  1  2  Table 6.4. Continued.  Gene name serS sodB speA  b no.  b0893 bl656 b2938  Fold change 2.78 3.13 2.29  Call stationary log phase phase P P A  P P P  Description  seryl-tRNA synthetase superoxide dismutase (FE) biosynthetic arginine decarboxylase (ADC) lipoprotein spr precursor stringent starvation protein A ' , regulator of transcription stringent starvation protein B ' 2-oxoglutarate dehydrogenase ' E l component , (decarboxylase component) 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase) succinyl-CoA synthetase beta chain ' , beta subunit succinyl-CoA synthetase alpha chain ' , alpha subunit survival protein surA precursor (peptidylprolyl cis-trans isomerase surA) (PPIase) (rotamase C ) transaldolase B ' propionate kinase , putative kinase keto-acid formate acetyltransferase (ketoacid formate-lyase)probable formate acetyltransferase 3 queuine tRNA-ribosyltransferase aspartokinase I / homoserine dehydrogenase I threonine synthase threonyl-tRNA synthetase trigger factor tolA protein tolB protein precursor outer membrane protein tolC precursor thiol peroxidase (scavengase p20) tRNA (guanine-nl)-methyltransferase (m 1 g-methyltransferase) tryptophan synthase alpha chain tryptophan synthase beta chain anthranilate phosphoribosyltransferase / anthranilate synthase component II 1  1  1  spr sspA  b2175 b3229  2.86 2.04  P P  P P  1  1  2  1  2  2  sspB sucA  b3228 b0726  2.68 3.25  P P  P P  1 2  1  sucB  b0727  2.64  P  P  2  1  sucC  b0728  2.99  P  P  sucD  b0729  3.51  A  P  surA  b0053  2.68  P  P  1 2  1 2  1  talB tdcD tdcE  b0008 b3115 b3114  3.17 2.11 2.79  P P A  P P P  tgt thrA  b0406 b0002  2.28 2.49  P P  P P  1  2  1  2  1  1  thrC thrS tig tolA tolB tolC tpx trmD  b0004 bl719 b0436 b0739 b0740 b3035 bl324 b2607  2.46 3.85 3.93 2.70 3.14 2.42 2.24 5.93  P P P P P P A P  P P P P P P P P  trpA trpB trpD  bl260 bl261 bl263  9.26 8.90 5.08  A A A  P P P  1  1  1  1  1  1  1  1  1  1  173  Table 6.4. Continued.  Gene name trpL trpT truB tsf tsr  b no.  M265 b3761 b3166 b0170 b4355  Fold change 2.41 4.71 2.24 7.31 3.67  Call stationary log phase phase P A A P P  P P P P P  Description Trp operon leader peptide tryptophan t R N A ' tRNA pseudouridine 55 synthase ' elongation factor EF-Ts methyl-accepting chemotaxis protein I (MCP-I) (serine chemoreceptor protein) elongation factor EF-Tu elongation factor EF-Tu ribonuclease R valyl-tRNA synthetase protease ecfE undecaprenyl pyrophosphate synthetase unknown protein from 2d-PAGE precursor (spots m62/m63/o3/o9/t35) hypothetical 10.2 kD protein in aroM-araJ intergenic region hypothetical 11.9 kD protein in tgt-secD intergenic region (ORF 12) 10.9 kD protein in cydB-tolQ intergenic region(ORFD) hypothetical 28.2 kD protein in pal-lysT intergenic region precursor hypothetical 19.3 kD protein in rne-rpmF intergenic region (G30K) hypothetical 24.5 kD protein in add-nth intergenic region putative proton-dependent oligopeptide transporter hypothetical 26.4 kD protein in ruvC-aspS intergenic region hypothetical 4.2 kD protein in prc-prpA intergenic region hypothetical 8.1 kD protein in sbcB-hisL intergenic region hypothetical 49.8 kD transport protein in sbcB-hisL intergenic region hypothetical 36.2 kD protein in ndk-gcpE intergenic region , putative membrane protein 1  1  1 2  1  1  tufA tufB vacB valS yaeL yaeS yaeT  b3339 b3980 b4179 b4258 b0176 b0174 b0177  2.55 3.25 2.22 2.29 2.45 2.25 2.51  P P P P P P A  P P P P P P P  1  1  1  1  1  1  1  yaiE  b0391  2.87  P  P  1  yajC  b0407  2.23  A  P  1  ybgE  b0735  2.58  P  P  1  ybgF  b0742  3.70  P  P  1  yceD  bl088  3.92  P  P  1  ydgQ  bl632  2.33  A  P  1  ydgR  bl634  2.33  P  P  1  yebC  M864  2.06  P  P  1  yebJ  bl831  2.83  P  P  1  yeeD  b2012  4.58  A  P  1  yeeF  b2014  2.36  P  P  1  yfgA  b2516  2.15  P  P  1  174  Table 6.4. Continued. Gene name yfgB  b no.  b2517  Fold change 1.99  Call stationary log phase phase P P  Description  hypothetical 43.1 kD protein in ndk-gcpE intergenic region , orf, hypothetical protein hypothetical tRNA/rRNA methyltransferase yfiF hypothetical 23.2 kD protein in prpB-rpoS intergenic region , putative epimerase/aldolase hypothetical 29.2 kD protein in mutSrpoS intergenic region ( 0 2 5 8 ) o r f , hypothetical protein hypothetical 40.2 kD protein in kduI-lysS intergenic region , putative carbamoyl transferase putative diaminopropionate ammonialyase (diaminopropionatase) hypothetical 44.8 kD protein in kduI-lysS intergenic region , putative deacetylase hypothetical 24.9 kD protein in tolC-ribB intergenic region (ORFD) ( 0 2 3 4 ) o r f , hypothetical protein hypothetical 16.3 kD protein in exuRtdcC intergenic region , orf, hypothetical protein hypothetical 10.8 kD protein in ftsJ-greA intergenic region (097) , orf, hypothetical protein hypothetical 35.9 kD protein in pmra-fis intergenic region (ORF1) \ putative dehydrogenase hypothetical 38.6 kD protein in cysG-trpS intergenic region (F361) \ orf, hypothetical protein 33.2 kD protein in dinD-rph intergenic region (ORF X ) \ putative alpha helix protein GTP-binding protein TypA/BipA (tyrosine phosphorylated protein A) \ putative GTP-binding factor 1  2  yfiF  b2581  2.13  A  P  1  ygbL  b2738  3.86  A  P  1  ygbM  b2739  2.26  P  P  2  ygeW  b2870  5.88  A  P  1  2  ygeX  b2871  4.18  A  P  1  ygeY  b2872  6.33  A  P  1  ygiB  b3037  2.00  P  P  2  2  yhaR  b3113  3.47  A  P  1  2  yhbY  b3180  2.50  P  P  1  2  yhdG  b3260  2.58  P  P  yhfS  b3376  2.02  A  P  2  yicC  b3644  1.99  P  P  2  yihK  b3871  2.86  P  P  2  175  Table 6.4. Continued. Gene name yjdA  b no.  b4109  Fold change 2.43  Call stationary log phase phase P A  Description  hypothetical 84.2 kD protein in phnAproP intergenic region (ORF742) , putative vimentin hypothetical 31.1 kD protein in eaeHbetA intergenic region , carbamate kinase , putative kinase hypothetical 5.4 kD protein in speA-metK intergenic region ( F 4 8 ) o r f , hypothetical protein hypothetical 8.1 kD protein in speA-metK intergenic region (071) \ orf, hypothetical protein high-affinity zinc transport system substrate-binding protein l  2  ykgG  b0308  2.02  P  P  yqeA yqgB  b2874 b2939  4.43 2.23  A P  P P  1  1  2  2  yqgC  b2940  2.04  P  P  2  znuA  bl857  2.35  P  P  1  2 3 4  http ://www. genome, ad.j p/kegg/ Affymetrix gene chip data base P= Present (gene was detected) A = Absent (gene was not detected)  176  CHAPTER SEVEN G E N E R A L DISCUSSION, G E N E R A L CONCLUSION AND RECOMMENDATIONS FOR FUTURE STUDIES  177  7.1  General discussion  7.1.1  Effect of V M on E. coli cells In the present work an attempt was made to verify whether the lethal effect of vacuum  microwave on microorgnisms was entirely due to heat or whether other effects were associated with microwave radiation. For this purpose E. coli was chosen as a model microorganism because of its relatively simple structure, and well known physiology and genome sequence. E. coli kinetic parameters under lethal and sub-lethal conditions were determined, followed by a study of the E. coli transcriptional response to sub-lethal treatment with vacuum microwave and convective heating. The larger D-values for V M , at lower temperatures and small D-values at higher temperatures compared to conventional heat treatments, provided evidence of a factor(s) involved in V M other than heat in E. coli inactivation. One possible source of the difference in lethality was the difference in heating rates of bacteria in the microwave treatment compared to the water bath under vacuum treatment. Kaur and colleagues (1998) studied the effect of heating rate on the survival of E. coli at 60°C for 40s. They reported that for heating rate of 1 °C/min the mean number of survivors was 1.4 log CFU/ml while for heating rate 10 °C /min it was 2.6 log CFU/ml. They concluded that this might be due to exposure to potentially lethal temperatures for longer during heating period. Therefore higher inactivation with slower heating rate would be expected. Another hypothesis for lower destruction rates at lower temperatures is that direct heating of microorganisms with microwaves enhances the production of heat shock proteins, thereby increasing t heir r esistance c ompared t o t he c ontrol. O ther r esearchers a Iso r eported t hat h eat resistance of some bacteria increases upon exposure to temperatures slightly higher than their  178  optimum (Foster & Spector 1995; Kaur et al. 1998). Kusukawa & Yura (1988) proposed that this higher resistance is due to expression of heat shock proteins. The result of the present work on E. coli transcriptional response at 50°C did not support this hypothesis and showed no significant difference on the expression of heat s hock genes between vacuum microwave and water bath under vacuum treatment. During the kinetic study at 50°C, E. coli cells were exposed to V M for 20-25 minutes, whereas the transcriptional response was determined after three minutes of exposure to vacuum microwave. It is possible the expression of heat shock genes and production of heat shock proteins might happen after a longer exposure time. Thus, here we can only say that heat shock genes were not significantly expressed as a result of short-time exposure to sub-lethal vacuum microwave treatment. At the same time, more down-regulation in 5S and 16S rRNA due to water bath treatment showed that ribosomal subunits in V M treated cells were affected less and thus were more stable. This could be a reason for less destruction at 50°C in V M treatment compared to water bath treatment. Activation energies showed that V M treated cells needed higher levels of energy for destruction, compared to the water bath under vacuum treatment.  Since activation energy  represents the minimum kinetic energy that must be possessed by a molecule in order to react, it can be concluded that destruction of E. coli under V M treatment occurs by a different mechanism than under the convection heat treatment. Dielectric loss tangent and loss factor were higher for the centrifuged E. coli pellet compared to peptone water. Therefore, when the mixture of culture and peptone water was exposed to microwave radiation, E. coli produced more heat than the surrounding liquid environment (peptone water). This may cause a slight local temperature increase inside the cells. This lends credence to the selective heating theory, one of the four predominant theories of  179  nonthermal inactivation of microwave (Kozempel et al. 1998). The selective heating theory hypothesizes that microorganisms are heated more effectively by microwaves than their surrounding medium and therefore can be killed more rapidly (Datta & Davidson 2000). The presence of evidence for the existence of a factor or factors other than heat associated to V M led to further investigation. To search for these factors at the molecular level, E. coli cell transcriptional responses to sub-lethal V M and water bath treatment at 50°C for 3 minutes were studied. The number of genes altered through water bath treatment was higher than V M , indicating that water bath treatment had a greater impact. Since the D value for water bath treatment at 50°C was shorter than for V M , more severe changes in cells exposed to water bath treatment would indeed be expected. Some differences in E. coli responses at transcriptional level to V M compared to water bath treatment were observed, such as the effects on genes involved in cell membrane and cell transport systems. The cysWand ybaR genes related to copper and sulfate transport respectively and the ompF gene encoding porins and responsible for dipeptide permease, were significantly altered during both treatments.  Simultaneously the yejE, btuC, exuT, ycjO, ydiQ, yfcC and  b0878 genes involved in membrane transport of peptide, vitamin B i , galacturonate and 2  glucuronate, putative S-transferase, multiple sugar and A B C transporter were all down-regulated while fecA encoding for ferric citrate outer membrane receptor protein was up-regulated in V M treated E. coli compared to the water bath treated E. coli.  This suggests that due to V M ,  transcription for genes involved in ion transfer were increased while transport for larger molecules including peptides, multiple sugars and vitamins was decreased.  Effect of  electromagnetic field on periplasm-binding protein-dependent transport system were previously reported. Nascimento and colleagues (2003) showed higher amounts of glucose transported into  180  the E. coli cells exposed to electromagnetic field (60 Hz, 8 hours, 28°C), and Liburdy and coworkers (1985) reported an increase in sodium passive transport at the membrane of rabbit erythrocytes e xposed t o microwaves (2450 M Hz, 4 00 m W/g) w ithin a n arrow r ange (17.7 t o 19.5°C) of temperature. In addition fimC, fimD and fimG genes related to outer membrane protein, periplasmic chaperone and morphology of fimbriae and fliG encoding for the flagellar motor switch were expressed more in V M treated E. coli compared to water bath treated E. coli. The murG gene encoding for an enzyme involved in peptidoglycan biosynthesis was expressed less in V M compare to water bath treated E. coli.  Peptidoglycan in the cell wall is responsible for  mechanical strength and maintaining the shape of the cell (Singleton & Sainsbury 2000). Thus it could suggest that while genes related to membrane structure and transport systems were affected in both treatments, the effect of the V M treatment was greater than conventional heat treatment. This may lend credence to dielectric cell-membrane rupture theory.  This theory  hypothesizes that an external electric field induces an additional trans-membrane electric potential in addition to the normal potential of the cell which in turn results in a voltage drop across the cell membrane sufficient for membrane rupture (Datta & Davidson 2000; Kozempel et al. 1998; Zimmermann et al. 1974) or pore formation, increased permeability, and loss of cell integrity (Brunkhorst et al. 2000, Kozempel et al. 2000). The other difference noted in this study was in tRNA synthesis. Although genes related to tRNAs specific to glutamine, tryptophan and leucine were up-regulated by both treatments compared to untreated E. coli cells, genes e ncoding for g lutamine synthetase and g lutaminyltRNA synthetase were significantly higher in V M compared to water bath treated E. coli. This indicates that glutamine synthesis was more active in E. coli after V M treatment.  181  Higher expression of the gene responsible for ubiquinone biosynthesis and lower expression of the gene involved in menaquinone synthesis, along with increased expression of genes involved in flagellar motility in V M compare to water bath treated E. coli are signs of aerobic respiration in V M treated cells.  It has been reported that mutation in the quinone  biosynthesis pathway gives rise to immobility and lack of flagellum (Poole & Ingledew 1987). At the same time, in water bath treated cells, genes involved in energy metabolism through oxidative phosphorylation and nitrogen metabolism were not detected or were expressed less compared to untreated samples while these genes remained unchanged after V M treatment. Simultaneously, transcription levels for genes related to copper and sulfate ions functioning as electron acceptors in anaerobic respiration were shown to be up-regulated for both treatments while gene for ferric ion were up-regulated in V M treated E. coli.  This may suggest that  although E. coli showed signs of anaerobic respiration in both treatments, the transition to anaerobic respiration was more advanced in water bath treated E. coli than in V M treated E. coli. In this study rrlD gene related to 23 S ribosomal R N A in the rrnD operon was upregulated about 250 and 300 fold in water bath and V M treated E. coli respectively. This could be explained as an exposure to stress conditions as reported by other researchers for Saccharomyces cerevisiae (Lopez et al. 2002) and S. typhimurium (Tolker-Nielsen et al. 1997). At the same time the expression of 5S rRNA genes were down-regulated in both treatments. But the number of down-regulated genes (6 in water bath treated E. coli and 4 in V M treated E. coli) as well as the average fold change (42.5 in water bath compare to 17.5 in V M ) was higher for the water bath treated E. coli. In addition, one gene related to 16S r R N A showed down-regulation during water bath treatment while the level of gene expression remained unchanged in V M treated E. coli compared to untreated E. coli. Therefore, the effect of sub-lethal V M treatment  182  on ribosomal R N A was less pronounced than water bath under vacuum treatment.  Since  ribosomes are responsible for translation of messenger RNAs into proteins (Madigan et al. 2003), this could suggest that the process of translation for protein production was affected less by V M treatment than by water bath heat treatment.  7.1.2  Effect of growth phase on E. coli transcriptome In the present work, kinetic studies and gene transcription studies were of stationary cells  while most gene expression studies use cells from exponential stages of growth. Thermal death kinetics of bacteria are typically studied on stationary phase cells, for bacteria cells in this stage are in their most resistant form. Thus to close the loop, the effect of growth phase on E. coli transcription was examined. The gene expression of E. coli cells from mid-stationary phase were compared with that of late-log phase cells. The expression profiles of genes involved in transcription and translation, including the major subunits of R N A polymerase, ribosomal proteins, and translation factors were downregulated in mid-stationary phase.  A decrease in the overall translation activity or protein  synthesis was previously reported to occur during transition from the exponential growth to the stationary phase in E. coli cells (Wada et al. 1990). In addition, the csg genes required for curli synthesis (Chirwa & Herrington 2003) were induced in mid-stationary phase cells. Curli, one of the adhesive organelles in E. coli, promote clumping of bacterial cells in culture and are important for biofilm formation (Vidal et al. 1998; Austin et al. 1998; Prigent-Combaret et al. 2000). Since curli synthesis was started in stationary phase cells, the expression of rpoS, ompR and cpx genes reported to be responsible for csg genes expression (Arnqvist et al. 1994; Prigent-Combaret et al., 2001; Chirwa & Herrington 2003)  183  expected to be up-regulated. At the present work the expression of csg genes either displayed no significant change between late-log and mid-stationary E. coli or were not detected in neither phase of the growth, which indicated that other regulatory systems could be involved in the activation of csg genes. As curlin contains high amounts of glycine, Chirwa & Herrington (2003) proposed that up-regulation of glyA is an essential response for curli formation. But in this study, although glyA was detected in all samples, its transcription level did not show any significant change between the two stages of growth. It is possible that glyA was already sufficiently expressed in the late-log phase to produce amino acid necessary for rapid cell growth, and its expression continued in the stationary phase to provide necessary glycine for curli synthesis, where other genes involved in amino acid biosynthesis were down-regulated. In cells at mid-stationary phase of growth, 70.5% of the genes required for flagellar synthesis, chemotaxis and subsequent motility were down-regulated or were not expressed at all. These E. coli cells in the mid-stationary phase of growth would have less capacity for motility. Some unexpected changes in regulatory events were observed. Fis and Rpos are two regulatory proteins which co-ordinately control the expression of some of the genes during late log and stationary phase. While Fis expression is said to be at its maximum in the early-to-mid log condition, the expression of Rpos is believed to be turned on in late exponential and stationary phase and is required for expression of genes important under starvation conditions or stationary phase (Xu & Johnson 1995). In this experiment the expression of fis gene was 2.25 fold higher in late-exponential phase, but interestingly, although the rpoS gene was detected as present in both phases, its expression did not show any significant change between the two stages of growth. X u & Johnson (1995) reported that the products of xylF and mglA are required  184  for growth under nutrient-poor conditions, in which fis levels are low.  The result of this  experiment was in agreement with X u & Johnson (1995) for expression of xylF gene, which was 1.57 fold higher in mid-stationary phase but was in disagreement for the mglA gene. The mglA gene showed a higher expression in late-exponential growth where the fis level of expression was not reduced. X u & Johnson (1995) also reported that the presence of rpoS reduced the expression of xylF, mglA and sdhA. The present study showed that while the expression oirpoS did not change, the expression of these three genes were altered between the two stages of growth. Perhaps other regulatory genes are involved in the induction or repression of xylF, mglA and sdhA in different stages of growth. On the other hand, most of reported changes in literature were detected at the protein level, and gene transcript and protein activity do not have a linear correlation, thus some discrepancies would be expected. A comparison between the expression of previously known genes involved in early stationary phase and their expression in this study, mid-stationary phase, showed some interesting results. In the present work, the transcription of hupA, hupB, hlpA, himA and himD was 2.42, 3 .33, 3 .26, 2.68 and 1.81 fold down-regulated respectively i n mid-stationary phase cells while other researchers reported a higher transcription for these genes upon entry into stationary phase (Dersch et al. 1993; Weglenska et al. 1996; Claret & Rouviere-Yaniv 1997). The transition to stationary phase has been described as a general stress response in E. coli cells (Hengge-Aronis 1996, 1999). Thus early stationary phase could be viewed as a short-term response to the external stress factor while the gene expression at mid and late stationary phase could be referred to as a long-term stress response and difference between short-term and longterm stress response would be expected.  185  7.2  General conclusion The present work revealed that although there is much similarity between conventional  heat treatment and V M , there is evidence for the presence of an inactivation mechanism other than heat associated with V M . While temperature within experiments and between treatments, was kept constant, slower inactivation at temperatures less than 53°C and higher reduction in microbial population at temperatures above 53°C for V M treated E. coli was observed, along with significant differences in activation energy and temperature sensitivity between V M and water bath treated E. coli. The impact of temperature on lethal rate of E. coli was different when microwaves were the medium of heat transfer and the destruction mechanism of V M was therefore different from that of water bath heating. Thus the presence of factor(s) other than heat involve in microwave under vacuum was established. At 50°C, V M had a larger effect on transcription of genes related to membrane structure and membrane transport system, as well as genes related to metabolism of carbohydrates, lipids and amino acids than the water bath treatment.  On the other hand, the effect of conventional  water bath treatment on ribosomal subunits was greater. Interestingly, although both treatments included equal vacuum and signs of anaerobic respiration would be expected, water bath treated E. coli shown more evidence for the start of anaerobic respiration at transcriptional level than V M treated E. coli. In addition, this work identified some differences in transcriptional response of E. coli in late-log and mid-stationary phase of growth. In mid-stationary phase, genes encoding for energy metabolism as well as amino acids and carbohydrate metabolism were down-regulated. In addition, the E. coli response in transcriptional level showed lower expression for genes involved  186  in cell motility and higher espression for genes involved in curli synthesis in mid-stationary E. coli compared to late-log cells. Interestingly some genes reported by other researchers to upregulate upon entry into stationary phase showed down-regulation in mid-stationary phase cells suggesting that the mechanisms involved in cell behaviour are not only different between lag, log and stationary phase of growth but may differ in early, mid and late stationary phase.  7.3  Proposed theories The data presented in this study is not sufficient to elucidate the mechanism of E. coli  destruction upon exposure tornicrowave r adiation under v acuum. N onetheless, b ased o n t he present findings the following theories can be proposed: This study showed that V M treatment affected the transcription of genes related to membrane structure. This could indicate that V M damages the E. coli cell membrane. Findings on t he c hanges i n t ranscription o f g enes related to t he c ell m embrane t ransport s ystem c ould suggest that cell transportation systems are disturbed. E. coli could then be faced with a lack of essential substrate or excess of unnecessary substrate. This imbalance of material could affect the cell function and result in cell destruction.  Changes in transcription of genes related to  aerobic respiration due to V M treatment could act to favour cell growth or not depending on the environmental conditions. If the environmental conditions require oxidative respiration, E. coli with up-regulated aerobic respiration would have more chance to survive, but i f environmental conditions require anaerobic respiration, E. coli cells with more tendency for aerobic respiration will have less chance to survive.  187  7.4  Recommendations for future studies There i s n o d oubt t hat m ore s tudies a re n eeded t o r each a comprehensive e xplanation  about the effect of microwaves on living bacterial cells.  The following are some  recommendation for future directions:  •  Investigate the expression of rrlD gene under different stress conditions such as acid stress, starvation and or cold stress to find that whether this gene can be recognized as an stress indicator in E. coli.  •  Investigate the E. coli response to longer exposure time for example 6 and 9 minutes, to microwave radiation and conventional heat treatment to monitor the changes in transcription of genes related to membrane structure and transport system.  •  Investigate the effect of microwave on E. coli cells on membrane genes at translation level using a proteomic approach.  •  Investigate the effects of microwaves on E. coli cells from the exponential growth phase. Cells in this stage are more active and less resistant to any stress, therefore the cell response to microwave radiation as a stress factor would be expected to be greater.  In addition, since more pathways are active in this stage, more  changes could be expected. •  Investigate the effect of microwave radiation (2450 M H z and/or 915 MHz) on the gene expression of human epithelial cells, as the primary tissue of humans exposed to microwaves.  188  •  Any of the above studies could be conducted with prolonged, repeated exposure to check for a cumulative effect of microwave radiation.  •  A comparison of genes involved in different stages of stationary phase (early, mid and late) using D N A microarray would be informative  •  A study of the genes involved in early, mid and late exponential phases of E. coli growth would be informative.  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Results  Test Gram stain Lactose fermentaion (Lauryl Sulfate Tryptose Broth) Lactose fermentation (Brilliant Green Bile Broth) Glucuronidase activity (Violet Red Bile M U G Agar) Beta-galactosidase and Beta-glucoronidase activity (Chromocult Coliform Agar) ' Kornacki & Johnson 2001. Linton etal. 1997. Alonsoetal. 1998. Finney et al. 2003. 1  1  2  4  2  3  4  213  Small rod shape cell, Pink colour Gas formation Gas formation Red colonies surrounded by turbid zone + scatter blue light under U V Dark blue to violet colonies  9.2  Appendix II: Continuous Vacuum System: Schematics and suppliers  1. Microwave oven (General Electric- JE435, Mississauga, Canada) or water bath 2. Dessiccator (Pyrex brand-Fisher Scientific), I.D. 160mm modified by Sandfire Scientific Ltd. Mission, BC) (Figures 9.1-9.6) 3. Micropump (Pressure-loaded compact low-flow pump head without canister, Micropump, Inc. W A , USA) 4. Vacuum pump (SIHI pumps Ltd. Guelph, Canada) 5. Stainless steel Tubing & Connectors (Columbia Valve & Fitting Ltd., North Vancouver, Canada)  214  MICROWAVE OVEN OR WATER BATH CONNECTORS STAINLESS STEEL STAINLESS STEEL TU1ING ID = 6,82nn •D = 9.5E nn  519  THERMOCOUPLE CONNECTED TD DATA LODGER FDR MEASURING TEMPERATURE  cm  Figure 9.1. Overview of Continuous Vacuum System  215  ,=3  GUAGE  ID= 7.51 nn •D= 12.81 nn  <nzz  £85 cn 27.2 cn (SIDE ARM)  Figure 9.2. Glass vacuum chamber body; Side-view.  Figure 9.3. Glass vacuum chamber body; Inside-view.  216  PYREX  TUBING  19,8 cn  Figure 9.4. Glass vacuum chamber body; Top-view  217  Figure  9.5. Glass vacuum Chamber lid; Top-view  218  Figure 9.6. Glass vacuum Chamber lid; Side-view.  219  9.3  Appendix III: Microwave power determinations  The power is calculated from the following formula: P = 70 x (ATi + AT )/ 2  Eq(9.1)  2  Where  P = power (W) ATi and AT2 = Temperature rise of the water in the two beakers (°C).  Table 9.2. Microwave power determined using IMPI2-Liter test (Buffler 1993). Microwave Oven setting  Microwave Power (W)  mean ± stdv  10  700.00  700.00  735.00  711.67 ±20.21  9  682.50  656.25  638.75  659.17 + 22.02  8  507.50  507.50  516.25  510.42 + 5.05  7  393.75  376.25  376.25  382.08+ 10.10  6  253.75  253.75  245.00  250.83 ± 5.05  5  201.25  210.00  218.75  210.00 ±8.75  4  175.10  192.50  192.50  186.70 ± 10.05  3  87.50  87.50  87.50  87.50 ± 0 . 0 0  2  70.00  70.00  70.00  70.00 ± 0 . 0 0  1  35.00  35.00  35.00  35.00 ± 0 . 0 0  220  9.4  Appendix IV: Micro pumpflowrate determinations  T a b l e 9.3.  The flow rate of micro pump was determined under normal atmosphere and vacuum  (22, 24 and 26 inHg). Each value is the mean of three measurements. Pump setting  Flow rate ml/min no vacuum  26 inHg  24 inHg  22 inHg  10  626.3  582.5  602.5  642.5  9  620.0  580.0  593.3  633.3  8  495.0  435.0  485  583.3  7  410.0  422.2  396.7  453.3  6  286.3  257.5  316.7  340.0  5  220.0  233.3  220.0  203.3  221  9.5  Appendix V : Thermocouple calibration  H  10 0 H 0  1  1  1  1  1  1  1  1  10  20  30  40  50  60  70  80  90  ASTM Thermometer (C) Figure 9.7. Regression equation for temperatures from the data logger versus recorded temperatures from the A S T M thermometer(ASTM l c , -20/150 CP, VWRbrand, VWR), as a correction factor for T type thermocouple.  222  9.6  Appendix VI: Survival curves for E. coli plated on PCA and PCA-BS  1 0  \ 0  1  1  1  1  1  20  40  60  80  100  ;  120  1  140  Time (second) Figure 9.8. Differential counts of E. coli on P C A — A —and P C A - B S — • — during vacuum microwave (711W) at 58.43°C.  223  1 0 -I 0  1  25  1  1  1  50  75  100  1  125  r—  150  Time (second) Figure 9.9. Differential counts of E. coli on P C A — A —and P C A - B S — • — during vacuum microwave (510W) at 58.19°C.  224  Figure 9.10. Differential counts of E. coli on P C A — A —and P C A - B S — • — during water bath treatment under vacuum at 58.62°C.  225  1 0  "I 0  1  !  1  1  1  1  1  1  50  100  150  200  250  300  350  400  Time (second) Figure 9.11. Differential counts of £ coli on P C A — • —and P C A - B S — • — during vacuum microwave (711W) at 51.84°C.  226  8  A  CF /ml  7  n  4 -  CD O _J  3 -  5 -  2 1 0 50  100  150  200  250  300  Time (second) Figure 9.12. Differential counts of E. coli on P C A — • —and P C A - B S — • — durin vacuum microwave (510W) at 50.21°C.  227  225  Time (second) Figure 9.13. Differential counts of E. coli on P C A — A —and P C A - B S — • — during water bath treatment under vacuum at 50.5°C.  228  /  9.7 A p p e n d i x V I I : G e n e s a l t e r e d less t h a n t w o f o l d b e t w e e n l a t e - l o g a n d m i d - s t a t i o n a r y cells (p<0.05). Table  9.4.  Genes up-regulated less than 2 fold in mid-stationary phase cells compared to  late-log phase E. coli cells (p<0.05). Gene  citC csgF cvpA dmsA gapC_2 glcB  gigs  pheL pheV phoH rhaR rhoL rspA sip soxS syd tl50 tehA ubiX wcaD xylF yadQ yadT yaeE ybcl ycdQ yceE ycfQ ycjZ ydjZ yebF yeeT yfaA yfcC yffB  b no.  b0618 M038 b2313 b0894 bl416 b2976 b3049 b2598 b2967 M020 b3906 b3782 M581 b3506 b4062 b2793 b3558 bl429 b2311 b2056 b3566 b0155 b0158 b0198 b0527 bl022 M053 bllll bl328 bl752 bl847 b2003 b2230 b2298 b2471  F o l d change  Call stationary  log  phase  phase  P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P  A P P P A A P P P A A P P P A P A A P P A A P P P A P P P A P A A A P  1.40 1.42 1.40 1.42 1.44 1.73 1.76 1.72 1.38 1.47 1.59 1.35 1.54 1.59 1.93 1.37 1.65 1.31 1.50 1.46 1.57 1.51 1.36 1.65 1.54 1.18 1.30 1.23 1.63 1.58 1.37 1.43 1.66 1.84 1.24  J  229  4  Table 9.4. Continued. Gene  yfiP ygaC yghK yhdM yhdU yhd yhdY yheL yiaG yicE yicO yidB yihG yjhR yjjP ynfL yohJ yqgD yrbL yrfG ytfli  4  b no.  b2583 b2671 b2975 b3292 b3263 b326 b3270 b3343. b3555 b3654 b3664 b3698 b3862 b4308 b4364 bl595 b2141 b2941 b3207 b3399 b4212 bl680 b2596 b3050 bl675 b2372 b0832 b3051 b2375 b0919 b2666 b0539 b0964  Fold change 1.30 1.59 1.63 1.51 1.55 1.28 1.45 1.35 1.48 1.51 1.66 1.19 1.57 1.75 1.42 1.48 1.17 1.53 1.52 1.43 1.90 1.84 1.75 1.73 1.73 1.64 1.61 1.41 1.41 1.39 1.39 1.27 1.26  Call stationary phase P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P  P= Present (gene was detected) A = Absent (gene was not detected)  230  log phase P A P P P P A P P P P P P P P P A P P P P A P P A A P P P P P P A  Table  9.5. Genes down-regulated less than two fold in mid-stationary phase cells compared to  late-log phase E. coli cells (p<0.05). Gene  b no.  accB accD argC aroB artQ atpl basS bcp bisC carB cbl ccmH crp err damX dep dctA deoR dksA did dppD dppF dsbA edd efP fdol fimF fnr focA ftsL fumA gcpE glgP glnL gloB  giys greA  b3255 b2316 b3958 b3389 b0862 b3739 b4112 b2480 b3551 b0033 bl987 b2194 b3357 b2417 b3388 M538 b3528 b0840 b0145 b2133 b3541 b3540 b3860 bl851 b4147 b3892 b4318 bl334 b0904 b0083 M612 b2515 b3428 b3869 b0212 b3559 b3181  Call  Fold change  stationary  log  phase  phase  P P A P P P A P A P P A P P P A A P P A A P P P P P A A P P P P P P P P P  P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P  1.73 1.77 1.86 1.85 1.51 1.59 1.68 1.86 1.25 1.94 1.47 1.73 1.88 1.88 1.95 1.75 1.65 1.42 1.95 1.88 1.61 1.34 1.45 1.40 1.90 1.77 1.77 1.77 1.58 1.89 1.86 1.82 1.86 1.54 1.59 1.71 1.75  j  231  4  Table 9.5. Continued. Gene  guaA guaC hemC hemE hemX himD hisA hisF hybA hybG ilvC imp kdsA kdsB lexA manY map mdh mltA mltB modB motB mraY mrcA mreC msbB mtlA murA nagA nagE nemA nrdA nrdB nrfB nuoK nusG ogrK oppC panD parE  b no.  b2507 b0104 b3805 b3997 b3803 b0912 b2024 b2025 b2996 b2990 b3774 b0054 bl215 b0918 b4043 bl818 b0168 b3236 b2813 b2701 b0764 bl889 b0087 b3396 b3250 bl855 b3599 b3189 b0677 b0679 M650 b2234 b2235 b4071 b2279 b3982 b2082 bl245 b0131 b3030  Fold change 1.69 1.84 1.80 1.63 1.42 1.81 1.71 1.83 1.77 1.47 1.56 1.79 1.87 1.48 1.66 1.41 1.54 1.48 1.64 1.37 1.54 1.73 1.81 1.27 1.79 1.74 1.24 1.67 1.55 1.90 1.57 1.72 1.82 1.70 1.78 1.76 1.43 1.66 1.60 1.79  Call stationary phase A P P P P P P P P P P P P P P P P P P A A A P P P A A A P A A A P A P P A P P A  232  log phase P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P  Table 9.5. Continued.  Gene  b no.  Fold change  pepB pepD pepQ Pgk PgpA pheS phnE pinO pitA phB pncB pntB ppa ppsA proB pta ptsA ptsH putA pyrE recC rfaC rffG rhlB rho rnpA sbcB sdhB sdhC sdhD secE serA slpA slyA slyD speB spoT sseB suhB tbpA thiG  b2523 b0237 b3847 b2926 b0418 bl714 b4104 b3322 b3493 b4041 b0931 M602 b4226 bl702 b0242 b2297 b3947 b2415 bl014 b3642 b2822 b3621 b3788 b3780 b3783 b3704 b2011 b0724 b0721 b0722 b3981 b2913 b0028 bl642 b3349 b2937 b3650 b2522 b2533 b0068 b3991  1.57 1.94 1.54 1.76 1.20 1.54 1.31 1.13 1.53 1.67 1.90 1.75 1.62 1.74 1.47 1.65 1.28 1.89 1.88 1.55 1.12 1.29 1.62 1.40 1.71 1.93 1.67 1.92 1.85 1.92 1.85 1.79 1.81 1.83 1.43 1.52 1.70 1.80 1.34 1.71 1.74  Call stationary log phase phase P P A P P P P A P A P P P P P P P P A P P P P P P P P P P P P A A P P P P P P A P  233  P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P  Table 9.5. Continued. Gene  tldD torA ubiA uspA uvrC wecB xthA yadG yaeQ ybaS ybeA ybiC ybiS ybiT ybjT ycbE ycjN ycgR ychH ychN yehT yeiO yeiP yfaO yfcE yfhJ yfhO ygdD ygdH ygfQ yhbC yhbG yhbZ yhcG yhjH yi5A yiaF yijC yjcE  b no.  b3244 b0997 b4040 b3495 bl913 b3786 M749 b0127 b0190 b0485 b0636 b0801 b0819 b0820 b0869 b0933 bll06 bll94 bl205 bl219 b2125 b2170 b2171 b2251 b2300 b2524 b2530 b2807 b2795 b2884 b3170 b3201 b3183 b3220 b3525 b3557 b3554 b3963 b4065  Fold change 1.41 1.79 1.70 1.38 1.63 1.47 1.43 1.68 1.36 1.27 1.62 1.33 1.62 1.70 1.61 1.42 1.66 1.48 1.32 1.47 1.36 1.43 1.49 1.86 1.83 1.71 1.66 1.73 1.68 1.62 1.82 1.92 1.74 1.65 1.38 1.63 1.75 1.45 1.34  Call stationary phase P P P P A P P A A P A P P A A P P P P P P A A A A P P A A P P P A A A P P P A  234  log phase P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P  T a b l e 9.5.  Gene  Continued. b no.  Call  Fold change  stationary  log  phase  phase  P P P P A A P A P P A P P A A P P P P P P P P P P P A A  P P P P P P P P P P P P P P P P P P P P P P P P P P P P  1.32 b4121 yjdF 1.38 b4161 yjeQ 1.53 b4233 yjfG 1.63 b4243 yjgF 1.32 b4262 yjgQ b4391 1.59 yJJK 1.37 b4377 yjju 1.84 b0307 ykgF 1.57 b0838 ylU 1.42 b3033 yqiB 1.18 b3198 yrbl 1.34 b4220 ytjM 1.85 b0955 b2340 1.76 1.76 bl832 1.76 bl840 1.72 b3838 1.64 b2290 1.58 b2511 1.49 b2875 1.39 b2817 1.37 b2899 1.23 bl007 1.21 b0762 1.37 b0105 bl448 1.35 1.68 bl809 1.34 bl647 P= Present (gene was detected) A = Absent (gene was not detected) 4  235  

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