Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A study of endogenous respiration in Pseudomonas aeruginosa Gronlund, Audrey Florence 1964

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1964_A1 G76.pdf [ 9.16MB ]
Metadata
JSON: 831-1.0105256.json
JSON-LD: 831-1.0105256-ld.json
RDF/XML (Pretty): 831-1.0105256-rdf.xml
RDF/JSON: 831-1.0105256-rdf.json
Turtle: 831-1.0105256-turtle.txt
N-Triples: 831-1.0105256-rdf-ntriples.txt
Original Record: 831-1.0105256-source.json
Full Text
831-1.0105256-fulltext.txt
Citation
831-1.0105256.ris

Full Text

A STUDY OF ENDOGENOUS RESPIRATION IN PSEUDOMONAS AERUGINOSA V AUDREY FV° GRONLUND B.Sc, University of British Columbia, 1959 M.Sc, University of British Columbia, 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in Agricultural Microbiology in the Division of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1964 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia,, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study, I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission* Department of • < ^ « ^ ^ c € G i ^ ? ^ ^ The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8 5 Canada The U n i v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY B . S c , The U n i v e r s i t y of B r i t i s h Columbia 1959 M.Sc, The U n i v e r s i t y of B r i t i s h Columbia 1961 FRIDAY, MAY 1, 1964, at 3:30 P.M. IN ROOM 0, AGRICULTURE BUILDING COMMITTEE IN CHARGE Chairman: F.H. Soward E x t e r n a l Examiner: J.F. Wil k i n s o n U n i v e r s i t y of Edinburgh Medical School of AUDREY F. GRONLUND J.J.R. Campbell D.C.B. Duff B.A. Eagles D.P. Ormrod W.J. Po l g l a s e J . J . Stock / A STUDY OF ENDOGENOUS RESPIRATION IN PSEUDOMONAS AERUGINOSA ABSTRACT The nature of the reserves of Pseudomonas aeruginosa that are o x i d i z e d during endogenous r e s p i r a t i o n was stu d i e d by f o l l o w i n g the changes i n the chemical c o n s t i -tuents and i n the d i s t r i b u t i o n of r a d i o a c t i v i t y of s t a r v i n g c e l l s that had been grown on C^-^-labeled sub-s t r a t e s . The t o t a l p r o t e i n and n u c l e i c a c i d of c e l l suspensions decreased during s t a r v a t i o n . Deoxyribonuc-l e i c a c i d increased s l i g h t l y , whereas r i b o n u c l e i c a c i d decreased. C^ -^ 02 was evolved from endogenously r e s p i r i n g c e l l s s p e c i f i c a l l y l a b e l e d i n the n u c l e i c a c i d f r a c t i o n and from c e l l s s p e c i f i c a l l y l a b e l e d i n the p r o t e i n f r a c -t i o n . Chemical f r a c t i o n a t i o n of C ^ - l a b e l e d c e l l s showed a decrease i n hot t r i c h l o r o a c e t i c a c i d - s o l u b l e and i n s o l u b l e compounds, i n d i c a t i n g that the C^ -^ 02 arose from the degradation of RNA and p r o t e i n and not from f r e e pool compounds. A decrease i n ribosomal.RNA and p r o t e i n was evident from p h y s i c a l f r a c t i o n a t i o n s of starved l a b e l e d c e l l s . An enzyme resp o n s i b l e f o r the i n i t i a t i o n of ribosomal degradation was found to be as s o c i a t e d w i t h the ribosome f r a c t i o n and was i d e n t i f i e d as p o l y n u c l e o t i d e phosphory-l a s e . The enzyme was i n a c t i v e i n h i g h magnesium concen-t r a t i o n s but was a c t i v e under c o n d i t i o n s which allowed the d i s s o c i a t i o n of the large ribosomal u n i t s i n t o 50S and 30S components. P o l y n u c l e o t i d e phosphorylase was not s o l u b i l i z e d by the d i s s o c i a t i o n of the 70S ribosomes but remained f i r m l y attached to the 50S subunit. The o x i d a t i o n of exogenous substrates r e s u l t e d i n va r y i n g degrees of suppression of the o x i d a t i o n of endo-genous RNA and t h i s suppression was a t t r i b u t e d to the r e l a t i v e s t a b i l i z i n g e f f e c t that the exogenous sub-s t r a t e s exerted on the ribosomes. The o x i d a t i o n of endogenous p r o t e i n was depressed during the o x i d a t i o n of exogenous glucose, a s p a r t i c a c i d and adenosine and was increased during the o x i d a t i o n of - k e t o g l u t a r i c a c i d . The response of endogenous r e s p i r a t i o n to the o x i d a t i o n of exogenous substrates appeared to be r e l a -ted to a requirement f o r ammonium ions f o r a s s i m i l a t i o n of carbon. GRADUATE STUDIES F i e l d of Study: A g r i c u l t u r a l M i c r o b i o l o g y Intermediary Metabolism Seminar J.J.R. Campbell G.I. Drummond A.R.P. Paterson S.H. Zbarsky J.J.R. Campbell Related Studies: Advanced Organic Chemistry I n t r o d u c t i o n to Genetics B a c t e r i a l Genetics Enzymology Biochemistry of St e r i o d s and Hormones Molecular S t r u c t u r e and B i o l o g i c a l Function G.G.S. Dutton D.E. McGreer Kathleen Cole J.E. Bismanis W.J. Po l g l a s e V.J. 0'Donne11 P.H. J e l l i n c k W.J. Pol g l a s e G.M. Tener G.I. Drummond R.H. Pearce P.D. Bragg PUBLICATIONS 1. Warren, R.A.J., A.F. E l l s , A.F. Gronlund and J.J.R. Campbell, 1960. Endogenous r e s p i r a t i o n of Pseudomonas aeruginosa. J . B a c t e r i d , 79.: 875. 2. Gronlund, A.F., and J.J.R. Campbell, 1961. N i t r o -genous compounds as substrates f o r endogenous r e s p i r a t i o n i n microorganisms. J . B a c t e r i o l . 81:721. 3. Campbell, J.J.R., A.F. Gronlund and M.G. Duncan, 1963. Endogenous metabolism of Pseudomonas. Ann N.Y. Acad. S c i . 102: 669. 4. Gronlund, A.F., and J.J.R. Campbell, 1963. Nitrogenous substrates of endogenous r e s p i r a t i o n i n Pseudomonas aeruginosa. J . B a c t e r i o l . 8j>: 58. - l i -ABSTRACT The nature of the reserves of Pseudomonas aeruginosa that are oxidized daring endogenous respiration was studied by following the changes in the chemical constituents and in the distribution of radioactivity of starving cells that had been grown on C^-labeled substrates. The total protein and nucleic acid of cell suspensions decreased during starvation. Deoxyribonucleic acid increased slightly, whereas ribonucleic acid decreased. C^ Cv, was evolved from endogenously respiring cells specifically labeled in the nucleic acid fraction and from cells specifically labeled in the protein fraction. Chemical fractionation of C14-labeled cells showed a decrease in hot trichloroacetic acid-soluble and insoluble compounds, indicating that the C^Og arose from the degradation of RNA and protein and not from free pool compounds. A decrease in ribosomal KNA and protein was evident from physical fractionations of starved labeled cells. An enzyme responsible for the initiation of ribosomal degradation was found to be associated with the ribosome fraction and was identified as polynucleotide phosphorylase. The enzyme was inactive in high magnesium concentrations but was active under conditions which allowed the dissociation of the large ribosomal units into 5°S and 30S components. - i i i -Polynucleotide phosphorylase was not solubilized by the dissociation of the 70S ribosomes but remained firmly attached to the $0S subunit. 1 The oxidation of exogenous substrates resulted in varying degrees of suppression of the oxidation of endogenous RNA and this suppression was attributed to the relative stabilizing effect that the exogenous substrates exerted on the ribosomes. The oxidation of endogenous protein was depressed during the oxidation of exogenous glucose, aspartic acid and adenosine and was increased during the oxidation of oc-ketoglutaric acid. The response of endogenous respiration to the oxidation of exogenous substrates appeared to be related to a requirement for ammonium ions for assimilation of carbon. J.J.R. Campbell ACKNOWLEDGEMENT I would like to express my sincere gratitude to Dr. J.J.R. Campbell for his encouragement and criticisms during the course of this study and to the National Research Council of Canada for financial assistance during a part of this study. -Iv-TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF THE LITERATURE 2 I. Energy of Maintenance 3 II, Nature of Endogenous Reserves 4 1. Lipid 6 2. Polysaccharide 11 3. Polyphosphate 17 4. Sulfur 25 III. Biochemical Differentiation 26 1. Induced enzyme synthesis 26 2. Sporulation 28 3. Turnover of cellular constituents 30 IV. Nitrogenous Substrates of Endogenous Respiration 33 V. The Influence of Exogenous Substrates on Endogenous Respiration 37 MATERIALS AND METHODS 42 I. Cultural Conditions and Radioactive Labeling of the Organism 42 1. Cultural conditions 42 2. Radioactive labeling of cells 43 V-Page II. Manometrlc Procedures 43 1. Oxygen uptake 43 2. Evolution of radioactive carbon dioxide 44 III. Analytical Procedures 45 1. Protein 45 2. Ammonia 45 3. Deoxyribonucleic acid 45 4. Ribonucleic acid 46 5. Inorganic phosphate 46 6. Polyamines 47 7. ultraviolet spectra 47 8. Paper electrophoresis 47 9. Viable cell counts 47 IV. Preparation of Cell-free Extracts 48 1. Lysozyme treatment 48 2. Hughes* press 48 3. French pressure cell 49 V. Chemical Fractionation of Whole Cells 50 VI. Physical Fractionation of Cell-free Extracts 51 VII. Sucrose Gradients 52 1. Preparation of gradients 52 2. Preparation of cell-free extracts and conditions of centrifugation 54 Page 3. Distribution of ribosomal material 55 4. Ultracentrifuge analyses of ribosomal material 55 VIII. Ribosome Degradation 56 1. Assay for the presence of a degradative enzyme 56 2. Identification of the enzyme 57 3 . Ribosomal location of the enzyme 58 IX. Radioactivity Measurements 60 EXPERIMENTAL RESULTS AND DISCUSSION 62 I, Preliminary Experiments 62 1. Cell viability during endogenous respiration 62 2. The effect of starvation on glucose oxidation 64 3. The Influence of cell concentration on endogenous oxygen consumption 66 II. Studies with Non-radioactive Cells 68 1. The appearance of UV absorbing material in Warburg supernatant fluids 68 2. Changes in cellular constituents 71 3 . Oxidation of purines and pyrimidines 7* v i i -Page 4. The influence of spermine on the release of UV absorbing material 7& 5. The effect of magnesium and phosphate ions on the release of UV absorbing material, oxygen consumption and ammonia evolution 81 III. Studies with C14-labeled Cells 84 1. Specificity of incorporation of radioactive proline and uracil 84 2. Evolution of radioactive carbon dioxide 86 3. Distribution of radioactivity in cell fractions 89 a. Chemical fractions 89 b. Physical fractions 93 IV. Oxygen Consumption and Ammonia Evolution by Various Cell Fractions 96 V. Sedimentation Analyses of the Ribosomal Components of Cell-free Extracts 102 1. Sedimentation coefficients of ribosomal particles 105 2. Labeling of ribosomal RNA by the "shift upH technique 108 3. Changes in ribosomal components during endogenous respiration 111 v i i i -Page VI. The Detection, Identification and Localization of an Enzyme Present in the Ribosomal Fraction and Responsible for Rlbosome Degradation 121 1. Detection of the enzyme 121 2. Identification of the enzyme 123 3. Ribosomal location of the enzyme 124 VII. The Influence of Exogenous Substrates on Endogenous Respiration 129 1. The effect of growth conditions, an excess of carbon and energy supply, substrate concentration and ammonium ions 130 2. Influence on RNA and protein oxidation I36 3. Influence on the release of UV absorbing material 146 GENERAL DISCUSSION 151 I. Ribosomal Material as an Endogenous Substrate 151 1. In P. aeruginosa 151 2. As a general phenomenon in microorganisms 1~4 II. The Influence of Exogenous Substrates on Endogenous Respiration 1~»8 SUMMARY 161 BIBLIOGRAPHY I63 -ix-LIST -OF FIGURES Figure Page 1. The effect of starvation on glucose oxidation 65 2. The influence of cell concentration on endogenous oxygen uptake 67 3. Increase in UV absorbing material in Warburg supernatant fluids of endogenously respiring cells 69 4. Oxidation of purines, pyrimidines (A) and purine degradation products (B) 75 5. The influence of spermine on the release of UV absorbing material 78 6. The influence of magnesium and phosphate ions on the release of UV absorbing material 82 7. Oxygen uptake by various cell fractions 98 8. The influence of ribosomes and RNase on oxygen consumption by whole cells 101 9. The effect of heat on the ability of ribosomes to stimulate oxygen uptake 103 10. Patterns of UV absorbing material from sucrose gradients 106 11. "Shift up" labeling of ribosomal RNA 110 Figure Page 12. The effect of starvation on optical density of 70S, 50S and 30S ribosomes 112 13. The effect of starvation on the C^4 content of 70S, 50S and 30s ribosomes 113 14. Change in optical density in 50S and 30S ribosomes during endogenous respiration 115 15. Change in C 1 4 content in 50S and 30S ribosomes during endogenous respiration 117 16. Decrease in optical density from ribosomal RNA during starvation 119 17. Decrease in content of ribosomal RNA during starvation 12G 18. Enzymatic release of UV absorbing material from ribosomes 122 19. Release of UV absorbing material from isolated ribosomes 125 20. Oxygen uptake with various exogenous substrates 138 21. Influence of exogenous substrates on the release of UV absorbing material 148 INTRODUCTION Numerous reports in the literature have suggested that microorganisms generally store non-nitrogenous carbonaceous materials which may be utilized to supply energy of maintenance during the course of endogenous respiration. However, i t was shown that when Pseudomonas  aeruginosa was grown in a glucose-ammonium salts medium, no accumulation of carbonaceous reserves could be detected but cell viability was maintained for considerable lengths of time during starvation. Ammonia and carbon dioxide were found to be the end products of endogenous respiration and amino acids were implicated as the source of this ammonia (Warren, Ells and Campbell, i 9 6 0 ) . It has been the intent of this study to elucidate the nature of the nitrogenous compound or compounds oxidized during starvation by following the changes in chemical constituents and the changes in the cellular distribution of radioactivity, when P. aeruginosa was grown on C^ labeled substrates and subsequently allowed to respire endogenously. As the status of endogenous respiration in the presence of exogenous substrates has not been clearly established, i t was further intended to determine the nature of the influences exerted by exogenous substrates on endogenous respiration. REVIEW OF THE LITERATURE Endogenous metabolism has been defined by Lamanna (1963) as being the sum of a l l the biochemical a c t i v i t i e s carried on i n an organism i n the absence of e x t r a c e l l u l a r sources of the carbonaceous and nitrogenous materials es s e n t i a l f or energy production and growth. In the absence of external energy and growth substrates, microorganisms may remain viable f o r considerable lengths of time with t h e i r metabolism being carried on at a greatly diminished rate. This metabolic a c t i v i t y of starving c e l l s i s essential f or t h e i r s u r v i v a l and the capacity of an organism to survive i n the absence of exogenous energy supplies i s of prime importance to i t i n the ever-changing natural environment. The a b i l i t y of Aerobacter aerogenes to survive under starvation conditions has been dealt with competently by Postgate and Hunter (1962) and Strange, Dark and Ness (1961). These workers found that p r i o r conditions of growth, the growth phase, concentration of c e l l s during starvation, pH, presence of cations, buffer, temperature and i l l u m i n a t i o n a l l had pronounced effects on the rate at which v i a b i l i t y was decreased. - 3 I. Energy of Maintenance C e l l s r e s p i r i n g endogenously must expend energy to maintain osmotic regulation, solute permeability, m o t i l i t y , resynthesis of macromolecules degraded during the process of turnover and biochemical d i f f e r e n t i a t i o n ; that i s , synthesis of induced enzymes or new st r u c t u r a l macromolecules as occurs during endotrophic sporulation. The energy required by starving microorganisms i n order to sustain v i a b i l i t y has been termed energy of maintenance. The requirement for energy of maintenance has been established conclusively for higher animals and suggested for higher plants, however, most work with microorganisms has yielded generally inconclusive r e s u l t s (Mallette, 1 9 6 3 ) . McGrew and Mallette (1962) observed that repeated additions of low levels of glucose to starving Escherichia  c o l i did not support an increase i n c e l l numbers but did prolong the v i a b i l i t y of the c e l l s for a considerably greater period of time than that which was observed with "unfed" controls. They concluded that E. c o l i s p e c i f i c a l l y used the exogenous supply of glucose for maintenance, thus preserving i t s i n t r a c e l l u l a r endogenous substrates. Using a k i n e t i c approach to the problem of energy of maintenance, Marr, Nilson and Clark (1963) l i m i t e d the growth of E. c o l i by a steady state concentration of carbon source i n an attempt to determine a value f o r s p e c i f i c maintenance. -4-As the greater portion of s p e c i f i c maintenance i s probably-required to provide energy for resynthesis of macromoleeules during turnover, Marr et a l . assumed that there would be c e r t a i n reactions requiring an expenditure of energy/ml of culture/unit time that would be independent of growth. They demonstrated that a portion of the metabolism of the carbon source was diverted from c e l l synthesis over a wide range of growth rates. From an assumed value f o r the rate of turnover for a l l c e l l u l a r constituents, Marr e£ a l . calculated that s p e c i f i c maintenance was independent of growth rate i n carbon l i m i t e d steady state cultures. As the rate of turnover i s maximal i n starving c e l l s and almost n e g l i g i b l e during unrestricted growth, Marr et a l . therefore concluded that the required energy of maintenance i n starving c e l l s would be considerably greater than i n c e l l s undergoing either l i m i t e d or unrestricted growth. I I . Nature of Endogenous Reserves The a b i l i t y of microorganisms to survive under starvation conditions f or various time i n t e r v a l s has stimulated i n t e r e s t i n the nature of the endogenous reserve materials capable of supplying the required energy and perhaps carbon intermediates. Attention has been focused mainly on the possible existence, i n bac t e r i a , of storage compounds analogous to those found i n higher plants and animals such as starch, glycogen and creatine phosphate. Wilkinson (1959) has suggested that the following c r i t e r i a should apply to an energy storage compound, with the greatest emphasis being placed on the l a s t r e q u i s i t e : (a) the compound must accumulate at a time when the exogenous energy supply i s i n excess of the requirements of the c e l l (b) the accumulated reserve must be used by the c e l l when the exogenous energy supply has been depleted to a l e v e l which i s no longer capable of sustaining the organism (c) the reserve material must be broken down to a u t i l i z a b l e form of energy which gives the c e l l a b i o l o g i c a l advantage over c e l l s which do not have a si m i l a r storage compound. Wilkinson admits, however, that any endogenous compound that i s capable of being broken down to intermediates and energy may have an i n d i r e c t storage function. Wilkinson's c r i t e r i a imply that i f the growth of a b a c t e r i a l culture i s l i m i t e d by a deficiency of nitrogen, a required amino a c i d , phosphate or any required nutrient other than the source of carbon and energy, endogenous reserves w i l l accumulate within the c e l l . This, i n f a c t , appears to be the s i t u a t i o n since the capsular size and i n t r a c e l l u l a r poly-saccharide content of K l e b s i e l l a aerogenes have been found to be minimal i n carbon and energy lim i t e d cultures whereas, polysaccharide accumulated when nitrogen was the growth - 6 -limiting factor. K. aerogenes also accumulated volutin under conditions of nitrogen deficiency. A high glycogen content was found in E. coli in the presence of limiting nitrogen and low concentrations were found i f the carbon source was deficient. Bacillus species have been shown to accumulate poly-(3 -hydroxybutyrate in limiting nitrogen whereas a limiting carbon and energy source was unfavorable for accumulation of the polymer (Wilkinson, 1959> Duguid and Wilkinson, 1 9 6 1 ) . 1. Lipid The presence of refractile cytoplasmic inclusion granules, with an affinity for fat soluble dyes such as Sudan Black, has been noted in certain bacterial species. The growth conditions which support the synthesis of these lipid granules indicate that their role is that of a storage product. While studying the prolonged resistance to starvation of the tubercle bacillus, Andrejew (194-8) observed that during the oxidation of glycerol in the growth medium, the respiratory quotient (R.Q.) was O.85. When the glycerol of the medium was depleted, the R.Q. dropped to 0.7. Andrejew suggested that when the growth medium was depleted of its carbon source, the tubercle bacillus, well known for its high lipid concentration, was able to oxidize its own lipid, thus accounting for the organism's ability to survive for long -7-periods in the absence of an energy source. Edson ( 1 9 5 1 ) demonstrated that the amount of lipid which accumulated in Mycobacterium tuberculosis and in the fast-growing saprophytes was dependent upon the carbon source used during growth. The rate of endogenous oxygen uptake was considerably greater with the saprophytes than with M. tuberculosis and in both cases the oxygen consumption was believed to be due to the oxidation of fatty acids. Stickland (195&) noted that a strain of baker's yeast, with a polysaccharide content of up to 30 per cent of its dry weight, showed no measurable decrease in polysaccharide content during endogenous respiration. The endogenous R.Q. was approximately O.85 while the R.Q. during glucose oxidation was 1.02. From the R.Q. values and from Inhibitor studies it was concluded that the endogenous substrate was not poly-saccharide but was lipid in nature. The intra- and extracellular lipid production of yeasts was examined by Deinema ( 1 9 6 1 ) , who found that Lipomyces  starkeyi and Rhodotorula gracilis produced only intracellular lipids, whereas, Rhodotorula graminis, Rhodotorula glutinis and Candida bogoriensis produced both intra- and extracellular lipids. The lipid production was nearly proportional to glucose consumption and the accumulation of lipid generally began when nitrogen sources were exhausted. An increase In nitrogen content of the growth medium resulted in a decrease in the lipid content of the yeasts. When the yeasts were - 8 -Incubated without a carbon or nitrogen source, both intra-and extracellular lipids were degraded. The polymer poly- (i -hydroxybutyrate has been suggested as being an ideal storage compound for carbon and energy as it is insoluble in water and, therefore, can accumulate in the cell in large concentrations (Wilkinson and Duguid, i 9 6 0 ) . Williamson and Wilkinson (1958) were able to isolate apparently morphologically intact lipid inclusion granules from Bacillus species by a sodium hypochlorite digestion of the cells. The lipid granules contained approximately 89 per cent poly-(i-hydroxybutyrate and 11 per cent ether soluble lipid. The molecular weight of the polymer suggested a chain length of approximately sixty residues. In the presence of an excess carbon and energy supply, Bacillus cereus and Bacillus megaterium accumulated poly- (3-hydroxybutyrate to the extent that i t comprised up to 40 per cent of their dry weight. Added ammonium ion caused some inhibition of polymer accumulation, probably a result of some carbon intermediates and energy being diverted into synthesis of cellular constituents. The (3-hydroxybutyrate polymer was rapidly degraded in the absence of an external carbon source and organisms with a high concentration of the polymer had a slower rate of autolysis than those with a low concentration. It was suggested that poly-(3-hydroxybutyrate acts as a reserve carbon and energy source and this storage product, together with polysaccharide, will provide the carbon Intermediates and energy required for sporulation in organisms (McCrae and Wilkinson, 1958). Slepecky and Law ( i960) examined the correlation between synthesis and degradation of poly- p -hydroxybutyrate and sporulation in B. megaterium. Under growth conditions favorable for polymer accumulation, poly- (b -hydroxybutyrate represented 50 per cent of the dry weight of the cell. The peak concentration occurred at the entry into the stationary phase and then the polymer decreased probably due to endogenous utilization. No spores were formed under these conditions. With growth conditions considerably less favorable for polymer formation, the peak polymer concentration occurred during the log phase. Sporulation occurred following a rapid decrease in poly-/3-hydroxybutyrate. The results suggested to Slepecky and Law that i f spore forming micro-organisms concentrate large amounts of poly- /3 -hydroxybutyrate or i f conditions are unfavorable for rapid degradation of the polymer, the cells slowly utilize the polymer as a reserve energy and carbon source and are not prone to sporulate. However, if the polymer is present in a low concentration or if conditions for its rapid utilization are favorable, the organisms can use this source of endogenous nutrient in the energy requiring sporulation process. Clifton and Sobek ( 1 9 6 1 ) have implicated accumulated poly-(3-hydroxybutyrate in B. cereus as an endogenous reserve. -10-The distribution of poly-(5-hydroxybutyrate amongst a number of Gram-negative aerobic bacteria was examined by growing the organisms with a high concentration of either glycerol or glucose for 36 to 89 hours. The polymer was present in Chromobacter violacium, Azotobacter vinelandii, a Rhizobium species, Pseudomonas solanacearum and Pseudomonas  antemycetica. Poly- (3-hydroxybutyrate was not present, how-ever, in the green pigmented pseudomonads and species of Streptomyces, Lactobacillus, Xanthomonas and several other organisms. The role of the lipid as an energy and carbon storage compound again was indicated (Forsyth, Hayward and Roberts, 1958J Hayward, Forsyth and Roberts, 1959). The sudanophilic cytoplasmic granules in Pseudomonas  saccharophila and Rhodospirillum rubrum, similar to those in t n e Bacillus species, were found to consist of poly-(3-hydroxybutyrate. In the absence of an exogenous organic carbon source, 90 VeT cent of the polymer was degraded in twelve hours, demonstrating its ability to serve as a sub-strate for endogenous metabolism (Doudoroff and Stanier, 1959). In nitrogen limiting media a Hydrogenomonas species and Azotobacter chroococcum accumulated poly- (3 -hydroxybutyrate. When ammonium ions were added to resting cells, the cell nitrogen increased at the expense of the polymer. It was assumed, therefore, that in these two organisms poly-(3-hydroxybutyrate is an endogenous storage product which can -11-support metabolism in the absence of exogenous carbon sources and can be used for protein synthesis in the presence of ammonia (Sehlegel, Gottschalk and Von Bartha, 1961). Poly-(5-hydroxybutyrate also accumulated in Pseudomonas pseudomallel (Levine and Wolochow, i 9 6 0 ) , Micrococcus halodenitrifleans (Smithies, Gibbons and Bayley, 1955) and in the filamentous sheathed bacterium (Sphaerotilius natans (Rouf and Stokes, 1962). A large variety of microorganisms have been shown to accumulate poly-(5-hydroxybutyrate when grown in carbon and energy rich media. The polymer is degraded in starving cells and in resting cells in the absence of an exogenous carbon supply. In the presence of ammonium ions the degraded polymer may be incorporated into nitrogenous cellular constituents. Therefore, the compound fulfils at least the first two of Wilkinson's requisites for an endogenous storage compound. 2. Polysaccharide Duguid and Wilkinson (1953) observed that most of the intra- and extracellular polysaccharide of K. aerogenes was formed after cessation of growth due to the depletion of nitrogen, phosphate or sulfur. When growth was limited by exhaustion of the carbon and energy supply, a much lower polysaccharide to nitrogen ratio was realized. K. aerogenes -12-was unable to degrade and utilize its extracellular poly-saccharide and, therefore, the significant rate of endogenous respiration was considered to be the result of the metabolism of the accumulated intracellular polysaccharide (Wilkinson, 1958). A stimulation of glycogen production in the enteric bacteria Salmonella* Shigella, Aerobacter and Escherichia as detected by infra-red spectroscopy, was promoted by supplementing nutrient agar with 1 per cent glucose. The glycogen content reached a peak and then declined on further incubation (Levine et al., 1953). From studies involving the use of different carbon sources and various concentrations of carbon sources in growth media, Dagley and Johnson (1953) were able to conclude that E. coli stored both lipid and polysaccharide. An increase In polysaccharide concentration was brought about by increasing the glucose concentration in the growth medium and was accompanied by a fa l l in lipid content. The addition of acetate to the glucose medium enhanced lipid storage and decreased polysaccharide accumulation. Palmstierna (195&) detected an accumulation of a glycogen-like alkali stable polyglucose during the lag phase of growth in E. coli. The polysaccharide decreased during the log phase when the nitrogenous compounds of the cell increased at their highest rate under the conditions of culture. The effect of various growth limiting nutrients on glycogen formation in E. coli was investigated by Holme and Palmstierna ( 1 9 5 6 a, b). When a nitrogen deficiency limited growth, glycogen accumulated rapidly, however, the addition of ammonium chloride to the culture resulted in a high rate of synthesis of nitrogenous cellular constituents accompanied by a sharp decrease in the glycogen store. During carbon starvation, a slight decrease in nitrogenous cell materials was noted. The accumulation and utilization of glycogen reserves appeared to be correlated with the synthesis of the nitrogen containing compounds of the cell. Glycogen accumulated during phosphate starvation but to a considerably lesser extent than during nitrogen starvation. The addition of phosphate to the starved cells gave rise to an immediate increase in the rate of glycogen synthesis illustrating that the previous lack of phosphate was responsible for the relatively low accumulation. Following the addition of phosphate, a lag in the synthesis of nitrogenous constituents was evident. At the termination of the lag period nitrogen containing compounds were synthesized at the expense of the glycogen reserve. Holme and Palmstierna (1956 c) selectively labeled the glycogen of E. coli by adding glucose to a nitrogen deficient culture. When the glycogen labeled cells were transferred to a medium containing ammonium chloride but no - 1 4 -carbon source, glycogen was degraded and some protein was synthesized. When the glycogen concentration reached approximately 75 per cent of i t s o r i g i n a l value, the l a b e l i n the polysaccharide had disappeared i n d i c a t i n g that the glucose residues l a s t incorporated into glycogen were the f i r s t to be removed during carbon starvation. The studies with labeled glycogen indicated that the glycogen i n nitrogen d e f i c i e n t cultures did not undergo degradation simultaneously with synthesis. Washed suspensions of E. c o l i harvested from the exponential phase of growth, contained 5-6 per cent of t h e i r dry weight as carbohydrate. After aeration for one hour the t o t a l carbohydrate content was reduced by approximately one-h a l f and the glycogen content was n e g l i g i b l e (Dawes and Ribbons, 1$62 a ) . C e l l s harvested during the stationary phase from a nitrogen d e f i c i e n t medium had a carbohydrate content of 17-20 per cent of t h e i r dry weight and the carbo-hydrate content decreased markedly during aeration. I t was concluded by Dawes and Ribbons (1962 b) that glycogen serves as a primary endogenous substrate i n E. c o l i . In starving c e l l s of Saccharomyces cerevisiae, the CO2 evolution and O2 consumption decreased with time, however, the R.Q. value remained at 1.0. The addition of exogenous substrates at various times during starvation resulted i n high rates of r e s p i r a t i o n and indicated that there was no decline i n enzymatic a c t i v i t y during starvation. The rate -15-of endogenous respiration, therefore, was concluded to be limited by the concentration of endogenous substrate which was believed to be carbohydrate in nature (Stier and Stannard, 1936). During the fermentation of glucose by yeast, added ammonia reduced trehalose and glycogen formation whereas, the absence of ammonia caused the accumulation of both compounds within the cells (Trevelyn and Harrison, 1956)• Chester (1959) observed a decrease in trehalose and glycogen reserves in S. cerevisiae under both aerobic and anaerobic conditions of starvation. The degradation of endogenous substrates under anaerobic conditions led to the accumulation of ethanol. Aerobically, ethanol accumulated briefly and then disappeared. Later work showed that yeast grown anaerobically for 4-8 hours, with an excess of carbon and energy supply, contained 40 per cent of their dry weight as trehalose and glycogen. Aerobically grown cells, under otherwise identical conditions, contained only 10 per cent of their dry weight as carbohydrate. Cells grown aerobically had a very slow rate of endogenous respiration compared to cells grown anaerobically and the difference in endogenous respiration coincided with the difference in concentration of carbohydrate reserves (Chester, 1963). laton (i960) has indicated that three endogenous substrates exist in S. cerevisiae—two distinct glycogen pools and a pool of the non-reducing disaccharide trehalose. -16-One pool of glycogen has a d e f i n i t e requirement for oxygen for degradation while the other glycogen pool may be degraded either a e r o b i c a l l y or anaerobically. The trehalose pool was found to decrease only under anaerobic conditions. Panck (1963) , however, found that the trehalose which accumulated i n resting c e l l s of baker's yeast was ra p i d l y consumed when c e l l s were reincubated i n a growth medium. Trehalose u t i l i z a t i o n occurred while c e l l s were i n the log phase regardless of the nature of the exogenous carbon source. The disaccharide pool did not give r i s e to other storage carbohydrates. Panck considered the disaccharide to be used only c a t a b o l i c a l l y , however, no degradation took place during starvation. I t was suggested that trehalose functions as an energy reservoir for the energy requiring reactions preceding the d i v i s i o n of yeast c e l l s . The accumulation of polysaccharides i n the presence of excess carbon and energy and t h e i r subsequent degradation i n the absence of carbon and energy also has been shown to occur i n other bacteria such as M. tuberculosis var avium (Chargaff and Moore, 1944), Arthrobacter species (Mulder et a l . , 1962) and Agrobacterium tumefaciens (Madsen, 1963) . As w e l l as poly-(3-hydroxybutyrate and other l i p i d s , polysaccharides appear to serve adequately i n the ro l e of endogenous reserve materials. -17-3 . Polyphosphate Metachromatic or volutin granules, which have a marked affinity for basic dyes, are considered to be a microscopic morphological characteristic of certain groups of micro-organisms. The exact nature of these metachromatic granules is, as yet, unknown, however, the presence of polymers of high energy anhydride linked phosphates has been established. The phosphate polymer has been referred to variously as polyphosphate and metaphosphate. The former indicates an open-chain polymer whereas the latter is indicative of a cyclic polymer differing from polyphosphate only by the removal of a molecule of water (Schmidt, 1 9 5 1 ) . As the phosphate residues in the phosphate polymer found in volutin are numerous and the cyclic structure of only t r i and tetra metaphosphate have been proven, i t is likely that the term polyphosphate is the more accurate (Kornberg, Romberg and Simms, 1 9 5 6 ) . From histochemical studies, Widra ( 1 9 5 9 ) concluded that the volutin granules of Corynebacterium xerose, h.' aerogenes and possibly other microorganisms, contain lipoprotein linked to polyphosphate. In addition, ribonucleic acid (RNA) appears to be linked to both protein and polyphosphate, with linkages probably involving magnesium ions. - 1 8 -Wiame ( 1 9 4 9 ) found two physiologically distinct pools of polyphosphate in S. cerevisiae. One pool could be extracted from the cells with 10 per cent cold trichloroacetic acid (TCA) whereas the other could be extracted from the cells only with 5 per cent hot TCA or alkali. Both types of polyphosphate gave a metachromatic reaction with toluidine blue. The insoluble phosphate polymer could be transformed into orthophosphate rapidly and reversibly. When cells that had accumulated polyphosphate were transferred to a growth medium minus phosphate, nucleic acid synthesis was accompanied first by the disappearance of the insoluble phosphate polymer and then continued at the expense of the soluble polyphosphate. In growing yeast cells the soluble polyphosphate fraction maintained a steady state concentration from generation to generation while the insoluble polyphosphate concentration varied during the growth stages of the organism. It was suggested (Katchman and Fetty, 1955) that the differing metabolic activities of the polyphosphate fractions may be due to the differences in chain length. As polyphosphate forms complexes with mono- and polyvalent cations, it was suggested also that it may function as a growth and division regulator by controlling the availability of essential cations. Bacterium aerogenes. Bacterium cloacae, Bacterium  friedlanderi and Serratia marscens produced volutin in a -19-medium with a low phosphate content when acid production limited growth. Volutin was not produced when the original pH of the medium was maintained (Duguid, Smith and Wilkinson, 1954). Later investigations illustrated that volutin formation was greatly influenced by the limiting nutrient in the growth medium. Volutin was produced when growth was limited by exhaustion of nitrogen or sulfur but was not produced i f carbon, phosphate or potassium were limiting. Accumulation of volutin was inhibited by azide and dinitrophenol which act by interfering with synthesis of energy-rich phosphate bonds. Polyphosphate is probably an intermediate metabolite formed in the course of phosphate assimilation and normally used for cell growth so rapidly that it does not accumulate. The compound accumulates only when its synthesis is not affected and when its utilization is hindered by limiting growth (Smith, Wilkinson and Duguid, 1954). When wild type A. aerogenes was induced to form polyphosphate, subsequent transfer of the organism to a complete medium minus phosphate resulted in the synthesis of PiNA and deoxyribonucleic acid (DNA) at the expense of the polyphosphate store. Mutants of A. aerogenes blocked in the accumulation of polyphosphate did not demonstrate any obvious physiological disabilities (Harold and Harold, 1963). Sail, Mudd and Davis (195&) demonstrated the deposition of polyphosphate in resting cells of - 2 0 -Corynebacterium diphtherlae i n the presence of various oxidizable t r i c a r b o x y l i c acid cycle compounds. The accumula-t i o n was enhanced by the addition of potassium or ortho-phosphate. S a i l , Mudd and Takagi (1958) found that, i n synchronously dividing c e l l s of C. diphtherlae, polyphosphate accumulated immediately p r i o r to the increase i n c e l l numbers and then decreased following c e l l d i v i s i o n . I t was thought that polyphosphate was used for nucleic acid synthesis and c e l l growth. Harold ( i960) noted the storage of soluble polyphosphate i n wild type and mutants of Neurospora crassa when grown with l i m i t i n g nitrogen or l i m i t i n g amounts of a required amino ac i d . I f growth stopped due to a nitrogen d e f i c i e n c y , RNA was degraded and nucleic acid phosphate as well as exogenous phosphate appeared i n polyphosphate. In view of these observations, Harold suggested that RNA degradation i s probably important i n polyphosphate accumulation. Mycobacterium chelonel and Mycobacterium thamnopheos Incorporated p32 -inorganic phosphate into polyphosphate when grown i n the presence of the radioactive isotope and on transfer to a new growth medium, the la b e l appeared i n RNA, Stored polyphosphate, therefore, could be used for nucleic acid synthesis i n these organisms. In r e s t i n g c e l l s , both RNA and polyphosphate were depleted. When rest i n g c e l l s were incubated with glucose and potassium, polyphosphate decreased and RNA phosphate increased. I f malate was the exogenous -21-substrate RNA phosphate decreased and polyphosphate increased. Net RNA synthesis and turnover were more active i n the presence of glucose. In the presence of malate, RNA turnover was less active and the adenosine triphosphate (ATP) generated from the oxidation of the exogenous substrate was converted to polyphosphate. Dinitrophenol, which i n h i b i t s oxidative phosphorylation, prevented polyphosphate formation i n the presence of malate (Mudd, Yoshida and Koike, 19^8). These workers expressed the opinion that polyphosphate acts i n the capacity of a store of orthophosphate and phosphate bond energy and probably functions i n a manner analogous to phosphoarginine and phosphocreatine i n animal tissues. An enzyme has been obtained from Corynebacterium  xerosis which hydrolyzes high molecular weight b a c t e r i a l or synthetic polyphosphate to orthophosphate and does not attack or produce short chain phosphate polymers. The enzyme did not catalyze the transfer of orthophosphate from polyphosphate to adenosine diphosphate (ADP) to form ATP and did not synthesize polyphosphate from orthophosphate i n the presence of ATP or other nucleotides. A l l metal ions tested i n h i b i t e d the enzymatic hydrolysis of polyphosphate whereas ethylene-diaminetetraacetate (EDTA), i n low concentrations, stimulated the reaction. During phosphate s t a r v a t i o n , P^ 2 from P^2-polyphosphate ra p i d l y appeared i n most phosphate fractions of the c e l l . I t was suggested that polyphosphate may be simply -22 a source of phosphate which accumulates when the c e l l s are given an excess of inorganic phosphate and energy, and growth i s l i m i t e d hy a nitrogen or other deficiency. Muhammed, Rodgers and Hughes (1959) concluded that polyphosphate i s not necessarily an energy store which can phosphorylate other compounds i n the absence of ATP. The green su l f u r bacterium Chlorobium t h i o s u l f a -tophilum deposited large metachromatic granules of poly-phosphate when grown with a normal concentration of phosphate (Hughes, Conti and F u l l e r , 1963) . In c e l l - f r e e extracts, an ADP dependent, l i g h t independent release of orthphosphate from polyphosphate occurred. I f c e l l s with the metachromatic granules were transferred to a phosphate-free medium, growth ensued and the polyphosphate reserve disappeared. Afte r successive transfers through phosphate d e f i c i e n t media, c e l l -free extracts of the organism were unable to release phosphate from added synthetic polyphosphate. I t was suggested that phosphate was released by the following mechanism. (polyphosphate) n + ADP >ATP + (polyphosphate) n-l (1) ATP >ADP + P i (2) These investigators did not f e e l that i t was clear whether polyphosphate i s a phosphagen or merely a phosphate store which c e l l s accumulate under abnormal conditions where growth i s i n h i b i t e d but energy production proceeds. 23-An enzyme that synthesizes long chain lengths of polyphosphate from a polyphosphate primer and ATP has been isolated from E. coli. The enzyme catalyzes the following reaction XATP + ( P 0 3 ~ ) n > XADP + (P03")n+x (3) primer Only the terminal phosphate of ATP Is added to the primer and ADP inhibits the reaction (Kornberg et al., 1956). Later the enzyme reaction was found to be reversible and ATP was synthesized from polyphosphate in the presence of ADP. ( p o 3~)n + nADP * * nATP (4) The terminal phosphate of ATP was derived solely from the phosphate polymer. If reaction (4) was coupled with hexokinase, p 3 2 from labeled polyphosphate could be recovered quantitatively from glucose-6-phosphate (Kornberg, 1957). Winder and Denneny (1957) demonstrated that cell-free extracts of Mycobacterium smegmatis contained an inorganic polyphosphatase which was stimulated by magnesium ions. Another enzyme which was termed "polyphosphate-AMP-phosphotransferase" and synthesized ATP from AMP and inorganic polyphosphate, was also present in the cell-free extracts. Using AMP and polyphosphate, the cell-free extract was able to phosphorylate glycerol. So two systems for degrading polyphosphate appear to be present in the mycobacteria. 24-In 1962, Nishi established the presence of poly-phosphate, mainly in an acid insoluble form, in dormant spores of Aspergillus niger. Polyphosphate and phospholipid were found to play an important role in the initiation of spore germination. The diminution of both phosphate compounds was observed during the initial stages of germination and the two compounds were considered to act in the capacity of phosphagens. Harold (1962) noted that the accumulation of ATP within the mycelia of N. crassa preceded polyphosphate synthesis in phosphate starved mycelia, thus supporting the view that ATP is the metabolic precursor of polyphosphate. When mycelia were suspended in a medium deficient in phosphate, the previously accumulated polyphosphate was degraded and net synthesis of nucleic acids and phospholipids occurred. The direct formation of ATP from polyphosphate could not be demonstrated. Harold has suggested that while ATP generated either by fermentation or respiration is the likely immediate precursor of polyphosphate, the formation of ATP seems to be excluded as the major route of polyphosphate utilization in N. crassa. A more likely pathway for degradation of the polymer appears to be a stepwise hydrolytic cleavage with the ultimate formation of orthophosphate. The synthesis of polyphosphate under conditions of excess carbon and energy has been established. The function - 2 5 -of the polymer as a phosphate reserve, which may subsequently be utilized for nucleic acid and phospholipid synthesis under conditions of phosphate starvation, has been demonstrated. Adenosine triphosphate has been implicated as the precursor to polyphosphate formation and the concept has met with general approval. However, the role of polyphosphate as an energy reserve or phosphagen is, as yet, controversial. The formation of ATP from polyphosphate in cell-free extracts and in isolated enzyme systems from a limited number of micro-organisms , lends considerable support to the phosphagen theory. The fact that some microorganisms apparently support only a hydrolytic cleavage of polyphosphate to orthophosphate suggests that the polymer may not serve the dual function of a phosphate and an energy store. Further work is required in this area to establish conclusively the ability of volutin to supply energy to cells respiring endogenously. 4. Sulfur A photosynthetic purple sulfur bacterium of the Thiorhodaceae species produced hydrogen sulfide, carbon dioxide and acetic acid from endogenous fermentation when previously grown with thiosulfate. The origin of the hydrogen sulfide was suggested by Hendly (1955) as being elemental sulfur which had been enzymatically reduced in a reaction coupled with the oxidation of reserve organic materials. The addition of colloidal sulfur to cells without - 2 6 -an internal sulfur store stimulated the endogenous production of carbon dioxide and acetic acid. Hydrogen sulfide was not liberated endogenously in the absence of stored sulfur. The endogenous production of carbon dioxide from baker's yeast underwent a 10 fold stimulation in the presence of added colloidal sulfur. Apparently the endogenous reserves of sulfur can support a limited respiration with sulfur rather than oxygen serving as the oxidant. In the absence of sulfur H+ acts as the electron acceptor and H2 is evolved. Hendly did not examine the nature of the carbonaceous material oxidized during endogenous respiration. In this organism, the sulfur store does not act directly as the endogenous energy substrate but rather it acts indirectly by controlling the rate of endogenous respiration and hence the rate at which endogenously produced energy becomes available to the cell. III. Biochemical Differentiation 1. Induced enzyme synthesis The ability of resting cells to synthesize induced enzymes prior to the degradation of an exogenous substrate is well known. Halvorson and Spiegelman (1952) concluded that the substrate induced enzyme formation in non-growing S. cerevisiae involved the utilization of internal free amino - 2 7 -acids. S. eerevisiae, induced with maltose in the absence of exogenous nitrogen, showed a decrease in pool amino acids. The rate of maltase synthesis decreased as the amino acid pool decreased. The extent of depletion of the amino acid pool was greater than the decrease in maltase synthesis suggesting that during... nitrogen starvation intracellular protein was broken down to replenish the depleted amino acid pool (Halvorson and Spiegelman, 1 9 5 3 ) * Eisenstadt and Klein ( 1 9 5 9 ) resuspended washed cells of P. saccharophila in buffer containing starch and s35 and also in a complete medium with The concentration of amylase in the supernatant fluid was followed for four hours and i t was found that the total amount of enzyme released was the same under both conditions. The rate of enzyme release, however, was greater in the complete medium. Fo appeared in the amylase produced by cells incubated with buffer and starch whereas the amylase produced by cells in the complete medium was labeled. Eisenstadt and Klein concluded that the cells incubated in buffer, used endogenous sulfur sources exclusively during the experiment whereas those endogenous sources were not drawn upon during incubation in a complete medium. Chloramphenicol completely inhibted induction and a two hour period of starvation prior to induction prevented subsequent amylase formation in the absence of an exogenous nitrogen supply. These workers suggested that the organism contains endogenous reserves 2 8 -such as amino acids and peptides that are available for general protein synthesis. During growth these reserves flow into cellular material at a rapid rate and are replenished from the external medium. No growth occurred with the cells incubated in buffer, therefore, the endogenous pool was not exhausted by synthesis of cellular protein and was available for synthesis of induced enzymes. This concept did not exclude turnover where the rate of degradation may have exceeded the rate of resynthesis. The turnover of protein required for amylase production from cells resuspended in buffer was calculated (Schiff, Eisenstadt and Klein, 1959) and found to be extremely high. For the four hour experimental period approximately 85 per cent of the cellular protein would have been involved in turnover. Klein (1963) found the actual rate of protein turnover in the organism to be between 5 an<* 7 per cent and concluded that some pre-existing endogenous material was used for amylase synthesis. 2 . Sporulation Under starvation conditions, cells of the slime mold Dictyostelium discoideum aggregate to form multicellular organisms and undergo an elaborate sporulation process. This multicellular differentiation is not compatible with the presence of an exogenous energy supply. Initially the pseudoplasmodium forms are unable to produce cellulose from endogenous material but after approximately fifteen hours of - 2 9 -starvation they begin to synthesize the polysaccharide very r a p i d l y at the culmination stage of d i f f e r e n t i a t i o n . This change i n a b i l i t y to use endogenous materials indicates a marked change i n the biosynthetie pathways of the organism, undoubtedly involving the synthesis of new enzyme systems. There i s a sequential u t i l i z a t i o n of nitrogenous endogenous materials during the course of d i f f e r e n t i a t i o n i n the slime mold. The amino acid pool drops to approximately 30 per cent of i t s i n i t i a l value, the ethanol soluble protein to 60 per cent and the ethanol insoluble protein to about 80 per cent of i t s i n i t i a l value. The organism depends on amino acids as an energy source and as building blocks for enzyme synthesis (Wright and Anderson, i 9 6 0 ; Wright, 1963) . Sporulation i n microorganisms appears to be induced either by substances that accumulate i n the growth medium or by repressors of sporulation that disappear from the medium and i s not an obligatory consequence of p r o l i f i c vegetative development. P r i o r to and during sporulation extensive modifications i n the biosynthetie capacity of the c e l l take place (Halvorson, 1962) . When harvested during vegetative growth, B. cereus lacks a functional t r i c a r b o x y l i c acid c y c l e . The enzymes required for the completion of th i s cycle are synthesized during the t r a n s i t i o n from growth to sporulation (Hanson, Srinivason and Halvorson, 1963). -30-The depletion of poly-(i-hydroxybutyrate reserves during sporogenesis indicates that cellular material as well as exogenous nutrients are essential parts of the energy and nitrogen sources required for the biochemical differentiation (Murrell, 1 9 6 D . 3. Turnover From tracer studies Sehoenheimer and Rittenberg (1940) concluded that a l l biological substances in the animal body were constantly undergoing degradation and resynthesis in the presence or absence of growth. This process of turnover has been examined in bacterial systems. Borek, Ponticorvo and Rittenberg (1958) observed that protein was continually degraded and resynthesized during methionine starvation of a methionine requiring mutant of E. coli. Mandelstam (1958) a, b) labeled a leucine requiring mutant of E. coli with c l 4 _ i e u c i n e ? depleted the free amino acid pool and observed that during nitrogen starvation amino acids, including c l 4 _ i e u c i n e appeared in the amino acid pool, and therefore must have arisen from protein degradation. The rate of turnover in growing and non-growing E. coli was investigated by using amino acid requiring mutants previously grown with the labeled amino acid. The labeled cells were transferred to a complete medium and to a complete medium minus both the required amino acid and ammonium ions. - 3 1 -Mandelstam noted a negligible rate of turnover of protein in growing cells and a balanced degradation and resynthesis of protein in non-growing cells at the rate of approximately 4-5 per cent/hour. The addition of ammonium ions to non-growing cells resulted in a substantial decrease in protein degradation. Mandelstam suggested that the increase in protein turnover in non-growing cells keeps an adequate supply of amino acids in the amino acid pool which then is available for the synthesis of new enzymes that may be required. Ribosomal and soluble protein were degraded at approximately the same rate in nitrogen starved E. coli. Ribosomal RNA was also degraded at about the same rate as protein but there was a lower rate of resynthesis (Mandelstam and Halvorson, i 9 6 0 ). In stationary phase populations of E. coli, RNA was actively degraded and the instability of RNA, as protein, appeared to be a consequence of cessation of growth (Mandelstam, i 9 6 0 ) . Brba (1959) followed protein turnover in growing and non-growing cells of B. cereus in a manner analogous to that used by Mandelstam. She found a balanced degradation and resynthesis in non-growing cells of approximately 7 per cent/ hour. Less than one per cent of the total catalase of the non-growing cells was found in the suspending fluid at the termination of the two hour experiment, thus demonstrating that the observed protein degradation was not a result of cell lysis. - 3 2 -The rate of protein degradation in S_. cerevisiae was at least twenty-three times greater in resting cells than in growing cells. There was a loss of purines from RNA of non-growing cells but there was no appreciable quantity of nucleic acid degradation in growing cells (Halvorson, 1958 a). Cells subjected to prolonged nitrogen starvation were able to derive their nitrogen for induced enzyme synthesis from ammonia formed from adenine or guanine thus demonstrating a flow of nitrogen from nucleic acid to protein (Halvorson, .1958 b). The turnover of protein in resting cells ensures an adequate supply of pool amino acids which may be used for induced enzyme formation. This allows microorganisms to adjust to changing environmental conditions and possibly to prolong survival. The turnover of RNA provides the cells with nucleic acid residues necessary for RNA synthesis—a likely prerequisite to induced enzyme formation. IV. Nitrogenous Substrates of Endogenous Respiration Lipid, polysaccharide and volutin appear to have become generally accepted as the "normal" endogenous sub-strates of microorganisms. This implies firstly, that micro-organisms "normally" store such a reserve product and secondly that i f an organism does not store one of these reserve compounds or, if an organism is grown in a carbon and energy limiting medium, its ability to survive under starvation - 3 3 -conditions would be diminished considerably. There are indications i n the l i t e r a t u r e that many microorganisms u t i l i z e nitrogenous materials exclusively or i n conjunction with carbonaceous materials while r e s p i r i n g endogenously. Bernheim, De Turk and Pope (1953) found that suspensions of M. tuberculosis produced ammonia when r e s p i r i n g endogenously. During endogenous r e s p i r a t i o n of Sarcina lutea, ammonia, was lib e r a t e d and a s i g n i f i c a n t decrease i n carbo-hydrate or l i p i d could not be detected. As the l e v e l of amino acids i n the pool decreased, the decreased to a n e g l i g i b l e l e v e l . Certain amino acids were implicated as the endogenous substrate (Dawes and Holmes, 1958). More recently the endogenous r e s p i r a t i o n of S. lutea was followed over an extended period of time (Burleigh, Dawes and Ribbons, 1963) . During the i n i t i a l period of starvation, the amino acid pool was depleted to one-half of i t s o r i g i n a l concentration, then a slower depletion of amino acids occurred up to ten hours. Ammonia evolution correlated with the decrease i n the amino acid pool for the f i r s t ten hours and a f t e r that time i t became evident that other substrates were also y i e l d i n g ammonia. Throughout the starvation period RNA declined s t e a d i l y and 260 mu absorbing material and pentose were released into the suspending f l u i d . There was no decrease i n c e l l numbers u n t i l a f t e r a 4-5 hour period of starvation when a rapid loss of v i a b i l i t y ensued. The presence of assimilated -34-polysaccharide in the cells did not retard the release of ammonia. The evolution of ammonia and the ratio of oxygen consumption to ammonia evolution in endogenously respiring cells implicated amino acids and protein as the endogenous substrates of Pseudomonas aeruginosa. The endogenously produced ammonia was reassimilated by the cells in the presence of exogenous glucose. No decrease in viable cell count or total carbohydrate was observed during the period of starvation (Warren, Ells and Campbell, i 9 6 0 ). It had been shown previously, that prolonged aeration had little effect in reducing the rate of endogenous respiration in P. aeruginosa (Norris, Campbell and Ney, 1949) . This work was extended to cover a variety of microorganisms and it was found that Pseudomonas fluorescens, Achromobacter species, E. coli, Bacillus subtilis, S. cerevisiae and Streptococcus  faecalls produced ammonia during endogenous respiration. With the exception of S. faecalls all the organisms assimilated the endogenously produced ammonia when exogenous glucose was added. The ratio of oxygen consumption to ammonia evolution varied considerably and indicated that some organisms simultaneously oxidized nitrogenous and non-nitrogenous compounds. The low O2SNH3 found with B. subtilis and S. faecalis suggested that if amino acids were oxidized, they were either incompletely oxidized or basic amino acids were the substrate (Gronlund and Campbell, I 9 6 I ) . -35-^ Clifton and Sobek (1961) noted that ammonia was released daring the endogenous respiration of B. cereus. Cells grown on nutrient agar showed a decrease in ammonia evolution relative to oxygen uptake with time. Cells grown on nutrient agar supplemented with one per cent glucose showed an increase in ammonia evolution relative to oxygen consumption with time. Ramsey (1962) demonstrated that the carbohydrate content of agar grown Staphylococcus aureus did not decrease during endogenous respiration. Ammonia was evolved and the decrease in Cl4 labeled glutamic acid from the amino acid pool could not account for the total observed oxygen uptake. This suggested that either amino acids other than pool glutamic acid were oxidized or that protein was degraded. If the organism was grown in a medium with an increased amino acid content, the rate of endogenous respiration was high. This is in opposition to the "usual" situation where a high carbo-hydrate content in the growth medium results in a high rate of endogenous activity. The mold Aspergillus sojae appeared to use different endogenous substrates depending on the conditions under which the organism was grown. When the mold was grown in a medium with a high carbon:nitrogen value, carbohydrate or lipid apparently was the main substrate of endogenous respiration during the initial stage. Nitrogenous materials were utilized -36-later. If the organism was grown in a medium with a low carbon:nitrogen value pool amino acids, protein and nucleic acids were the major endogenous substrates (Mizunuma, 1963). Ammonia was released from the mold Merulius lacrymans during endogenous respiration (Goksoyr, i960) and also from the marine bacterium Flavobacterium species (Ayers, I962). Cosgrove (1959) noted the evolution of ammonia from a trypanosomid flagellate and concluded that the endogenous substrate was nitrogenous in nature. Ribbons and Dawes (1963) compared the endogenous respiration of E. coli when grown in different media and when harvested from different growth phases. If growth conditions supported glycogen accumulation, no ammonia was evolved from the cells until the glycogen store had been depleted and i t was concluded that the presence of glycogen spared the nitrogenous reserves. Cells that did not contain glycogen immediately released ammonia on starvation. The endogenous Q02 values of E. coli correlated with the glycogen content of the cells. Nitrogenous cellular constituents are used as endogenous substrates by a number of microorganisms. Nitrogenous reserves appear to be used exclusively by some organisms, whereas in others, they appear to be used only when non-nitrogenous reserves have been depleted. Ammonia evolution has been the criterion used to measure degradation -37-of nitrogenous constituents and this has an obvious dis-advantage when studying organisms that oxidize lipid or carbohydrate reserves and are able to assimilate ammonia. v* The Influence of Exogenous Substrates on Endogenous  Respiration The status of endogenous metabolism in the presence of an exogenous substrate has long been the concern of those primarily interested in employing manometry in studies of carbohydrate metabolism. Consequently most observations in this area have been of secondary interest to the investigators While studying oxidative assimilation in the algae Prototheca zopfii, Barker (1936) used varying concentrations of exogenous substrates with a fixed concentration of cells. By comparing the percentage of theoretical oxygen uptake values with and without endogenous values subtracted, Barker was able to conclude that in the presence of easily oxidizable exogenous substrates the endogenous respiration may be completely suppressed. However, if the exogenous substrate is oxidized relatively slowly the endogenous respiration would not be negligible and would have to be corrected for. Norris, Campbell and Ney (194-9) used an approach similar to Barker's to follow the effect of exogenous sub-strates on endogenous respiration in P. aeruginosa. Norris et al. varied substrate concentrations with a standard cell -38-concentration and also used a standard substrate concentration with varying cell concentrations. If endogenous oxygen uptake values were not subtracted, low substrate concentrations showed greater than 100 per cent of theoretical oxygen uptake. When endogenous values were subtracted from tests with varying substrate concentration, approximately the same percentage of theoretical oxygen consumption was obtained. With increasing cell concentrations, the subtraction of endogenous oxygen uptake again gave approximately the same percentage of theoretical. On the basis of these experiments Norris et al. concluded that the endogenous respiration of P. aeruginosa was unaffected by the presence of oxidizable substrates. Wiame and Doudoroff ( 195D followed the assimilation of C14" labeled oxidizable exogenous substrates by P. saccharonhila and concluded that the presence of substrates largely inhibited endogenous respiration unless the assimila-tion of substrates was prevented by dinitrophenol. P. 14 14 fluorescens was grown on C -glucose and C 02 evolution was measured in the presence and absence of exogenous substrates (Gibbs and Wood, 1952) . The rate of C 1 4 0 2 evolution was the same or slightly accelerated in the presence of exogenous substrates. During oxidative assimilation studies with Hydrogenomonas facilis, i t was observed that the oxidation of acetate, lactate, pyruvate, fumarate and glucose did not suppress endogenous respiration (Marino and Clifton, 1955)• - 3 9 -Moses and Syrett (1955) labeled organisms with C14" and followed production from cells incubated in the presence and absence of exogenous substrates. Glucose suppressed the 14 C O2 evolution by approximately 10 per cent in the green ^Sae Chlorella vulgaris but stimulated endogenous respiration in baker's yeast. Succinate and acetate caused a suppression of 10 per cent and glucose had no effect on the C O2 evolution from Zygorhynehus moelleri. The rate of C 1 402 release from C 1 4 labeled Penicillium  chrysogenum was not measurably affected by glucose whereas acetate almost completely suppressed endogenous respiration (Blumenthal, Koffler and Goldschmidt, 1 9 5 2 ) . Vegetative cells of P. chrysogenum were Incubated with increasing concentrations of exogenous substrates and when endogenous oxygen uptake values were corrected for, a f a i r l y constant value for per-centage of theoretical oxygen consumption was obtained. The 14 evolution of C 0 2 from labeled cells Indicated that neither glucose nor acetate affected the endogenous respiration of glucose grown c e l l s . Acetate, however, inhibited the endogenous respiration of acetate grown cel l s and glucose had no effect (Blumenthal, Koffler and Heath, 1957). Midwinter and Batt ( i 9 6 0 ) labeled Nocardia corallina with C ^ "-propionate, -acetate or -glucosej washed the cells and incubated them with and without exogenous substrates. In a l l cases C 1 4 0 2 evolution from the labeled cells was increased -40-in the presence of substrate. Using the procedure of varying substrate concentration with a standard cell concentration, Wilner and Clifton (1954) concluded that glucose, succinate, fumarate and pyruvate did not suppress endogenous respiration of B. subtllis. The utilization of glucose appeared to have little effect on the endogenous respiration of B. megaterium as measured by C-L4'02 evolution from labeled cells. Pyruvate and acetate caused a slight depression of endogenous respiration. From these results Clifton (1963) arrived at the general conclusion that exogenous substrates only slightly repress endogenous respiration. From oxidative assimilation studies with B. cereus, Clifton and Sobek ( I 9 6 D suggested that a 16-40 per cent suppression of endogenous respiration occurs in the presence of exogenous glucose. Clifton (1963) observed that exogenous glucose slightly increased endogenous respiration in E. coli whereas Ribbons and Dawes (I963) noted that exogenous glucose caused a partial depression of the endogenous respiration of the glycogen reserves. It is not possible to draw any general conclusions regarding the influence of exogenous substrates on endogenous respiration from the available literature. Reports are often contradictory and conclusions drawn by several investigators have not been substantiated by adequate data, in some instances no supporting data has been offered. Considerable work is -41-required in this area to clarify the interrelationships between exogenous and endogenous substrates. -42-MATERIALS AND METHODS I. Cultural Conditions and Radioactive Labeling of the  Organism 1. Cultural conditions Pseudomonas aeruginosa ATCC 9027, the organism used throughout these studies, was maintained in a glucose ammonium salts medium (Warren et al., i 9 6 0 ). Stock cultures were stored at 6 C after a twenty-four hour growth period in the liquid medium. Periodically, the stock cultures were sub-cultured onto Plate Count agar and examined for both colonial and cellular morphology. Cells required for experimental procedures were obtained by inoculating Roux flasks, containing 100 ml of the glucose-ammonium salts medium, with a 1 per cent inoculum from a fresh twenty-four hour stock culture transfer. Incubation was carried out at 30 C for twenty hours and cells were harvested by centrifugation at 5»000 x g for 10 min at 6 C. For whole cell studies, the organisms were washed twice with 0.85$ sodium chloride (pH 7.4) and resuspended in tris (hydroxymethyl)-aminomethane (tris) buffer at the required cell concentration. -43-2 . Radioactive labeling of cells Cells were labeled with C-1-4 by adding either glucose-U-C14 (90 uc/100 ml), uracil -2-C 1 4 (70 uc/100 ml, 4 . 8 6 or 9.1 rag/100 ml), or proline-U-C14 (70 uc/100 ml, with carrier L-proline to give a concentration of 5 mg/ml) to growing cultures at twelve hours and continuing incubation until twenty hours. After harvesting, the cells used for whole cell studies were washed three times with 0.85$ sodium chloride (pH 7 . 4 ) . For sucrose gradient studies, ribosomal RNA was labeled with uracil -2-C 1 4 by the "shift up" technique in which the RNA of the cell increases rapidly during the initial incubation period (Kjeldgaard, I 9 6 D . The following sterile solutions were added to twenty hour cells in 100 ml of glucose-ammonium salts medium, 2 ml of 20$ glucose, 2 ml of 20$ yeast extract and 30 uc of uracil-2-C 1 4. After incubation for 90 min at 30 C, the cells were harvested and washed. II. Manometric Procedures 1. Oxygen uptake The oxygen consumption of cells respiring endogenously and respiring in the presence of exogenous substrates was measured at 3° C by means of a conventional Warburg respirometer. A typical endogenous reaction mixture was as follows, 2 . 0 ml of cells in 0 . 0 5 M tris buffer (pH 7 .4) at -44-1 5 times the growth concentration (approximately 7.5 mg dry weight/ml), 1.0 ml d i s t i l l e d water and 0.15 ml of 20$ KOH i n the center w e l l . The oxidation of exogenous substrates was examined according to the following example. Endogenous Substrate C e l l s i n 0.05 M t r i s buffer (pH 7 . 4 ) , 5 mg dry weight/ml 1.00 ml 1.00 ml 0.05 M t r i s buffer (pH 7 . 4 ) 1.00 ml 1.00 ml Substrate ( 2 5 um/ml) - 0.20 ml D i s t i l l e d water 1.00 ml 0.80 ml KOH (center well) 0.15 ml 0.15 ml 2. Evolution of radioactive carbon dioxide Radioactive carbon dioxide was released from the reaction mixture i n Warburg vessels by the addition of 0.2 ml of 1.5 N HC1 from a side arm and was c o l l e c t e d i n the center well which contained 20$ KOH and a folded s t r i p of f i l t e r paper. Incubation of the reaction mixtures was continued f o r 20 min and at the end of that time the KOH and f i l t e r paper were removed. The center well was washed three times with d i s t i l l e d water and the pooled radioactive material was d i l u t e d to an appropriate volume with d i s t i l l e d water and plated d i r e c t l y . -45-III. Analytical Procedures 1. Protein Protein concentrations were measured according to the method of Lowry et al. ( 1 9 5 D > which depends upon the reduction of the phosphomolybdic-phosphotungstic reagent by copper treated proteins in an alkaline solution. The sensitivity of the method is 25-500 ug of protein/ml and standard curves were prepared from crystalline egg albumin. 2. Ammonia The presence of ammonia in Warburg supernatant fluids was determined by the Conway microdiffusion technique (Conway, 1 9 5 0 ) . The particular technique employed was adequate for measuring ammonia concentrations between 0-26 ug. 3 . Deoxyribonucleic acid The diphenylamine procedure of Schneider ( 1 9 5 7 ) ^as used for DM determinations. The standard curves were prepared from purified DNA-1- and the range of sensitivity of the assay was 25-500 ng DNA/ml. •^Purified calf thymus DNA was supplied by Dr. N. Tomlinson, Pacific Fisheries Technological Station. —46— 4. Ribonucleic acid Determinations of RNA concentrations were carr i e d out according to a modification of the method described by Schneider ( 1 9 5 7 ) ? which measures purine bound pentose. The reagents used were 0.2$ FeCl^^R^O i n 100 ml of concentrated HC1 and 1.0 gm of o r c i n o l i n 10 ml of 95$ ethanol. The reagents were combined immediately before use and 1.5 ml of the combined reagent was added to 1.5 ml of standard or test solutions. The tube contents were mixed, the tubes were covered, heated i n a b o i l i n g water bath for 4 5 min and cooled. The o p t i c a l density at 6 6 5 mp. was recorded with a Beckman model B spectrophotometer. Standard curves were prepared with p u r i f i e d RNA2 and the assay was sensitive between 10-100 ug RNA/ml. As DNA gives a s i g n i f i c a n t r e a c t i o n with the o r c i n o l reagent, DNA standard curves were prepared with each RNA assay. A f t e r the DNA concentration had been determined for each test sample by the diphenylamine reagent, the interference i n the o r c i n o l reaction due to the presence of DNA could be corrected f o r . 5. Inorganic phosphate Inorganic phosphate (Pi) concentrations were measured by the method of King ( 1 9 3 2 ) . 2 P u r i f i e d RNA was supplied by Dr. G. Tener, Department of Biochemistry, University of B r i t i s h Columbia. -4?-6 . Polyamines The polyamine content of starved and unstarved cells was determined by a procedure of Rosenthal and Tabor (1956) which involved the extration of amines from an alkaline salt mixture into t-butyl alcohol followed by color development with 2,4-dinitrofluorobenzene. Standard curves were prepared from spermine at concentrations between 2-20 ug/ml. 7. Ultraviolet spectra All ultraviolet (UV) spectra were determined with either a Beckman model DU spectrophotometer or a Bausch and Lomb double beam recording spectrophotometer. 8. Paper electrophoresis Paper electrophoresis of Warburg supernatant fluids were carried out using Whatman no. 1 paper, formic-acetic buffer (pH 2; Smith, i 9 6 0 ) and a flat plate water-cooled electrophoresis apparatus with a RSCo model 1911 power supply. 9 . Viable cell counts Samples of endogenously respiring cell suspensions were removed from a large Warburg vessel at specified time intervals and appropriately diluted with either O.O33 M phosphate buffer (pH 7.4) or O.O33 M phosphate buffer with 0 . 8 $ NaCl and 0.1% gelatin. The number of viable cells/ml was determined by the -48-plate count procedure. IV. Preparation of Cell-free Extracts 1. Lysozyme treatment The procedure used was similar to that described by Strasdine ( I 9 6 I ) . Approximately 12 mg dry weight of cells were suspended in 0 . 9 ml of distilled water and 0 . 2 ml of 0 . 1 M tris buffer (pH 8 . 0 ) , 0 . 6 ml of EDTA (20 mg/ml) and 0 .2 ml of a commercial preparation of lysozyme (4 mg/ml) were added to the suspension. The reactants were mixed by means of a magnetic stirrer for 5 min at room temperature. Following the addition of 0.2 ml of 0 . 2 M MgCl2, 0 . 1 ml deoxyribonuclease (DNase; 1 mg/ml) and 0.5 ml of distilled water, the reaction mixture was stirred for 1 min at room temperature. After centrifugation for 10 min at 5 c and at 5>000 x g to precipitate whole cells, the supernatant fluid (cell-free extract) was removed. This method of cell breakage was used during early experiments as it was applicable to small numbers of cells. The procedure, however, was not entirely satisfactory as the percentage of cell breakage was relatively low due to the necessarily short incubation period with lysozyme. 2 . Hughes' press Harvested cells were washed twice with 0.85$ NaCl and once with a 0 .2 mg$ aqueous solution of glutathione. The cell -49 pellet was packed into open-ended pyrex vials with an inside diameter of 15 mm. The vials were sealed tightly with rubber stoppers and the cells were frozen quickly by placing the vials in an ethanol dry-ice bath for 30 min. If not used immediately, the frozen cells were stored at -18 C. The cell-free extracts were prepared by crushing the frozen cells in a Hughes' press (Hughes, 1 9 5 D by applying pressure of approximately 12,000 lbs with a Carver hand-operated hydraulic press. The assembled Hughes' press was stored at -18 C for at least 24 hr prior to preparing the extracts. The frozen cell extract was placed in a teflon Potter homogenizer and a mixture of cold M tris buffer (pH 7.4) and diluent, in a ratio of 1 : 6 . 6 6 , was added to the extract to yield an approximate concentration of 35 mg dry weight of cells/ml. After homogenization, the viscosity of the cell-free extract was decreased by a 60 sec period of treatment in a 10-kc Raytheon sonic oscillator. The suspension was centrifuged at 6 C at 5>000 x g for 10 min and the supernatant fluid (cell-free extract) was removed. The diluent, for cell-free extracts prepared by this technique, was composed of 0 . 0 5 M glycylglycine, 0 . 0 5 M tris and 500 mg# of egg albumin in distilled water. 3 . French pressure cell Cells were suspended in 0 . 0 1 M tris buffer (pH 7.4) at an approximate concentration of 20 mg dry weight of cells/ml - 5 0 -and the cell suspension was added to the chamber of the French pressure cell (Milner, Lawrence and French, 1950) , which previously had been stored at 6 C. The cell-free extract was obtained by passing the material dropwise through the orifice of the pressure cell while the pressure was maintained at 10,000 to 15,000 lbs with a Carver hydraulic press. An appropriate volume of DJTase (l mg/ml) was added to the extract to decrease the viscosity and the preparation was centrifuged to remove unbroken cells. Viable cell counts of the preparations prior to centrifugation indicated that 90-95$ cell breakage was achieved by this procedure. V. Chemical Fractionation of Whole Cells 14 C -labeled and non-labeled cells generally were fractionated according to a modification of the procedure of Roberts et al. (1955) as follows. Cell pellets containing approximately 10 mg dry weight of cells were resuspended in 2 ml of distilled water by means of a Vortex mixer and 2 ml of 10$ cold TCA were added. The material was held in ice for 20 min and then centrifuged for 15 min at 7»500 x g at 6 C. The supernatant fluid (cold TCA soluble fraction) was removed and 4 ml of 75$ acid-ethanol (pH 2 . 5 with H 2 S O 4 ; Clifton and Sobek, 1962) were added to the pellet. The mixture was heated for 30 min at 45 C, cooled, centrifuged and the supernatant fluid (acid-ethanol soluble fraction) was removed. Four ml of 5$ TCA were added to the pellet and the suspension was heated -51-at 90 C for 9 min. After cooling, centrifligation and removal of the supernatant fluid (hot TCA soluble fraction), the pellet was dissolved in 0.1 N NaOH by using low heat (hot TCA insoluble fraction). To establish the cellular distribution of -uracil, a modification of the Schmidt-Thannhauser technique was used to separate RNA and DNA by alkaline hydrolysis of the RNA. A typical fractionation procedure was as follows! cell pellets containing approximately 5 mg dry weight/ml were resuspended in 0.5 ml of distilled water and 0.5 ml of 1 N NaOH was added. The material was incubated at 35.5 C for 17 hr and then neutralized with 3 N HC1 (Hutchison, Downie and Munro, 1962). Trichloroacetic acid was added to bring the final TCA concentration to 5$ and the material was held in ice for 20 min. After centrifugation, the supernatant fluid (RNA) was removed, 2 ml of 5$ TCA was added to the pellet which was subsequently heated at 100 C for 30 min (Roodyn and Handel, i960). Following cooling, centrifugation and removal of the supernatant fluid (DNA), the pellet (hot TCA insoluble fraction) was dissolved in 1 ml of 0.5 N NaOH. VI. Physical Fractionation of Cell-free Extracts Cell-free extracts were prepared by one of the previously mentioned techniques and cell fractions were obtained by a procedure generally similar to that described by - 5 2 -Campbell, Hogg and Strasdine (1962). Centrifugation of cell-free extracts at 25,000 x g for 3 0 min yielded a "membrane" pellet and centrifugation of the 25,000 x g supernatant fluid for 2 hr at 140,000 x g in a Spineo model L preparative ultracentrifuge yielded a "ribosome" pellet. The following fractions, therefore, could be obtained by this procedure: 25,000 x g pellet—"membrane" fraction, 25,000 x g supernatant— soluble cytoplasm plus ribosomes, 140,000 x g pellet— "ribosome" fraction, 140,000 x g supernatant—soluble cytoplasm. , This method for the physical fractionation of cell-free extracts was employed for a variety of experiments such as manometric studies, distribution of radioactivity and purification of ribosomes. Although the centrifugation process was not altered, the suspending fluid for membrane and ribosome pellets and the original suspending fluid for whole cells prior to breakage and differential centrifugation, with particular reference to magnesium ion concentration, were dependent upon the individual system under study. These alterations in conditions will be mentioned in the text. VII. Sucrose Gradients 1. Preparation of gradients The change in the ribosome complement of P. aeruginosa after a 3 period of starvation was followed by means of the sucrose gradient technique. Linear sucrose gradients from 20 to 5$ were prepared by a method analogous to that of B r i t t e n and Roberts (i960). The apparatus used for the formation of the gradients consisted of a Plexiglas chamber divided down the center to produce two compartments. Narrow bent glass tubing led from the bottom of one compartment to a 5 ml Lusteroid centrifuge tube. Twenty per cent sucrose (2.5 ml) was added to the compartment with the outlet and 5% sucrose (2.5 ml) was added to the second compartment. The two compartments were connected by a small U tube containing 5$ sucrose and adequate mixing of the solutions was provided by a magnetic s t i r r e r . This technique r a p i d l y and reproducibly yielded 4.8 ml of a l i n e a r gradient. A 1 ml tuberculin syringe was f i t t e d with a no. 18 hypodermic needle from which the bevel had been removed and the end bent into the shape of a U. The open end of the needle was placed s l i g h t l y below the surface of the sucrose gradient and 0.2 ml of c e l l - f r e e extract (20 mg dry weight/ml) was c a r e f u l l y dispensed from the syringe. The samples were centrifuged at 37,500 RPM for the appropriate length of time i n a SW 39 rotor i n a Spinco model L preparative u l t r a c e n t r i f u g e . Following centrifugation, the Lusteroid tubes were placed i n a s p e c i a l l y constructed holder^ which allowed the bottom of the tube to be punctured with a no. 22 hypodermic needle. The needle was ^The apparatus f o r preparing sucrose gradients and c o l l e c t i n g f r a c t i o n s were made by Dr. R. Stace-Smith, Canada Department of Ag r i c u l t u r e . -54-inserted a distance of approximately 1 mm from the bottom of the tube and the tube contents were dispensed dropwise into 1 ml centrifuge tubes. Approximately 25 equal f r a c t i o n s were collected i n t h i s manner. 2. Preparation of c e l l - f r e e extracts and conditions of centrifugation P r i o r to breakage, 2.8 ml of c e l l s at 20 times the growth concentration were removed from a Warburg vessel and resuspended i n 1.4 ml of t r i s buffer (pH 7.4), a f t e r the Warburg supernatant f l u i d had been removed by centrifugation. C e l l - f r e e extracts were prepared with a French pressure c e l l using c e l l s at approximately 20 mg dry weight/ml. The conditions of centrifugation for sucrose gradients were s i m i l a r to those used by McCarthy, B r i t t e n and Roberts (1962) and are indicated below. Buffer for c e l l resuspensions Centrifugation time and sucrose solutions at 37,500 RPM 0.01 M t r i s buffer (pH 7.4) containing 10~2 M MgClg 90 min 0.01 M t r i s buffer (pH 7.4) containing 10~4 M MgCl2 150 min 0.01 M t r i s buffer (pH 7.4) containing 0.5$ sodium monolauryl sulfate 3&0 min (Duponol) -55-3 . B i s t r i b u t i o n of ribosomal material The d i s t r i b u t i o n of ribosomal material i n the c o l l e c t e d f r a c t i o n s was followed by d i l u t i n g the samples 1:9 with d i s t i l l e d water and measuring the o p t i c a l density at 260 mu with a Beckman model DU spectrophotometer. The d i s t r i b u t i o n of r a d i o a c t i v i t y i n the various f r a c t i o n s was measured as follows: 0.2 ml of 1.4 N perchloric a c i d (PCA) was added to each f r a c t i o n , the tube contents were mixed and the tubes were held i n i c e f o r 20 min. A f t e r centrifugation, the supernatant f l u i d was removed and discarded and 0.05 ml of 0.2 N NaOH was added to the cold PCA insoluble material. The tube contents were mixed by means of a Vortex mixer and also a Pasteur pipette. The a l k a l i n e s o l u t i o n was plated d i r e c t l y onto a s t a i n l e s s s t e e l planchet, the 1 ml centrifuge tubes were rinsed twice with approximately 0.025 ml of d i s t i l l e d water and the washings were added to the planchet. 4. Ultracentrifuge analyses of ribosomal material The sedimentation c o e f f i c i e n t s of the ribosomal material found i n the major peaks of the sucrose gradients were determined. 4 - One f r a c t i o n , containing the greatest amount of 260 mu absorbing material for a single peak, was 4The u l t r a c e n t r i f u g a t i o n procedures and the c a l c u l a t i o n of sedimentation c o e f f i c i e n t s were carr i e d out by Dr. M.E. Reichmann, Canada Department of Ag r i c u l t u r e . dialyzed f o r 5^  hr at 6 C against 0.01 M t r i s buffer (pH 7.4) containing either 0.01 M or 10~ 4 M MgCl 2. Analyses of the dialyzed f r a c t i o n s were carried out with the a i d of a Spinco model E ultracentrifuge using both S c h l i e r n and UV optics. The UV negatives were scanned with a Spinco A n a l y t r o l model R A . VIII. Ribosome Degradation 1. Assay for the presence of a degradative enzyme C e l l - f r e e extracts were prepared from 20 hr c e l l s by means of a Hughes' press and the extract was d i l u t e d with 0.05 M t r i s buffer (pH 7.4). The "ribosome" f r a c t i o n was obtained by the previously described d i f f e r e n t i a l centrifuga-t i o n procedure and the "ribosome" p e l l e t was suspended i n 0.05 M t r i s buffer (pH 7.4). at 150 times the growth concentration by means of a t e f l o n Potter homogenizer. Reaction mixtures were prepared i n duplicate as follows: 0.3 ml of the ribosome suspension was added to 0.1 ml of 0.5 M t r i s buffer (pH 7.4) and the volume was adjusted to 1.0 ml with d i s t i l l e d water. Samples of the ribosome suspension were also incubated i n the presence of 0.06 M Mg++, 0.06 M P04= and EDTA at a concentration of 1 mg/ml of reaction mixture. Enzymatic a c t i v i t y was stopped at zero time and a f t e r a 60 min incubation period at 30 C by the addition of 1 ml of 1.4 N PCA. Reaction mixtures were held i n ice for 10 min, centrifuged and the increase i n UV absorbing material -57-in the supernatant fluid, over the zero time value, was measured with a Beekman model DU spectrophotometer. 2. Identification of the enzyme Preliminary evidence indicated that the enzyme associated with the ribosomal fraction and responsible for the initiation of its degradation was polynucleotide phosphorylase (Grunberg-Manago and Ochoa, 1955)* To differentiate conclusively between ribonuclease (RNase) and polynucleotide phosphorylase, an assay similar to that described by Littauer and Kornberg (1957) was used. If polynucleotide phosphorylase was the enzyme involved, charcoal absorbable P^2 would be obtained as a result of the reaction RNA + nP 3 20 4 N * nXPP32 (5) where XPP represents purine and pyrimidine nucleoside diphosphates. If RNase was the degradative enzyme, however, no charcoal absorbable P32 would be recovered. Harvested, washed 20 hr cells were resuspended in 0.05 M tris buffer (pH 7.4) at 40 times the growth concentration and a cell-free extract was prepared with a French pressure cell. The ribosome fraction was obtained by the centrifugation procedure and i t was resuspended in 0.05 M tris buffer (PH 7.4) to give 60 times the growth concentration. To 0.2 ml of 0.5 M tris buffer (pH 7.4) were added 0.6 ml of the ribosome suspension, 0.24 ml of 0.5 M P O 4 =, 0.27 ml of P 3 2 58 (approximately 11.18 nc/ml) and O.69 ml of distilled water. The enzymatic reaction was stopped at zero time and after 60 min incubation at 30 C by the addition of 2 ml of 1.4 N PGA. The test and control mixtures were held in ice for 10 min and at the end of this period the tube contents were adjusted to approximately pH 4 by the addition of 0.49 ml of 5 N KOH. Following centrifugation, 20 mg of formic acid washed charcoal (Baddiley et al., 1956) were added to 3 ml of both test and control supernatant fluids and the tube contents were incubated at room temperature for 60 min while mixing was carried out by means of a magnetic stirrer (Smith, i 9 6 0 ) . At the end of the incubation time, the charcoal was recovered by centrifugation and was washed twice with 3 *B1 volumes of distilled water. The charcoal was then suspended in 3 ml of etbanol-ammonia-water (10:l :9j Baddiley et al., 1956) and incubated for 60 min at 35.5 C (Smith, i 9 6 0 ) . Following incubation, the supernatant fluid was obtained by centrifugation and the radioactivity and UV absorbing material in the test and control charcoal eluates were measured. 3. Ribosomal location of the enzyme Washed 20 hr cells were resuspended at a concentration of 20 mg dry weight/ml in 0.05 ^  tris buffer (pH 7.4) containing 0.01 M MgCl . A cell-free extract was obtained with a French pressure cell and ribosome pellets were acquired - 5 9 -by means of the differential centrifugation method. The ribosomal material was washed once with 0 .05 M tris buffer (pH 7.4) containing 0 .01 M MgCl2 and one half of the material (A) was resuspended by homogenization in 2 ml of 0 . 0 1 M tris buffer (pH 7.4) containing 0 .01 M MgCl2. The other half of the ribosomal material (B) was resuspended in 0 . 0 1 M tris buffer (pH 7.4) containing IO"* M MgCl2. One ml of the ribosome suspensions was equivalent to 220 times the ribosome content of 1 ml of the growth concentration of 20 hr cells. Three-tenths of a ml of the ribosome suspension (A) were applied to the top of each of three sucrose gradients containing 4.7 ml of 5-20$ sucrose in 0 . 0 1 M tris buffer (pH 7.4) and 0 . 0 1 M MgCl2. The tubes were centrifuged at 37,5QO RPM for 90 min and fractions were collected from each preparation in the manner already described. The optical density at 260 mu was determined on 1:99 dilutions of the fractions. The two fractions from the 70S peak (see RESULTS AND DISCUSSION) which contained the greatest amount of UV absorbing material were pooled from each preparation to give a total of six fractions. This material was dialyzed with stirring for 90 min at 6 C against 0 .05 M tris buffer (pH 7.4) with 0 . 0 1 If MgCl2. The last or top three fractions from each of these sucrose gradients were pooled to give a total of nine fractions. These pooled fractions, designated as 70S supernatant, were dialyzed for 90 min at 6 C against - 6 o -0.05 M t r i s buffer (pH 7 . 4 ) with IO - 4 M g C l 2 . Three-tenths of a ml of ribosome suspension (B) were applied to the top of each of three sucrose gradients containing 4.7 ml of 5-20$ sucrose i n 0 . 0 1 M t r i s buffer with -4 10 M MgCl 2. Centrifugation was carried out for 150 min at 37,500 BJPM. By using the procedure for obtaining 70S ribosomes, 5°S and 30S ribosomes were recovered; dialysis, however, was against 0.05 M t r i s buffer with 1 0 " 4 M MgCl 2. A 50S-3°S supernatant was acquired in the same manner as the 70S supernatant. Assays mixtures i n 1 ml volumes, contained 0.05 M t r i s buffer, 0 . 2 0 ml of the appropriate ribosome preparation and 0.06 M Pj_. Also, 0.5 ml of the 70S supernatant was incubated with 0 . 2 ml of RNA ( 5 mg/ml) and the 5OS-3OS supernatant was incubated with RNA, 50S or 3OS ribosomes. One ml of 1.4 N PCA was added to the zero time controls and to the tests after a 60 min incubation at 30 C. After centrifugation, the UV spectra of the control and test supernatant fluids were obtained with a Bausch and Lomb spectrophotometer. IX. Radioactivity Measurements Duplicate samples of whole cells and c e l l fractions were plated at in f i n i t e thinness on stainless steel planchets and dried with an Infra-red lamp. Radioactivity was measured with a model l 8 l Nuclear Chicago scaler with a gas-flow -61-automatic counter, having a thin end-window Geiger tube. Corrections were made for background and for coincidence as required. Radioactive areas on electrophoretograms were located by passing strips through a Nuclear Chicago model C 100 B Actigraph II with a gas-flow counter, a model 1620 B Analytical Count Ratemeter, and Chart Recorder. •-62*" EXPERIMENTAL RESULTS AND DISCUSSION I. Preliminary Experiments 1. Cell viability during endogenous respiration Harrison (i960) suggested that products of dead or dying bacteria are utilized by the remaining living organisms thus allowing them to maintain their viability. To determine if a significant amount of cell lysis occurred during the 2 to 3 hr periods of starvation used throughout this experimental work, the total cell count of a dense population of endogenously respiring P. aeruginosa was followed for 3 hours. Samples of cells respiring in 0.05 M tris buffer (pH 7«4) were removed from a Warburg vessel at hourly intervals, suspended in various buffers and diluted to a suitable range for plate counts. The cells suspended in gelatin-NaCl-phosphate buffer exhibited an over-all decrease in viability of 36 per cent in 3 hr, whereas a portion of the same starved-cell suspension diluted with O.O33 M phosphate buffer showed no decrease in viability (Table 1). These results are consistent with those obtained by Warren et al. (i960), who found no decrease in viable cells of less dense populations of P. aeruginosa after a 2 hr interval of starvation. It is concluded that cell lysis does not offer a significant -63-contribution to the support of cell viability during the 3 hr test period used in this and subsequent experiments. The organism, therefore, is capable of remaining viable by virtue of the energy gained from the degradation of intracellular constituents. Table 1. Effect of endogenous respiration on cell viability Diluent Time of starvation Gelatin-NaCl- Phosphate phosphate buffer buffer Hr Viable cells/ml x lCf 9 0 28.3 27.5 1 21.7 25.8 2 20.5 30.7 3 18.2 28.5 S. lutea did not show a decrease in cell numbers until after 45 hr of starvation (Burleigh, Dawes and Ribbons, 1963) and aqueous suspensions of A. aerogenes exhibited a decrease in viable cells of only 11 per cent after storage at 37 C for 69 hr (Strange, 1961). The effects of various physical and 64. chemical conditions during starvation as well as previous growth conditions, on the survival of bacteria, has been stressed by Postgate and Hunter (1962). 2. The effect of starvation on glucose oxidation The effect of starvation on enzyme activity was examined by adding 5" of glucose to cells (14 mg dry weight) at zero time and after 1, 2 and 3 n r of starvation and then following oxygen uptake by means of a Warburg respirometer. No lag in oxygen consumption was observed when glucose was added to cells that had respired endogenously for the time intervals indicated. Also, there was not an obvious decrease in the rate at which the exogenous substrate was oxidized (Figure 1). Similar results have been obtained with endogenously respiring yeast cells (Stier and Stannard, 1936). It has been shown previously that after storage of P. aeruginosa at refrigeration temperature for 4 days or after aeration at 3° c for 8.5 hr, the rate of glucose oxidation was not affected to any extent (Norris, Campbell and Ney, 1949). It can be concluded, then, that starvation of the organism does not cause a decrease in intracellular enzyme levels or activities, at least with the enzymes concerned in glucose oxidation. These data also support the previous results which showed that cell lysis is not an important factor in studies of endogenous respiration in this organism under the conditions employed. FIGURE I. The effect of starvation on glucose oxidation - 6 6 -3 . The influence of cell concentration on endogenous oxygen consumption As diverse analyses, requiring various cell concentra-tions, were to be made on starved and unstarved cells, the effect of cell density on oxygen uptake was determined in an effort to establish the validity of comparing the changes that occurred during endogenous respiration in cells suspensions of different densities. Cells were resuspended in 0.05 M tris buffer (pH 7.4) at 10, 20, 40, and 50 times the growth concentration of 20 hr cells. As a cell suspension at 10 times the growth concentration is equivalent to approximately 5 mg dry weight of cells/ml, the corresponding cell concentrations on a dry weight basis were 5> 10, 20 and 25 mg/ml respectively. The endogenous oxygen uptake of 1 ml of the cell suspensions during a 2 hr period of starvation is shown in Figure 2. The amount of oxygen consumed by 5 mg dry weight of cells was assigned on arbitrary value of 1.0 and the increase in oxygen uptake by the other cell concentrations, relative to this value, was calculated. From Table 2 it can be seen that cell populations of P. aeruginosa between 5 and 25 mg dry weight/ml do not exhibit a significant change in the proportion of oxygen consumed/mg dry weight of cells during endogenous respiration. It will be assumed, therefore, that within the range of cell densities examined, there is not a population-dependent change in the pattern of endogenous respiration. 67-mg^ml 0 25 60 90 120 MINUTES F I G U R E 2. The inf luence of cell concent ro t i on on endogenous oxygen uptake -68-Table 2. The influence of cell density on endogenous oxygen consumption Cell concentration Oxygen uptake Increase in O2 uptake relative to 5 mg dry weight/ml mg dry weight/ml uM/2 hr 5 5.90 1.00 10 11.95 2.03 20 24.10 4.07 25 29.50 5.07 II. Studies with Non-radioactive Cells 1. The appearance of UV absorbing material in Warburg supernatant fluids The appearance of increasing amounts of UV absorbing material in Warburg reaction mixture supernatant fluids of endogenously respiring cells was noted (Figure 3)« During the first hour of starvation, the UV absorbing material increased approximately 265 per cent over the zero time value, an additional 56 per cent during the second and 40 per cent during the third hour. This material showed maximal absorption at 258-260 mu, minimal absorption at 240 mu, reacted positively with the orcinol reagent and negatively with diphenylamine. FIGURE 3- Increose in UV absorbing material in Warburg supernatant fluids of endogenously respiring cells -70-The material gave a slight increase in optical density at 260 mu when incubated with RNase and no increase when incubated with.DNase plus Mg++ and therefore, was concluded to consist primarily of RNA and/or RNA degradation products as the properties mentioned are consistent with these materials. After electrophoresis, actigraphs of Warburg supernatant 14 fluids from glucose-U-C labeled cells, followed by elution of radioactive areas and UV spectroscopy, confirmed the presence of high molecular weight 260 mu absorbing material that did not migrate in an electric field. In addition, nucleotides, nucleosides and possibly free bases were detected. In a like manner, the presence of apparently lesser quantities of "protein" and free amino acids was established. The 280/260 ratio of the material released from the cells decreased during the 3 hr incubation period indicating that, with time, the increase in RNA in the Warburg supernatant fluids was proportionately greater than the increase in protein. The release of both nucleic acids and amino acids by a variety of microorganisms, respiring either endogenously or In the absence of some required nutrient, has been reported by many workers. Hemophilus parainfluenzae, suspended in tris buffer, released RNA (Herbst and Doctor, 1959)> B. megaterium liberated hypoxanthine and RNA in phosphate buffer (Delamater, Babcock and Mazzanti, 1959) and RNA degradation -71-products were found in the suspending fluid of starved A. aerogenes (Strange, Wade and Hess, 1963). S. cerevisiae released polynucleotides, oligonucleotides and mononucleotides while suspended in citrate buffer with 2 per cent sucrose (Higuchi and Uemura, 1959)> E. coli excreted RNA during glucose starvation (Borek, Ryan and Rockenbach, 1955) and non-growing B. cereus released amino acids into the suspending fluid (Urba, 1959) as did nitrogen starved E. coli (Mandelstam, 1958). It is generally accepted that this release of amino acids and RNA or RNA degradation products is not dependent on cell lysis but rather is dependent on the intracellular degradation of RNA and protein. In view of the results of the viable cell counts, It must be concluded that this is also the case with P. aeruginosa, and the release of UV absorbing material, then, is related to endogenous respiration in this organism. The release of RNA and amino acids from microorganisms appears to be, in general, a manifestation of starvation conditions. 2. Changes in cellular constituents Previous work with P. aeruginosa has shown that during a 2 hr period of starvation there was no decrease in the total carbohydrate content of the cell whereas the intracellular amino acid concentration decreased and the extracellular amino acids increased yielding a total increase in free amino acids. The evolution of ammonia during starvation indicated - 7 2 -the nitrogenous nature of the material oxidized and amino acids and protein were implicated as the endogenous substrate (Warren et al., i960). No change in either protein or nucleic acid content could be detected under the previous conditions of starvation probably because any change that may have occurred was within the experimental limitations of the chemical assays employed. In an effort to overcome this possible difficulty, the cell concentration was increased three-fold, the starvation period was lengthened by one hour and the various assays were carried out on physical and chemical cell fractions as well as on whole cells. The total changes in the chemical fractions of the cells after a 3 hr period of starvation on a Warburg respirometer showed an increase in extracellular proteins and RNA and a net decrease in total protein and RNA. Deoxy-ribonucleic acid increased slightly but there was a net decrease in total nucleic acids (Table 3)• The increase in organic phosphate coincided very closely, on a uM basis, with the decrease in RNA. The small increase in DNA was characteristic of all test samples analyzed and is probably a manifestation of the organisms progression towards reproduction. An increase in DNA at the apparent expense of RNA has been reported in phosphate-deficient E. coli (Horiuchi, 1959). Table 3' Changes in cellular constituents during endogenous respiration RNA DNA Total nucleic acid Pi Protein ug/ml Control Intracellular 1213 410 1623 - 3390 Extracellular 0 50 - 268 Total 1263 410 1673 40.6 3658 Test (3 hr starvation) Intracellular 926 445 1371 - 2944 Extracellular 158 0 158 - 408 Total 1084 445 1529 52.4 3352 Test minus control -179 +35 -144 +11.8 -306 These results suggested that both protein and RNA probably serve as endogenous substrates in this organism. A decrease in both of these constituents during starvation has been observed with A. aerogenes (Strange, I961), a decrease in amino acids and protein with S. lutea (Dawes and Holms, 1958) and a slime mold (Wright and Anderson, i960), and a decrease -74-in RNA in E. coli (Dawes and Ribbons, 1962). 3 . Oxidation of purines and pyrimidines The feasibility of RNA serving as a source of energy during starvation was investigated by following oxygen consumption and ammonia evolution by P. aeruginosa when incubated with nucleosides, purines and pyrimidines or their degradation products. Warburg vessels contained 2 ml of a 10 times the growth concentration of cells and 5 uM of substrate. Incubation was carried out for 4 hr (Table 4). There was no evident oxidation of a number of nucleoside-51-monophosphates tested; this, however, may have been the consequence of a permeability phenomenon. All of the other compounds that were added to the Warburg vessels as substrates were oxidized without a lag period (Figure 4). Bachrach (1957) has reported that unless certain pseudomonads were grown on uric acid they required an induction period before the oxidation of uric acid or allantoin could proceed. With P. aeruginosa 9027, all of the compounds listed in Table 4 were oxidized by constitutive enzymes which supports the previous evidence that implicated RNA as an endogenous substrate. O C 2) m •o o c X O. 5* o 5" a. 3 * «o"i O o -^i a. a c — » on ine •o on o Q. •o C o -"I Vt | " a! 5' (B ttt > o 3 a. JJLITERS OXYQEN CONSUMED — ro w O o o O Q O Q «0 C O - I x O ? ° o O (/) too 1 \ \ K mo \ \\ X • o I w C O « X 3 3 <J. i s CA -76-Table 4. Oxidation of purine and pyrimidine compounds Oxygen uptake Ammonia produced uM/uM substrate* Adenosine 2.72 2.66 Adenine 2.84 3.96 Guanosine 2.14 2.35 Xanthine 1.98 3.75 Uric Acid 2.39 2.06 Allantoin 2.94 3.22 Uridine 1.42 -O.38 Uracil 1.35 -0.45 Cytidlne 1.55 O.72 Cytosine 0.23 O.90 Ribose O.87 -O.36 •Endogenous oxygen and ammonia values have been subtracted. 4. The influence of spermine on the release of UV absorbing material Herbst and Doctor (1959) noted that orcinol positive 260 mu absorbing material was released from H. parainfluenzae when a cell concentration of 1 mg dry weight/ml was incubated at 37 c in tris buffer. When a spermine concentration of -77-0.5 mM was added to the starving cells, the polyamine demonstrated a pronounced effect in inhibiting RNA degradation. As spermine is a required growth factor for the organism, i t was suggested that the effect of the polyamine as an inhibitor of RNA breakdown was indicative of an essential role of the growth factor in nucleic acid metabolism. The effect of spermine on the release of 260 mu absorbing material from P. aeruginosa was followed by adding 50 uM of the polyamine and 15 mg dry weight of cells to a Warburg vessel. The amount of UV absorbing material in a 2*7 dilution of Warburg supernatant fluids was measured at zero time and after a 3 hr period of incubation in the presence and absence of spermine. The results (Figure 5) were similar to those obtained by Herbst and Doctor in that at 3 n r an 82.6 per cent decrease in the amount of UV absorbing material was noted in the presence of spermine. It was also evident, however, that the presence of the polyamine promoted a marked increase in both oxygen consumption and ammonia evolution (Table 5). Spermine was, in fact, an oxidizable exogenous substrate which was capable of supplying a source of both carbon and nitrogen to the cells. Herbst and Doctor did not measure either oxygen consumption or ammonia evolution from H. parainfluenzae when incubated with spermine. The influence of exogenous substrates on the release of UV absorbing material from P. aeruginosa will be discussed more fully in another section. -78-F I G U R E 5. The influence of spermine on the release of UV absorbing mater ia l -79"" Table 5». The influence of spermine on oxygen uptake and ammonia production Oxygen uptake Ammonia production urn/Warburg vessel Endogenous 2 9 . 8 7.7 plus spermine 6 8 . 0 19.5 A s t r a i n of P. aeruginosa has been found to oxidize a number of polyamines and 2 uM of /3 alanine were produced from the oxidation of 1 uM of spermine. S. marscens also was found to oxidize a variety of polyamines (Razin, Gery and Bachrach, 1959) . The e f f e c t s of polyamines and Mg++ on ribosome components of c e l l - f r e e extracts of E . c o l i were examined by Cohen and Lichtenstein ( i 9 6 0 ) who found that both types of compounds contributed to the s t a b i l i t y of ribosomes. A combination of spermidine and Mg++ permitted a more extensive aggregation of 45S and $0S components than that produced by either substance alone. In view of these r e s u l t s , Cohen and Lichtenstein suggested that Mg++ and spermidine not only act as cations i n n e u t r a l i z i n g anions but also are the n a t u r a l l y occurring l i n k s of the complex ribosomal components exis t i n g -80-in bacteria. Approximately 15 per cent of the total polyamine concentration of E. coli was found to be associated with the ribosomal fraction. Spahr (1962) found that 0.4 per cent of the weight of E. coli ribosomes can be attributed to polyamines. From amino acid analyses, Spahr established that ribosomal proteins are basic in nature containing an excess of 6.92 moles of basic residues per 100 moles of amino acids in the 70S ribosomes, in comparison with cytoplasmic proteins which contain an excess of 1.33 moles of acidic residues per 100 moles of amino acids. The ribosomal proteins, however, are less basic than the histones which contain an excess of 18.3 moles of basic residues per 100 moles of amino acids. It would appear from these data that the basic nature of the ribosomal proteins is considerably more important in the neutralization of anions and stabilization of ribosomes than the very low concentration of polyamines associated with the ribosomes. The effect of starvation on the intracellular concentration of polyamines in P. aeruginosa was followed in an attempt to determine i f polyamines are one of the sources of the ammonia that is evolved during endogenous respiration. At zero time 5 mg dry weight of cells contained 15.74 ug of polyamines, expressed as spermine, and the value decreased to 14.41 ug following a 3 hr period of starvation. The assay -81-procedure employed was not entirely specific for polyamines as both adenine and histidine gave a slight color development with the reagents. This may mean that the apparent slight decrease in polyamine concentration after starvation was, in actuality, the result of the decrease in protein and RNA. From these results it is concluded that the polyamines in P. aeruginosa do not play a significant role in the endogenous metabolism of the organism. 5. The effect of magnesium and phosphate ions on the release of UV absorbing material, oxygen consumption and ammonia evolution Bowen, Dagley and Sykes (1959) reported that a ribonucleoprotein component of E. coli was influenced considerably by environmental conditions. When cells were incubated in phosphate buffer, the ribonucleoprotein component was depleted but was reformed on the addition of magnesium Ions. The addition of either 3.33 x IO"2 M phosphate or magnesium to endogenously respiring P. aeruginosa caused a decrease in the total amount of UV absorbing material appearing in the supernatant fluids in 3 hr. The presence of magnesium ions exhibited a greater repression on the release of UV absorbing material than did the presence of phosphate ions (Figure 6). In both instances there was a continual -82-endogenous F IGURE 6. T h e inf luence of mognesium and phosphate ions on the re l ea se of U V absorbing ma te r i a l -83-increase in this material with time, the greatest increase occurring during the first hour of incubation. The influence of these ions on oxygen consumption and on ammonia production was followed at hourly intervals (Table 6 ) . The presence of inorganic phosphate stimulated oxygen uptake but slightly depressed ammonia production. Table 6. Effect of magnesium and phosphate ions on endogenous respiration Time r Control Plus Mg++ Plus P0 4s Oxygen uptake 0 - 1 hr 4.75 4.15 5.17 uM/ml 1 - 2 hr 4.00 3-72 3.86 2 - 3 hr 3.19 3.23 3.40 Total in 3 hrs 11.94 11.10 12.43 Ammonia production 0 - 1 hr 0.93 1.03 0.98 uM/ml 1 - 2 hr 0.80 0.82 0.66 2 - 3 hr 1.19 0.71 0.93 Total in 3 nrs 2.92 2.55 2.57 02/NH3 At 3 hrs 4.10 4.35 4.84 -84. Primarily, this was probably due to a slight shift in the ratio of oxidation of RNA to oxidation of protein. The depression of endogenous activity in the presence of magnesium ions is in accord with ribonucleoprotein serving as an endogenous substrate, as magnesium has a major stabilizing influence on ribonucleoprotein. Wade (1961) demonstrated the inhibitory effect of magnesium ions on the endogenous degradation of the RNA of E. coli. III. Studies with Cl4-labeled Cells 1. Specificity of incorporation of radioactive proline and uracil To confirm the oxidation of cellular protein or RNA, cells were labeled with uracil-2-C 1 4, proline-U-C14, or glucose-U-C14" with the intent of measuring radioactivity in the carbon dioxide evolved during endogenous respiration. In view of the ability of P. aeruginosa to utilize a large number of individual nitrogenous compounds as the sole source of carbon and nitrogen, the specificity of the labeling of nucleic acid with uracil-2-C-1-4' was determined by a modification of the Schmidt-Thannhauser technique and the specificity of the labeling of protein, when proline-U-C^4, was substrate, was determined by the modification of the procedure of Roberts et al. (1955). -85-Cells labeled with uracil^-C^ 4 retained only 2 .7 per cent of the radioactivity in the "protein" residue (Table 7 ) • This is well within the experimental limitations of the fractionation procedure, as has been demonstrated by Hutchison et al. (1962); in the interpretation of subsequent results of fractionation procedures, radioactive uracil has been considered to be confined solely to the nucleic acid fraction. The appearance of approximately 30 per cent of the total radioactivity in the DNA fraction illustrates the probable ability of this organism to interconvert pyrimidine nucleotides (Potter, i 9 6 0 ) . Table 7 . Distribution of radioactivity in cells grown on uraeil-2-Cl4 % of Fraction Counts/min/ml x 10""3 total C Cold TCA soluble extract of NaOH hydrolysate (RNA) 130.0 69.7 Hot TCA soluble extract (DNA) 51.75 27.7 Hot TCA residue (Protein) 5.11 2.7 -86-Proline-U-C'*"4 labeled cells retained only 3«4 per cent of the total radioactivity in the nucleic acid fraction and again this has been considered as contaminating radioactivity (Table 8). It would appear, therefore, that the proline-U-C-1-' was incorporated exclusively into protein. Table 8. Distribution of radioactivity in cells grown on proline-U-C14 . Fraction Counts/min/ml x 10""3 % of total C 1 4 Cold TCA soluble 24.7 2.5 Acid-ethanol soluble 104.0 10.5 Hot TCA soluble 33-5 3.4 Hot TCA residue 825.4 83.5 2. Evolution of radioactive carbon dioxide The relative amount of radioactivity evolved as C1402 by endogenously respiring cells labeled with glucose-U-Cl^", uracil-2-C14, or proline-U-C14 was determined and expressed as per cent of total radioactivity in the Warburg flask (Table 9)• After zero time values were subtracted from the results 14 obtained with glucose-U-C -labeled cells, 3.31 per cent of - 8 7 -the total radioactive carbon was recovered as c l 4 0 2 . An even greater percentage of radioactive CO2 was evolved from cells labeled with C^4-proline or -uracil. Because of the specificity of labeling with these two compounds, this clearly illustrates that both nucleic acid and protein were oxidized and, therefore, must serve as endogenous substrates in P. aeruginosa. Table 9. Radioactivity evolved as C 1 4 0 2 Cells labeled with Zero time Endogenous after Increase in control 3 hr starvation C l 4 o 2 in 3.hr Counts/min/ml x 10""3 % ©f total C 1 4 Glucose-U-C14 C 0 2 23.72 Cells* 2 , 8 7 8 . 0 115.3 2 , 6 2 5 . 0 3.31 Proline-U-C1* C 0 2 19.4-5 Cells 1 ,529.0 88.7 1 ,586.0 4-.Q5 u r a c i i - 2 - c i 4 co 2 3 .85 Cells 204.0 15.24 188.5 5.64 •Cells plus supernatant fluid 88** From Table 3 i t can be seen that RNA decreased during endogenous r e s p i r a t i o n whereas DNA increasedj further, the UV-absorbing material i n Warburg supernatant f l u i d s was i d e n t i f i e d as RNA. Therefore, i t may be concluded that the r a d i o a c t i v i t y i n C 1 4 ^ from uracil-2-Cl4 labeled c e l l s arose s o l e l y from RNA. As 70 per cent of the t o t a l l a b e l appeared i n the RNA f r a c t i o n (Table 7), the apparent amount of RNA oxidized would be 8.05 per cent rather than 5»64 per cent. This value may be lower than the actual percentage of RNA oxidized, for the rate of oxidation of pyrimidines i s somewhat slower than that of purine and purine degradation products (Figure 4). An analogous s i t u a t i o n may e x i s t with pro l i n e -U-C 1 4 labeled c e l l s . The complete oxidation of a l l the ribosomal RNA of uniformly labeled c e l l s would r e s u l t i n the evolution of 13•5 per cent of t h e i r r a d i o a c t i v i t y . Therefore, under the experimental conditions where 8.05 per cent of the RNA was oxidized, one may suggest that the oxidation of t h i s amount of ribosomal RNA would y i e l d 1.08 per cent of the t o t a l radio-14 a c t i v i t y of uniformly labeled c e l l s as C 02. In the same way, the C 1 40 2 from the proline-U-C 1 4 labeled c e l l s constituted 4.05 per cent of the t o t a l c e l l u l a r protein which, i f the c e l l s had been uniformly labeled, would have contributed 2.32 per cent of the c e l l u l a r C 1 40 2 released. The c e l l s grown with 14 glucose-U-C may be considered to be uniformly labeled, and the C1402 released from these c e l l s as a r e s u l t of the - 8 9 -oxidation of c e l l u l a r protein and ribosomal RNA would, according to the above reasoning, amount to 3'40 per cent. The experimental value obtained was 3 .31 per cent, thus confirming the absence of carbohydrate endogenous reserves. To e s t a b l i s h that the r a d i o a c t i v i t y i n the CO2 released from uracil - 2-C^" 4 and p r o l i n e - U - C 1 4 labeled c e l l s was, i n f a c t , a v a l i d c r i t e r i o n of endogenous r e s p i r a t i o n i n t h i s organism and did correlate with oxygen uptake, the C^ C v , evolution and oxygen consumption of the labeled c e l l s were followed f o r 5 hrs. I t was thought that i f the C 1 4 0 2 evolution was due, at l e a s t i n part, to the oxidation of labeled u r a c i l or proline present as exogenous materials from i n s u f f i c i e n t washings of harvested c e l l s , then the r a t i o of 14 C 0 2 evolution to oxygen uptake would decrease with time. From Table 10 i t can be seen that t h i s r a t i o did not decrease, but remained f a i r l y constant, during the endogenous r e s p i r a t i o n of u r a c i l - 2 - C 1 4 labeled c e l l s . With proline-U-C^ 4 labeled c e l l s , i d e n t i c a l r e s u l t s were obtained i n that no s i g n i f i c a n t change i n the r a t i o of c l 4 0 2 evolution to oxygen uptake was observed (Table 11 ) . 3 . D i s t r i b u t i o n of r a d i o a c t i v i t y i n c e l l f r a c t i o n s (a) Chemical f r a c t i o n s To ensure that the r a d i o a c t i v i t y i n from p r o l i n e -U-C-*-4 and uracil - 2-C^- 4-labeled c e l l s did not a r i s e merely from free i n t r a c e l l u l a r "pools" of proline or u r a c i l , whole c e l l s -90-Table 10. Oxygen consumption and cl4o 2 evolution from uracil - 2 - C l 4 labeled c e l l s Time % c 1 4 o 2 * u l 02 taken up % C 1 40 2/ul 02 min x 10~2 30 0.78 0.37 2.10 60 1.72 0.81 2.12 120 3.08 1.43 2.15 180 4.06 I.96 2.08 240 5.22 2.34 2.21 300 6.15 2.81 2.18 •Expressed as per cent of t o t a l C 1 4 i n the Warburg vessel. were fractionated according to the modified procedure of Roberts et a l . (1955) at zero time and a f t e r 3 hr of starvation. The change i n d i s t r i b u t i o n of r a d i o a c t i v i t y i n the various f r a c t i o n s , as well as the influence of 3.33 x 10~2 M magnesium and phosphate on this change was determined (Table 12). As chemical f r a c t i o n a t i o n could not be ca r r i e d out on c e l l s used for C1402 determinations, Table 12 i s a composite of two separate experiments. -91-Table 11. Oxygen consumption and c ! 4 o 2 evolution from proline-U-Cl4 labeled cells Time % c 1 4 o 2 * ul 02 taken up % c ! 4 o 2/ul 0 2 min x IO"2 3 0 0.95 0.51 1.86 60 1.78 0.90 1.97 120 3.04 U55 1.96 180 3.81 2.01 1.89 240 4.20 2.30 1.82 300 5.17 2.60 1.96 •Expressed as per cent of total 0 in the Warburg vessel. Under all test conditions there was a significant decrease in the radioactivity in the "protein residue" and the hot trichloroacetic acid soluble fraction. This confirms that the C 1 4 0 2 from uracil-and proline-labeled cells was not a result of oxidation of "pool" material but was due to the degradation of both protein and RNA. It would appear that protein and RNA are degraded more rapidly than they can be oxidized and, therefore, are excreted into the suspending fluid. In each test sample, the "free amino acid pool" decreased during endogenous respiration, whereas the "nucleic - 9 2 -Table 12. Distribution of C 1 4 in chemical fractions of whole cells after 3 hr starvation Cells labeled Fraction with radio-active Zero time Endoge nous Plus Mg++ Plus P04= % O f Total radioactivity C02 Glucose - 3.32 3 .29 3.21 Uracil - 5.64 4.97 5.02 Proline - 4.05 4.67 4 . 5 5 Warburg Glucose 10.80 14.70 7.34 11.92 supernatant 10.65 7.64 fluid Uracil 3.29 4.22 Proline 4.89 7.94 4.50 7.42 Cold TCA Glucose 3.34 2.58 3.36 3 .78 soluble 8.90 8 .35 Uracil 7.20 11.05 Proline 3 . 3 5 2 f 70 3.24 2.21 Acid-ethanol Glucose 7.73 8.39 6.70 10.09 soluble Proline 9.45 10.25 9.15 9 .30 Hot TCA Glucose 14.10 11.70 13.42 10.70 soluble 78.10 Uracil 90.40 74.20 79.70 Hot TCA Glucose 6 6 . 9 0 59.90 , 65.90 62.00 insoluble 76.90 Proline 82.60 74.40 78.80 - 9 3 -acid precursor pool" increased. This suggests a preference for oxidation of amino acids or a greater ability of the organism to oxidize amino acids. From a comparison of the amount of RNA and protein in the cell and the apparent percentage of these components oxidized as indicated by C 1 40 2 data, approximately twice as much protein as RNA was oxidized. In the presence of both magnesium and phosphate, relatively less RNA was degraded, and with magnesium the degradation products were retained, to a greater extent, in the cold trichloroacetic acid-soluble pool rather than being excreted into the suspending fluid. From the C1402 values, it can be seen that phosphate and magnesium ions have a slight inhibitory effect on RNA oxidation and a slight stimulatory effect on protein oxidation. (b) Physical fractions Variously labeled cells were disrupted by the lysozyme-EDTA technique at zero time and after respiring endogenously for 3 hr, and were fractionated by differential centrifugation (Table 1 3 ) . Both the commercial DNase and lysozyme preparations contained RNase as a contaminating enzyme, as was shown by the release of nucleotides from commercial RNA incubated with either DNase or lysozyme. The ribonuclease accounts, in part, for the abnormally low percentage of radioactivity in the ribosome fraction and the abnormally high percentage of radio-activity in the supernatant fluid (140,000 x g). The -94-majority of the radioactivity in the supernatant fluid (140,000 x g) of uracil-2-C14-labeled cells was cold t r i -chloroacetic acid-soluble, indicating that it was low molecular weight material (see Table 14). This is in contrast to the data obtained from the chemical fractionation of whole cells in which the lysozyme treatment was omitted. From Table 12 i t can be seen that the majority of the radioactivity from the chemical fractionation procedure was contained in material insoluble in cold trichloroacetic acid. All test samples showed a significant decrease in the protein and the RNA of the ribosome fraction, demonstrating the occurrence of ribosomal degradation during endogenous respiration. While following the turnover of protein and RNA in E. coli, Mandelstam and Halvorson ( i 9 6 0 ) found that the ribosomes contributed almost half of the amino acids and essentially all of the ribonucleotides passing through the free pool during starvation. However, no reference was made to the oxidation of these compounds. The increase in protein in the supernatant fluid (140,000 x g) of cells incubated in the presence of magnesium ions and the apparently only slight decrease in RNA may be attributed to the solubilization of particulate material accompanied by a lower level of excretion of these materials into the Warburg suspending fluid. The appearance of nucleic acid in the "membrane" fraction can be accounted for by the presence of a small number of "bound" ribosomes. This -95-Table 13. Distribution of C1^ in physical fractions of cells after 3 hr starvation Cells labeled with radio- Zero Endoge Plus Plus Fraction active time nous Mg++ PO4S % of total radioactivity CO2 Glucose - 3.32 3.29 3.21 Uracil - 5.64 4.97 5.02 Proline - 4.05 4.65 4.55 Warburg Glucose 10.80 14.77 7.34 11.92 supernatant 10.65 7.64 fluid Uracil 3.29 4.22 Proline 4.89 7.97 4.50 7.42 "Membranes" Glucose 16.10 10.55 8.79 16.90 Uracil 7.22 6.24 7.10 5.61 Proline 10.91 8.92 8.86 12.41 "Ribosomes" Glucose 2.28 1.18 1.36 1.41 Uracil 1.92 1.11 0.93 1.44 Proline 4.79 3.14 1.97 2.38 140,000 x g Glucose 64.80 66.10 76.30 66.20 supernatant 87.80 fluid Uracil 75.50 82.50 77.50 Proline 77.20 75.90 81.40 71.50 -96-ribosomal material can be dissociated from the membrane fraction by incubating the membrane fraction with EDTA or by washing the fraction with tris buffer. The chemical fractionation of the 140,000 x g super-natant fluid shows an increase in radioactivity in the hot TCA insoluble residue of a l l test samples demonstrating the solubilization of particulate protein during endogenous respiration,(Table 14). A decrease in the nucleic acid in the soluble cytoplasm of a l l the test samples also is evident from the values obtained for the cold TCA and hot TCA soluble fractions. IV* Oxygen Consumption and Ammonia Evolution by Various  Cell Fractions The observed decrease in RNA and protein from chemical analyses of cells and the decrease in C 1 4 from labeled RNA, protein and ribosomes promoted a further attempt to substantiate the data relating to the cytological location of the endogenous substrates in P. aeruginosa. Cells were crushed by means of a Hughes' press and were fractionated into their cytological components by differential centrifugation. The oxygen uptake and ammonia production of the various components, incubated singly and combined, were measured during a 4 hr Warburg experiment. The cell fractions in the Warburg vessels were at a concentration of 140 times the growth concentration of 1 ml of 20 hr cells. - 9 7 -Table 14. Chemical distribution of radioactivity in the soluble cytoplasm Fraction Cells labeled with radio-active Zero time Endoge nous - Plus Mg++ Plus P04= % of total radioactivity in the Warburg vessel Cold TCA soluble Glucose Uracil 24.90 77.00 19.60 68.40 24.60 68.10 15.40 66.05 Acid-ethanol soluble Glucose 2.83 2.53 2 . 8 3 3.77 Hot TCA soluble Glucose Uracil 3.34 9.70 2.74 11.22 3.84 14.50 3.74 II .38 Hot TCA insoluble Glucose 33.50 39.10 4 2 . 9 0 40.40 The addition of "membranes" to the cell-free extract caused a small but significant increase in both oxygen and ammonia values whereas the addition of ribosomes to the cell-free extract resulted in a 33 Per cent increase in oxygen uptake and a 92 per cent increase in ammonia production. The removal of membranes from the extract (25,000 x g supernatant) effected a decrease in both values proportional to the increase JJ LITERS OXYGEN CONSUMED O o c a m o «< A C T3 cr < Q - i o c w o A o o o 3 (A O ro c 3D CO 0» o 1-O 0> O n u t ) £ b c O x o © • 3 _ o • 3 tfl O " 3 2. 3 •  3 a A A o o X o X 30 CO O © # w h O © I W \ © • I \ \ \ O © • \ \ \ x • © o 3 » cr 3 c c c o cr • • cr o o 2 . 3 * A * o a 4c ** •» A 3 » o ? o o 3 a * ^ A 3 O CA 3 3 00 »x - 8 6 --99-observed on the addition of "membranes" to the cell-free extract (Figure 7 A, Table 15). Table 15. Endogenous activities of cell fractions Oxygen uptake NH3 produced uM/3 ml Cell free extract 5-58 9.36 + membranes 6.16 11.01 + ribosomes 7.43 18.0 25,000 x g supernatant 5.14 7.86 100,000 x g supernatant 1.92 4.0 + membranes 3.78 7.32 + ribosomes 5.14 13.38 Similar but more pronounced results were obtained when the soluble cytoplasm replaced the cell-free extract in this experiment. The addition of membranes to the soluble cytoplasm resulted in an increase of oxygen uptake of 97 per cent and an increase in ammonia production of 83 per cent. The addition of ribosomes again augmented oxygen consumption 100-and ammonia evolution to a greater extent than did the presence of membranes; these increases being 168 per cent and 235 per cent respectively (Figure 7 B, Table 15). The apparent increase i n endogenous r e s p i r a t i o n i n c e l l - f r e e extracts and soluble cytoplasm stimulated by the presence of added "membrane" f r a c t i o n once more may be attributed primarily to a small quantity of contaminating ribosomal material i n t h i s f r a c t i o n . From the preceding data i t i s evident that endogenous oxygen consumption and ammonia evolution i n c e l l - f r e e extracts are associated e s s e n t i a l l y with the ribosomal f r a c t i o n . The influence of ribosomal material on whole c e l l s was followed by incubating c e l l suspensions with ribosomes or ribosomes plus commercial BJase and measuring oxygen uptake. The addition of RNase to the whole c e l l s increased the t o t a l oxygen uptake by 30 per cent i n a 2 hr period. In a l l p r o b a b i l i t y t h i s increase was due to the presence of an oxidizable material i n the commercial enzyme preparation. When added to the c e l l suspensions, ribosomes stimulated oxygen uptake by 73 P e r cent whereas ribosomes and RNase effected a 163 per cent increase i n oxygen consumption (Figure 8). The increase i n oxygen uptake with c e l l s plus ribosomes, but no RNase, suggested that either the ribosomal f r a c t i o n was contaminated with free amino acids, purines pyrimidines or nucleosides or that the ribosomal f r a c t i o n -101-30 60 90 120 MINUTES FIGURE 8. Tht influence of ribosomes and RNost on oxygtn consumption of whole ctlls - 1 0 2 -contained an enzyme or enzymes responsible for ribosome degradation. A preliminary experiment to indicate the presence of a degradative enzyme in the ribosome fraction was carried out by heating ribosomal material in a boiling water bath for 3 min, adding the heated material to soluble cytoplasm and following oxygen uptake. From Figure 9 it can be seen that the heat treatment of the ribosomes completely destroyed the ability of this fraction to stimulate the oxygen consumption of the soluble cytoplasm. From these results it is concluded that an enzyme associated with the ribosomes is responsible for the initiation of degradation of this cellular fraction during endogenous respiration. Wade (1961) found that most of the RNA depolymerase activity of E. coli was present in the ribosome fraction and high concentrations of magnesium ions inhibited RNA degradation. Two routes of RNA degradation were evident, one was stimulated by orthophosphate and the other by EDTA. V. Sedimentation Analyses of the Ribosomal Components of  Cell-free Extracts The first indication of the presence of high molecular weight, distinct particles in bacterial cytoplasm was found by Schachman, Pardee and Stanier (1952) who noted that the cell-free extracts of several species of bacteria produced similar patterns in the ultracentrifuge. Since that time a considerable -103-o UJ Z D (0 z o o UJ o > X o </> tc t i l 3. 160 L • soluble cytoplasm 0 plus ribosomes 0 plus heated ribosomes 120 8 0 40 2 HOURS FIGURE 9. The effect of heat on the ability of ribosomes to stimulate oxygen uptake -103a-amount of work has been carried out in an effort to elucidate the nature and function of these ribosomal particles. A direct relationship between ribosomes and protein synthesis was established by McQuillen, Roberts and Britten in 1959 and this relationship has been at least partially clarified by Rich (1963) who has found that polysomes, or aggregates of ribosomes, joined by strands of messenger RNA, are the actual sites of protein synthesis. Ribosomes constitute 30-40 per cent of the cell mass and contain approximately 90 per cent of the cellular RNA and 25 per cent of the cellular proteins (Luria, i960). The basic nature of E. coli ribosomal protein was established by Spahr (1962) who found an excess of 6.92 moles of basic residues per 100 moles of amino acids. Tissieres et al. (1959) observed that in cell-free extracts of E. coli, 7 0 S ribosomes predominated in a magnesium concentration of IO""2 M, whereas in a magnesium concentration of 10~4 M, the 70S particle dissociated reversibly to form 50S and 30S units. Magnesium concentrations of less than IO"*4' M cause an irreversible cleavage of RNA and protein (Bonner, 1961). The ribonucleoprotein particles are bound through magnesium ions which interact with the phosphodiester groups of the RNA, a fully saturated particle contains one Mg++ per three phosphodiester groups. The association of ribosomal RNA and protein through hydrogen bonding is evident from the fact that the dissociation of the two is rapidly accomplished by 104-reagents which attack hydrogen bonds (Bonner, 1961). By incubating ribosomal material with Duponol Kurland (i960) was able to i s o l a t e 23? and l6S RNA components, with molecular weights of 1.1 x 10 and 5«6 x K K respectively. From u l t r a c e n t r i f u g a t i o n analyses McCarthy (i960) was able to conclude that the ribosome content of a b a c t e r i a l c e l l i s c h a r a c t e r i s t i c of the physiological state of the c e l l . The absolute quantity of ribosomes i n E. c o l i varied according to growth conditions and there was a change i n the proportions of the various ribosomal components from the growing to the re s t i n g state. Further studies with E. c o l i (McCarthy, 1962) showed that long periods of magnesium starvation, i n an other-wise complete medium, resulted i n a loss of ribosomes but no loss of v i a b i l i t y . A f t e r a 20 hr period of starvation no ribosome peak could be detected i n the c e l l s , however, the addition of magnesium to the c e l l s at 24 hr caused an exponential increase i n ribosomes. McCarthy suggested that the ribosomes were degraded to structures of low S numbers during starvation but that the RNA was not broken down to small molecules. In view of the previous evidence which has established that ribosomal material i s an endogenous substrate i n P. aeruginosa, i t was thought that a sedimentation analysis of c e l l - f r e e extracts might reveal that during starvation of the organism a p r e f e r e n t i a l u t i l i z a t i o n of a p a r t i c u l a r ribosomal -105-coraponent occurred. 1. Sedimentation coefficients of ribosomal particles Ribosomal material was isolated for sedimentation coefficient determinations as previously outlined and Figure 10 illustrates the patterns of UV absorbing substances obtained under the various conditions of centrifugation. The S value of the material in fraction 9, Figure 10 (A) was found to be 70.5 at 20 C, fraction 10 (B) was 49.6 and fraction 14 (B) was 33. Fairly sharp boundaries were evident for the 70S and 50S peaks after scanning the UV negatives whereas the boundary for the 33S peak was not sharp indicating the heterogeneity of the fraction. The presence of contaminating polydisperse material in the 33s peak no doubt accounts for the high sedimentation coefficient obtained for this ribosomal fraction which will be referred to as 30S. If cell-free extracts containing IO"*4 M Mg++ were centrifuged at 37>500 x g for 90 min instead of 150 min, the 50S and 30S peaks appeared in the same fractions as the two small peaks in Figure 10 (A), therefore, fraction 13 contained 50S ribosomes and fraction 17 contained 30S ribosomes. The S values of the RNA peaks in Figure 10 (C) were not determined but the sedimentation pattern is identical to that observed by McCarthy, Britten and Roberts (1962) and thus it may be assumed that fraction 11 contains 23S RNA and fraction 15 contains l6S RNA. - io6-C 23S 37,500 RPM A 90 MIN, 10-2 M Mg 6 150 MIN, 10-4 M Mg C 360 MIN, 0.5 % DUP0N0L FRACTION NO. FIGURE 10. Potttrns of UV absorbing material from sucrose gradients -107-The ratio of 280 mu to 260 mu absorbing material in the isolated ribosomes was compared with the 280/260 ratios of various cell fractions in order to provide an indication of the purity of the fractions (Table 16). The 280/260 ratio Table 16. The ratio of 280 to 260 mu absorbing material in cell fractions Fraction 280/260 Ribosomes 70S 0.515 50S 0.533 30S 0 . 5 7 6 RNA 23S 0 . 4 9 8 l 6 S O.496 Fraction number 3 0.757 25 0.660 Cell-free extract 0.599 "Membranes" 0 . 7 5 0 Washed membranes O.890 "Ribosomes" 0.575 Soluble cytoplasm O.626 - 1 0 8 of the 30S ribosome was s i g n i f i c a n t l y higher than that of either the 50S or 70S ribosomes, thus suggesting that the 30S peak was probably contaminated with protein. Under a l l conditions of centrifugation of sucrose gradients, f r a c t i o n 3 i n v a r i a b l y contained a small peak of UV absorbing material and from a comparison of 280/260 r a t i o s i t would appear that th i s may be attributed to the presence of small fragments of c e l l membrane. The l a s t or top fractions from the sucrose gradients appeared to contain the soluble cytoplasm of the c e l l s and the r a t i o s observed for the 23S and l6S RNA peaks were t y p i c a l of i s o l a t e d RNA. The 280/260 r a t i o of the "ribosome" f r a c t i o n was i d e n t i c a l to that of the 30S peak which suggests that i t also may contain a small amount of contaminating protein. The difference between the r a t i o s of "membranes" and washed membranes exemplifies the previously mentioned contamination of the membrane f r a c t i o n by ribosomal material. 2 . Labeling of ribosomal RNA by the " s h i f t up" technique Short term labeling of ribosomal RNA by the " s h i f t up" technique, followed by a comparison of the changes i n radio-a c t i v i t y and the changes i n o p t i c a l density that occurred during starvation i n the various f r a c t i o n s c o l l e c t e d from sucrose gradients, was used to detect a possible preference for the u t i l i z a t i o n of "old" or "new" ribosomal material as an endogenous substrate. I t was necessary, therefore, to - 1 0 9 -sstablish conditions for labeling cells such that an adequate 14 incorporation of uracil-2-C ^  into ribosomal RNA would be achieved before a significant increase in cell numbers occurred. The extent of labeling of ribosomal RNA was followed by measuring the increase in radioactivity in the cells, the decrease in cold TCA soluble radioactivity (expressed as percentage of total C^4 incorporated by the cells) and the increase in the optical density of the cell suspensions during incubation. From Figure 1 1 it can be seen that a 9 ° min period of incubation of the cells in the growth medium containing uracil-2-C-1-4 resulted in a suitable uptake of radioactivity into cold TCA insoluble material, as determined by the difference in radioactivity in the cold TCA soluble fraction and the whole cells, prior to a marked increase in cell numbers. Although prolonging incubation for an additional 3 0 min provided a considerably greater incorporation of radioactivity into the cold TCA insoluble material, cell multiplication had commenced by this time. A 9 0 min period of incubation of cells in growth medium containing C-1-4-uracil, therefore, was employed for the following experimental work. % OF TOTAL C 1 4 IN COLD TCA SOLUBLE F R A C T I O N — — ro us o cn o I I I -on-- 1 1 1 -3* Changes In ribosomal components daring endogenous respiration The changes that occurred in the 70S, JOS and 30S ribosomes of P. aeruginosa during a 3 hr interval of starvation on a Warburg respirometer were determined by preparing sucrose gradients in various Mg++ concentrations from cell-free extracts of starved and unstarved cells. In the presence of 1 0 " 2 M Mg++ there was a decrease in both radioactivity and optical density in the 70S ribosomes and an increase in the 50S and 30S ribosomes (Figure 12, Figure 13). A further illustration of the changes in the 70S and 5QS particles is shown in Table 17 where the total radioactivity and total optical density have been calculated for these peaks. As the conditions for preparing sedimentation analyses of starved and unstarved cells were identical, and as these conditions promote the aggregation of 50S and 30S particles, the decrease in the 70S fraction must be considered to be a result of endogenous respiration and to be a reflection of an alteration in the 50S and 3 0 S components such that aggregation is less favored. Only a small percentage of the total ribosomal RNA in the cells was labeled under the experimental conditions used, therefore, i f newly formed ribosomes were selectively degraded during endogenous respiration the ratio of the radio-activity in the 3 hr 70S component, relative to the zero hr - 1 X 2 -70S 5 10 15 20 FRACTION NO. FIGURE 12. The effect of starvation on optical density of 70s, 50s and 30s ribosomes -113-70S FRACTION NO. FIGURE 13. Tht efftct of ftorvotlon on tht C 1 4 content of 70s, 50s ond 30s ribosomes 114-Table 17. The changes in radioactivity and optical density of the 70S and 50S ribosomes in IO**2 M ffig++ after starvation Ribosome Total counts/min per peak x 10-3 Total optical density per peak 70S 50S 70S/50S 0 hr 6.56 7.88 3 hr* 36.83 8.90 4.15 3 hr/0 hr 0.714 1.35 0 hr 2.85 0.37 7.80 3 hr 2.12 0.41 5.20 3 hr/0 hr 0.744 1.12 •Length of starvation period 70S component, would be considerably lower than the ratios of 260 mu absorbing material. However, from Table 17 i t is obvious that there was no substantial difference in these two ratios indicating that there was a random degradation of 7 0 S ribosomes during starvation. The change in 5QS ribosomes relative to 30S ribosomes — 4 was demonstrated by preparing sucrose gradients in 10 M Mg++ thus dissociating the 70S component. Prom optical density measurements, i t appeared that both the $0S and 30S ribosomes decreased during starvation (Figure 14) whereas - I N -FRACTION NO. FIGURE 14. Chongt in optical density in 50s and 30s ribosomes during endogenous respiration -116. radioactivity values indicated a decrease in the 50S component and an increase in the 30S component (Figure 15). The apparent increase in the 3OS fraction may be an anomaly of the experimental procedure. As seen previously with the 70S ribosomes, there was no apparent preferential degradation of wold M or new ribosomes (Table 18). Both optical density Table 18. The influence of starvation on the optical density and radioactivity in 50S and 30S ribosomes Ribosome 50S 30S 50S/30S Total couhts/min per peak x 10-3 0 hr 3 hr* 40.70 35.88 17.10 19.18 2.38 1.87 3 hr/0 hr 0.884 1.120 Total optical density per peak 0 hr 3 hr 3.06 2.55 1.46 1.35 2.08 I.89 3 hr/0 hr O.834 0.930 *Length of starvation period and radioactivity changes observed for the 5°S and 3OS ribosomes during three hr of starvation revealed a much more pronounced utilization of the 50S than of the 30S component. -117-50S 5 10 15 20 FRACTION NO. FIGURE 15. Changt in C 1 4 content in 508 and 308 ribosomes during endogenous respiration -118-Both 23S and l6S RNA decreased during the 3 hr starvation period (Figure 16, Figure 17) and 23s RNA, the RNA present in 50S ribosomes, decreased to a greater extent than did l6S RNA (Table 19). Table 19. Decrease in radioactivity and optical density from 23S and l6s RNA following starvation RNA 23S l6S 23S/16S Total counts/min 0 hr 40.40 21.60 I.87 per peak x 10-3 3 ^ 2 9 ? Q ^ Total optical 0 hr 3.28 1.84 I.78 density per peak 3 h r 2 ^ 1 ^ 1 / ? Q •Length of starvation period These data demonstrate that the $0S ribosomes are more susceptible to degradation during endogenous respiration in P. aeruginosa than are the 30S ribosomes and, further, there is no obvious preferential use of either newly formed or previously formed ribosomal material as the endogenous substrate. - I N -FRACTION NO. FIGURE 16. Decrease in optical density of ribosomal RNA during starvation -120-5 10 15 20 FRACTION NO. FIGURE 17. Decrease in C 1* content of ribosomal RNA during starvation 121-V I« The Detection, Identification and Localization of an  Enzyme Present in the Ribosomal Fraction and  Responsible for Ribosome Degradation 1. Detection of the enzyme From the earlier experiment that demonstrated the destructive effect of heat on the ability of ribosomes to stimulate oxygen uptake of the soluble cytoplasm, i t was concluded that an enzyme was associated with the ribosomes and was responsible for their initial degradation during endogenous respiration. When the ribosome fraction was incubated in tris buffer (pH 7 .4) there was an increase in cold PCA soluble material which showed maximum absorption at 260 mu. EDTA completely inhibited this increase in UV absorbing material which tends to exclude the presence of a ribonuclease and the "VM route of ribosomal breakdown as described in E. coli (Wade, I96D, as RNase is activated by EDTA (Wade and Robinson, 1963). The addition of inorganic phosphate to the reaction mixture resulted in a 21-fold increase in activity, strongly Implicating polynucleotide phosphorylase (Grunberg-Manago and Ochoa, 1955) as the degradative enzyme (Figure 18). Almost complete inhibition of the release of cold PCA soluble 260 mu absorbing material was brought about by a magnesium concentration of 6 x 10 M . As ribonuclease is inhibited by Mg++ (Wade, I96D and as -221--123-polynucleotide phosphorylase requires Mg++ for activity (Grunberg-Manago and Ochoa, 1955)> these results appeared to be conflicting. 2. Identification of the enzyme In order to finalize the identity of the enzyme as either RNase or polynucleotide phosphorylase, ribosomes were incubated with radioactive inorganic phosphate. The cold PCA soluble UV absorbing material was examined for the presence of charcoal absorbable P32 which would be present as purine or pyrimidine nucleoside diphosphates, with label in the terminal phosphate group. The presence of the labeled nucleoside diphosphates would occur only i f poly-nucleotide phosphorylase was the enzyme involved. A comparison of the amount of radioactivity and optical density found in the charcoal eluates is shown in Table 20 and demonstrates that the enzyme was, ln fact, Table 20. Radioactivity and UV absorbing material in charcoal eluates Optical Counts/min/ml density/ml Control After 3 hr starvation 300 800 1.00 9-45 124-polynucleotlde phosphorylase. This enzyme has been reported previously to be associated with the ribosomal fraction of P. aeruginosa (Strasdine, Hogg and Campbell, 1962). The breakdown of endogenous RNA by polynucleotide phosphorylase has been shown to occur in other organisms (Wade and Lovett, 196lj Ogata, Imada and Nakao, 1962), and this, rather than the biosynthesis of RNA, may be the true function of the enzyme. Wade and Robinson (1963) found polynucleotide phosphorylase to be present in, and RNase to be absent from, the ribosomal fraction of P. fluorescens. 3. Ribosomal location of the enzyme The 70S, 50S and 30S ribosomes were isolated and examined for polynucleotide phosphorylase activity to determine whether or not the enzyme was associated with a particular ribosomal component. The soluble cytoplasm (see Table 16) was also examined, under conditions where 70S ribosomes were isolated and under conditions where they were dissociated Into 50S and 30S ribosomes, in order to detect a possible solubilization of the enzyme during the dissociation of the 70S particle. The release of cold PCA soluble UV absorbing material by the various ribosomes when incubated with Pi is shown in Figure 19. The 50S ribosomes and the 70S ribosomes, when incubated without added Mg++, exhibited marked polynucleotide phosphorylase activity whereas the 30s OPTICAL DENSITY p ro o p 01 o c JO m - H CD O 3 * — 2. O O Q s A O. O ro o ro o ro oo o | » U o • x : 8 < s _ o -"J ^ o o CO CO • o c «? < s §. 3 % • o cr 3 Q -Set-126-component demonstrated only slight activity. No increase in 260 mu absorbing material was found when either the 5OS-3OS or the 70S soluble cytoplasm was Incubated with RNA. As the ribosome suspensions had been dialyzed against either IO""4 M or IO*"2 ffi Mg++ to maintain the integrity of the desired ribosomal particle, the magnesium concentration in the various test reaction mixtures was not constant. Table 21 records Table 21. Magnesium concentration of reaction mixtures and change in optical density at 260 mu of released cold PCA soluble material Reaction mixture Mg++ cone. A O.D. at 260 mu 70S ribosome 2 x 10-3 0.285 8.8 x 10-3 0.000 50S ribosome 2 x io"? 0.658 30S ribosome 2 x IO"? 0.086 50S + 30S ribosomes 4 x i o -* 0.702 70S supernatant + RNA 5 x 10-5 -0.084 50S + 3OS supernatant + RNA 5 x io-5 -0.100 + 50S ribosome 3 x io-5 0.546 + 30S ribosome 3 * 10 ' 0.073 -127-the magnesium concentration and the change in the release of cold PCA soluble 260 mu absorbing material for the test solutions employed. The 70S ribosome exhibited no enzyme activity in 8.8 x 1G"3 M Mg++ whereas a significant increase in optical density was evident at the lower Mg++ concentration which tended to allow dissociation of the 70S component. The small amount of activity associated with the 30S ribosome, in all probability, may be regarded as a contamina-tion of this ribosomal peak. The soluble cytoplasm did not exhibit polynucleotide phosphorylase activity either under conditions such that the 70S ribosome was isolated intact or under conditions where i t was allowed to dissociate. This shows that the enzyme is attached to the 70S intact unit in an inactive form and on dissociation of this unit the enzyme is not solubilized but remains firmly bound to the JOS subunit, the RNA of which serves as a substrate. These data are consistent with the results obtained from sedimentation analyses of cell-free extracts of starved and unstarved cells, which demonstrated a greater decrease in the JOS component than In the 30S component during endogenous respiration. These data also clarify the previous seemingly anomalous results obtained when the ribosome fraction was incubated in a high magnesium concentration. The incubation of either RNA or ribosomes with the soluble cytoplasm in all cases resulted in an apparent decrease -128. in the release of UV absorbing material. The soluble cytoplasm of P. aeruginosa, however, contains an active adenosine deaminase as measured by the decrease in optical density at 2^8 mn of reaction mixtures containing soluble cytoplasm and adenosine. The adenosine deaminase may be responsible for the observed decrease in optical density found in the presence of the soluble cytoplasm. Bison (1959) showed that essentially all of the RNase activity and some of the DNase activity of cell-free extracts of E. coli were associated with the ribonucleoprotein. The enzymes were inactive while ribonucleoprotein remained intact but were activated through their release from ribosomes by either trypsin or urea treatment of the ribonucleoprotein particles. When the 70S ribosomes were allowed to dissociate, nearly a l l of the RNase activity was associated with the 30S ribosomes and the low RNase activity in the 50S components was attributed to the presence of small amounts of contaminating 30S ribosomes (Elson and Tal, 1959* Spahr and Hollingworth, I 9 6 I ) . The ribosomal DNase of E. coli, approximately 10 per cent of the total DNase of the cell, was found to be associated with 100S and 70S ribosomes but not 30S or 50S ribosomes. Dissociation of the large ribosomal particles, as effected by a lowered magnesium concentration, caused the release of DNase, some protein and RNA from the particles. When the magnesium concentration was increased, the dissociated -129-ribosomes reassoeiated and the previously released RNA, protein and DNase became attached to the ribosomes (Tal and Elson, 196lj Tal and Elson, 1963). Spahr and Hollingworth (I96I) demonstrated that a l l of the activity of an acid phosphatase, which cleaved inorganic phosphate from (^-glycerol phosphate and fruetose-1,6-diphosphate, was in the pure ribosomes of E. coli. The phosphatase was associated with both the 3OS and the 5QS ribosomes with twice as much activity/mg of ribosome in the 30S component as in the $0S component. VII. The Influence of Exogenous Substrates on Endogenous  Respiration The endogenous metabolism of microorganisms supplies the energy required for maintenance of viability under starvation conditions. In conditions of nitrogen starvation, it has been shown that endogenous turnover of RNA and protein supplies amino acids and nucleotides that may be utilized for biochemical differentiation (Mandelstam and Halvorson, i960); but, theoretically, under these conditions essentially a l l the energy is provided by the easily accessible exogenous carbon source. Wilkinson ( I 9 6 3 ) stated that cells that are in the stationary phase due to a nitrogen deficiency, are under conditions equivalent to those found in washed cell suspensions provided with a suitable carbon and energy source. This Implies either that during oxidation of exogenous energy -130' substrates no net decrease in RNA or protein takes place or that during turnover in resting cells, there is, in fact, a net decrease in these cellular constituents. It should be noted that, in general, studies involving RNA and protein turnover in resting cells have not included measurements of C14-02 evolution from the Cl^-labeled amino acids, purines or pyrimidines used as indicators of turnover. The effect of exogenous substrates on the endogenous respiration of P. aeruginosa was followed by labeling cells with C*4* growth substrates and measuring C1402 evolution from organisms respiring in the presence and absence of exogenous substrates. 1. The effect of growth conditions, an excess of carbon and energy supply, substrate concentration and ammonium ions Washed cells, previously grown with glueose-U-cl4 were resuspended at the growth concentration of 20 hr cells in 0.1 M tris buffer (pH 7.4). Cells were also resuspended in concentrated glucose-ammonium salts medium which would support two complete cell divisions, and in 0.1 M tris buffer (pH 7.4) containing the same amount of glucose as the concentrated growth medium (33 uM/ml). The cell suspensions were placed in large Warburg vessels and Cl402 evolution was determined after a 2 hr period of respiration. The Cl40o evolution, expressed as per cent of total label in the -131-Warburg vessel, is shown in Table 22. Table 22. Release of c l 4 02 from cells respiring in various suspending fluids C 1 4 0 2 as % of Suspending fluid cl4(>2* endogenous c l 4 02 0.1 H tris buffer (pH 7.4) 2.44 Glucose-salts medium 0.16 6.84 0.1 M tris buffer (pH 7.4) plus 33 uM glucose/ml 0.80 32.80 •Expressed as per cent of total c l 4 in the Warburg vessels Under the conditions which would support growth, a decrease in Cl 40o evolution of 93 P©r cent, as compared with the endogenous value, was observed indicating that during growth of this organism, the endogenous substrates contribute only a minor amount of oxidizable materials. With an excess of carbon and energy but no nitrogen source, the cells released one-third as much labeled CO2 as did the cells respiring endogenously. This suggests that although the cells were provided with an adquate carbon and energy supply, the nitrogenous endogenous substrates of the organism were oxidized possibly as the result of the release of a control -132-mechanism subsequently supplying the ammonia required for the assimilation of glucose intermediates as nitrogenous compounds. The assimilation of endogenously produced ammonia in the presence of exogenous glucose has been shown to occur with P. aeruginosa (Warren et al., i960). The amount of glucose used in these experiments was considerably In excess of the concentrations of substrates generally employed in manometrlc studies involving oxidation of carbohydrates. The effect of various concentrations of exogenous glucose was examined as well as the Influence of ©<-ketoglutaric acid. Oxidative assimilation studies with this organism have suggested that ammonia is the factor which determines the rate of assimilation of carbon into the cell since c<-ketoglutarate accumulated in the supernatant fluids during oxidation of glucose, but did not accumulate when ammonia was added. Also the organism was shown to contain a strong glutamic dehydrogenase indicating that carbon was assimilated by way of glutamic acid (Duncan and Campbell, 1962). Substrates were added to small Warburg vessels containing 1 ml of cells which had been uniformly labeled by growth on glucose-U-C14*. The cells were suspended at 10 times the growth concentration of 20 hr cells and C 1 4 ^ evolution was measured after a 2 hr period of respiration. Increasing glucose concentrations caused a depression of the oxidation of endogenous cellular constituents (Table 23). •133-Table 23. Influence of ot-ketoglutarate and Increasing concentrations of glucose on endogenously produced Cl4()2 M . C l 4 ° 2 as % J J Substrate Cr 02* endogenous C^M^ Endogenous 3»38 Glucose 5 uM 2.90 85.8 15 uM 2.48 73.4 25 UM 2.41 71.3 <X-ketoglutarate 15 uM 3.67 109.0 Glucose 5 uM plus 5 uM cHcetoglutarate 3.3O 97.6 •Expressed as per cent of total C 1 4" in the Warburg vessels These results are contrary to the conclusions drawn by Norris et al. (1949) who measured oxygen uptake using a standard cell concentration and varying glucose and acetate concentra-tions, also using a standard glucose concentration and varying cell concentration. They calculated the percentage of theoretical oxygen uptake before and after subtracting endogenous oxygen consumption and found that with low substrate concentrations, values in excess of 100 per cent of theoretical were obtained i f endogenous oxygen uptake was not corrected for. 134-However, if endogenous values were subtracted similar results for percentage of theoretical were realized for all substrate concentrations. Norris et al. concluded, therefore, that endogenous respiration functions normally in the presence of exogenous substrates. On reexamination, however, their data showed that as the exogenous substrate concentration increased, lower values were obtained for the percentage of theoretical oxygen uptake which demonstrated, in fact, that as substrate concentration increased, endogenous respiration decreased. Using 5 uM of glucose as substrate and increasing cell concentrations, Norris and coworkers actually recorded increasing values for percentage of theoretical oxygen consumption. This procedure served to dilute the amount of substrate/cell and thus showed that decreasing exogenous substrate concentrations coincided with increases in endogenous respiration. When <*-ketoglutarate was used as the exogenous substrate, an increase in endogenously produced C^02 was observed (Table 23). Oxidation of c<-ketoglutarate by P. aeruginosa occurs in two stages: the first involves a significant but slow rate of oxidation, that is, a lag period, and the second stage involves a considerably more rapid rate of oxidation. The Initial lag period is a manifestation of induced permease synthesis (von Tigerstrom and Campbell, unpublished data). The increase in endogenous respiration in the presence of added o^-ketoglutarate, then, may be a result -135-of the increase in energy required for synthesis of the permease, or a result of the release of a control mechanism, previously suppressing ammonia production, but now allowing the keto acid to be assimilated or, more likely, a combination of both processes. The presence of <*-ketoglutarate and glucose together as exogenous substrates gave results in between those obtained with the substrates individually. These results demonstrate that endogenous respiration in P. aeruginosa is influenced by both the nature and the concentration of the exogenous substrates. The effect of added ammonia and of glucose and <*-ketoglutarate, with and without added ammonia, on endogenous respiration was shown by incubating cells, which had been grown on glucose-TJ-C14, with 15 pM of exogenous substrate In the presence and absence of 15 uM of ammonia. Ammonium ion itself depressed endogenous C l 4 ^ evolution by 11 per cent but promoted an even greater decrease when Incubated in conjunction with either glucose or -ketoglutarate (Table 24). These results illustrate that when exogenous substrates are present, endogenous respiration functions, in part, to supply ammonium ions required for carbon assimilation but i t also must function in order to supply energy essential to the cells. This implies either that the concentration of the exogenous energy source was not sufficient to f u l f i l the energy requirements of the cells or that the exogenous -136-Table 24. The effect of ammonia on endogenous cl402 production in the presence and absence of exogenous glucose and «-ketoglutarate G1402 as % of C1402 as % _ endogenous °f endogenous Substrate C 1 40 2* C14Q2 plus NH3 C 1 40 2 Endogenous 3.62 Endogenous + NH3 3.22 89.O Glucose 2.50 69.O Glucose + NH3 1.99 55.0 62.0 o<-ketoglutarate 4.08 113.0 o(-ketoglutarate + NH3 3.31 91.5 103.0 •Expressed as per cent of total CI 4 in Warburg vessels substrates initially impose additional energy requirements in the form of enzyme synthesis and possibly messenger RNA synthesis. 2. Influence on RNA and protein oxidation Cells were labeled with uracil-2-Cl 4 or proline-!!- C l 4 during growth and subsequent C i 4 0o evolution was measured during the 2 hr period of incubation to establish i f the depression of endogenous respiration due to exogenous glucose, 137-and the increase in endogenous respiration due to the presence of c<-ketogluturate, were concerned with the endogenous substrates in general or selectively affected either the oxidation of RNA or of protein. Aspartic acid and adenosine were also used as exogenous substrates and 5 yM of the substrates were added to 1 ml of cells (5 mg dry weight/ml). Several Warburg vessels containing identical reaction mixtures were prepared so that any changes that occurred in the oxidation of endogenous substrates, with time, could be observed during the incubation period. The oxygen consumption of cells incubated with the various exogenous substrates is illustrated in Figure 20. Table 25 shows that glucose caused a 6 per cent depression of protein oxidation and a 47 per cent inhibition of RNA oxidation at 2 hr. It has often been suggested that exogenous substrates may appear to inhibit the oxidation of endogenous reserves merely by a dilution effect i f common intermediates are involved. As the intermediates in amino acid degradation are, in general, tricarboxylic acid cycle components, then by the above reasoning a greater suppression of protein oxidation than RNA oxidation would be expected, however, the reverse situation occurred. The suppression of RNA degradation, therefore, was the consequence of some other sequence of events. A considerable increase in protein oxidation was evident during the first 10 min of substrate oxidation and, -138-FIGURE 20. Oxygen uptake with various exogenous substrates Table 25. The influence of exogenous glucose on the oxidation of endogenous protein and RNA Cells labeled with Proline-U-C14 Uracil-2-C 1 4 Time Endogenous Glucose % of endogenous C I ?0 2 Endogenous Glucose % of endogenous c»o 2 min % cl4-02* % c i 4 o 2 % Cl 40 2 % c i 4 o 2 0 - 1 0 O.38 0.61 l60 O.76 O.36 47 0 - 3 0 1.20 0.97 81 1.66 0.76 46 0 - 6 0 I.89 1.48 78 3.05 1.81 59 0 - 120 3.29 3.10 94 4.80 2.55 53 0 - 1 0 O.38 0.61 160 O.76 O.36 47 10 - 30 0.82 O.36 44 O.90 0.40 44 30-60 O.69 0.51 74 1.39 1.05 75 60 - 120 1.40 1.62 116 1.75 0.74 42 I H KM vO I •Percentage of total C 1 4 in the Warburg vessel 140-as previously mentioned, this may have been the result of increased ammonia production or enzyme synthesis. It should be mentioned that during this 10 min interval, there was a decrease in total W absorbing material in the Warburg supernatant fluid, which contained both amino acids and RNA degradation products (see Table 12), to a value below that of the zero time level. The amount of protein oxidized showed a marked drop at 10-30 min, at which time glucose was being rapidly oxidized, and then showed a gradual increase relative to the endogenous C 1 40 2 at 30-120 min, at which time glucose was depleted and <<-ketoglutarate was being oxidized (Figure 20; Duncan and Campbell, 1962). The oxidation of RNA, while being consistently suppressed, did show some changes in the rate of oxidation with time. When oC-ketoglutarate was the exogenous substrate, endogenous C1402 evolution showed a 19 per cent elevation in amino acid oxidation and a 29 per cent suppression of RNA oxidation in 2 hr (Table 26). The depression of RNA oxidation was significantly less than that observed during glucose oxidation. The interval during which the oxidation of RNA was at its highest was the same as that interval during which the oxidation of amino acids was at its lowest. The observed increase in the amino acid oxidation was fairly constant with time and oxygen consumption was also fairly constant indicating that the exogenous substrate was not depleted during the 2 hr incubation. Table 26. The Influence of exogenous «-ketoglutarate on the oxidation of endogenous protein and RNA Cells labeled with Proline-U-C14 Uracil-2-C 1 4 Time Endogenous •< -keto-glutarate % of endogenous C K 0 2 Endogenous «-keto-glutarate % of endogenous 0^02 min % C 1 40 2* % C 1 40 2 % C 1 40 2 % C1402 0 - 1 0 0.50 0.55 110 0.43 0.20 47 0 - 30 1.00 1.16 116 0.93 0.57 61 0 - 6 0 2.18 2.26 104 2.14 1.62 76 0 - 120 3.10 3.68 119 3.46 2.46 71 0 - 1 0 0.50 0.55 110 0.43 0.20 47 10 - 30 0.50 0.61 122 0.50 0.37 74 30 - 60 1.18 1.10 93 1.21 1.05 87 60 - 120 0.92 1.42 155 1.32 0.84 64 •Percentage of total C1* in the Warburg vessel 142 It was thought that i f an amino acid was used as an exogenous nitrogenous substrate, a significant inhibition of endogenous protein oxidation would be evident. At the end of the incubation time, i t was found that the CJl^ Og evolution from proline-U-C'1-4' labeled cells was depressed by 16 per cent in the presence of aspartic acid. The greatest suppression occurred during the first 1G min interval (Table 27). As this organism contains aspartase (von Tigerstrom, unpublished data) i t is suggested that this initial decrease may be caused, in part, by the presence of an adequate ammonia supply in the form of the exogenous substrate. This situation was opposed to that observed with glucose, where endogenous protein oxidation Initially was markedly stimulated probably by the removal of ammonium ions which repress proteolysis. As aspartic acid enters the tricarboxylic acid cycle as fumarie acid and thus supplies an intermediate common to that provided by endogenous amino acid degradation, it may be argued that this is merely a "dilution effect" and not a true suppression of endogenous respiration. However, if any exogenous substrate depresses oxidation of endogenous materials by dilution through a common intermediate this does mean, in fact, that less endogenous materials will be oxidized since the exogenous substrate exhibits a sparing effect on the endogenous substrate and this, therefore, must be considered as a true suppression of endogenous respiration. Table 27. The influence of exogenous aspartic acid on the oxidation of endogenous protein and RNA Cells labeled with Proline-U-C14 Uracil -2-C 1 4 Time Endogenous Aspartate % of endogenous Cl4o 2 Endogenous Aspartate $ of endogenous cl4o 2 min % C 1 4 02* % c i 4 o 2 % C 1 4 0 2 % C 1 4 G 2 0 - 10 0.43 0.26 61 0.43 0.34 79 0 - 30 0.95 0.71 75 0.93 0.64 69 0 - 60 1.76 1.45 82 2.16 1.04 49 0 - 120 2.68 2.26 84 3.46 2.60 75 0 - 10 O.43 0.26 61 0.43 0.34 79 10 - 30 O.52 0.45 87 0.50 0.30 60 30 - 6o 0.81 0.74 91 1.21 0.40 30 60 - 120 O.92 0.81 88 1.32 1.56 118 •Percentage of the total Cl4 in the Warburg vessel - 1 4 4 -The total decrease in RNA oxidation was similar to that found when «*-ketoglutarate was substrate, i t should be noted, however, that with aspartate, a continual decrease in RNA degradation was evident during the first 60 min of incubation. Between 60 and 120 min, when the exogenous substrate was depleted (Figure 20) the RNA oxidation was greater than that observed in the absence of substrate. The interval at which RNA oxidation was at its lowest (30-60 min) coincided with the interval at which protein oxidation was at its highest. The pattern of protein oxidation in the presence of adenosine was similar to that observed during glucose oxidation in that a total depression of 8 per cent occurred during 2 hr and a marked stimulation was evident during the 0-10 min interval. The oxidation of RNA was depressed by 24 per cent and the amount of RNA oxidized showed a considerable variation at different time intervals when compared with endogenous Cl^Qg evolution in the absence of substrate. Again there appeared to be an interrelationship between the amount of RNA and the amount of protein oxidized at a given time (Table 28). Adenosine exhibited the least suppression of RNA oxidation but also supported the lowest rate of oxygen consumption. It is concluded, then, that during the oxidation of exogenous substrates by P. aeruginosa, endogenous respiration Table 28. The influence of exogenous adenosine on the oxidation of endogenous protein and RNA Cells labeled with Proline-U-C 14 Uracil-2-C 1 4 Time Endogenous Adenosine % of endogenous ci*o 2 Endogenous Adenosine % of endogenous C l 40 2 min % C 1 40 2* % C 1 40 2 % C 1 40 2 % C 1 40 2 0 - 1 0 0.24 0.37 154 0.24 0.17 71 0 - 3 0 O.83 0.73 88 0.78 0.72 92 0 - 6 0 1.56 1.43 94 1.72 1.20 70 0 - 120 2.61 2.41 92 3.08 2.34 76 0 - 1 0 0.24 G.37 154 0.24 0.17 71 10 - 30 0.59 0.36 61 0.54 0.55 102 30 - 60 0.73 0.70 96 0.94 0.48 51 60 - 120 1.05 O.98 93 1.36 1.14 84 •Percentage of GL* in the Warburg vessel -146 is not carried on at its normal rate. In general, RNA oxidation is suppressed and the degree of suppression appears to be correlated with the oxygen uptake, and hence energy production, provided by the exogenous substrate. The relative stability of RNA is no doubt directly proportional to the stability of the ribosomal particles which would be dependent on both the amount of available energy and the requirement of ribosomal structures for protein synthesis. The effect of exogenous substrates on endogenous protein oxidation appears to be more concerned with the nature of the exogenous material being oxidized, particularly with regards to the demand for ammonium ions essential for carbon assimilation. The amount of endogenous substrate oxidized varied considerably during various time intervals and an apparent correlation between the amount of protein or amount of RNA oxidized for a given time was noted, that is, a decrease in protein oxidation was accompanied by a relative increase in RNA oxidation. 3« Influence on the release of UV absorbing material The increase in UV absorbing material in Warburg supernatant fluids of cells respiring endogenously and in the presence of 5 of glucose, tA-ketoglutarate and aspartic acid was followed over a 2 hr interval. When glucose was the exogenous substrate, the UV absorbing material in the supernatant fluid decreased during the first 10 min and then, at 2 hr, returned to a value slightly higher than that -147-observed at zero time (Figure 21). It was during this 10 min period that the increase in C^Og evolution from C^-proline labeled cells was noted. As has been mentioned previously, the increase in UV absorbing material released from P. aeruginosa while respiring endogenously shows a decrease in the 280/260 ratio with time indicating that a greater proportion of RNA than of protein is released during starvation. In the presence of exogenous glucose only a slight decrease in this ratio was demonstrated (Table 29). This substantiates the C1402 data which showed that ribosomal material was relatively stable during glucose oxidation either because of the amount of energy gained from substrate oxidation and/or because of the requirement for ribosome structures for protein synthesis. Table 29. The influence of exogenous substrates on the 280/260 ratio of UV absorbing material in Warburg supernatant fluids Substrate Incubation time 280/260 ratio min Endogenous 0 0.650 120 0.464 Glucose 120 0*633 ot-ketoglutarate 120 0.510 Aspartate 120 O.58I -148-FI6URE 21. Influence of exogenous substrates on the release of UV absorbing material 149-With oOketoglutarate as the exogenous substrate, there was an increase in release of 260 mu absorbing material, slightly in excess of that of the 2 hr endogenous control and the 28G/26G ratio dropped to 0.510. As has been shown, ON-ketoglutarate stimulates endogenous protein oxidation and suppresses, to some extent, the oxidation of RNA. The RNA, however, is obviously subjected to a greater degree of degradation than is found during glucose oxidation. This again may be correlated with the low oxygen consumption and thus, energy production, demonstrated with cX-ketoglutarate. In the presence of aspartic acid, there was no increase in UV absorbing material in the Warburg supernatant fluids and the UV spectra of the fluids at various time intervals were generally similar, but not identical, to the zero time control as the 280/260 ratio decreased to O.58I at 2 hr. Even though the oxygen uptake, in the presence of this nitrogenous substrate, was relatively low i t is evident that RNA was more stable during oxidation of this substrate than during oxidation of <*-ketoglutarate. The polyamine, spermine, was shown to provide some inhibition of the release of 260 mu absorbing material. As spermine was found to be an oxidizable exogenous substrate, it is most probable that the apparent influence exerted on ribosomal stability should not be attributed to the basieity of the compound but rather to the fact that the compound was a carbon, nitrogen and energy source. 150. The influence of exogenous substrates on the release of endogenous UV absorbing material from P. aeruginosa varies considerably with the nature of the exogenous substrates. It is suggested, however, that those substrates which provide both a significant energy source and carbon intermediates that may be assimilated into nitrogenous cellular constituents will exhibit the greatest influence on the stability of ribosomes and, hence, of RNA. -151-GENBRAL DISCUSSION I. Ribosomal Material as an Endogenous Substrate 1. In P. aeruginosa As microorganisms are generally considered to oxidize carbohydrate, lipid or volutin as normal endogenous reserves, the oxidation of ribosomal material, at first, appears to represent a very abnormal situation. It would appear to be uneconomical from the point of view that the energy to be gained from the oxidation of RNA and protein is relatively less than that to be gained from the oxidation of carbonaceous reserves. Also, it would appear to be physiologically unsound in that it suggests that a loss of viability would possibly be encouraged by the degradation of the "essential" cellular constituents. As ribosomal degradation is initiated by polynucleotide phosphorylase, the immediate products of the degradation are nucleoside diphosphates. P. aeruginosa contains an active adenylate kinase (Strasdine, 1961) which effectively removes ADP by the following reaction 2 ADP^=^AMP + ATP (6) thus supplying the organism with an available high energy compound. The non-specific interconversions of nucleotides -152-by transphosphorylation reactions is known to occur in micro-organisms. By such a mechanism the purine and pyrimidine nucleoside diphosphates would be converted to nucleoside monophosphates and nucleoside triphosphates, thus stimulating polynucleotide phosphorylase activity by removing the products of the degradative enzyme. The sequential activity of nucleotidases and nucleosidases would yield free bases, ribose and inorganic phosphate—with inorganic phosphate stimulating polynucleotide phosphorylase activity. The organism contains a ribokinase which would allow the pentose to enter the pentose phosphate cycle (Gronlund, 1961). The enzymes for nucleoside, purine and pyrimidine oxidation were shown to be constitutive in the organism and the presence of enzymes concerned with the oxidation of purines has been shown in other strains of P. aeruginosa (Campbell, 1955; Franks and Hahn, 1955; Bergmann et al., 1962). The degradation of pyrimidines in P. aeruginosa 9027 was demonstrated also by the release of G14"02 from uracil-2-C 1 4, probably either through hydrolysis of @-ureidopropionic acid or through barbituric acid with subsequent hydrolysis of urea. No attempt was made to determine i f transfer or messenger RNA served as endogenous substrates is. P. aeruginosa. Midgley and McCarthy (1962) have shown that messenger RNA has an average lifetime of 2.5 min and that degradation products may be incorporated into both DNA and -153-transfer MA. It was estimated that messenger RNA accounts for one per cent of the total RNA present in exponentially growing cells. It is suggested, then, that messenger RNA may play a small role as an endogenous substrate but i t may be more intimately concerned with the observed increase in DNA during endogenous respiration. The presence of polynucleotide phosphorylase firmly bound to the 50S ribosomes is, no doubt, a control mechanism. Under conditions where the need for structural ribosomes is at a minimum and the cell does not have an adequate energy source, the 70S particles dissociate and consequently activate the degradation of ribosomal material which provides either energy and/or required nitrogenous intermediates. While particulate ribosomal protein was shown to decrease during starvation, an increase in the protein content of the soluble cytoplasm was noted, indicating that ribosomal protein had been solubilized. Therefore, conclusive evidence has not been presented to substantiate the suggestion that the ribosomes provided the endogenous protein substrate. It is reasonable to suggest, however, that ribosomal protein is at least a part of the proteinaceous endogenous substrate. The data obtained from incubating variously labeled cells with exogenous glucose showed that RNA oxidation was suppressed to a considerably greater extent than was protein oxidation, suggesting a relative stability of ribosomes. This may indicate that cellular proteins, other than ribosomal -154-protein, may serve as endogenous substrates. Preliminary data, not presented here, has demonstrated that an enzymatic autodegradation of the isolated membrane fraction takes place fairly readily. Brown (1962) also has shown the proteolytic autolysis of the isolated "cell envelopes*1 of a marine pseudomonad. 2. As a general phenomenon in microorganisms Numerous reports in the literature have shown that UV absorbing material is released into the suspending fluid during the endogenous respiration of microorganisms and, in many Instances, ammonia evolution has been noted. A decrease of RNA and/or protein has been shown to occur in several microorganisms during starvation. Bacterial cytologists have noted that a partial starvation of bacteria depletes the cytoplasmic RNA (Braun, 1957; Wilkinson and Duguid, i960). Hoagland (i960) stated that there is a direct association between nucleic acid and protein synthesis in that the rate of protein synthesis is dependent upon the number of ribosomes present in the cells. Kjeldgaard (1961) found that in Salmonella typhimurlum, the ribosomal RNA showed a very pronounced variation with the growth rate. Ecker and Schaechter (1963) demonstrated that the ribosome concentration in S. typhimurium, expressed as the ratio of ribosomes to soluble protein, was a direct and linear function of the growth rate. Induced enzyme formation in A. aerogenes 155-proceeded at rates proportional to the concentration of ribosomal RNA rather than to that of DNA or protein (Kennell and Magasanik, 1962). When cells respire endogenously a situation of absolute minimal protein synthesis must exist. As the rate of protein synthesis is proportional to the number of ribosomes, under starvation conditions ribonucleoprotein is in excess and presents a logical endogenous substrate capable of supplying the maintenance energy requirements of the cell. The ribosome concentration would appear to be more than adequate to serve this function as well as acting as a source of free amino acids and nucleotides required for the synthesis of enzymes. Mandelstam and Halvorson (i960) concluded that the protein and RNA of non-growing E. coli undergoes a balanced degradation and resynthesis at about 5 per cent per hour. These workers did not detect a net loss of either protein or RNA, however, no measurements of Cl -^Cv) evolution were made. Generally, conditions of nitrogen starvation have been employed in experiments involving the measurement of turnover of cellular constituents. Under these same conditions, the "normal" endogenous reserves such as carbohydrate and lipid have been found to accumulate. Therefore, i t may be assumed that during the accumulation of these reserves turnover of RNA and protein also occurs. 156-Herbert (I96D stated that i f resting cells are transferred to a fresh medium, an exponential increase in dry weight begins almost immediately but there is no increase in cell numbers for a considerable length of time. During this lag period there is a rapid increase in cellular RNA content until the value reaches that of log phase cells and then dry weight and cell numbers increase at the same rate. This infers that in resting cells, where protein synthesis is minimal, the ribosome content is reduced considerably. Cells of B. megaterium, obtained from spores germinating in nitrogen deficient media, were enlarged, contained refractile inclusions, were deficient In total nitrogen and RNA and were high in saponlfiable lipid (Sail, Mudd and Payne, 1957)* Mutants of N. crassa grown with limiting essential nitrogenous nutrients degraded RNA when growth was halted by exhaustion of the required nutrient (Harold, i960). Resting cells of S. cerevisiae, from a nitrogen deficient medium, showed no distinct sedimenting boundaries of microsomes. It appeared that the microsomes present in resting cells underwent an appreciable degradation which was probably related to the excretion of peptides and other nitrogenous materials. It was suggested that the lag in the onset of active growth on reincubating resting cells in fresh medium may be associated with the depletion of the microsomes (Cooper, Harris and Neal, 1962). 157-The preceding data strongly suggest that during the accumulation of "normal" endogenous reserves, turnover of RNA and protein takes place and the individual cells become deficient in these nitrogenous cellular constituents, with no apparent loss of total RNA or protein from the cultures. The balanced turnover of RNA and protein at 5 per cent/hr may mean that an actual increase in cell numbers of 5 per cent/hr takes place with the increase in turbidity of cultures being attributed solely to the accumulation of carbonaceous reserves. Under conditions of prolonged nitrogen starvation where endogenous carbon reserves may account for 50 per cent of the dry weight of the cells, the ribosome content and the total nitrogen/cell would be at a minimum level. When subjected to starvation, such organisms would be unable to maintain viability after their carbonaceous reserves had been depleted as the most readily oxidizible nitrogenous cellular constituents already would have been depleted by dilution. It is suggested that in microorganisms capable of oxidizing endogenous RNA and protein the accumulation of large amounts of carbonaceous endogenous reserves does not give the organisms a biological advantage in survival under starvation conditions. Such an accumulation, in fact, would be a biological disadvantage in that induced enzyme formation would not be favored due to the previous exhaustion of nitrogenous reserves. If carbonaceous reserves accumulate in microorganisms under conditions where the nitrogenous 158. constituents of the cells do not become deficient, then such an accumulation would be advantageous under starvation conditions. As ribosomes constitute 30-40 per cent of the cell mass (Luria, i960) and as the cell's requirement for protein synthesis is at a minimum during starvation, it is suggested that ribosomal material presents an adequate energy source for energy of maintenance during endogenous respiration. Further, ribosomes represent an excellent endogenous reserve as, in addition to energy, they provide the cells with amino acids and nucleotides which allow a high degree of adaptability of organisms in their ever-changing natural environment. The degradation of endogenous nitrogenous compounds has been reported to occur in mammalian tissue slices. The degradation of protein by rat liver and kidney slices has been noted (Simpson, 1953; Steinberg and Vaughan, 1956) and the evolution of endogenously produced ammonia from guinea pig brain cortex slices was observed (Takagakl, Hirano and Tsukada, 1957). II. The Influence of Exogenous Substrates on Endogenous  Respiration The instability of ribosomes during endogenous respiration is, no doubt, partially due to the conditions of magnesium starvation imposed on the organism. When C14-labeled cells were allowed to respire endogenously in the -159-presence of 3.33 x IO"*2 M Mg++, less UV absorbing material •was excreted into the suspending fluid but only a 12 per cent reduction in C l ^ release from uracil-2-C 1 4 labeled cells was observed. Therefore, magnesium starvation played a small but significant role in the dissociation of ribosomes under the conditions employed. The suppression of RNA oxidation by exogenous substrates, in general, appea_red to be related to the amount of energy made available to the cells through the oxidation of the exogenous substrate, the degree of ribosome stability being directly proportional to the amount of energy produced. The effect of exogenous substrates on endogenous protein oxidation appeared to be correlated with the nature of the exogenous substrate and the ammonium ion requirement for carbon assimilation. The increase in protein oxidation observed during the oxidation of exogenous °<-ketoglutarate may possibly be explained by the removal of endogenously produced ammonia through the reductive amination of <<-ketoglutarate by glutamic acid dehydrogenase and the subsequent decrease in the inhibitory effect of ammonium ions on proteolytic enzyme activity. P. aeruginosa also carries out a broad range of transamination reactions Involving o<-ketoglutarate (McQuillan, 1958) so that through transamination and assimilation, the exogenous substrate may have been spared at -160-the expense of endogenous amino acids. A further factor contributing to the increase in endogenous protein oxidation may be the energy required for the synthesis of the induced permease for o\>ketoglutarate. It is suggested that exogenous substrates which undergo patterns of metabolism generally similar to those of the substrates studied would produce the same overall influence on endogenous respiration in P. aeruginosa. However, i f an organism uses carbonaceous reserves as its primary endogenous substrate, then the influence of exogenous substrates on the oxidation of these reserves would be dependent, firstly, on the amount of energy gained from oxidation of the exogenous substrates and secondly, on the interrelationships of the metabolic pathways involved. -161-Su"MMARY Chemical analyses of starved cells of P. aeruginosa revealed a significant decrease in RNA, a slight increase in DNA and inorganic phosphate and a net decrease in total nucleic acid and protein. During endogenous respiration, radioactive carbon dioxide was evolved from cells labeled specifically in protein with proline-U-C14 or in RNA with uracil-2-C 1 4, thus supporting the chemical evidence for the oxidation of RNA and protein. Starved cells showed a decrease in radioactivity in hot TCA-soluble material and hot TCA-insoluble material indicating that the C^Oo arose from the degradation of high molecular weight compounds and not from "free pools'* of radio-active substrates. The presence of magnesium or phosphate ions during starvation created a slight change in the ratio of protein to RNA oxidized. Physical fractions of variously labeled starved cells showed a substantial decrease in ribosomal protein and RNA implicating the ribosomes as a major endogenous substrate. Sedimentation analyses of cell-free extracts of starved cells demonstrated a net decrease in ribosomal material. Oxygen consumption and ammonia evolution from cell-free extracts was found to be attributed mainly to the ribosome fraction, thus -162-confirming the oxidation of ribosomal material during endogenous respiration in P. aeruginosa. The polynucleotide phosphorylase associated with the ribosomal fraction of this organism was shown to be inactive under conditions in which the 70S particles remained intact. When the 70S ribosomes were dissociated, the enzyme was found to be firmly bound to the 50S subunit, the RNA of which served as a substrate for enzyme activity. During the oxidation of exogenous glucose, <*-keto-glutarate, aspartic acid and adenosine, varying degrees of suppression of endogenous RNA oxidation were observed and attributed to the stabilizing effect of the exogenous substrates on the ribosomes. Protein oxidation in the presence of <<-ketoglutarate was increased and was decreased in the presence of the other three exogenous substrates. The influence of exogenous substrates on protein oxidation appeared to be related, at least in part, to the ammonium ion requirements imposed on the cell by the individual exogenous substrate. 163-BIBLIOGRAPHY Andrejew, A. 194-8. L'utilisation par oxidation de quelques substances azbtees par le bacille de Koch. Ann. Inst. Pasteur 74:464. Ayers, W.A. 1962. The influence of cobamides on the endogenous and exogenous respiration of a marine bacterium. Can. J. Microbiol. 8:86l. Bachrach, U. 1957. The aerobic breakdown of uric acid by certain pseudomonads. J. Gen. Microbiol. 17:1. Baddiley, J., J.G. Buchanan, B. Carss, A.P. Mathlas, and A.R. Sanderson. 1956. The isolation of cytidine diphosphate glycerol, cytidine diphosphate ribltol and mannitol-1-phosphate from Lactobacillus arabinosus. Biochem. J. 64:599. Barker, H.A. 1936. The oxidative metabolism of the colorless alga, Prototheca zopfli. J. Cell. Comp. Physiol. 8:231. Bergmann, F., H. Ungar-Waron, H. Kwietny-Govrin, H. Goldberg, and S. Leon. 1962. Some specific reactions of the purine-oxidizing system of Pseudomonas aeruginosa. Biochim. Biophys. Acta 55*5l2"T Bernheim, F., W.E. DeTurk, and H. Pope. 1953. The nitrogen metabolism of certain strains of Mycobacterium tuberculosis J. Bacteriol. 6^ :544. Bluraenthal, H.J., H. Koffler, and E.P. Goldschmidt. 1952. The rate of endogenous respiration as affected by the oxidation of exogenous substrates. Science 116:475. Blumenthal, H.J., H. Koffler, and B.C. Heath. 1957. Biochemistry of filamentous fungi. V. Endogenous respiration during concurrent metabolism of exogenous substrates. J. Cell. Comp. Physiol. 50*471. Bonner, J. 1961. Structure and origin of the ribosome, P« 323-335. In R.J.C. Harris (ed.), Protein biosynthesis Academic Press, Inc., London. Borek, E., L. Ponticorvo, D. Rittenberg. 1958. Protein turnover in microorganisms. Proc. Natl. Acad. Sci. U. S. 44:364. -164-Borek, E., A. Ryan, and J. Rochenbach. 1955. Nucleic acid metabolism in relation to the lysogenic phenomenon. J. Bacterid. 69:460. Bowen, T.J., S. Dagley, and J. Sykes. 1959. A ribonucleo-protein component of Escherichia coll. Biochem. J. 72*419. — Braun, W. 1957. Bacterial genetics, p. 38. W. B. Saunders Co., Philadelphia. Britten, R.J., and R.B. Roberts, i960. High resolution density gradient sedimentation analysis. Science 131*32. Brown, A.D. 1962. The peripheral structures of Gram-negative bacteria. III. Effects of cations on proteolytic degradation of the cell envelope of a marine pseudomonad. Biochim. Biophys. Acta 62:132. Burleigh, I.G., E.A. Dawes, and D.W. Ribbons. 1963. Endogenous metabolism and survival of Sarcina lutea. Biochem. J. 88:30 p. Campbell, L.L. 1955. Oxidative degradation of uric acid by cell extracts of a Pseudomonas. Biochim. Biophys. Acta 18:160. Campbell, J.J.R., L.A. Hogg, and G.A. Strasdine. 1962. Enzyme distribution in Pseudomonas aeruginosa. J. Bacteriol. 83:1155. Chargaff, E., and D.H. Moore. 1944. On bacterial glycogen: the isolation from avian tubercle bacilli of a poly-glucosan of very high particle weight. J. Biol. Chem. 155:493. Chester, V.E. 1959. Endogenous metabolism of freshly harvested cells of a brewer*s yeast. Nature 183:902. Chester, V.E. 1963. The dissimilation of the carbohydrate reserves of a strain of Saccharomyces cerevisiae. Biochem. J. 86:153. Clifton, C.E. 1963 a. Oxidative assimilation by Bacillus  megaterium. J. Bacteriol. 85:1365. Clifton, C.E. 1963 b. Influence of growth medium on assimilation activities of Escherichia coli. J. Bacteriol. 85:1371. 165< Clifton, C.E., and J.M. Sobeck. 1961. Endogenous respiration of Bacillus cereus. J. Bacteriol. 82:252. Clifton, C.E., and J.M. Sobek. I96I. Oxidative assimilation of glucose and endogenous respiration of Bacillus cereus. J. Bacteriol. 81:284. Cohen, S.S., and J. Lichenstein. i960. Polyamines and ribosome structure. J. Biol. Chem. 235:2112. Conway, E.J. 1950. Microdiffusion analysis and volumetric error, 3 r <* ed. Crosby, Loekwood and Sons Ltd., London. Cooper, A.H., G. Harris, and G.E. Neal. 1962. The incorporation of labeled amino acids into subcellular fractions from brewer's yeast. Biochim. Biophys. Acta 61:573. Cosgrove, W.B. 1959. Utilization of carbohydrates by the mosquito flagellate, Crithidia faciculata. Can. J. Microbiol. jf:573-Dagley, S., and A.R. Johnson. 1953. The relation between lipid and polysaccharide contents of Bacterium coll. Biochim. Biophys. Acta 11:158. Dawes, E.A., and W.H. Holms. 1958. Metabolism of Sarcina  lutea. III. Endogenous metabolism. Biochim. Biophys. Acta 30:278. Dawes, E.A., and D.W. Ribbons. 1962. Endogenous metabolism of Escherichia coll. Biochem. J. 82:49 p. Dawes, E.A., and D.W. Ribbons. 1962. Effect of environmental conditions on the endogenous metabolism of Escherichia  coll. Biochem. J. 84:97 p. Deinema, M.H. 1961. Intra- and extra-cellular lipid production by yeasts. Meded. Landbouwhogeschool, Wageningen 6l:l. DeLamater, E.D., K.L. Babcock, and G.R. Mazzanti. 1959. On leakage of cellular material from Bacillus megaterium. J. Bacteriol. 2Z :?13-Doudoroff, M., and R.Y. Stanier. 1959. Role of poly- (5-hydroxybutyric acid in the assimilation of organic carbon by bacteria. Nature 183:1440. Duguid, J.P., I.W. Smith, and J.F. Wilkinson. 1954. Volutin production in Bacterium aerogenes due to development of an acid reaction. J. Pathol. Bacteriol. 6£:289. -166-Duguld, J.P., and J.F. Wilkinson. 1953. The influence of cultural conditions on polysaccharide production by Aerobacter aerogenes. J. Gen. Microbiol. %}UJ4-. Duguid, J.P., and J.F. Wilkinson. 1961. Environmentally induced changes in bacterial morphology. Symp. Soc. Gen. Microbiol. 11:69. Duncan, M.G., and J.J.R. Campbell. 1962. Oxidative assimilation of glucose by Pseudomonas aeruginosa. J. Bacteriol. 84:784. Eaton, N.R. i960. Endogenous respiration of yeast. 1. The endogenous substrate. Arch. Biochem. Biophys. 88:17. Ecker, R.E., and M. Schaechter. 1963. Ribosome content and the rate of growth of Salmonella typhimurium. Biochim. Biophys. Acta 2§*275. Edson, N.L. 1951. The intermediary metabolism of the mycobacteria. Bacteriol. Rev. 15:14-7. Eisenstadt, J.M., and H.P. Klein. 1959. Sulfur incorporation into the tA-amylase of Pseudomonas  saccharophila. J. Bacteriol. 77:66l. Elson, D. 1959. Latent enzymic activity of a ribonucleo-protein isolated from Escherichia coli. Biochim. Biophys. Acta ^ 6:372. Elson, D., and M. Tal. 1959. Biochemical differences in ribonucleoprotein. Biochim. Biophys. Acta 36:281. Forsyth, W.G.C., A.C. Hayward, and J.B. Roberts. 1958. Occurrence of poly- 0-hydroxybutyric acid in aerobic Gram-negative bacteria. Nature 182:800. Franke, W., and G.E. Hahn. 1955* Bacterial degradation of purines. II. Degradation of amino-, hydroxy-, and methyl-purines by Pseudomonas aeruginosa. Z. Physiol. Chem. 299:15. Gibbs, M., and W.A. Wood. 1952. The effect of substrate on the endogenous respiration of Pseudomonas fluorescens. Bacteriol. Proc, p. 137. Goksoyr, J. i960. Studies on the metabolism of Merulius  lacrymans. II. The respiration of washed mycelium from shake-cultures. Physiol. Plant 2d:559. -167-Gronlund, A.F. 1961. Pathways of glucose dissimilation in Pseudomonas aeruginosa. M. Sc. Thesis, University of B . C . , V a n c o u v e r . Gronlund, A.F., and J.J.R. Campbell. 1961. Nitrogenous compounds as substrates for endogenous respiration in microorganisms. J. Bacteriol. 81:721. Grunberg-Manago, M., and S. Ochoa. 1955* Enzymatic synthesis and breakdown of polynucleotides; polynucleotide phosphorylase. J. Am. Chem. Soc. 77:3165. Halvorson, H. 1958 a. Studies on protein and nucleic acid turnover in growing cultures of yeast. Biochim. Biophys. Acta 27:267. Halvorson, H. 1958 b. Intracellular protein and nucleic acid turnover in resting yeast cells. Biochim. Biophys. Acta 27:255. Halvorson, H. 1962. Physiology of sporulation, p. 223-264. In I.C. Gunsalus and R.Y. Stanier (ed.), The bacteria, vol. 4. Academic Press, Inc., New York. Halvorson, H.O., and S. Spiegelman. 1952. The inhibition of enzyme formation by amino acid analogues. J. Bacteriol. 64:207. Halvorson, H.O., and S. Spiegelman. 1953. The effect of free amino acid pool levels on the induced synthesis of enzymes. J. Bacteriol. 65:496. Hanson, R.S., V.R. Srinivasan, and H.O. Halvorson. 1963. Biochemistry of sporulation. II. Enzymatic changes during sporulation of Bacillus cereus. J. Bacteriol. 86:45. Harold, F.M. i960. Accumulation of inorganic polyphosphate in mutants of Neurospora crassa. Biochim. Biophys. Acta 45:172. Harold, F.M. 1962. Depletion and replenishment of the inorganic polyphosphate pool in Neurospora crassa. J. Bacteriol. 8^ :1047. Harold, R.L., and F.M. Harold. 1963. Mutants of Aerobacter  aerogenes blocked in the accumulation of inorganic polyphosphate. J. Gen. Microbiol. 31:241. -168-Harrison, A.P. i960. The response of Aerobacter aerogenes when held at growth temperature in absence of nutriment; an analysis of survival. Bacteriol. Proc, p. 191. Hayward, A.C., W.G.C. Forsyth, and J.B. Roberts. 1959. Synthesis and breakdown of poly-(5-hydroxybutyric acid by bacteria. J. Gen. Microbiol. 20:510. Hendley, D.D. 1955. Endogenous fermentation in Thiorhodaceae. J. Bacteriol. 70:625. Herbert, D. 1961. The chemical composition of microorganisms as a function of their environment. Symp. Soe. Gen. Microbiol. 11:391. Herbst, E.J., and B.P. Doctor. 1959. Inhibition of ribonucleic acid degradation in bacteria by spermine. J. Biol. Chem. 234:1497. Higuchi, M., and T. Uemura. 1959. Release of nucleotides from yeast cells. Nature lo4:138l. Hoagland, M.B. i960. The relationship of nucleic acid and protein synthesis as revealed by studies in cell-free systems, p. 349-408. In E. Chargaff and J.N. Davidson (ed.), The nucleic acicTs, vol. VIII. Academic Press, Inc., New York. Holme, T., and H. Palmstierna. 1956 a. Changes In glycogen and nitrogen-containing compounds in Escherichia coli B during growth in deficient media. 1. Nitrogen and carbon starvation. Acta Chem. Scand. 10:578. Holme, T., and H. Palmstierna, 1956 b. Changes in glycogen and nitrogen-containing compounds in Escherichia coli B during growth in deficient media. II. Phosphorus and sulphur starvation. Acta Chem. Scan. 10:1553. Holme, T., and H. Palmstierna. 1956 c. On the glycogen in Escherichia coll B; its synthesis and breakdown and its specific labeling with 1 4 c . Acta Chem. Scand. 10:1557. Horiuchi, T. 1959. RNA degradation and DNA and protein synthesis of E. coll in a phosphate deficient medium. J. Biochem. (TokyoT~46:1467. Hughes, D.E. 1951. A press for disrupting bacteria and other microorganisms. Brit. J. Exptl. Pathol. 32:97. -169-Hughes, D.E., S.F. Conti, and R.C. Fuller. 1963. Inorganic polyphosphate metabolism in Chlorobium thlosulfatophllum. J. Bacteriol. 8 5 * 5 7 7 ~ ~ Hutchison, W.C., E.D. Downie, and H.N. Munro. 1962. Factors affecting the Schneider procedure for estimation of nucleic acids. Biochim. Biophys. Acta 55*56l. Katchman, B.J., and W.O. Fetty. 1955. Phosphorus metabolism in growing cultures of Saccharomyees cerevisiae. J. Bacteriol. 69*607. Kennell, D., and B. Magasanik. 1962. The relation of ribosome content to the rate of enzyme synthesis in Aerobacter aerogenes. Biochim. Biophys. Acta 55*139. King, E.J. 1932. The colorimetrie determination of phosphorus. Biochem. J. 26:292. Kjeldgaard, N.O. 1961. The kinetics of ribonucleic acid and protein formation in Salmonella typhlmurium during the transition between different states of balanced growth. Biochim. Biophys. Acta 49:64. Klein, H.P. 1963. Synthesis of enzymes in resting cells. Ann. N.Y. Acad. Sci. 102:637. Kornberg, A., S.R. Kornberg, and E.S. Simms. 195&. Meta-phosphate synthesis by an enzyme from Escherichia coli. Biochim. Biophys. Acta 40:215. Kornberg, S.R. 1957. Adenosine triphosphate synthesis from polyphosphate by an enzyme from Escherichia coll. Biochim. Biophys. Acta 26:294. Kurland, C.G. i960. Molecular characterization of ribonucleic acid from Escherichia coli ribosomes. 1. Isolation and molecular weights. 3". Mol. Biol. 2:83. Lamana, C. 1963. Studies of endogenous metabolism in bacteriology. Ann. N.Y. Acad. Sci. 102:517. Levine, S., H. Stevenson, E.C. Tabor, R.H. Bordner, and L.A. Chambers. 1953. Glycogen of enteric bacteria. J. Bacteriol. 66:664. Levine, H.B., and H. Wolochow. i960. Occurrence of poly- -hydroxybutyrate in Pseudomonas pseudomallel. J. Bacteriol. 79*305. -170 Littauei*, U.Z., and A. Kornberg. 1957. Reversible synthesis of polyribonucleotides with an enzyme from Escherichia  coli. J. Biol. Chem. 226:1077. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. Luria, S.E. i960. The bacterial protoplasm: composition and organization, p. 1-34. In I.C. Gunsalus and R.Y. Stanier (ed.), The bacteria, vol. 1. Academic Press, Inc., New York. Macrae, R.M., and J.F. Wilkinson. 1958. Poly- -hydroxybutyrate metabolism in washed suspensions of Bacillus cereus and Bacillus megaterium. J. Gen. Microbiol. 1^ :210. Madsen, N.B. 1963. The biological control of glycogen metabolism in Agrobacterium tumefaciens. Can. J. Biochem. Physiol. 41:5617 " Mallette, M.F. 1963. Validity of the concept of energy of maintenance. Ann. N. Y. Acad. Sci. 102:521. Mandelstam, J. 1958 a. The free amino acids in growing and non-growing populations of Escherichia coli. Biochem. J. 69:103. Mandelstam, J. 1958 b. Turnover of protein in growing and non-growing populations of Escherichia coli. Biochem. J. 62:110. Mandelstam, J. i960. The intracellular turnover of protein and nucleic acids and its role in biochemical differentia-tion. Bacteriol. Rev. 24:289. Mandelstam, J., and H. Halvorson. i960. Turnover of protein and nucleic acid in soluble and ribosome fractions on non-growing Escherichia coli. Biochim. Biophys. Acta 40:43. Marino, R.J., and CE. Clifton. 1955. Oxidative assimilation in suspensions and cultures of Hyrogenomonas facills. J. Bacteriol. 69?l88. Marr, A.G., E.H. Nilson, and D.J. Clark. 1963. The maintenance requirement of Escherichia coll. Ann. N. Y. Acad. Sci. 102:536. McCarthy, B.J. i960. Variations in bacterial ribosomes. Biochim. Biophys. Acta 39*563. -171-McCarthy, B.J. 1962. The effects of magnesium starvation on the ribosome content of Escherichia coli. Biochim. Biophys. Acta 55s880. McCarthy, B.J., R.J. Britten, and R.B. Roberts. 1962. The synthesis of ribosomes in E. coll. III. Synthesis of ribosomal RNA. Biophys. JT 2:57. McGrew, S.B., and M.F. Mallette. 1962. Energy of maintenance in Escherichia coll. J . Bacteriol. 83:844. McQuillan, A.M. 1958. Transamination in Pseudomonas aeruginosa. M. Sc. Thesis, University of B.C., Vancouver. McQuillan, K., R.B. Roberts, and R.J. Britten. 1959. Synthesis of nascent protein by ribosomes in Escherichia  coll. Proc. Natl. Acad. Sci. U. S. 4£:1437. Midgley, J . , and B.J. McCarthy. 1962. The synthesis and kinetic behavior of deoxyribonucleic acid-like ribonucleic acid in bacteria. Biochim. Biophys. Acta 61:696. Midwinter, G.G., and R.D. Batt. i960. Endogenous respiration and oxidative assimilation in Nocardia corralllna. J . Bacteriol. 7j2*9. Milner, H.W., N.S. Lawrence, and C.S. French. 1950. Colloidal dispersion of chlorophyll material. Science 111:633-Mizunuma, T. 1963. Studies on the metabolism of Aspergilll. Part 1. Substrates of endogenous respiration. Report of the Noda Institute for Scientific Research, No. 7, p. 15. Moses, V., and P.J. Syrett. 1955. Tne endogenous respiration of microorganisms. J . Bacteriol. 70:201. Mudd, S., A. Yoshida, and M. Koike. 1958. Polyphosphate as accumulator of phosphorus and energy. J . Bacteriol. 75:224. Muhammed, A., A. Rodgers, and D.E. Hughes. 1959. Purification and properties of a polymetaphosphatase from Corynebacterlum xerosis. J . Gen. Microbiol. 20:482. Mulder, E.G., M.H. Deinema, W.L. Van Veen, and L. Zevenhuizen. I962. Polysaccharides, lipids and poly- (3-hydroxybutyrate in microorganisms. Recueil 81:797. 172-Murrell, W.G. 1961. Spore formation and germination as a microbial reaction to the environment. Symp. Soc. Gen. Microbiol. 11:100. Nishi, A. 1961. Role of polyphosphate and phospholipid in §erminating spores of Aspergillus niger. J. Bacteriol. _1:10. Norris, F.C., J.J.R. Campbell, and P.W. Ney. 194-9. The intermediate metabolism of Pseudomonas aeruginosa. I. The status of endogenous respiration. Can. J. Research 22:157. Ogata, K., A. Imada, and Y. Nakao. 1962. Formation of 5' nucleotides by degradation of endogenous RNA in a Bacillus by its own polynucleotide phosphorylase. Agr. Biol. Chem. (Tokyo) 26:586. Palmstierna, H. 1956. Glycogen-llke polyglucose in Escherichia coli B during the first hours of growth. Acta Chem. Scand. 10:567. Panek, A. 1963. Function of trehalose in baker's yeast (Saccharomyces eerevisiae). Arch. Biochem. Biophys. 100:422. Postgate, J.R., and J.R. Hunter. 1962. The survival of starved bacteria. J. Gen. Microbiol. 29:233. Potter, V.R. i960. Nucleic acid outlines. Vol. 1, Structure and metabolism, p. 166. Burgess Publishing Co., Minneapolis. Ramsey, H.H. 1962. Endogenous respiration of Staphylococcus aureus. J. Bacteriol. 83:507. Razin, S., I. Gery, and U. Bachrach. 1959. The degradation of natural polyamines and diamines by bacteria. Biochem. J. 71:551. Ribbons, D.W., and E.A. Dawes. 1963. Environmental and growth conditions affecting the endogenous metabolism of bacteria. Ann. N. Y. Acad. Sci. 102:564. Rich, A. I963. Polyribosomes. Sci. Am. 209:44. Roberts, R.B., P.H. Abelson, D.B. Cowie, E.T. Bolton, and R.J. Britten. 1955. Studies of biosynthesis in Escherichia coli. Carnegie Inst. Wash. Publ. No. 607. -173-Roodyn, D.B., and H.G. Mandel. i960. A simple membrane fractionation method for determining the distribution of radioactivity in chemical fractions of Bacillus cereus. Biochim. Biophys. Acta 41:80. Rosenthal, S.M., and C.W. Tabor. 1956. The pharmacology of spermine and spermidine distribution and excretion. J. Pharm. Exptl. Therapeutics 116:131. Rouf, M.A., and J.L. Stokes. 1962. Isolation and identifica-tion of the sudanophilic granules of Sphaerotilis natans. J. Bacteriol. 8^ :343. Sail, T., S. Mudd, and J.G. Davis. 1956*. Factors conditioning the accumulation and disappearance of metaphosphate in cells of Corynebacterium diphtherlae. Arch. Biochem. Sail, T., S. Mudd, and J.I. Payne. 1957. Changes in the composition and morphology of Bacillus megaterium on Sail, T., S. Mudd, and A. Takagi. 1958. Polyphosphate accumulation and utilization as related to synchronized cell division of Corynebacterium diphtherlae. J. Bacteriol. 26i640. — ~ Schachman, H.K., A.E. Pardee, and R.Y. Stanier. 1952. Studies on the macromolecular organization of microbial cells. Arch. Biochem. Biophys. 38:245. Schiff, J.A., J.M. Eisenstadt, and H.P. Klein. 1959. <*-amylase formation in growing and non-growing cells of Pseudomonas saccharophila. J. Bacteriol. 7°:124. Schlegel, H.G., G. Gottschalk, and R. Von Bartha. 1961. Formation and utilization of poly- (5 -hydroxybutyric acid by Knallgas bacteria (Hydrogenomonas). Nature 191:463. Schmidt, G. 1951. The biochemistry of inorganic pyrophosphates and metaphosphates, p. 443-475. In W.D. McElroy and B. Glass (ed.), Phosphorous metabolism, vol. VI. The Johns Hopkins Press, Baltimore. Schneider, W.C. 1957. Determination of nucleic acids in tissues by pentose analysis, p. 680. In S.P. Colowick and N.O. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press, Inc., New York. nitrogen deficient medium. -174-Schoenheimer, R., and D. Rittenberg. 1940. The study of intermediary metabolism of animals with the aid of isotopes. Physiol. Rev. 20:218. Simpson, M.V. 1953• The release of labeled amino acids from the proteins of rat liver slices. J. Biol. Chem. 201:143. Slepecky, R.A., and J.H. Law. 1961. Synthesis and degradation of poly-P-hydroxybutyrlc acid in connection with sporulation of Bacillus megaterium. J. Bacteriol. 82:37. Smith, I. (ed.). i960. Chromatographic and electrophoretic techniques. Vol. 2, Zone electrophoresis, p. 170. Inter-science Publishers, Inc., New York. Smith, I.W., J.F. Wilkinson, and J.P. Duguid. 1954. Volutin production in Aerobacter aerogenes due to nutrient imbalance. J. Bacteriol.Tb:450. Smithies, W.R., N.E. Gibbons, and S.T. Bayley. 1955. The chemical composition of the cell and cell wall of some halophilic bacteria. Can. J. Microbiol. 1:605. Spahr, P.F. 1962. Amino acid composition of ribosomes from Escherichia coll. J. Mol. Biol. 4:395. Spahr, P.F., and B.R. Hollingworth. I96I. Purification and mechanism of action of ribonuclease from Escherichia coli ribosomes. J. Biol. Chem. 236:823. Strange, R.E. 1961. Induced enzyme synthesis in aqueous suspensions of starved stationary phase Aerobacter  aerogenes. Nature 191:1272. Strange, R.E., F.A. Dark, and A.G. Ness. 1961. The survival of stationary phase Aerobacter aerogenes stored in aqueous suspensions. J. Gen. Microbiol. 2J"f:6l. . Strange, R.E., H.E. Wade, and A.G. Ness. 1963. The catabolism of proteins and nucleic acids in starved Aerobacter  aerogenes. Biochem. J. 86:197. Strasdine, G.A. 1961. A study of oxidative phosphorylation in Pseudomonas aeruginosa. Ph. D. Thesis, University of B.C., Vancouver. Strasdine, G., L.A. Hogg, and J.J.R. Campbell. 1962. A ribosomal polynucleotide phosphorylase in Pseudomonas  aeruginosa. Biochim. Biophys. Acta 55:231. -175-Steinberg, D., and M. Vaughan. 1956* Intracellular protein degradation "in vitro". Biochim. Biophys. Acta 19*584. Stickland, L.H., 1956. Endogenous respiration and poly-saccharide reserves in baker's yeast. Biochem. J. 64:498. Stier, T.J.B., and J.N. Stannard. 1936. A kinetic analysis of the endogenous respiration of baker's yeast. J. Gen. Physiol. 19*461. Takagaki, G., S. Hirano, and Y. Tsukada. 1957. Endogenous respiration and ammonia formation in brain slices. Arch. Biochem. Biophys. 68:196. Tal, M., and D. Elson. 1961. The reversible release of protein, ribonucleic acid and deoxyribonuclease from ribosomes. Biochim. Biophys. Acta 53:277* Tal, M., and D. Elson. 1963. The reversible release of deoxyribonuclease, protein and ribonucleic acid from ribosomes. Biochim. Biophys. Acta 72:439. Tissieres, A., J.D. Watson, D. Schlessinger, and B.R. Hollingworth. 1959. Ribonucleoprotein particles from Escherichia coli. J. Mol. Biol. 1:221. Trevelyn, W.E., and J.S. Harrison. 1956. Studies on yeast metabolism. 7. Yeast carbohydrate fractions. Separation from nucleic acid, analysis, and behaviour during anaerobic fermentation. Biochem. J. 63:23. Urba, R.C. 1959. Protein breakdown in Bacillus cereus. Biochem. J. 71:513. Wade, H.E. 1961. The autodegradation of ribonucleoprotein in Escherichia coli. Biochem. J. 2§*457. Wade, H.E., and S. Lovett. I96I. Polynucleotide phosphorylase in ribosomes from Escherichia coli. Biochem. J. 81:319. Wade, H.E., and H.K. Robinson. 1963. Absence of ribonuclease from the ribosomes of Pseudomonas fluorscens. Nature 200: 661. Warren, R.A.J., A.F. Ells, and J.J.R. Campbell, i960. Endogenous respiration of Pseudomonas aeruginosa. J. Bacteriol. 79*875. Wiame, J.M. 1949. The occurrence and physiological behavior of two metaphosphate fractions in yeast. J. Biol. Chem. 178:919. -176-Wiame, J.M., and M. Dbudoroff. 1951. Oxidative assimilation by Pseudomonas saccharophila with Cl4-iabeled substrates. J. Bacteriol. 62:lb7. Widra, A. 1959. Metachromatic granules of microorganisms. J. Bacteriol. 78:664. Wilkinson, J.F. 1958. The extracellular polysaccharides of bacteria. Bacteriol. Rev. 22:46. Wilkinson, J.F. 1959. The problem of energy-storage compounds in bacteria. Exptl. Cell Research, Suppl. 2:111. Wilkinson, J.F. 1963. Carbon and energy storage in bacteria. J. Gen. Microbiol. 22:171. Wilkinson, J.F., and J.P. Duguid. i960. Influence of cultural conditions on bacterial cytology, p. 1. In G.H. Bourne and J.F. Danielli (ed.), International review of cytology, vol. IX. Academic Press, Inc., New York. Williamson, D.H., and J.F. Wilkinson. 1958. The isolation and estimation of the poly- -hydroxybutyrate inclusions of Bacillus species. J. Gen. Microbiol. 13:198. Wilner, B., and C.E. Clifton. 1954. Oxidative assimilation by Bacillus subtilis. J. Bacteriol. 67:571. Winder, F.G., and J.M. Denneny. 1957. The metabolism of inorganic polyphosphate in Mycobacteria. J. Gen. Microbiol. 1£:573. Wright, B. 1963. Endogenous substrate control in biochemical differentiation. Bacteriol. Rev. 22:273. Wright, B.E., and M.L. Anderson, i960. Protein and amino acid turnover during differentiation in the slime mold. 1. Utilization of endogenous amino acids and proteins. Biochim. Biophys. Acta 43:62. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0105256/manifest

Comment

Related Items