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Isolation and regulation of expression of the Rhodobacter capsulatus suca gene encoding the E1o component… Dastoor, Farahad 1991

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ISOLATION AND REGULATION OF EXPRESSION OF THE RHODOBACTER CAPSULATUS SUCA GENE ENCODING THE Elo COMPONENT OF THE oc-KETOGLUTARATE DEHYDROGENASE ENZYME COMPLEX by FARAHAD DASTOOR B.Sc, McGill University, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 17,1991 © Farahad Dastoor, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ii ABSTRACT The citric acid cycle (CAC) is an extremely wide-spread metabolic pathway involved in energy production and intermediary metabolism. One of the key enzymes of the C A C is the a-ketoglutarate dehydrogenase (KGD) enzyme complex. In facultative bacteria, activity of the K G D enzyme complex appears to vary in keeping with the primary function of the C A C , in response to growth conditions (energy production or biosynthesis). Therefore, under anaerobic conditions synthesis of the K G D enzyme complex is repressed in some species and carbon flow through some C A C steps is reversed converting the C A C into a branched pathway that functions primarily with intermediary metabolism. The purple photo synthetic bacterium Rhodobacter capsulatus relies on the C A C for energy production under aerobic growth conditions on minimal media containing organic acids such as malate, succinate, and pyruvate. However, when growing under anaerobic photo synthetic conditions R. capsulatus produces energy by photosynthesis, although intermediates of the C A C are still required for the biosynthesis of bacteriochlorophyll and amino acids. It is believed that R. capsulatus does not operate an anaerobic branched version of the C A C , as in other facultative bacteria, but a less active cyclic one. Therefore, it is of great interest to understand the mechanism by which KGD enzyme activity is regulated and how this affects the operation of the C A C in R. capsulatus in response to fluctuating environmental conditions. I have isolated the R. capsulatus sue A gene encoding the E lo enzyme component of the K G D enzyme complex. Pulse labelling of R. capsulatus cultures growing aerobically and photosynthetically indicated that the sue A gene was transcribed at a higher level under aerobic growth conditions than under photosynthetic growth conditions. This increased iii level of transcription resulted in a 7-fold increase in the steady state levels of K G D mRNA, and a 9 to 13-fold increase in K G D enzyme specific activity in crude cell extracts under aerobic growth conditions, compared to anaerobic photosynthetic growth conditions. This regulation of the sue A gene in response to oxygen is different from any other R. capsulatus gene studied, and is probably part of a larger network of processes that takes place to regulate control of metabolic pathways in cells that shift between two environmental conditions. iv T A B L E OF CONTENT Abstract ii Table of Contents iv List of Tables vi List of Figures vii Abbreviations and Symbols ix Acknowledgements xi Introduction 1 Materials and Methods 15 1. Bacterial Strains and Plasmids 15 2. Media and Growth of Bacteria 15 3. Measurement of Bacterial Growth 17 4. In Vitro DNA Techniques 17 5. Transformation and Conjugation 18 6. Plasmid Purification 18 7. Chromosomal DNA Isolation 20 8. DNA Sequence Analysis 20 9. Southern Blots and Hybridizations 21 10. RNA Isolation 22 11. RNA Blotting and Hybridization 23 12. oc-Ketoglutarate Dehydrogenase Enzyme Assay 24 13. Density Scans of RNA Blot Autoradiograms 26 14. Pulse Labelling of KGD RNA 26 Results 29 1. Screening of R. capsulatus gene bank for a gene that complemented the KGD 11 mutation 29 2. Localization of the DNA segment responsible for complementation of the KGD-phenotype of KGD11. . . . 29 V 3. Sequencing of a portion of the DNA segment responsible for rescue of the wild type phenotype in KGD 11 by recombination 41 4. Southern hybridization of R. capsulatus DNA 49 5. RNA blot hybridizations of RNA prepared from anaerobic photosynthetic and aerobic respiratory cultures of R. capsulatus BIO 53 6. KGD enzyme activities in extracts of R. capsulatus cultures grown under anaerobic photosynthetic and aerobic respiratory conditions 63 7. KGD RNA transcription in R. capsulatus BIO cultures grown under high aeration or anaerobic photosynthetic conditions , 63 Discussion 66 Conclusions 76 References 77 vi LIST OF TABLES Table I: Plasmids Used 16 Table II: Data from Complementation/Recombination of the KGD" Phenotype of KGD 11 with the 12 kb Sstl Fragment of pFDI Cloned in pRK415 40 Table LTI: Data from Expression Experiment with the 12 kb Sstl fragment of pFDI Cloned in the Expression Vector pJAJ9 44 Table IV: Data from Complementation/Recombination Experiment with Portions of the 12 kb Sstl Fragment 46 Table V: KGD Specific Activity in R. capsulatus Strain BIO Cell Extracts Grown Aerobically and Photosynthetically in RCV Medium 64 Table VI: Hybridization of Pulse Labelled RNA to a K G D mRNA-Specific Probe to Evaluate the Frequency of Initiation of Transcription of the KGD Gene under Aerobic or Anaerobic Growth Conditions 65 vii LIST OF FIGURES Figure 1: The Citric Acid Cycle 2 Figure 2: Branched Version of the Citric Acid Cycle in E. coli under anaerobic growth conditions . 6 Figure 3: Organization of KGD Genes in E. coli, B. subtilis, A. vinelandii, and P. fluorescens 11 Figure 4: Cosmid Clones pFDI and pFDII 30 Figure 5: Outline of Cloning of 4 kb, 2 kb, 1.8 kb, 1.5 kb, and 1 kb EcoRl Fragments from pFDI into pRK415 . . . 33 Figure 6: Outline of Complementation/Recombination Procedure 35 Figure 7: Outline of Construction of pRK415::14a andpRK415::14bPlasmids . . 38 Figure 8: Outline of Construction of pJAJ9::14a and pJAJ9::14bPlasmids 42 Figure 9: Partial Restriction Site Map of the 15 kb EcoRI Fragment 45 Figure 10: Nucleotide Sequence of Regions (a) and (b) 47 Figure 11: Alignment of R. capsulatus Translated (a) and (b) Sequences with the E. coli Elo Protein 50 Figure 12: Alignment of R. capsulatus Translated (a) and (b) Sequences with the A. vinelandii Elo Protein . . . . 51 Figure 13: Alignment of the 15 kb EcoRI Fragment that Complemented in KGD 11 with the E. coli sucAB Operon \ 52 Figure 14: Autoradiogram of a Southern Blot of R. capsulatus DNA 54 Figure 15: Autoradiogram of a Northern Blot of R. capsulatus RNA 57 viii Figure 16: Autoradiogram of a Northern Blot mf Guanidine Isothiocyanate Isolated R. capsulatus RNA 59 Figure 17: Autoradiogram of a Northern Blot of Guanidine Isothiocyanate Isolated R. capsulatus RNA Treated with DNase I 61 ix ABBREVIATIONS AND SYMBOLS aceE structural gene encoding Elp of the PDH complex in E. coli aceF structural gene encoding E2p of the PDH complex in E. coli ccKG oc-ketoglutarate Ap (or Ap1) ampicillin resistance structural gene ATP adenosine 5'-triphosphate CAC citric acid cycle cfu colony forming units cpm counts per minute CoA-SH reduced form of coenzyme A DNA deoxyribonucleic acid Elo oc-ketoglutarate dehydrogenase enzyme component of the KGD enzyme complex E2o dihydrolipoyl succinyltransferase enzyme component of the KGD enzyme complex E3 dihydrolipoamide dehydrogenase enzyme component of the KGD and PDH enzyme complexes Elp pyruvate dehydrogenase enzyme component of the PDH enzyme complex E2p dihydrolipoyl succinyltransferase enzyme component of the PDH enzyme complex FAD flavin adenine dinucleotide (oxidized form) FADH2 reduced form of FAD kb kilobase pairs KGD oc-ketoglutarate dehydrogenase enzyme complex Km (or Km1) kanamycin resistance structural gene Ipd structural gene encoding the E3 component of the PDH and KGD enzyme complexes LipS2 oxidized form of lipoic acid Lip(SH)2 reduced form of lipoic acid NAD + nicotinamide adenine dinucleotide (oxidized form) NADH reduced form of NAD+ PDH pyruvate dehydrogenase enzyme complex odhA B. subtilis sucA analogue odhB B. subtilis sucB analogue psi pounds per square inch pufB structural gene of light harvesting I (3 peptide in R. capsulatus X pufQ gene of unknown function required for bacteriochlorophyll synthesis in R. capsulatus RNA ribonucleic acid RPM revolutions per minute sue A structural gene encoding the Elo component of the KGD enzyme .complex sucB structural gene encoding the E2o component of the KGD enzyme complex Tc (or TcO tetracycline resistance structural gene TPP thiamine pyrophosphate xi ACKNOWLEDGEMENTS I would like to thank Dr. J.T. Beatty for his guidance, encouragement, enthusiasm, and seemingly endless patience during the course of my research. I would also like to acknowledge my present and former lab colleagues Tim Lilburn, Heidi LeBlanc, Danny Wong, Joanna Zilsel, Cheryl Wellington, Mimi Mah, and Andrew Taggart for creating a very stimulating and fun working environment and without whom morning coffee would be a dull experience. 1 INTRODUCTION The citric acid cycle (CAC) is a wide-spread and highly conserved metabolic pathway involved in energy production and intermediary metabolism, such that the C A C , or segments thereof, have been found in virtually all organisms tested (Fig. 1). Since the C A C was proposed as the principal pathway for the terminal steps in the oxidative breakdown of carbohydrate to carbon dioxide by Hans Krebs, researchers have developed a fuller appreciation of the role the C A C plays in other aspects of cell metabolism (Krebs and Johnson 1937). Although the C A C is often the main source of aerobic energy (ATP and NADH) in organisms that oxidize, carbohydrates, fats, and amino acids to carbon dioxide, it also produces intermediates required in the biosynthesis of many essential cell components. For example, the intermediate a-ketoglutarate (ocKG) is a precursor in the biosynthesis of the amino acid glutamate (and subsequently proline and glutamine), as are oxaloacetate for the biosynthesis of aspartate (and related amino acids asparagine, lysine, methionine, threonine, and isoleucine), and succinyl-CoA for the biosynthesis of bacteriochlorophyll and cytochromes. A third, more subtle, function of the C A C is the regulation of associated pathways by cycle intermediates. For example, the ratio of ocKG:glutamine regulates the activity of the enzyme glutamine synthetase (involved in nitrogen assimilation) in Escherichia coli (Magasanik 1977). The conversion of fructose 6-phosphate to fructose 1,6-diphosphate catalyzed by the enzyme phosphofructokinase in the glycolytic pathway is regulated directly by citrate, and indirectly by the abundance of ATP (produced via the CAC) (Lehninger 1982). Additionally, the metabolic pathway leading to the biosynthesis of fatty acids requires citrate as a positive modulator (Lehninger 1982). 2 Figure 1: The Citric Acid Cycle and associated reactions. Heavy lines outline the pathway proposed by Krebs, dashed lines indicate associated reactions subsequently elucidated, and essential cell components produced from cycle intermediates are boxed. The enzymes involved in the C A C reactions are: 1, citrate synthase; 2, aconitase; 3, isocitrate dehydrogenase; 4, a-ketoglutarate dehydrogenase; 5, succinyl-CoA synthetase; 6, succinate dehydrogenase; 7, fumarase; 8 malate dehydrogenase. The reactions catalyzed by enzymes 3, 4, 6 and 8 result in the production of reduced cofactors that serve as electron shuttles between the C A C and electron transport phosphorylation pathways. The conversion of succinyl-CoA to succinate, catalyzed by succinyl-CoA synthetase, results in a substrate-level phosphorylation. Pyruvate Malate 7 Fumarate 6 Succinate li t iarats \Aspartate Oxaloacetate t • - Acetyl-CoA Succinyl-CoA Citrate 2 cis-Aconitate 2 Isocitrate V a-Ketoglutarate | Bacteriochlorophyll | Cytochromes Glutamate 4 The C A C has been studied in the hope of improving our understanding of the regulation of carbon flow required to maintain the bioenergetic, biosynthetic, and regulatory functions of this pathway. However, analysis of the C A C in organisms that require this pathway for the generation of energy is complicated; hence, researchers have turned their attention to microorganisms that contain the pathway but do not rely on it for energy production under all growth conditions. Contrary to the situation in mitochondria, where a complete C A C operates in the oxidative direction (clockwise in Fig. 1), some microorganisms may lack portions of this pathway, operate the cycle in the reductive direction (counterclockwise in Fig. 1) or regulate the extent of carbon flow through the pathway. The methanogen Methanobacterium thermoautotrophicum does not produce isocitrate dehydrogenase and Methanosarcina barkeri lacks a-ketoglutarate dehydrogenase (KGD) and the CAC-related enzyme fumarate reductase (Balch, et al. 1979). Oxaloacetate is converted reductively to succinyl-CoA which is then carboxylated by ocKG synthase yielding ocKG required for biosynthesis in M. thermoautotrophicum. M. barkeri, on the other hand produces ocKG oxidatively from oxaloacetate, but is believed to lack all subsequent oxidative steps of the C A C . The bacterium Chlorobium limicola assimilates carbon dioxide under photo synthetic growth conditions to produce the biosynthetic precursors ocKG, oxaloacetate, succinyl-CoA, and pyruvate through a C A C operating in the reductive direction (Evans, et al. 1966). In Bacillus subtilis many C A C enzyme activities (aconitase, KGD, succinate dehydrogenase, fumarase, and malate dehydrogenase) are known to increase during the transition from vegetative growth to sporulation (Ohne 1975). The biological significance of these increases in enzyme activities is not yet known , however an increase in ATP synthesis and 5 oxygen consumption have been observed during the early stages of sporulation (Ohne 1975). The C A C has been studied extensively in E. coli where the highest levels of C A C enzyme activities are found during aerobic growth and the lowest during anaerobic growth. In fact, during anaerobic fermentative growth, K G D enzyme activity is severely reduced and the C A C is converted from a cyclic to a branched pathway (Fig. 2). However, both aerobic and anaerobic levels of C A C enzyme activities are affected by numerous other factors. Cells grown aerobically on a complex medium containing glucose have lower C A C enzyme activities than cells grown aerobically on a minimal medium with glucose. It is assumed that in a complex medium the cells obtain enough energy from glycolysis and most nutrients from the medium and, therefore, do not need maximal C A C activity. When grown in a minimal medium cells must rely on the C A C for production of both energy and all biosynthetic precursors (Gray, et al. 1966). The K G D enzyme complex is one of three types of 2-oxoacid dehydrogenase complexes: branched-chain oxoacid dehydrogenase, PDH, and K G D . These enzymes catalyze the oxidative decarboxylation of branched-chain oxoacids (oxidative deamination products of branched-chain amino acids), pyruvate, and ccKG, respectively, to their acyl-CoA derivatives. The K G D enzyme complex has been purified from such diverse sources as pig heart muscle (Massey 1960), cauliflower florette mitochondria (Karam and Bishop 1989), and E. coli (Koike, et al. 1960). In all cases, KGD activity is associated with a large multienzyme complex with a molecular weight of several million kilodaltons. The E. coli complex has been separated into its component proteins: E l o (cc-ketoglutarate dehydrogenase), E2o (dihydrolipoyl succinyltransferase), and E3 (dihydrolipoamide dehydrogenase) (Reed and Mukherjee 1969). The three proteins form a complex with the 6 Figure 2: The branched C A C believed to operate in E. coli growing under anaerobic conditions ensures that there are sufficient levels of oxaloacetate, succinyl-CoA, and oc-ketoglutarate for biosynthetic purposes. The conversion of fumarate to succinate is catalyzed by the anaerobically induced enzyme fumarate reductase and is part of an anaerobic electron transport chain where fumarate is the terminal electron acceptor. The enzymes involved in these pathways are: 1, citrate synthase; 2, aconitase; 3, isocitrate dehydrogenase; 4, malate dehydrogenase; 5, fumarase; 6, fumarate reductase; 7, succinyl-CoA synthetase. The conversion of oxaloacetate to a-ketoglutarate occurs oxidatively and the conversion of oxaloacetate to succinyl-CoA occurs reductively. 7 Glucose Phosphoenolpyruvate Pyruvate Oxaloacetate Malate I' Fumarate i' Succinate i Succinyl-CoA Acetyl-CoA Citrate 2 Aconitate 2 ; i t Isocitrate cc-Ketoglutarate 8 stoichiometry 12Elo:24E2o:12E3, and the holoenzyme catalyzes the oxidative decarboxylation of a K G to succinyl-CoA as outlined below. 1. H0 2 C(CH2) 2 COC0 2 H + TPP-Elo --> H02C(CH2)2CH(OH)-TPP-Elo + C 0 2 2. H02C(CH2)2CH(0H)-TPP-E lo + LipS2-E2o -> H0 2C(CH 2) 2CO-SLipSH-E2o + TPP-E lo 3. H0 2C(CH 2) 2CO-SLipSH-E2o + CoA-SH --> Lip(SH)2-E2o + H0 2C(CH 2) 2CO-SCoA 4. Lip(SH)2-E2 + FAD-E3 --> LipS2-E2o + FADI^-ES 5. FADH 2 -E3 + NAD+ --> FAD-E3 + NADH + H + Sum: H0 2 C(CH2) 2 C0C0 2 H + CoA-SH + N A D + --> HO^CCrl^CO-SCoA + C 0 2 + NADH + H + The Elo enzyme, which contains thiamine pyrophosphate (TPP), catalyzes the oxidative decarboxylation of a K G to produce a succinylated E2o protein (which contains lipoic acid (LipS2) as an electron carrier). The E2o protein then transfers to coenzyme A (CoA-SH) the succinyl group produced by the first reaction. The flavoprotein (FAD)-linked E3 enzyme, in turn, catalyzes the reoxidation of the dihydrolipoyl group of the E2o enzyme. Much of the research conducted on the PDH and K G D enzyme complexes is focused on the mechanisms by which these large multiprotein complexes are assembled and how the genes encoding the various protein components are coordinately expressed to maintain sufficient numbers of complexes to satisfy both energy production and biosynthesis under all conditions of growth. These studies have demonstrated that the core of the PDH and KGD enzyme complexes is composed of an E2 polypeptide (specific for each complex). The E2 protein can be considered to have three segments: a lipoyl binding domain at the amino terminal, a central region containing a binding site for E3 proteins, and an E l and E2 protein binding site along 9 with the E2 catalytic domain at the carboxy terminal (Perham, et al. 1987). The genes that code for the Elo , E2o, and E3 proteins (sueA, sucB, and Ipd, respectively) have been cloned and sequenced in E. coli (Darlison, et al. 1984; Spencer, et al. 1984; Stephens, et al. 1983). The sucA and sucB genes form an operon which is located near other C A C enzyme genes at about 17 minutes on the E. coli linkage map (Darlison, et al. 1984). However, the Ipd gene is adjacent to the aceEF operon that codes for the E lp and E2p protein components of the PDH complex (at about 3 minutes on the E. coli linkage map) (Stephens, et al. 1983). The Ipd gene is at the promoter distal end of the aceEF operon, is transcribed either as part of an aceEF-lpd transcript or independently from its own promoter, and its gene product is shared by both the KGD and PDH enzyme complexes (Stephens, et al. 1983). Transcription studies of E. coli C A C genes have demonstrated that transcription of the sucAB genes is more sensitive to repression by glucose and anaerobic growth conditions than is transcription of the aceEF and Ipd genes. However, independent transcription of the Ipd gene is closely coupled to transcription of the sucAB genes (Miles and Guest 1987). Promoter studies have revealed sequence similarities around the -35 region of both the Ipd and the sue promoters, a factor which may prove important in the coregulation of these genes (Spencer and Guest 1987). The genetic organization and/or regulation of C A C genes and gene products have been studied in B. subtilis (Carlsson and Hederstedt 1989), Azotobacter vinelandii (Schulze, et al. 1990; Westphal and de Kok 1990; Hanemaaijer, et al. 1988) and Pseudomonas fluorescens (Benen, et al. 1989). These species all have some KGD genes that are strikingly similar in genetic organization and nucleotide sequence to those observed in E. 10 coli, as well as K G D gene products that have similar amino acid sequences. From this, one could speculate that in these species of bacteria the enzyme complex formed by the three protein subunits is similar in higher order structure to the E. coli KGD enzyme complex. The sucA and sucB genes and their homologues in the above mentioned bacteria are part of an operon similar in structure to the E. coli sucAB operon, although there is some variation with respect to the location of the Ipd gene and promoters (Fig. 3). In the species A. vinelandii and P. fluorescens, the Ipd gene is linked to the sucAB operon whereas in E. coli and B. subtilis, the Ipd gene is at an unlinked locus (the B. subtilis Ipd gene is assumed to be associated with the genes encoding the PDH proteins, as in E. coli). As in E. coli, it is believed that the Ipd gene product is shared between the PDH and KGD complexes in these species. The P. fluorescens Ipd promoter has not yet been located and gene expression is assumed to be dependent on transcription from the sucAB promoter. However, the A. vinelandii Ipd gene is believed to be preceded by a promoter and, therefore, can be transcribed independently of the sucAB genes (Westphal and de Kok 1990). It has been suggested that differences in location of Ipd genes are reflections of differences in fundamental metabolic processes. In organisms where carbohydrate metabolism is a dominant source of energy generation (eg. E. coli), the PDH complex plays a central role. Hence Ipd gene expression would be closely linked to PDH gene expression. However, in organisms in which nitrogen assimilation through the glutamate-aKG couple is important (eg. A. vinelandii and P. fluorescens), the K G D complex would play a central role and Ipd gene expression would be closely associated with expression of KGD genes (Westphal and de Kok 1990). The purple photosynthetic bacterium Rhodobacter capsulatus is 11 Figure 3: Organization of K G D genes in the bacteria E. coli, B. subtilis, A. vinelandii, and P. fluorescens. The first gene in each operon codes for the protein Elo (oc-ketoglutarate dehydrogenase), the second for E2o (dihydrolipoyl succinyltransferase), and the third for E3 (dihydrolipoamide dehydrogenase). The E. coli sucAB promoter (indicated by the arrow) is located 200 bp upstream of the sue A translation initiation codon while the location of the odhAB promoter (indicated by the arrow) in B. subtilis has been narrowed down to the 800 bp region upstream of the odhA translation initiation codon. The A. vinelandii and P. fluorescens promoters have not been mapped. The size of the P. fluorescens sue A gene is not known and nucleotide sequence of the sucB gene has not been published but is reported to be very similar in amino acid sequence to the E2o protein of E. coli and A. vinelandii. Numbers within boxes indicate percent amino acid identity of the deduced amino acid sequence to the corresponding E. coli protein (Carlsson and Hederstedt 1989; Schulze, et al. 1990; Westphal and de Kok 1990). E. coli suck. sucB B. subtilis 56% odhA odhB A. vinelandii 59% H 63% 50% sucA sucB Ipd P. fluorescens H 42% sucA sucB Ipd 13 able to grow by aerobic respiration (when it produces energy via the CAC), anaerobic photosynthesis (generating energy by photosynthesis), fermentation of sugars, or by a number of alternative ways making it one of the most versatile microorganisms known (Beatty and Gest 1981a). It may be predicted that as a facultative anaerobe, R. capsulatus might repress the synthesis of the K G D enzyme complex during anaerobic growth and convert the C A C into a branched pathway. However, researchers have been unable to detect the enzyme fumarate reductase, necessary to produce succinate reductively from oxaloacetate (see Fig. 2) (Beatty and Gest 1981b). Therefore, it is believed that R. capsulatus must operate a cyclic C A C under anaerobic photosynthetic and fermentative growth conditions, but a less active one. Because of its extreme metabolic versatilities, and because of differences in regulation of the CAC, it was of interest to me to study the CAC in R. capsulatus. I hoped to improve our understanding of the mechanisms by which the C A C is converted from an energy producing pathway to one predominantly required for the generation of precursors for biosynthesis. The K G D enzyme complex of R. capsulatus seemed to be a good starting point to study regulation Of C A C activity, since K G D specific activity appears to be affected by the availability of molecular oxygen to the culture. The product of the KGD reaction, succinyl-CoA, is required under both aerobic and anaerobic growth conditions for the synthesis of bacteriochlorophyll and cytochromes (Cox, et al. 1983), and KGD specific activities in cultures grown under high aeration were reported to be 3 - 5 times higher than in cultures grown under anaerobic photosynthetic conditions. (I will show that under the growth conditions that I used, KGD specific activities in cell extracts of aerobically grown cultures were 9 - 1 3 times greater than in extracts of cultures grown under anaerobic photosynthetic conditions.) Cell extracts of R. capsulatus strain BIO grown photo synthetically under strict anaerobic conditions have been 14 reported to contain a KGD specific activity of 15 units (1 unit = 1 nmol NAD+ reduced/minute/mg protein) but when extremely low amounts of oxygen were introduced into the growth medium, KGD enzyme specific activity increased to 25 units (Beatty and Gest 1981a). Therefore, it appears that K G D enzyme specific activity is highly sensitive to the concentration of oxygen in the medium. Consequently, it would be expected that the amount and/or activity of the KGD enzyme complex must be efficiently controlled to maintain the cyclic bioenergetic or biosynthetic natures of the C A C under various conditions of growth. The focus of my thesis was to study the regulation of the KGD enzyme complex in R. capsulatus under aerobic respiratory and anaerobic photosynthetic conditions of growth. I hoped to determine if R. capsulatus has a huge multiprotein K G D enzyme complex as in mitochondria and E. coli, and the genetic organization of the KGD genes for comparison with the other species of bacteria discussed. I also did experiments to test if the higher K G D enzyme specific activity found under aerobic growth conditions is due to modulation of transcription of the K G D gene(s) from an oxygen-sensitive promoter. 15 MATERIALS AND METHODS 1. Bacterial Strains and Plasmids The laboratory wild type strain R. capsulatus BIO has been described previously (Weaver, et al. 1975). The mutant strain KGD11, derived by ethyl methane sulfonate mutagenesis of a BIO culture, lacks detectable levels of KGD activity (Beatty and Gest 1981b). Strain KGD 11 is capable of photosynthetic growth but cannot grow aerobically on substrates that require oxidation through the C A C (succinate, malate, pyruvate) (Beatty and Gest 1981a). E. coli strains C600 r"m+ (Bibb and Cohen 1982), JM83 (Yanisch-Perron, et al. 1985), SM10 (Simon, et al. 1983), and JM101 (Yanisch-Perron, et al. 1985) were plasmid DNA hosts and the latter strain was a host for plasmid DNA when producing single stranded DNA for sequencing. E. coli HB101(pRK2013) (Ditta, et al. 1985) was used as a mobilizing strain (herein referred to as the helper strain) in bacterial conjugations. The plasmids used in genetic cloning and sequencing are listed in Table I. 2. Media and Growth of Bacteria All E. coli strains were grown in LB medium, with the exception of JM101 which was grown in M9 medium (Maniatis, et al. 1982). Routinely, R. capsulatus BIO was grown either aerobically in darkness or photosynthetically under anaerobic conditions in minimal RCV or complex YPS medium (Weaver, et al. 1975). R. capsulatus strain KGD11 was grown photosynthetically in media from which as much dissolved oxygen as possible had been removed by storing in an anaerobic 16 Table I: PLASMLDS USED Plasmid Description and Source pLAFRl Tcr; unique EcoRl site, mobilizable, stable in many Gram negative hosts. (Friedman, et al. 1982) pFDI Cosmid pLAFRl containing a 15 kb partial EcoKL product of R. capsulatus BIO chromosomal DNA. (This study) pFDII Cosmid pLAFRl containing a 22 kb partial EcoJZI product of R. capsulatus BIO chromosomal DNA. (This study) pRK2013 Km r ; helper plasmid for mobilization of RK2 derived plasmids. (Ditta, et al. 1985) pRK415 Tcr; derivative of pRK404 with the pUC19 multiple cloning site; lacZa. (Keen, et al. 1988) pUC13 pJAJ9 Ap1"; lacZa. (Messing 1983) Tc r; expression vector contains the R. capsulatus puf operon promoter. (Johnson, et al. 1986) pTZ18U Apr; sequencing vector based on pUC18; lacZa. (Mead, et al. 1986) pTZ19U Ap r; sequencing vector; based on pUC19; lacZa. (Mead, et al. 1986) 17 environment. Plates of solid RCV medium and 17 or 21 ml screw-cap tubes filled with liquid RCV medium were supplemented with tetracycline and/or 5 mM succinate and sealed into BBL GasPak jars at least one day before use (Becton Dickinson and Co., Cockeysville, MD). Jars were made anaerobic with B B L GasPak disposable H 2 + C 0 2 generator envelopes. The plates and/or tubes were removed from the jar only when required. Once the tubes were inoculated with cells, they were filled to the top with similarly deoxygenated medium, sealed with the screw-cap, and incubated in a rack placed in an aquarium filled with water and illuminated with four 60 W Philips Lumiline II White tungsten filament incandescent lamps. Water temperature in the aquarium was controlled by a Haake DI water circulator/heater. The plates, once spread with bacteria, were sealed into B B L GasPak jars, and placed in this illuminated aquarium. Antibiotics were added to the following concentrations: ampicillin: 200 Ltg/ml, kanamycin: 10 Ltg/ml, and tetracycline: 10 Jig/ml (E. coli); tetracycline: 0.5 Ltg/ml (R. capsulatus). E. coli and R. capsulatus strains were grown at 37°C and 34°C respectively. 3. Measurement of Bacterial Growth Culture density was measured in a Klett-Summerson photometer fitted with a No. 66 (red) filter. 4. In Vitro DNA Techniques Agarose gel electrophoresis was performed in 0.5 X TBE buffer (0.089 M Tris base, 0.089 M boric acid, 0.002 M EDTA) (Maniatis, et al. 1982). Restriction endonucleases, Klenow, and T4 DNA ligase were purchased from Bethesda Research Laboratories, Gaithersburg, MD, calf 18 intestine alkaline phosphatase and DNase I (RNase-free) from Boehringer Mannheim (Laval, Canada), and used as recommended by the suppliers. The T 7 Sequenc ing™ kit and Deaza T 7 Sequenc ing™ mixes used for sequencing DNA were purchased from Pharmacia L K B Biotechnology, Baie d'Urfe, Canada. Radioactive compounds ((5,6-3H)-uridine and (oc-3 5S)dATP) were purchased from NEN-Dupont, Mississauga, Canada. The non-radioactive, Photogene Nucleic acid Detection System and biotin-11-dUTP were purchased from Bethesda Research Laboratories. 5. Transformation and Conjugation E. coli cells were made competent by the CaCl 2 method and transformed as described (Maniatis, et al. 1982). Conjugation of plasmid DNA into R. capsulatus strain KGD11 was accomplished by triparental mating (or biparental mating if plasmid DNA was in E. coli strain SM10) as follows: equal volumes of overnight stationary phase cultures of donor and recipient cells (and when necessary helper cells) were mixed, pelleted (30 seconds at 15,600 x g in an Eppendorf benchtop microcentrifuge), and resuspended in an equal volume of YPS medium. A 10 jil portion of the suspension was spotted onto filters (0.45 Jim pore size; Millipore Corporation, Bedford, MA) on RCV agar plates supplemented with 0.1% glucose and 5 mM succinate. Conjugation was allowed to occur overnight under anaerobic photosynthetic conditions in B B L GasPak jars as described in Media and Growth of Bacteria. 6. Plasmid Purification Routine plasmid analysis of cells was performed by the alkaline lysis method of Maniatis et al. (Maniatis, et al. 1982) with the following modifications. Lysozyme was omitted from the lysis solution, 3 M sodium acetate (pH 4.8) instead of the potassium acetate (pH 4.8) solution was used and the phenol/chloroform extraction step was omitted. 19 For large-scale plasmid purification, 800 ml of LB broth in 2 1 flasks supplemented with 9.6 mM K P 0 4 buffer (pH 6.8), 0.2% glucose, and appropriate antibiotic were inoculated with an overnight stationary phase culture of the desired E. coli strain and incubated in a shaking water bath (set at 150 RPM). Overnight cultures (16 - 18 hours) grown this way were usually between 300 and 400 Klett units (1.1 X 109 to 1.5 X 109 cfu/ml) when harvested. Plasmid DNA was routinely isolated from a total of 1600 - 3200 ml of culture using either the alkaline lysis protocol (with the above mentioned modifications) or the Triton X-100 (octyl phenoxy polyethoxyethanol; Sigma Chemical Company, St. Louis, MO) procedure (Maniatis, et al. 1982). In the latter procedure, cell pellets from 800 ml of culture were resuspended in 2.5 ml of a 10% sucrose-Tris solution on ice (10% sucrose, 0.05 M Tris-HCl, pH 8.0). To this suspension 0.5 ml of lysozyme (10 mg/ml in distilled H 2 0) were added followed by 0.5 ml of 0.5 M EDTA (pH 8.0). While swirling, 4.0 ml of a Triton X-100 lysis buffer were added (0.05 M Tris-HCl (pH 8.0), 10 mM EDTA, 2% Triton X-100). The lysed cells were immediately centrifuged at 15,000 RPM for 25 minutes in an SS34 rotor. To each ml of DNA solution obtained (from either of the above mentioned protocols) was added 1 g solid CsCl. This solution was split evenly between Beckman heat-seal ultracentrifuge tubes and 300 ul of a 10 mg/ml ethidium bromide (EtBr) solution were added to each (Beckman Instruments Inc, Mississauga, Canada). The tubes were topped with paraffin oil, balanced in pairs, heat sealed, and then placed into a Beckman VTi65.2 rotor and centrifuged at 62,000 RPM for five hours. The DNA was visualized with a U.V. light source while the CsCl gradient was dripped through the bottom of the tubes to collect the plasmid fraction. This fraction was added to a new heat seal tube, the volume increased to 5 ml with a 1 g/ml CsCl solution, and EtBr and paraffin oil were added as 20 above. The tubes were sealed and then centrifuged at 55,000 RPM for 18 hours (if the first centrifugation was at 55,000 RPM for 18 hours then the second was at 62,000 RPM for five hours). The plasmid fraction was isolated as described above, the EtBr removed by repeated extraction with isopropanol equilibrated with a TE solution (10 mM Tris, 1 mM EDTA, pH 8.0) saturated with CsCl (each time keeping the lower phase). The resultant DNA solution was dialyzed 3 times for 30 minutes against 2 1 of T E at 4 ° C , extracted twice with a 1:1 mixture of phenol and chloroform, once with chloroform, and precipitated with ethanol as described. The yield of DNA was estimated by either absorption at 260 nm or by comparing band intensities on EtBr stained agarose gels to intensities of known amounts of bacteriophage X DNA on the same gel. 7. Chromosomal DNA Isolation Chromosomal DNA was isolated from R. capsulatus strain B10 using the Triton X-100 lysis protocol outlined above with the following modifications. After the addition of 4.0 ml of Triton X-100 lysis buffer, the solution was heated to 6 5 ° C for 10 minutes. To each ml of the resulting solution 1 g solid CsCl was added. All the subsequent steps were as outlined for large-scale plasmid purifications. 8. DNA Sequence Analysis Nucleotide sequence determinations were performed using the dideoxy chain termination method of Sanger et al. (Sanger, et al. 1977). DNA fragments were cloned into the vectors pTZ18U and pTZ19U and sequenced using the Pharmacia T7 sequencing kit, the deaza T7 sequencing mixes, (oc- 3 5S)dATP, and universal M13 primers according to the supplier's specifications (with omission of urea extraction from gels prior to drying). Computer analysis of the sequence data was performed with the PCGENE (IntelliGenetics, Inc) software package. 21 9. Southern Blots and Hybridizations Five jig of chromosomal DNA and 4 ng of plasmid DNA, purified by CsCl density centrifugation, were digested with Hindlll, loaded in adjacent wells in a 0.7% agarose gel, and electrophoresed at 25 V in 0.5 X TBE buffer for about 16 hours at room temperature. The gel was then stained with 15 ill of a 10 mg/ml solution of EtBr (approximate EtBr concentration in staining solution = 0.3 ug/ml) (10-30 minutes), destained in distilled H 2 0 (20-30 minutes) and photographed on a U.V. transilluminator to determine the quality of electrophoresis. The DNA was denatured by soaking the gel twice in 20 gel volumes of 1.5 M NaCl, 0.5 M NaOH for 15 minutes at room temperature. The gel was neutralized by soaking twice in 20 gel volumes of 1.5 M NaCl , 1.0 M Tris (pH 7.5) for 15 minutes at room temperature. The neutralization step was repeated if the pH of the gel was > 7.8 (pH was checked by laying pH paper on the gel). DNA was then transferred to ICN Bio-Trans nylon membranes (wetted in 0.5 X TBE buffer) (ICN Biomedicals Canada Ltd., Mississauga, Canada) by electroblotting at 20 V in 0.5 X T B E buffer for about 16 hours, followed by 80 V for 2 hours in a BIO-RAD Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Richmond, CA). The buffer was cooled during the transfer with a water-circulation coil. The membrane was then soaked in 5 X SSPE (20 X SSPE: 3.6 M NaCl, 200 mM N a H 2 P 0 4 , 20 mM EDTA, pH 7.4) for 5 minutes at room temperature and photographed with U.V. illumination along with the gel to determine the efficiency of transfer. The DNA was fixed to the membrane by drying in a vacuum oven at 80°C for 2 hours. 22 Membranes were prehybridized for a minimum of 2 hours at 42°C in 20 ml of 5 X SSPE, 0.3% SDS, 50% formamide, and 100 Ltg/ml sheared salmon sperm DNA. The prehybridization buffer was replaced with 3 ml of fresh buffer to which was added approximately 50 ng of alkali denatured probe ( 3 2 P or biotin-labelled by the random oligonucleotide primer method using 50-100 ng of template DNA (Feinberg and Vogelstein 1983). Hybridization occurred overnight at 42°C, after which membranes were washed 3 times for 7 minutes in 200 ml 2 X SSPE, 0.1% SDS at 45°C, and then used to expose X-ray films for 1 to 26 hours at -80°C with intensifying screens. When the non-radioactive Photogene Nucleic Acid Detection System was used, the suggested protocol was followed, and X-ray films were exposed for 30 seconds to 15 minutes at room temperature with intensifying screens (X-Omat X-ray film; Eastman Kodak Company, Rochester, NY). 10. RNA Isolation RNA was isolated from R. capsulatus B10 cultures grown aerobically and photosynthetically in RCV medium by either the hot phenol extraction procedure (von Gabain, et al. 1983) or the guanidine isothiocyanate method (Carl Bauer personal communication). Anaerobic photosynthetic cultures were grown in 21 ml screw-cap tubes, whereas the aerobic cultures were grown in 250 ml Erlenmeyer flasks filled to 8% of their nominal volumes and shaken at 300 RPM in a water bath shaker as described in Media and Growth of Bacteria. Both types of cultures were harvested at the same cell density (approximately 220 Klett units or 8 x 108 cfu/ml) and growth phase (late log phase). In the guanidine isothiocyanate procedure, 100 ml cultures were chilled to < 4 ° C , cells were pelleted in a prechilled SS34 rotor by 23 centrifugation for 10 minutes at 10,000 RPM. Cell pellets were resuspended in 10 ml of a guanidine isothiocyanate solution (4.2 M guanidine isothiocyanate, 0.025 M Na-Citrate (pH 7.0), 0.1 M p-mercaptoethanol). Once resuspended, the cells were disrupted by passage through a French press (15,000 psi) and cell debris was removed by centrifugation for 10 minutes at 15,000 RPM in an SS34 rotor (using Corex tubes). To the supernatant liquid were added 0.025 volumes 1.0 N acetic acid and 0.75 volumes 95% ethanol (done in a fume hood), the resulting solution was chilled for 10 minutes at -20°C, and the RNA pelleted at 15 000 RPM in an SS34 rotor (-10°C, 10 minutes). The pellet was dissolved in 4.0 ml of the guanidine isothiocyanate solution, to which was added 2.0 ml of a 5.7 M CsCl solution and 2 jil of a 10 mg/ml EtBr solution. This RNA/CsCl solution was layered onto a 3.4 ml cushion of 5.7 M CsCl in Beckman 14 x 89 mm Ultra-Clear tubes. The tubes were balanced in pairs, placed in an SW41 rotor, and centrifuged at 32,000 RPM for 22 hrs (20°C). The supernatant fluid was carefully removed with a Pasteur pipette from the RNA, which was resuspended in 1.0 ml of sterile T E buffer. The RNA was extracted twice with TE-buffered phenolxhloroform (1:1) and once with chloroform, then precipitated with ethanol and recovered by centrifugation for 10 minutes at 15,000 RPM in an SS34 rotor. The RNA pellet obtained was dissolved in sterile distilled water and stored at -80°C. The quality of the RNA was determined by agarose gel electrophoresis, EtBr staining, and visualization of the RNA on a U V transilluminator. The quantity and purity of the RNA was determined by scanning its absorbance between 200 nm and 300 nm. Preparations had, on average, a 260/280 ratio of about 2.0 and typically 30 - 60 jig of RNA was isolated from one 20 ml culture. 11. RNA Blotting and Hybridization Samples containing 20 (ig of R. capsulatus RNA were ethanol 24 precipitated and resuspended in 10 ill of EtBr buffer (50% formamide, 20% formaldehyde, 1 X MOPS running buffer (see below), 1 u.g EtBr/ul) (Rosen, et al. 1990) to which was added 2 ul of loading buffer (1 X MOPS running buffer (see below), 50% formamide, 20% formaldehyde, 0.05% bromophenol blue, 0.05% xylene cyanol). The RNA was then denatured by heating at 65°C for 10 minutes and the samples were loaded into 1.4% agarose-formaldehyde gels (1.4 g agarose, 10 ml 10 X MOPS running buffer (see below), 72 ml sterile distilled H 2 0 , 18 ml 35.8 M formaldehyde). The RNA was subjected to electrophoresis for 3 hours at 100 V in 1 X MOPS running buffer (10 X MOPS: 0.2 M MOPS (3-[N-morpholinojpropane sulfonic acid), 250 mM EDTA, 2 M NaOAc, 5 M NaOH, pH 7.0) with buffer recircularization at room temperature. Gels were rinsed for 5 minutes in distilled H 2 0 , photographed on a U.V. transilluminator to determine the position of the various RNA species with respect to the molecular size markers and then equilibrated in two changes of 0.5 X T B E buffer for 25 minutes at room temperature. The RNA was transferred by electroblotting (see above) to nylon membranes, the membranes and gels were photographed on a U.V. transilluminator, soaked in 5 X SSPE, and the RNA was fixed to the membrane by baking in a vacuum oven at 80°C, followed by a 10 minute U.V. cross-linking step. Membranes were prehybridized and hybridized (with 32P-labelled probes) as outlined. X-ray film was exposed for 1 to 32 days at -80°C with an intensifying screen. 12. a-Ketoglutarate Dehydrogenase Enzyme Assay R. capsulatus cultures were grown as for RNA isolation. Cultures at 220 Klett units (approximately 8 x 108 cfu/ml) were centrifuged for 10 minutes at 10,000 RPM in an SS34 rotor to pellet the cells, which were frozen at -80°C until assayed. Cell pellets from 40 ml cultures were resuspended in 1.0 ml 50 mM K P 0 4 buffer (pH 7.0). Resuspended cells 25 were sonicated on ice with a Sonifier Cell Disruptor 350 (Branson Sonic Power Co.) equipped with a microprobe 3 times for 15 seconds at 0.3% of maximum power. Sonicated cells were centrifuged for one minute in an Eppendorf benchtop microcentrifuge to remove intact cells and large membrane particles, and the supernatant fluids were used for K G D enzyme activity assays and protein concentration assays. The enzyme assays were completed within five hours of sonication and the protein assays were completed within 24 hours of completion of the enzyme assays. K G D enzyme activity was assayed by measuring the rate of increase in absorbance at 340 nm due to the ocKG-dependent reduction of NAD+ using a Hitachi U2000 spectrophotometer. The KGD enzyme assay reaction mixture contained, in a total volume of 1 ml: 150 mM Tris-HCl (pH 8.5); 1 mM MgCl 2 ; 2.5 mM L-cysteine; 86 UM CoA-SH; 170 uM thiamine pyrophosphate; 2 mM N A D + ; 10"5 M rotenone; cell extract usually containing 109 - 436 u.g of protein (from aerobically grown cultures) or 305 - 651 Ltg of protein (from anaerobic photosynthetically grown cultures); 3 mM ocKG (pH 8.5). All solutions were made up in 0.01 M K P 0 4 buffer (pH 6.8) except for CoA-SH, rotenone, and a K G which were made up in 0.01 M K P O 4 buffer (pH 4.4), ethanol, and 0.01 M Tris-HCL (pH 8.5), respectively. The components were added in the order listed up to ocKG, the endogenous rate, if any, was determined, and the reaction was then started by addition of ocKG. Enzyme activities were calculated using the extinction coefficient of NADH of 6.22 m M _ 1 c m _ 1 (Dawson, et al. 1969). The protein contents of the extracts were determined by the modified Folin phenol method of Lowry (Peterson 1983). 26 13. Density Scans of RNA Blot Autoradiograms The intensities of the signals detected on RNA blot autoradiograms were determined by scanning over the area of each lane on X-ray films with a Molecular Dynamics Densitometer. The data were analyzed using the ImageQuant™ V.3.0 Fast Scan Software package with integration over the whole area of the lane. 14. Pulse Labelling of KGD RNA Two 250 ml Erlenmeyer flasks were filled to 8% of their nominal volume with R C V medium, inoculated to 50 Klett units (1.85 x 108 cfu/ml) with an overnight stationary phase aerobic culture of R. capsulatus strain BIO, and incubated in a shaking water bath under highly aerated conditions (300 RPM). Two 21 ml screw-cap tubes filled with R C V were inoculated to 50 Klett units (1.85 x 108 cfu/ml) with an overnight stationary phase culture grown under low oxygen conditions (50 ml Erlenmeyer flasks filled to 80% of their nominal capacity and shaken at 75 RPM) and incubated under anaerobic photosynthetic conditions as described in Materials and Methods. Both sets of cultures were incubated until they reached a cell density of ca. 200 Klett units (ca. 1A x 108 cfu/ml), allowed to incubate for a further 15 minutes, and then each culture was pulsed with 1.2 mCi of (5,6-3H)-uridine (37.6 mCi/mmol, 1.0 mCi/ml) for two minutes. Following the pulse, cultures were poured over ice, the cells pelleted, and RNA isolated using the hot phenol method. The quality, quantity, and purity of RNA preparations were determined as described in RNA Isolation (these preparations had an average 260/280 ratio of 2.3). The specific incorporation of labelled uridine into RNA was determined by precipitating 0.6 to 1.5 iig of RNA in ice-cold 10% trichloroacetic acid (TCA), collecting the precipitate on glass-fiber filters (#25; Schleicher & Schuell, Inc., Keene, N.H.), and counting radioactive 27 decay in a toluene-based scintillation fluid (Liquid scintillation solution; British Drug House, Inc., Toronto, Canada) (Maniatis, et al. 1982). Preparations had a specific activity of 2.9 x 104 to 2.2 x 105 cpm/jig RNA. Complementary strand DNA from pTZ18U::ZiCY>RV0-ZtamHI and non-complementary strand DNA from pTZ19U::£'coRV0-fiamHI were spotted onto Millipore 0.45 |im pore size filters at the following amounts: 0.5 jig, 1.0 jig, and 2.0 jig. The filters were allowed to air dry and then were baked in a vacuum oven at 80°C for 2 hours. Filters spotted with these three different amounts of complementary and non-complementary strand probe were placed into 19 mm x 65 mm screw-cap vials and prehybridized for 2 hours in 5 ml of hybridization buffer as described in Southern Blots and Hybridizations (except that sheared salmon sperm DNA was replaced with yeast tRNA). The prehybridization buffer was replaced with 2.1 ml of fresh buffer to which was added the entire labelled RNA preparation (24.3 to 59.4 jig total cellular RNA at a specific activity of 2.9 x 104 to 2.2 x 105 cpm/jig RNA). Hybridization occurred overnight at 42°C after which the filters were washed as described in Southern Blots and Hybridizations, air dried under a 150 W flood lamp for 6 hours, and radioactive disintegrations counted in a toluene-based scintillation fluid over a 10 minute period. Portions of 50 |il (before hybridization) and 10 jil (after hybridization) of the hybridization liquid were withdrawn and the RNA in these samples was TCA-precipitated, collected, and counted as described in the previous paragraph. The amounts of K G D mRNA hybridized to the probes were calculated by subtracting the radioactivity bound to pTZ19U::£c6>RV0-B a mill (non-complementary probe) filters from that bound to p T Z 1 8 U : : £ c 0 R V 0 - £ a m H I (complementary probe) filters, and expressing the result as a percentage of input TCA-precipitable radioactivity. Non-specific binding (radioactivity bound to non-complementary probe filters) ranged from 0.0001% to 0.0049% of input 28 TCA-precipitable radioactivity. 29 RESULTS 1. . Screening of R. capsulatus gene bank for a gene that complemented the KGD 11 mutation. R. capsulatus strain KGD11 lacks K G D enzyme activity, and is derived from the laboratory wild type R. capsulatus strain BIO. Strain KGD 11 is unable to grow aerobically on the organic acids succinate, malate, and pyruvate because the absence of KGD enzyme activity results in the loss of the catalytic, cyclic nature of the CAC. I used a gene bank of R. capsulatus DNA (15-25 kb size-fractionated EcoRI partial digest products of R. capsulatus chromosomal DNA cloned in the unique EcoRI site of the cosmid vector pLAFRl) to isolate two different cosmid clones, pFDI and pFDII, that complemented the mutation in KGD11 (i.e., restored the capacity for aerobic growth). This was done by mobilization of the cosmids by conjugation from E. coli into KGD11, and selection for cosmid recipients that were capable of aerobic (dark) growth on RCV medium. Because pFDI and pFDII complemented the KGD 11 mutation in trans, these cosmids were thought to possibly contain a promoter as well as the KGD structural gene mutated in KGD11. It was also possible that these cosmids contained a KGD regulatory gene that might have been mutated in KGD11. 2. Localization of the DNA segment responsible for complementation of the KGD" phenotype of KGD 11. The cosmid clone pFDI contains a 15 kb segment of R. capsulatus DNA, whereas pFDII contains a 22 kb insert (total cosmid clone sizes are 37 kb and 44 kb, respectively) (Fig. 4). Complete EcoRI digestion of these cosmids revealed that each contained six EcoRI fragments 30 Figure 4: Cosmid clones pFDI and pFDII from the R. capsulatus gene bank that complemented the K G D - phenotype of KGD11. The shaded EcoRI inserts in both clones (5 kb in pFDI and 12 kb in pFDII) are the segments unique to each; the clear EcoRI inserts in each contain the five other EcoRI fragments (4 kb, 2 kb, 1.8 kb, 1.5 kb, and 1 kb fragments). 31 EcoRI EcoRI 32 (excluding the vector); five of the six EcoRI fragments were the same size in both pFDI and pFDII, whereas the sixth was unique to each clone. At one end of the insert in pFDI is a unique 5 kb fragment whereas in pFDII there is a unique 12 kb fragment (Fig. 4). This indicated to me that the EcoRI inserts in pFDI and pFDII were overlapping segments of the R. capsulatus chromosome and that the gene responsible for complementing the K G D 11 mutant phenotype would probably be located in the region of overlap. Attempts to localize the D N A region responsible for complementation of the K G D - phenotype of K G D 11 were, therefore, limited to the region of overlap on the smaller cosmid clone, pFDI. Cosmid pFDI was digested to completion with EcoRI and the resultant fragments were cloned into the vector pRK415 (Fig. 5). Complementation of the mutant phenotype by these clones was tested by conjugation into KGD 11 and incubation of the exconjugants aerobically in the dark to select for the K G D + phenotype. Unfortunately none of the clones complemented the mutation in KGD11. Longer aerobic (dark) incubation of the plates spread with the exconjugants did not result in the growth of colonies, so I concluded that an EcoRI site was too close to the mutated sequence(s) in the KGD 11 allele to allow recombination. It was possible that during subcloning of some DNA fragments from pFDI the KGD structural gene would be truncated or separated from its promoter, and expression of the plasmid-borne gene(s) would be lost. However, as long as some wild type sequences of the KGD" allele were present on a plasmid it would be possible to obtain rescue of the KGD 11 phenotype by recombination. Therefore, it was necessary to develop an assay by which complementation, recombination, and reversion of the KGD 11 mutant phenotype could be differentiated easily. This assay is outlined in Fig. 6. The assay developed was based on the understanding 33 Figure 5: Outline of the cloning of the 4 kb, 2 kb, 1.8 kb, 1.5 kb, and 1 kb EcoRl fragments from the cosmid clone pFDI into the broad host range vector pRK415. Stippled EcoRl fragment in cosmid pFDI is the unique 5 kb fragment. The 4 kb, 2 kb, 1.8 kb, 1.5 kb, and 1 kb EcoRl fragments are contained within the clear part of the EcoRl insert in pFDI. 34 p4FR p2FR pl.8FR pl.5FR plFR 35 Conjugate Plasmid clone into KGD11 Select for Tc exconjugants (PS in liquid medium)' Spread defined number of cells onto solid medium and select for KGD+phenotype Observe number of colonies that develop on plates between the second and fifth day of incubation Second day t <2% ca. 100% A Fifth day No change 20-90% No change reversion recombination complementation Figure 6: Outline of the procedure designed to distinguish between complementation, recombination, and reversion used in the search for the DNA sequence that complemented the mutation in KGD11. Percent values represent colonies growing aerobically as a fraction of colonies growing photosynthetically. 36 that if a piece of DNA were to complement a mutation in trans it would do so in all cells in which it was located. If only recombination were possible, the phenomenon would only occur in a portion of the cells and the number of recombinants should increase with time. Reversion of KGD11 (frequency was measured as 2 - 12 x 10"6) would occur at the lowest frequency. Therefore, once tetracycline resistant exconjugants were selected (the pRK415 plasmid used carried a tetracycline resistance gene) photosynthetically in liquid medium, a known number of cells were spread on solid RCV medium with tetracycline, the plates incubated under aerobic conditions to select for the K G D + phenotype and duplicate plates were incubated photosynthetically to allow both KGD" and K G D + cells to grow. After two days of incubation, the colonies that grew aerobically on the plates were counted and compared to the number of colonies that grew photosynthetically. If the number of colonies on photosynthetically and aerobically incubated plates were approximately equal, then the clone being tested was said to have complemented the mutation in KGD11. If the number of aerobically competent cells (KGD + ) were much less than photosynthetically competent cells ( K G D + and KGD") but the proportion increased over a five day incubation period, then the clone being tested was credited with allowing recombination to take place. If < 2% of the cells spread formed colonies after two days and this number did not increase appreciably after five days, then it was assumed that neither recombination nor complementation was possible with the fragment being tested. As none of the cloned EcoRI fragments complemented (nor replaced by homologous recombination) the KGD" allele in KGD11, I decided to clone larger fragments of DNA that spanned EcoRI sites. An Sstl digest of pFDI produced a 12 kb fragment that was derived 37 exclusively of R. capsulatus DNA. This 12 kb Sstl fragment was cloned into pRK415 in both orientations (pRK415::14a and pRK415::14b (Fig. 7)). Complementation of the K G D 11 mutant phenotype by both pRK415::14a and pRK415::14b was tested as described previously. The results of this experiment indicated that the 12 kb Sstl fragment no longer complemented the mutation as only 0.61% and 1.7% of the photosynthetically competent cells of KGD1 l(pRK415::14a) and KGDll(pRK415::14b), respectively, grew aerobically after two days of incubation (Table II). However, the proportion of cells able to grow aerobically increased dramatically to 34.9% and 40%, respectively, by the fifth day of incubation indicating that the Sstl fragment was allowing recombination to take place. From this one can infer that the 12 kb Sstl fragment contains only a portion of the gene that was responsible for complementation, but this was sufficient to allow recombination to replace the corresponding mutated region of the KGD 11 chromosome. Alternatively, the Sstl fragment may have contained the entire KGD allele (or regulatory gene) mutated in KGD11, but may be missing upstream sequences required for efficient expression, such as a promoter. Therefore, I used an expression vector to attempt to drive transcription of sequences on the Sstl fragment. The expression vector used, pJAJ9, contains the R. capsulatus puf operon promoter, the pufQ gene, and the first 20 codons of the pufB gene' upstream of unique EcoRl, Sstl, BamHl, and Pstl restriction sites. One problem in using this expression vector to drive transcription of genes primarily involved in aerobic respiratory growth is that the puf operon promoter is regulated such that maximal expression occurs during anaerobic photosynthetic growth. Activity of the pJAJ9 puf promoter is believed to be four times higher in cultures grown under oxygen-limiting conditions relative to cultures grown under highly aerated conditions (Johnson, et al. 1986). 38 Figure 7: Outline of cloning of the 12 kb Sstl fragment from the cosmid clone pFDI into the broad host range vector pRK415. EI: EcoRI; S: Sstl; H: Hindlll; C: Clal; EV: EcoRV; B: BamHl. 39 40 Table II: Data from Complementation/Recombination Experiment of the K G D " Phenotype of K G D 11 with the 12 kb Sstl Fragment Cloned in pRK415. # Colonies on Plates After 2 3 4 5 Straina Growth0 Days of Incubation0 KGD 11 PS 430 O2 0 KGDll(pFDI) PS 473 O2 487 KGDll(pRK415::14a) PS 495 0 2 3 (0.61) 14 (2.8) 60 (12.1) 173 (34.9) KGDll(pRK415::14b) PS 472 O2 8 (1.7) 21 (4.4) 88 (18.6) 189 (40.0) a K G D l l and KGDll(pFDI) are included as controls to monitor the frequency of reversion and a positive control for complementation, respectively. D 0 2 refers to aerobic dark growth and PS refers to anaerobic photosynthetic growth of the cultures, respectively. cExcept where indicated, additional colonies were not detected over the incubation period. Numbers in parentheses express aerobically competent cells as a percentage of photosynthetically competent cells. 41 The 12 kb Sstl fragment was cloned downstream of the puf operon promoter on pJAJ9 at the Sstl site in two orientations (Fig. 8). These constructs were again screened for complementation in KGD 11 as described previously. The proportion of KGD1 l(pJAJ9::14b) photosynthetically competent cells that were able to grow aerobically increased from 4.6% to 88.6% over the five day incubation period compared to KGDll(pJAJ9::14a) cells which increased from 0.98% to 60.2% during the same period (Table Ul). These results suggested that the Sstl fragment was not missing the KGD promoter. Restriction enzyme mapping of the Sstl fragment in pUC13 provided useful information for further clonings (Fig. 9). The Sstl-EcoRY and Sstl-BamYR fragments were cloned into the vector pRK415 resulting in the clones pRK4\5::Sstl-EcoR\0 and pRK415::S.sfI-BamHl. These constructs were conjugated into KGD 11 and cells capable of wild type growth were selected as described above but neither was able to recombine into the K G D 11 chromosome (the pattern of growth observed with KGDll(pRK415::14a) and KGDll(pRK415::14b) was not repeated). However, an Sstl-Clal fragment cloned in pRK415 (pRK415::SM-C7<zl0) was able to recombine, restoring wild type KGD activity in KGD 11 (Table IV). These findings led me to conclude that the mutation on the K G D 11 chromosome is somewhere within (or very near to) the EcoRV-BamHl region (designated by "*" in Fig. 9). 3. Sequencing of a portion of the DNA segment responsible for rescue of the wild type phenotype in KGD 11 by recombination. The EcoRV-BamEl segment of pUC13::14b (highlighted by "*" in Fig. 9) was cloned into the sequencing vectors pTZ18U and pTZ19U and sequenced. This segment is about 900 bp long and I was able to sequence approximately 340 bases on each strand (Fig. 10). The sequences 42 Figure 8: Outline of cloning of the 12 kb Sstl fragment from the cosmid clone pFDI into the R. capsulatus expression vector pJAJ9. EI: EcoRI; S: Sstl; H: Hindlll; C: Clal; EV: EcoRV; B: BamHl; P: Pstl; Sm: Smal. 43 44 Table III: Data from Expression Experiment with the 12 kb Sstl fragment Cloned in the Expression Vector pJAJ9. # Colonies on Plates After 2 3 4 Straina Growth0 Days of Incubation0 KGD 11 PS 846 O2 22 KGDll(pJAJ9::14a) PS 508 O2 5 (0.98) 206 (40.6) 306 (60.2) KGDll(pJAJ9::14b) PS 395 O2 18 (4.6) 275 (69.6) 350 (88.6) a K G D l l is included as a control for reversion. 0 O 2 and PS refer to aerobic dark and anaerobic photosynthetic growth of the cultures, respectively. cExcept where noted, additional colonies were not observed over the incubation period. Numbers in parentheses express aerobically competent cells as a percentage of photosynthetically competent cells. 45 H E V B h S Figure 9: Partial restriction site map of the 15 kb EcoRl insert in pFDI. Region indicated by "*" is the segment cloned into the sequencing vectors pTZ18U and pTZ19U for D N A sequencing as well as the fragment used as the probe in Southern and Northern hybridization experiments. EI: EcoRl; S: Sstl; H: HindlU; B: BamHl; E V : EcoRV; C: Clal. 46 Table IV: Data from Complementation/Recombination Experiment with Portions of the 12 kb Sstl Fragment. # Colonies on Plates After 2 3 4 5 Straina Growth0 Days of Incubation0 KGDll(pFDI) PS 449 0 2 421 KGDll(pRK415::S-B) PS 518 O2 55 KGDll(pRK415::S-RV) PS 720 O2 60 KGDll(pRK415::S-C) PS 597 O2 0 8 (1.3) 60 (10.1) 120(20.1) KGDll(pRK415::14a) PS 589 O2 11 (1.9) 17 (2.9) 40 (6.8) 108 (18.3) a KGDll (pFDI) and KGDll(pRK415::14a) are included as positive controls for complementation and recombination, respectively. °PS and 02 refer to photosynthetic and aerobic growth, respectively. cExcept where noted, additional colonies were not observed over the incubation period. Numbers in parentheses express aerobically competent cells as a percentage of photosynthetically competent cells. S: Sstl; B: BamUl; RV: EcoRV0; C: Clal0. 47 Figure 10: Nucleotide sequence of regions (a) and (b) from the EcoRV-BamBI segment of the 12 kb Sstl fragment that allowed recombination to occur between the plasmid-borne wild type and chromosomal mutated KGD allele in KGD11. The (a) sequence shows the coding strand read 5' to 3' from the. BamHl restriction site and the (b) sequence shows the non-coding strand read 5' to 3' from the EcoRY restriction site. 10 20 30 40 50 60 I I I I I I CGCGCTCTTACGGTTTCACCGAAGCCGACATGGACCGGATGATCTTCATCGACAACGTGC 70 80 90 100 110 120 I I I I I I TGGGTCTGCAGGTCGCCTCGATGCGGCAGATCCTCGACGTGCTCAAGCGCACCTATTGCG 130 140 150 160 170 180 I I I I I I GCACCTTCGCGCTGCAATACATGCATATTTCGAACCCCCGAGGAAGCCGCCTGGCTGAAA 190 200 210 220 230 340 I I I I I I GAGCGGATCGAGGGCTACGGCAAGGAAATCGCCTTCACCCGCGAAGGCCGCCGCGCCATC 250 260 270 280 290 300 I I I I I I CTGAACAAGCTGGTCGAGGCCGAGGGCTTCGAGAAATTCCTGCATGTGAAATATACCGCA 310 320 330 340 I I I I CCAAGCGCTTCGCTGGATGGCGGCGAGGCGCTGATCCGCGA 10 20 30 40 50 60 I I I I I I GGATAGGGCGAGGTGCGGCTGAAATGCGGCGCCGTGGTGAAGCCGATCTGGTTGTTCACG 70 80 90 100 110 120 I - I I I I I ACGATATGGATGCAGCCGCCGGTGCGGTGCCCCTTGATGCCCGAAAGCTGCAGACATTCC 130 140 150 160 170 180 I I I I I I GCCACGATCCCCTGCCCGGCAAAGGCCGCATCGCCATGCAGCAGCACCGACAGAACCTGG 190 200 210 220 230 240 I I I I I I ACGTTGCGGTCCTCGTCATGGGCCTGATCCTGCTTGGCGCGGACCTTGCCCAGAACGACC 250 260 270 280 290 300 I I I I I I GGGTTCACCGCCTCAAGGGTGAGAGGGGGTTTCGCGGTCAGCGACAGGTGCACGGTGTGG 310 320 330 340 I I I I GCCGTCAAACGAGCGTCGACGAGGGCGCCAAGTGATATTTCACGTGC 49 do not overllp and were maintained as separate sequences (designated (a) and (b)). Using the DNA analysis algorithms of PCGENE, the sequences were translated and open reading frames were compared to the published sequences of the sue A and sucB gene products of E. coli and A. vinelandii. I was able to find an alignment with an internal segment of the sucA gene product Elo of these species of bacteria (Fig. 11 and 12, respectively). The translated sequences have 16.8% (a) and 46% (b) amino acid identity with amino acids 121-242 and 271-407, respectively, of the E. coli E lo protein sequence. These same R. capsulatus sequences have 23% (a) and 44.1% (b) amino acid identity with amino acids 125-249 and 277-412, respectively, of the A. vinelandii E lo protein sequence. Therefore, I believe that the regions sequenced are portions of the R. capsulatus sue A gene. This confirms that the gene mutated in KGD 11 is a gene encoding a KGD protein and not a regulatory one. 4. Southern hybridization. Since the 15 kb EcoRI partial digest fragment on pFDI complemented in trans the mutation in KGD 11 and the Sstl fragment only allowed recombination, it was possible that the KGD allele was intact on the 12 kb Sstl fragment but was missing a promoter. In aligning a partial restriction enzyme map of the R. capsulatus Sstl fragment with a map of the E. coli sucAB operon at the sites of (a) and (b) sequence homology, an interesting difference became apparent (Fig. 13). While the E. coli sucAB promoter is 200 bp upstream of the E lo translation initiation codon, the R. capsulatus promoter appears to be at least 3 kb upstream, located between the EcoRI and the Sstl restriction sites (region denoted by "#" in Fig. 13). The R. capsulatus gene bank was made by shot-gun cloning size-fractionated 15 - 25 kb EcoRI partial digest products into the cosmid 50 (a) ADLDPSFHDLTEADFQETFNVGSFASGKETMKLGELLEALKQTYCGPIGA -170 RSYGFTEADMDRMIFIDNV-LGLQVASMRQILDVLKRTYCGTFAL EYMHITSTEEKRWIQQRIESGRATFNSEEKKRFLSELTAAEGLERYLGAK -220 QYMHI SNPRGSRLAERA DRGLRQGNRLHPRRPPRHPEQAGRGRGLRE FPGAKRFSLEGGDALIPMLKEM -242 IPACEIYRTKRFAGWRRGADPR (b) LGKKPQDLFDEFAGKHKEHLGTGDVKYHMGFSSDFQTDGGLVHLALAFNP -320 HLHVCKLRSLTIQRSTAEDVTFGDVKYHLGASSDRSFDGHTVHLSLTANP SHLEIVSP WI GSVRARLDR-LDEP SSNKVXPI TI HGDAAVTGQGWQET - 3 6 9 SHLEAVNPVVLGKVRAKQDQAHDEDRNVQVLSVLLHGDEAFAGQGIVAEC LNMSKARGYEVGGTVRIVINNQVGFTTSNPLDARSTPY -4 0 7 LQLSGIKGHRTGGCIHIWNNQIGFTTA-PHFSRTSPY Figure 11: Alignment of R. capsulatus translated (a) and (b) sequences (from region essential for recombination; EcoRV-BamBI) with the corresponding region of the E. coli K G D Elo protein, (a) Alignment with amino acids 121-242 of the E. coli sequence. Identity in the sequence (:) is 16.8% with 0 and 2 gaps inserted in the E. coli and R. capsulatus sequences respectively, (b) Alignment with amino acids 271-407 of the E. coli sequence. Identity in the sequence (:) is 46% with 1 gap inserted in both the E. coli and R. capsulatus sequences. 51 (a) SDLSITHYGLTDADLDTPFRTGELYIGKEEATLREILQALQETYCRTIGA -174 RSYGFTEADMDRMIFIDNV-LGLQVASMRQILDVLKRTYCGTFAL EFTHIVDSEQRNWFAQRLESVRGRPVYSKEAKSHLLERLSAAEGLEKYLG -224 QYMHISNPRGSRLAERADRGLRQGNRLHPRRPPRHPEQAGRGRGLREIPA TKYP GTKRF GLE GGE SLVP WDE11 -249 CEIYRTKRFAGWRRGADPR (b) LGKNPRDLFDEFEGKHLVELGSGDVKYHQFSSNVMTSGG-EVHLAMAFNP -325 HLHVCKLRSLTIQRSTAEDVTFGDVKYHLGASSDRSFDGHTVHLSLTANP SHLEIVSPVVEGSVRARQDR-RVDATGEKVVPISIHGDSAFAGQGWMET -374 SHLEAVNPWLGKVRAKQDQAHDEDRNVQVLSVLLHGDEAFAGQGIVAEC FQMSQIRGYKTGGTIHIWNNQVGFTTSNPVDTRSTEY -412 LQLSGIKGHRTGGCIHIWNNQIGFTTAPHF-SRTSPY Figure 12: Alignment of R. capsulatus translated (a) and (b) sequences (from region essential for recombination; EcoRV -B amHI) with the corresponding region of the A. vinelandii K G D Elo protein, (a) Alignment with amino acids 125-249 of the A. vinelandii sequence. Identity in the sequence (:) is 23% with 0 and 1 gap inserted in the A. vinelandii and R. capsulatus sequences respectively, (b) Alignment with amino acids 277-412 of the A. vinelandii sequence. Identity in the sequence (:) is 44.1% with 2 and 1 gaps inserted in A. vinelandii and R. capsulatus sequences respectively. 52 Figure 13: Approximately scaled alignment of the E. coli sucAB operon encoding the E lo and E2o protein components of the K G D complex with the 15 kb EcoRI fragment of pFDI that complemented in KGD11 at the regions of sequence similarity in the R. capsulatus sequences (a) and (b). The E. coli sucAB operon promoter is located as indicated (200 bp upstream of the sucA translation initiation codon). The R. capsulatus sucA promoter is predicted to be located between the extreme left EcoRI and Sstl restriction sites (#). 1 cm = 0.64 kb. 53 vector p L A F R l . It is conceivable, though unlikely, that in this construction two chromosomally unrelated EcoRI fragments joined together to form the 15 kb insert in pFDI. Therefore, Southern blot hybridization was used to test the organization around the R. capsulatus sucA gene. The Sstl fragment in pUC13::14b digested with Hindlll releases a 10 kb fragment. Therefore, digestion of R. capsulatus chromosomal DNA with Hindlll should release a corresponding 10 kb fragment if a chromosomally continuous segment had been cloned to form pFDI. An autoradiogram of a Southern blot experiment designed to test this possibility is shown in Fig. 14. As can be seen, only one 10 kb band was detected in each lane, confirming that the organization of sequences on pFDI is likely to be the same as on the R. capsulatus chromosome. Although the mobilities of the bands detected by the 32P-labelled probe appear slightly different, those detected by the biotin-labelled probe do not. Therefore, I am confident that the bands detected by both probes are the same 10 kb Hindlll band, and slight differences in mobilities are due to slight variations in experimental conditions. Therefore, there appears to be some difference in organization between R. capsulatus and E. coli with regard to the distance between the sucA gene and its promoter. 5. R N A blot hybridizations of RNA prepared from anaerobic photosynthetic and aerobic respiratory cultures of R. capsulatus BIO. In order to evaluate if regulation of KGD enzyme activity could be due to changes in K G D mRNA accumulation, KGD mRNA levels and K G D enzyme specific activities under aerobic respiratory and anaerobic photosynthetic growth conditions were compared. RNA was isolated from photosynthetic and aerobic cultures of R. capsulatus BIO using the hot phenol method described in Materials and Methods. Equal amounts of total cellular RNA were electrophoresed on a 1.4% agarose-formaldehyde gel and probed with the radioactively labelled EcoRV-BamHl fragment 54 Figure 14: Autoradiogram of a Southern blot hybridization experiment of R. capsulatus BIO chromosomal DNA. Panel (a) shows a blot probed with a radioactively labelled EcoRV-BamBl fragment and panel (b) a blot probed with the same fragment labelled with biotin. In each panel, lane 1 contains 4 ng of pUC13::14b plasmid DNA digested with Hindlll and mixed with 5 jig of sheared salmon sperm DNA and lane 2 contains 5 jig of BIO chromosomal DNA digested with Hindlll. Molecular lengths are indicated in kb (bacteriophage X DNA digested with Mlul). The radioactive blot was used to expose X-ray film for 2 hours at -80°C with an intensifying screen and the non-radioactive blot for 1 minute at room temperature with an intensifying screen. 55 56 that had been used for sequencing (designated with "*" in Fig. 9). An autoradiogram developed from one such hybridization is shown in Fig. 15. It can be seen that there is several times more KGD-specific mRNA in the lane containing RNA derived from aerobic cultures than in that from photosynthetic cultures. Densitometer scanning of several such autoradiograms revealed that the density in the lanes containing RNA from aerobically grown cultures was about 7 times greater than that in lanes containing RNA from anaerobically (photosynthetically) grown cultures. Although the most intense signal was obtained from species in the range of 3 to 1 kb in length, there was faint hybridization extending up above the 10 kb length range. I conclude that sucA mRNA is very unstable and could be synthesized as a very large (> 10 kb) primary transcript. An attempt was made to obtain a full length KGD message using a different RNA isolation protocol (described in Materials and Methods). Unfortunately, the quality Of KGD mRNA obtained by both methods was very similar as demonstrated by the similarity in appearance of K G D -specific message detected in the autoradiograms (Fig. 16). However, a band migrating more slowly than the longest RNA ladder marker (9.5 kb) was detected in these RNA preparations. The length of this molecule was estimated to be 15 kb. However, when the RNA was treated with RNase free DNase I and hybridized to a sucA mRNA-specific probe, this high molecular weight band disappeared (conditions of DNase I digestion were those used for the hot phenol protocol, see Materials and Methods) (Fig. 17). Although this 15 kb band is more easily seen at the top of Fig. 16 than at the top of Fig. 17, the band is missing from the lanes containing DNase I treated RNA in the latter figure. Whether the signal was due to DNA contamination of the RNA preparation or an RNA molecules that were degraded by RNase contaminating the DNase I enzyme is analyzed in Discussion. However, the simplest interpretation is that this band was due to chromosomal DNA contamination. 57 Figure 15: Autoradiogram of a Northern blot hybridization of R. capsulatus BIO RNA. Lane 1 contained 20 u.g of RNA isolated from aerobically (dark) grown cultures and lane 2 contained 20 itg of RNA from photosynthetically (anaerobically) grown cultures. Molecular lengths are given in kb (0.24 kb - 9.5 kb RNA ladder, BRL). The probe used was the radioactively labelled EcoRY-BamHl fragment. The X-ray film was exposed for 32 days with an intensifying screen at -80°C. 9.5 7.5 4.4 2.4 1.4 0.24 59 Figure 16: Autoradiogram of a Northern hybridization blot of RNA isolated by the guanidine isothiocyanate procedure from R. capsulatus BIO cultures grown aerobically in the dark and photosynthetically (anaerobically). Lanes 1 and 2 contained 20 Ltg of aerobically and photosynthetically derived RNA, respectively. The probe used was the radioactively labelled EcoRV-Bamrll fragment. The X-ray film was exposed for 17 days at -80°C with an intensifying screen. Molecular lengths are indicated in kb (0.24 kb - 9.5 kb RNA ladder, BRL). 9.5 7.5 4.4 2.4 1.4 0.24 61 Figure 17: Autoradiogram of a Northern blot hybridization of RNA isolated by the guanidine isothiocyanate procedure from R. capsulatus BIO cultures that was subjected to DNase I digestion. Lane 1 contained 20 jig of DNase I treated RNA from aerobically (dark) grown cultures and lane 2 contained 20 |ig of DNase I treated RNA from anaerobically (photosynthetically) grown cultures; lanes 3 and 4 contained 20 jig of RNA (not treated with DNase I) from aerobically (dark) and anaerobically (photosynthetic) grown cultures, respectively. —> possible 15 kb RNA message. The probe was the radioactively labelled EcoRV-BamHl fragment and the X-ray film was exposed for almost 3 days at -80°C with an intensifying screen. Molecular lengths are indicated in kb (0.24 kb -9.5 kb RNA ladder, BRL). 9.5 7.5 4.4 2.4 1.4 0.24 63 6. K G D enzyme activities in extracts of R. capsulatus cultures grown under anaerobic photosynthetic and aerobic respiratory conditions. Cell extracts of R. capsulatus strain BIO grown photo synthetically and aerobically were prepared and assayed for KGD activity as described in Materials and Methods. Four to five assays were done on each of two independently derived cell extracts for each growth condition. The KGD specific activities in extracts of cells grown aerobically (56.8 ± 19 units) were 9 - 1 3 times greater than in extracts of photosynthetically grown cells (5.0 ± 0.79 units) (Table V). This is similar to the differences in density of detected KGD-specific message in the RNA hybridization blots. 7. K G D RNA transcription in R. capsulatus BIO cultures grown under high aeration or anaerobic photosynthetic conditions. Cultures of R. capsulatus strain BIO were grown and RNA was pulse labelled with (5,6-3H)-uridine and isolated from cells as described in Materials and Methods (Pulse Label of KGD RNA). The specific activities of the RNA preparations from aerobically grown cultures were 2.18 x 105 and 6.23 x 104 cpm/|ig RNA and from photosynthetically grown cultures were 2.88 x 104 and 5.66 x 104 cpm/u.g RNA. Qualitative analysis of the data in Table VI indicates that there is more K G D gene transcription initiated under aerobic respiratory than anaerobic photosynthetic growth conditions. There was an average of 28 times more radioactivity (% radioactive KGD message of the total input TCA-precipitable counts) detected per jig of probe used from aerobically grown cultures of R. capsulatus BIO (86.5 x 10"4 %/iig of probe) compared to anaerobically photosynthetically grown cultures (3.1 x 10"4%/|ag of probe). 64 Table V: K G D Specific Activity in R. capsulatus Strain BIO Cell Extracts Grown Aerobically and Photosynthetically in RCV Medium. Growth Modea Specific Activity0 Aerobic 56.8 (19.0) Photosynthetic 5.0 (0.79) aAerobic: aerobic respiratory growth in the dark; PS: anaerobic photosynthetic growth. °Specific activity expressed as nmol NADH produced/minute/mg protein (defined as units); values given are the mean of at least four assays in each of two separate experiments for a given growth condition; standard deviations are given in parentheses. 65 Table VI: Hybridization of Pulse Labelled RNA to a KGD mRNA-Specific Probe to Evaluate the Frequency of Initiation of Transcription of the KGD Gene Under Aerobic or Anaerobic Growth Conditions. Growth Conditionsa Probe Concentration K G D R N A b Average % KGD (x 10~4 %) Message detected per pg probe Aerobic 0.5 pg 36 Aerobic" 1.0 pg 87 Aerobic 2.0 pg 170 Aerobic 0.5 pg 65 Aerobic 1.0 pg 70 Aerobic 2.0 pg 150 86.5% Photosynthetic 0.5 pg 0 Photosynthetic 1.0 pg 4.2 Photosynthetic 2.0 pg 3.4 Photosynthetic 0.5 pg 2.7 Photosynthetic 1.0 pg 4.6 Photosynthetic 2.0 pg 5.7 3.1% aAerobic: aerobic respiratory growth (dark); PS: anaerobic photosynthetic growth. bvalues determined by subtracting radioactivity bound to a non-specific probe (pTZ19U::£'cY>RV0-5amHi) from radioactivity bound to a KGD mRNA specific probe (pTZlSV::EcoR\0-BamRT) and expressing the result as a percentage (x 10"4) of the total TCA-precipitable counts in the hybridization mixture. 66 DISCUSSION The similarity in genetic organization of the K G D operons and KGD gene products in the species E. coli,B. subtilis, A. vinelandii, and P. fluorescens suggested that a similar enzyme complex with a similar genetic organization might be present in R. capsulatus. In order to find the gene(s) that encodes a K G D protein(s), the K G D " mutant strain of the wild type strain BIO, KGD11 was used to screen a gene bank of R. capsulatus DNA. As mentioned in section 1 of Results, this approach yielded only two types of cosmid clones (pFDI and pFDII) that were able to rescue the wild type phenotype in KGD 11 by complementation in trans (see Fig. 4). Therefore, both these clones contained the wild type KGD allele mutated in KGD11 as well as its promoter. The results from complete EcoRI digestion of the clones indicated that the inserts were overlapping segments of the R. capsulatus chromosome (see Fig. 4). Because I was able to find only two types of cosmid clones that complemented the mutation in KGD11, I suspected that a large continuous segment of the chromosome was required to obtain expression of the cosmid-borne wild type KGD allele. Therefore, the K G D allele may be part of an operon or may require cis acting regulatory sequences that are quite distant from the KGD sequence(s). However, at the time, I hoped that the wild type KGD gene would be intact on an EcoRI fragment, and therefore tested the 4 kb, 2 kb, 1.8 kb, 1.5 kb, and 1 kb EcoRI fragments present in the region of overlap of the inserts in the two cosmid clones for complementation of the KGD" phenotype in KGD11. Unfortunately, none of the clones tested rescued the 67 wild type phenotype, suggesting that the KGD allele was not located entirely on a single EcoRl fragment. As previously mentioned, it was possible that the K G D allele required upstream sequences for expression. Therefore, even if it were intact on an EcoRl fragment it would fail to complement the KGD" phenotype of KGD11. If this were the case, then recombination could occur between the wild type and mutated KGD sequences resulting in rescue of the wild type phenotype. Therefore, the plates spread with exconjugants were left to incubate aerobically in the dark for two weeks. After that time, as no colonies grew aerobically, I concluded that an EcoRl site was probably too close to the mutated K G D sequence in KGD 11 to allow recombination. As a result of the lack of rescue of the wild type phenotype by the EcoRl clones, I decided to test larger fragments that spanned EcoRl restriction sites for complementation or recombination in KGD11. To easily distinguish complementation, recombination and reversion, I developed the assay outlined in Fig 6. Complementation of the KGD" phenotype in KGD 11 in trans would occur in all cells that contained all the sequences required for KGD expression. Rescue of the wild type phenotype in K G D 11 by recombination required that there be enough homology between the wild type and mutated KGD genes to allow a double cross-over event to occur. Therefore, if a defined number of cells were spread on plates of RCV medium + tetracycline and incubated aerobically in the dark (to select for the K G D + phenotype), the number of colonies that grew as a proportion of the number spread would distinguish complementation (if most of the cells grew aerobically) from recombination (if only a portion of the cells spread grew aerobically). A 12 kb Sstl fragment tested in this manner resulted in the rescue of the wild type phenotype in KGD11. Over the five day incubation 68 period, the number of aerobically competent KGDll(pRK415::14a) KGD+ colonies increased from 0.61% to 34.9% of the number of colonies that grew photosynthetically, and the number of aerobically competent KGDll(pRK415::14b) colonies increased from 1.7% to 40.0% (Table II). Therefore, the 12 kb Sstl fragment contained either a truncated K G D sequence or the entire promoterless KGD gene. In both cases there would be enough sequence homology for recombination with the mutated chromosomal allele. The results from the pJAJ9 expression experiment supported the hypothesis that maybe not all the structural gene (or c^s-active sequences) was present on the 12 kb Sstl fragment (Table III). However, due to the decreased activity of the puf operon promoter under aerobic conditions, the possibility that the KGD gene promoter is missing on the 12 kb Sstl fragment cannot be categorically excluded. With more detailed analysis of the 15 kb EcoRI fragment, it should be possible to design experiments to directly assay for the presence of a promoter. DNA segments upstream of the regions sequenced (containing the putative sucA promoter) could be cloned upstream of a promoterless p-galactosidase encoding gene and enzyme activity assayed under anaerobic and aerobic growth conditions. Recombination between the Sstl fragment and the KGD11 chromosome was required to rescue the wild type phenotype, whereas, the larger EcoRI partial digest fragment complemented the mutation in trans, suggesting that the wild type KGD gene was at one end of the Sstl fragment. Using the data obtained from restriction enzyme site mapping (Fig. 9), the 12 kb Sstl fragment was cut into two (an Sstl -EcoRV and an Sstl-BamHl fragment), the resulting fragments were cloned into pRK415 and their ability to allow recombination in KGD11 was assessed as described in Results (section 2). Neither clone recombined into the KGD 11 chromosome indicating that the region essential for recombination 69 was within (or near) the overlapping region of the two fragments. This is the EcoRV-Bamlil region designated by "*" in Fig. 9 (see Table IV). To test this hypothesis, a third fragment (Sstl-Clal, Fig. 9) was cloned into pRK415 and tested for recombination in KGD11. This last fragment allowed recombination to occur between the wild type plasmid-borne and mutated chromosomal alleles, and restored the ability for aerobic growth to K G D 11 (Table IV). This confirmed the hypothesis that the EcoRV-BamHl region was essential for recombination between the wild type and mutated KGD allele. Since the EcoRV-BamHl region was essential for recombination and, therefore, contained a portion of the wild type allele of the gene mutated in KGD11, I determined the DNA sequence of part of it to identify, if possible, which of the three known KGD genes was mutated in KGD11. Approximately 340 bases on each end of the EcoRV-BamHI fragment were sequenced (Fig. 10). Sequence similarity with the proper polarity with respect to the (a) and (b) translated sequences was found with an internal segment of the E. coli and A. vinelandii sucA gene products (Elo) (Fig. 11 and 12). The amino acid identity between the translated (a) sequence of R. capsulatus with amino acids 121-242 of the E. coli Elo protein was 16.8% with two gaps inserted in the R. capsulatus sequence, and the translated (b) sequence showed 46% amino acid identity with amino acids 271-407 when one gap was inserted in both the R. capsulatus and E. coli sequences. Amino acids 125 to 249 of the A. vinelandii Elo protein had 23% identity with the R. capsulatus translated (a) sequence with one gap inserted in the latter sequence, and the translated (b) sequence had 44.1% amino acid identity with amino acids 277 to 412 with one and two gaps inserted in the R. capsulatus and A. vinelandii sequences, respectively. Therefore, I concluded that the gene partially sequenced is the R. capsulatus sue A gene (not a KGD regulatory gene) and it is this gene on the KGD 11 chromosome that was mutated yielding the KGD" phenotype. 70 In aligning the partial restriction map of the 15 kb EcoRl insert on the pFDI clone at the region of (a) and (b) sequence homology with the E. coli sue A gene, it appeared that the distance between the R. capsulatus sue A gene and its promoter was different from that in E. coli (Fig. 13). Whereas the E. coli promoter is 200 bp upstream of the sue A translation initiation codon, the R. capsulatus K G D promoter is at least 3 kb upstream of the sequenced region. Therefore, the DNA region between the putative R. capsulatus sue A promoter and the sue A gene may be occupied by another gene, possibly the sucB or Ipd homologues. This indicates that the K G D genes in R. capsulatus may be expressed as an operon as in other organisms, but with a different gene order. Alternatively, this DNA region may be occupied by another gene required for aerobic growth. If so, this would suggest that there may be coordinate expression of KGD genes with other genes required for aerobic growth in R. capsulatus. Sequencing of the 15 kb EcoRl fragment has been initiated and could reveal open reading frames with homology to sucB, Ipd or some other C A C enzyme encoding genes. Confirmation of the genetic organization around the R. capsulatus sucA sequences by Southern blot hybridization additionally supports the hypothesis that the sucA promoter is at least 3 kb upstream of the gene. This is consistent with the observation that in R. capsulatus promoters are often quite distant from the genes they transcribe. Many of the R. capsulatus genes required for photosynthetic growth are organized into operons, the distal genes of which may be 10 kb downstream from their promoters (Wellington, et al. 1991). Some of these operons are known to be nested with significant read-through transcription into downstream operons and this arrangement has been found to be significant for adaptation to changes in environmental conditions (Wellington, et al. 1991). 71 The possibility that the R. capsulatus K G D genes exist in an operon with transcription initiated from a KGD promoter as well as from read-through from upstream sequences should be explored. One could speculate that read-through transcription would ensure a basal level of K G D enzyme complex formation to satisfy biosynthetic requirements during anaerobic growth, whereas transcription of the KGD genes from a KGD promoter would result in the increased levels of K G D complexes required for aerobic growth. This hypothesis suggests that the K G D promoter is active under aerobic growth conditions (i.e. the promoter is "turned on" by oxygen, or some other factor dependent on oxygen, and "turned off by the lack of it). Alternatively, regulation of the KGD genes may be more direct through a single promoter that is more active with the presence of oxygen in the culture medium or less inactive by the lack of it. These hypotheses could be tested by SI nuclease end mapping of the RNA molecules produced from the KGD promoter. However to do such experiments, the K G D promoter needs to be located, so specific DNA fragments could be used as probes for 5'-end mapping. The K G D promoter is being located at present. It is known that/?, capsulatus photosynthesis genes are transcribed at increased levels in response to decreasing availability of molecular oxygen (Clark, et al. 1984). One might, therefore, expect the transcription of genes involved primarily in aerobic growth to be regulated in a manner opposite to that of photosynthesis genes. Therefore, I measured the levels of KGD mRNA under these two growth conditions to compare with K G D specific activities in extracts of wild type R. capsulatus cultures grown aerobically in the dark and photosynthetically (anaerobically). The autoradiograms of the RNA blot hybridizations show clearly that there was more KGD-specific messages (or fragments of messages) 72 detected in lanes containing RNA obtained from aerobically grown cultures compared to lanes containing R N A from anaerobic photosynthetically grown cultures (Fig. 15 and 16). I was able to isolate roughly the same amount of RNA from equal volumes of aerobic respiratory and anaerobic photosynthetic cultures grown to the same cell density and growth phase, suggesting that cells grown under both conditions contain roughly the same amount of RNA. Therefore, the differences in KGD mRNA detected by the Northern hybridizations reflect the differences in the intracellular KGD mRNA levels under the conditions studied. However, it appeared that KGD mRNA was degraded too rapidly to allow detection of full length transcription products. Unfortunately, attempts made to isolate intact KGD mRNA using a different isolation procedure gave about the same results (compare Fig. 15 and 16). Northern hybridization blots of RNA isolated using a guanidine isothiocyanate protocol revealed the presence of a large hybridizing species (approximately 15 kb; see Fig. 16). As RNA isolated with the hot phenol method was treated with DNase I and RNA isolated with guanidine isothiocyanate was not, I decided to subject the latter RNA to DNase I digestion. RNA hybridization blots of these RNA samples did not show a 15 kb band (Fig. 17). Since the RNA was separated from DNA in the cell extract by CsCl density centrifugation, it seemed unlikely that there was DNA contaminating the RNA sample. However, it seems equally unlikely that the DNase I enzyme was contaminated with RNase. Therefore, as the DNase I treatment of RNA did not appear to greatly degrade the lower molecular size RNA species in my preparations, I concluded that the 15 kb band detected was the result of chromosomal DNA contamination. The sue A-specific RNA appeared to consist of approximately the same size range of molecules under the aerobic and anaerobic growth conditions studied, as seen by the similarity in the starting point and end of 73 the smears. It is difficult to estimate the starting points of the smears, but there are sue A -encoding mRNA molecules longer than that required for transcription products of the sucA gene (by analogy to other species of bacteria, the R. capsulatus sue A gene should be about 3 kb in length). Therefore, it is likely that the sue A gene is transcribed with at least one other gene, possibly sucB or Ipd. The similarity in the sucA mRNA smears also implies that the size and extent of degradation of the R. capsulatus sucA transcript is the same under aerobic and anaerobic growth conditions. Therefore, the differences in sue A mRNA detected in preparations from aerobically and photosynthetically grown cultures could be due to differences in either the frequency of transcription initiation of the sue A gene or to differences in the half-life of the message(s) under these conditions. The differences in the rates of RNA transcription under photosynthetic and aerobic growth conditions are discussed below. K G D enzyme activity under aerobic respiratory and anaerobic photosynthetic growth conditions appears to be regulated by the accumulation of K G D mRNA under these growth conditions. Under aerobic conditions, KGD specific activities in wild type R. capsulatus BIO cell extracts were found to be 56.8 ± 1 9 units. This is 9-13 times greater than in extracts of photosynthetically grown R. capsulatus BIO cultures which had K G D specific activities of 5.0 ± 0.79 units (Table V). This is similar to the 7-fold difference in density of detected SMCA -speci f ic messages between the lanes containing RNA from aerobically and photosynthetically grown cultures on RNA hybridization blots. The results of pulse labelling of R. capsulatus RNA and subsequent hybridization to sucA mRNA-specific probes indicated that there was 28 times more transcription of the sucA gene under aerobic respiratory conditions than under anaerobic photosynthetic conditions (Table VI). Equal volumes of photosynthetic and aerobic cultures were grown to the same cell density and pulsed with the same amount of labelled uridine, so 74 the differences in amount of radioactivity bound to the filters is a reflection of the differences in relative amounts of sucA mRNA in the cells in the two cultures. Therefore, it appears that transcription of the sucA gene occurs to a higher degree under aerobic respiratory growth conditions, relative to anaerobic photosynthetic growth conditions, and this is reflected by an increase in KGD specific enzyme activity. The initiation of transcription of the R. capsulatus sucA gene appeared to be 28 times higher under aerobic growth conditions compared to anaerobic growth conditions. However, the levels of K G D enzyme specific activities were measured to be 9 - 13 times greater under aerobic growth conditions compared to anaerobic growth conditions. As the differences in transcription of the sucA gene under the growth conditions studied are higher than the differences in enzyme activities, it appears that there might be additional levels of regulation of sucA gene expression. These may include a higher rate of mRNA degradation under aerobic growth conditions, differences in transcription of the sucB or Ipd genes influencing the rate of assembly of the KGD enzyme complex, or control of assembly of the holoenzyme. Recently a two-component regulatory system responsive to the levels of oxygen in the environment in R. capsulatus has been reported (Taremi and Marrs 1990). Upstream of the puf operon promoter is located an inverted repeat with complementary sequences designated IR-L and IR-R. Under low oxygen growth conditions, a membrane bound kinase phosphorylates a puf promoter binding protein (PPBP) which then binds to IR-R exclusively, and activates transcription of the puf operon. Under high oxygen growth conditions, PPBP is dephosphorylated and binds to both IR-L and IR-R, repressing transcription. Whether transcription of the sue A gene in R. capsulatus is also regulated by the same or a similar two-component system (but opposite in effect to that of the photosynthesis genes) needs to be studied. 76 CONCLUSIONS I have cloned the R. capsulatus sue A gene that encodes the Elo (oc-ketoglutarate dehydrogenase) component of the KGD enzyme complex. The R. capsulatus sue A gene product has great sequence similarity to its E. coli and A. vinelandii homologues. Therefore, the R. capsulatus KGD enzyme complex might likely be a huge multiprotein complex, as in E. coli and mitochondria. However, the genetic organization of the sue A gene of R. capsulatus appears to be different from that found in E. coli with respect to the location of the sucA promoter. It appears that the ratio of aerobic respiratory and anaerobic photosynthetic levels of KGD specific activity is paralleled by the ratio of sue A- specific mRNA, supporting the hypothesis that KGD specific enzyme activity is controlled by the availability of K G D mRNA. These differences in sucA mRNA levels appear to be due to control of transcription of the sue A gene cloned in response to oxygen in a manner different from any other R. capsulatus gene studied. The mechanism by which KGD gene expression is controlled could be part of an extensive process that takes place in cells that shift between two growth conditions, to regulate control of metabolic pathways as part of a multicomponent adaptive response. 77 REFERENCES Balch, W. E. , Fox, G. E. , Magrum, L . J., Woese, C. R. and Wolfe, R. S. 1979. Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 43: 260-296. Beatty, J. T. and Gest, H. 1981a. 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