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BACrERIOLOGICAL REVIEWS Vol. 28, No. 2, pp. 128-149 June, 1964 Copyright @ 1964 by the American Society for Microbiology Printed in U.S.A. SOME ASPECTS OF THE ENDOGENOUS METABOLISM OF BACTERIA E. A. DAWES AND D. W. RIBBONS Department of Biochemistry, University of Hull, Hull, England INTRODUCTION.................................................................................. 126 SUBSTRATES FOR ENDOGENOUS METABOLISM .................................................... 127 Role of PHB ................................................................................. 127 Biosynthesis of PHB ....................................................................... 130 Catabolism of PHB......................................................................... 131 Glycogenlike Reserves ......................................................................... 132 Amino Acid Pools............................................................................ 135 RNA Metabolism............................................................................. 136 Protein...................................................................................... 138 Other Potential Substrates..................................................................... 138 ENERGY OF MAINTENANCE...................................................................... 138 STARVATION AND SURVIVAL..................................................................... 142 Death........................................................................................ 143 Relationship Between Survival and Endogenous Substrates ...................................... 144 SUMMARY AND FUTURE OUTLOOK............................................................... 145 LITERATURE CITED............................................................................. 146 "One great use of a Review, indeed, is to make men wise in ten pages, who have no appetite for a hundred pages; to condense nourishment, to work with pulp and essence, and to guard the stomach from idle burden and unmeaning bulk." Sidney Smith Edinburgh Review, 1825 INTRODUCTION Current interest in the endogenous metab- olism of microorganisms has been reflected by two recent symposia (Focal Topic A7, Intern. Congr. Microbiol., 7th, Montreal, 1962; Ann. N.Y. Acad. Sci., vol. 102, art. 3, p. 515-793, 1963) and by our own previous survey (15). The present article is intended to be a more discursive review of the present situation in this area of study and to deal in greater detail with those aspects which were not previously elaborated. Endogenous metabolism may be defined as the total metabolic reactions that occur within the living cell when it is held in the absence of compounds or elements which may serve as specific exogenous substrates. Some products of endogenous metabolism may be released into the surrounding medium and are often utilized by the cells, sometimes resulting in regrowth, a phenomenon which is discussed later. It is im- portant to stress the viability and integrity of the cell in this context, because some authors have used the term "endogenous" loosely in relation to the activities of cell extracts (see, for example, 84). It must also be recognized that the reactions characteristic of endogenous metabolism may continue in the presence of exogenous substrates, and, since metabolism of the latter compounds is usually effected after they have been taken into the cell, in the final analysis the distinction between the metabolism of endogenous and exogenous substrates may become largely a matter of semantics, although problems of com- partmentation within the cell should be appre- ciated (22a). This is particularly the case where the oxidation of an exogenous substrate by a washed suspension of microorganisms leads to the assimilation of material which subsequently can be utilized as an endogenous substrate. Analysis of the status of the endogenous metabolism in the presence of added substrates presents many experimental problems, but these have already been adequately reviewed (6, 15). The possible significance of endogenous metab- olism in relation to the survival of microor- ganisms is a topic presently under study in several laboratories. One view which may be held is that endogenous metabolism occurs simply because the organism cannot help it, and it therefore bears no relationship, direct or other- wise, to the period of survival (22). The other extreme outlook is that the survival character- 126 ENDOGENOUS METABOLISM OF BACTERIA istics are related directly to the endogenous metabolic activities of the cell. The evidence available at the time of writing suggests that, for those organisms which have been studied, the truth lies somewhere between these extremes, as will be discussed later. The existence of an energy of maintenance for microorganisms has attracted considerable ex- perimental attention of late. The idea that a definite amount of energy must be expended to enable a microbial cell to maintain its integrity without growth or death occurring seems a reasonable concept, and appears now to be sup- ported by some evidence. The question of whether a cell can exist in a condition such that it is committed neither to growth nor to death is, however, still a debatable issue. The experi- mental problems posed by the death and regrowth of bacterial cultures make difficult a conclusive answer to this query at present. Aspects of endogenous metabolism concerning macromolecular turnover and cell differentiation into spores were focal points of the symposium published by the New York Academy of Sciences, and will not be elaborated here. A symposium on microbial reaction to environment has also directed attention to the considerable variation in composition and properties that can be pro- duced by alteration of experimental conditions, and additionally to the influence of the previous history of the cells. Although the effect of some environmental factors on endogenous metab- olism has been investigated (65), there are many other aspects of this same problem which have not been explored. Indeed, there are so many possible external influences on endogenous metabolism that considerable caution should be exercised in making generalized statements con- cerning the endogenous metabolic behavior of microorganisms. The present review will pay particular atten- tion to the role of poly-f3-hydroxybutyrate (PHB) as a substrate for endogenous metab- olism, because, as a storage compound unique to bacteria, it holds a position of considerable interest. Several groups of investigators have demonstrated the presence of the polymer and have studied its metabolism in various bacterial species, but there has so far been no attempt to correlate their findings in relation to endogenous metabolism. SUBSTRATES FOR ENDOGENOUS METABOLISM A consideration of possible substrates for endogenous metabolism naturally focuses atten- tion on energy-storage compounds. Wilkinson (93) described three main classes of compounds that could possibly act as energy-storage com- pounds. These are polysaccharides, lipids (includ- ing PHB), and polyphosphate; all occur in widely varying amounts dependent upon the particular species and the environmental condi- tions. However, it is now apparent that there are many other substrates for endogenous metabolism; these include ribonucleic acid (RNA) and protein (both are also subject to turnover) and free amino acid and peptide pools. Possible substrates not yet implicated in endoge- nous metabolism include deoxyribonucleic acid (DNA), cell-wall polymers, and cell-membrane materials. Role of PHB The elucidation of the role of PHB in bacterial endogenous metabolism is a fascinating story, for, although the polymer was originally dis- covered in 1927, a physiologicalrole for it was not convincingly demonstrated until some 30 years later. PHB was first isolated by chloroform extraction of an aerobic bacillus by Lemoigne (42), following his earlier discovery that (3- hydroxybutyrate was a metabolic product of the organism. Since that time, PHB has been demon- strated in a wide variety of bacterial species (Table 1), and in some instances has been im- plicated as an assimilatory product, from meas- urements of gaseous exchange during photo- metabolism of fatty acids and gross elemental composition, by Gaffron (cited in 78). The quan- tities of PHB within the bacterial cell vary enor- mously; contents of up to 50% of the dry weight have been recorded. It is a reserve that is peculiar to microorganisms, and its functions, formation, and synthesis have been studied extensively in recent years, mainly by Doudoroff and Stanier, Gibbons, Schlegel, Wilkinson, and their col- laborators. Sudanophilic granules present in bacteria were considered by Lemoigne, Delaporte, and Croson (43) to be composed of PHB; the data of Weibull (88) subsequently supported this proposition. However, it was not until the work of William- VOL. 28, 1964 127 DAWES AND RIBBONS son and Wilkinson (95) that the intracellular lipid granules of Bacillus cereus and B. mega- terium were demonstrated unequivocally to be composed mainly of PHB (about 90%), although neither this nor the remaining 10%, lipid is re- sponsible for the sudanophilic properties of the granules that are observed in situ. Macrae and Wilkinson (46, 47) also studied the effect of various cultural conditions on the synthesis of PHB in B. megaterium. When the glucose con- TABLE 1. Occurrence of poly-,3-hydroxybutyrate in bacteria* Species Reference Bacillus megaterium............... 43, 77, 95 B. cereus.......................... 43, 95 B. mycoides....................... 43 B. anthracis....................... 43 Azotobacter chroococcum ........... 43 A. agilis.......................... 21 A. vinelandii...................... 21 Rhizobium sp...................... 21 Vibrio sp.......................... 31, 32 Chromobacterium violaceum........ 21 Pseudomonas solanacearum........ 32 P. antimycetica ................... 32 P. methanica...................... 39 P. pseudomallei................... 44 P. saccharophila .................. 19 Pseudomonas AMi ................ 59 Micrococcus halodenitrificans ...... 75, 76 Sphaerotilus natans ............... 58, 69 Hydrogenomonas sp................ 73 Rhodospirillum rubrum............ 19, 78 Rhodopseudomonas spheroides.... . 10 Chromatiurn okenii................ 72 Spirillum itersonnii............... 54 S. anulus......................... 54 S. serpens......................... 32, 54 * This is not a complete list of the bacterial species that synthesize PHB. centration in the growth medium was raised, more of the polymer was synthesized; exhaustion of the nitrogen source in the presence of excess carbon and energy source permitted deposition of about four times the amount of PHB as was formed when glucose limited growth. Glucose, pyruvate, and f-hydroxybutyrate were suitable substrates for PHB production by washed sus- pensions, but acetate, although itself unable to effect synthesis (compare with Rhodospirillum rubrum), enhanced PHB formation when supple- menting other substrates. Anaerobic conditions prevented PHB synthesis, as did dinitrophenol. Concentrations of oxygen greater than 5% also inhibited the assimilatory process. B. cereus, but not B. megaterium, could effect PHB synthesis under hydrogen, although no net uptake of hydrogen was detected; PHB is not formed under nitrogen. When washed suspensions of these two bacilli were shaken under air or under nitrogen, in the absence of an exogenous carbon and energy source, stored PHB was metabolized. The anaerobic degradation of reserves was slower; e.g., in B. megaterium, 61 and 17% degradation of PHB occurred under air and nitrogen, respec- tively, within 8 hr. Metabolic products detected included f-hydroxybutyrate, acetoacetate, and acetate, although aerobically CO2 and water were the major products and only small amounts of acetoacetate accumulated. Some correlation was evident between the PHB content of cells and their endogenous respiration; e.g., cells with PHB-total N ratios of 0.83 and 3.27 had, respec- tively, endogenous Qo2 values of 169 and 536. Macrae and Wilkinson (46) also claimed that N-deficient cells with a high content of PHB are better able to withstand death and autolysis than those with a low PHB content. Autolysis was estimated by the total N content which, in 4 hr, fell by 12% in PHB-poor cells compared with 5% in PHB-rich cells; the PHB content of the latter cells decreased to the greatest ex- tent. The method of estimation of autolysis is not entirely satisfactory, because it has been shown in numerous cases that nitrogenous mate- rials are oxidized endogenously, releasing am- monia, and that substantial amounts of nitrog- enous compounds may diffuse from the cell without loss of viability. Specifically in the case of B. cereus, Clifton and Sobek showed that am- monia is produced endogenously under some conditions (12), and clearly B. megaterium utilizes an endogenous substrate other than, although concurrently with, PHB, since the oxygen consumption is in excess of that required for complete combustion of PHB; this other substrate is not polysaccharide (47). It is pos- sible, however, that autolysis may have occurred to a similar extent in both PHB-rich and PHB- poor cells, but the greater amount of reserve material in the PHB-rich cells permitted utiliza- tion of the liberated nitrogenous material and 128 BACTERIOL. REV. ENDOGENOUS METABOLISM OF BACTERIA some limited cellular synthesis occurred. Against this possibility may be set the authors' observa- tion that growth did not occur when PHB-rich cells were held in a medium lacking a carbon and energy source; growth was measured by total N so that in these bacilli PHB may serve as a reserve of energy but not as a source of carbon skeletons for synthesis. Doudoroff and Stanier (19) have made some interesting observations concerning the role of PHB in oxidative assimilation by Pseudomonas saccharophila and in photoassimilation by R. rubrum. They found with most substrates that a major portion of the assimilated carbon (60 to 90%) initially accumulates within the cells as PHB; when the exogenous substrate is removed, a rapid intracellular degradation of the polymer occurs, suggesting a physiological role as a re- serve material. When cells are subjected to standard fractionation procedures, the chemical properties of PHB result in its appearance in the hot trichloroacetic acid insoluble fraction (pro- tein fraction). This fact had led Wiame and Doudoroff (91) earlier to conclude that C14 is incorporated into nitrogenous materials during oxidative assimilation. Incubation of starved washed suspensions of R. rubrum with C'4-acetate allowed deposition of 70% of the assimilated C'4 into PHB, with no significant dilution. With C'4-butyrate, some dilution occurred, and the polymer contained only 58% of the assimilated C14. The fate of the polymer in the light over a period of 12 hr was studied under a variety of conditions, e.g., in the presence and absence of a source of organic sub- strates but in the presence of a N source and CO2. The absence of an exogenous organic carbon source led to the disappearance of more than 90% of the polymer, but much of the C14 of this material was redistributed into other cellular components. The authors did not study the fate of PHB in both nitrogen and carbon starvation, or in the dark, so its behavior under these con- ditions is not yet known. The rate of degrada- tion of PHB and its conversion to other cellular components wasdecreased when an exogenous source of butyrate was supplied. In marked con- trast, succinate, which is also metabolized under these conditions, was quite unable to prevent polymer breakdown and transfer of its C skele- tons to other cell constituents; these processes occurred to about the same extent as in the absence of an exogenous carbon source. The reason for this became apparent when it was shown that succinate is photoassimilated princi- pally to a glycogen-like polysaccharide, and the dry weight of the cells increased by 40%. Similar studies with P. saccharophila revealed that washed suspensions of glucose-grown cells incorporated 66% of the carbon assimilated from U-C14-glucose into PHB, and without appre- ciable dilution. An even greater amount (about 80%) of the assimilated carbon from acetate or butyrate appeared in the polymer. Although ex- perimental details were not given, the authors claimed that tracer experiments indicated the role of PHB as a substrate for endogenous metab- olism in the absence of an exogenous carbon source; its metabolism was reported to be much slower than in R. rubrum, and transfer of polymer carbon to other cell constituents could not be demonstrated. The use of PHB as an endogenous store of carbon skeletons for synthesis in R. rubrum was studied further by Stanier et al. (78), who showed that for conversion of stored PHB to other cell materials CO2 is essential. This was demon- strated with cells which had assimilated acetate and were then incubated in the presence of (i) NH4Cl and He, (ii) NH4Cl and He-CO2 mixture, and (iii) He-CO2 mixture in the absence of a nitrogen source. After 16 hr, very small changes in total dry weight had occurred and, in the absence of C02, the PHB content of the cells fell slightly but with no increase in the carbo- hydrate or nitrogen content. In the presence of CO2 without a nitrogen source, the PHB content fell by about 50% and the carbohydrate content showed a corresponding gain, but there was no change in nitrogen content. WNhen both nitrogen and CO2 were furnished, the PHB disappeared almost completely with concomitant increases in both carbohydrate and nitrogen content, includ- ing protein. None of the experiments was de- signed to test the possibility that PHB may serve as an energy store, as it does, perhaps, in B. megaterium and presumably also in P. sac- charophila. It is considered that PHB serves as a store of carbon and reducing power for further CO2 assimilation, which is essential for PHB utilization. The anaerobic utilization of PHB in the absence of CO2 and a nitrogen source would appear to be limited, but neither its formation nor degradation aerobically in the dark has been VO0L.- 28, 1964 129 DAWES AND RIBBONS studied, and here one might expect PHB to serve as a source of energy. The presence of PHB as an endogenous reserve in Hydrogenomonas is documented by the work of Schlegel, Gottschalk, and von Bartha (73). When chemolithotrophic growth, in an atmos- phere of H2-02-CO2 (60:30:10), is limited by nitrogen exhaustion (NH4Cl) in the medium, cells in the stationary phase continue to increase in size although no division occurs. The increase in dry weight is accounted for entirely by PHB forma- tion. Washed suspensions synthesize PHB from CO2 by oxidation of hydrogen, and the stoichi- ometry corresponds to: 25H2 + 802 + 4CO2 -- (C4H602) + 22H20 The rate of endogenous respiration of PHB- poor cells (about 10% of the dry weight) was considerably less than that of PHB-rich cells (about 50% of the dry weight). It was little affected by the addition of a nitrogen source (NH4Cl) which had a marked stimulatory effect on the respiration of the latter cells. Determina- tions of cellular carbon and total nitrogen during endogenous respiration, and during hydrogen oxidation in the absence of C02, were carried out with PHB-rich cells, both with and without a nitrogen source. In air, only about 8% of the PHB was consumed in 12 hr by endogenous metabolism, but with added NH4Cl the total cellular nitrogen increased significantly and the PHB decreased by some 73%. Under an at- mosphere of C02-free H2-02, the increase in cell nitrogen was much greater, although not much more PHB was utilized (76 %). These results suggest that in Hydrogenomonas stored PHB may serve as a carbon and energy source and can support protein synthesis in the presence of a suitable source of nitrogen. When hydrogen is provided as an additional energy source, the efficiency of protein synthesis is increased. Conditions for the accumulation of PHB in the halophile, Micrococcus halodenitrificans, were determined by Sierra and Gibbons (75). These authors further studied the role of PHB and its relationship to endogenous respiration and sur- vival of the organism (76). The RQ of the en- dogenous respiration of M. halodenitrificans was 0.87 i 0.05. (That required for complete com- bustion of PHB is 0.88.) Aeration of washed suspensions at 25 C reduced the PHB content slowly (from 55 to 29%G in 127 hr); under these conditions, some lysis of cells occurred and the endogenous Qo2 remained at 40 throughout this period. Some product of metabolism or lysis was inhibiting further endogenous respiration, since the inhibition could be relieved by washing the cells. Changes in the cell constituents of PHB-poor cells (containing 10% PHB) showed that 3 hr of starvation produced no changes in total N, either soluble material or carbohydrate, but almost half the PHB had been consumed. The endogenous Qo2 value decreased with poly- mer depletion. A similar but extended study was made with PHB-rich cells; after 96 hr of starvation had elapsed, the PHB content had diminished from 50 to 10% of the dry weight, and the endogenous Qo2 remained constant. Further starvation rapidly reduced the endogenous Qo2. From these and other data, it seems clear that the rate of PHB oxidation is slower during starvation experiments than in the respirometer: if, as appears to be the case, PHB is the sole substrate for respiration, cells containing 50% of their dry weight could respire for only 15 hr with a Qo2 of 40. Biosynthesis of PHB. In view of the unique occurrence of PHB as a storage polymer (or metabolic shunt product) in bacteria, it is some- what surprising that, at the time of writing, there is very little information regarding the metabolic routes and enzymes concerned with its biosynthesis. The only paper of significance is that of Merrick and Doudoroff (56). They demonstrated that polymer particles (obtained by differential centrifugation of lysozyme-treated bacteria) of B. megaterium KM were able to incorporate C'4-f-hydroxybutyryl-coenzyme A (CoA) into PHB. This was independent of the presence of the soluble fraction of the cell. Ap- proximately 40% of the total radioactivity be- came incorporated into the polymer, which cor- responded to the amount of thioester decomposed. A mixture of labeled g-hydroxybutyrate and un- labeled CoA did not allow incorporation of C14 into the polymer. Similar experiments by these authors were extended to the particulate fraction of R. rubrum which contained all the activity for the incor- poration of f-hydroxybutyryl-CoA into PHB. However, these particles also contained a very active depolymerase which could degrade the PHB. With crude extracts, supplemented with CoA, adenosine triphosphate (ATP) and reduced 130 BACTERIOL. REV. ENDOGENOUS METABOLISM OF BACTERIA nicotinamide dinucleotides, a small incorporation of C'4-acetate into polymer occurred. Activa- tion of f3-hydroxybutyrate could not be demon- strated. Although activation of ,3-hydroxybutyrate could not be demonstrated in extracts of R. ru- brum, whole cells of Hydrogenomonas incorporate l3-hydroxybutyrate into PHB (73). Crotonic acid can also be used as a substrate; in eithercase, the presence of hydrogen is not mandatory. Presumably, the oxidation of a portion of these substrates provides the energy necessary for polymerization. The utilization of crotonic acid is particularly interesting since there may exist a 2n ATP y 2n CH3CO2H 2n ADP 2n CH3CO-SCoA Acetyl GoA V HSCoA n CH3COCH2CO- SCoA Acetoacetyl CoA _______- - - V2n[H] CH3CHOHCHC0,H-I nCH3CHOHCH2CO' SCoA 4-Hydroxybutyrate 4-Hydroxybutyryl GoA ,, .4HSCoA CH3CH-CHCO2H----CHCH-CH-COSCIoA1 Crotonate Crotonyl CoA (CH3- H- CHaCO-)n I 9 Hydrogenomonas Poly-A-hydroxybutyrate FIG. 1. Biosynthesis of poly-f3-hydroxybutyrate. possible pathway of 13-hydroxybutyrate synthesis omitting acetoacetate. CH3-CH = CHC02H + H20 -* CH3 CHOHCH2 CO2H Reactions leading to PHB are schematically shown in Fig. 1. Catabolism of PHB. As with the biosynthesis of PHB, comparatively little is known of its degradation. There are, however, indications that the initial stages of depolymerization are extremely complex and appear to be bound up with the structural integrity of the polymer particles (56, 94). Thus, Merrick and Doudoroff (56) demonstrated that extracts of R. rubrum contain enzymes that degrade (i) native PHB from B. megaterium or (ii) boiled PHB particles from R. rubrum, but do not degrade the purified polymer. Sierra and Gibbons (76) demonstrated PHB esterase (depolymerase) activity in Ml. halode- nitrificans by the anaerobic release of CO2 from bicarbonate buffer. A stoichiometric release of CO2 by l3-hydroxybutyrate was recorded; this depolymerization is inhibited by the esterase inhibitor, diethyl-p-nitrophenyl phosphate (para- oxon). Further, the rate of release of f3-hydroxy- butyrate was identical with the rate at which oxygen is consumed for the complete combustion of PHB. For example, 0.76 umole of ,3-hydroxy- butyrate was liberated per hr by 2 mg of cells containing 50% of their dry weight as PHB; this requires an oxygen consumption of 3.4 Mmoles per hr for complete combustion. This is a figure realized by the recorded Qo2 values of 39 [(39 X 2)/22.4 Mmoles of 02 per 2 mg of cells per Poly - hydroxybutyrate I N PHB esterase(depolymerase) D- (-)-A - I ydroxybutyrate r NAD 4,- I4ydroxybutyrate NADH2 dehydrogenase Acetoacetate ATP, HSCoA, Mg2+ Acetyl GoA Oxaloacetate - 5 Citrate Tricarboxylic, Acid Cycle FIG. 2. Catabolism of poly-f3-hydroxybutyrate. hr = 3.48]. On this basis, the depolymerization appears to be rate-limiting. Sierra and Gibbons later showed that depolymerase activity is markedly dependent upon Nat and Lit ions (76, 76a). Cells washed and resuspended in KCl or water do not oxidize their PHB reserves unless Na+ or Li+ is added. Enzymes involved in the degradation of /3-hy- droxybutyrate were also demonstrated by Sierra and Gibbons (76). DL-f3-Hydroxybutyrate is oxidized by crude extracts to acetoacetate by a nicotinamide adenine dinucleotide (NAD)-spe- cific enzyme. Only the D(-)-f-hydroxybutyrate is utilized, and the acetoacetate is not further metabolized unless ATP, Mg2, CoA, and oxalo- VOL. 28, 1964 131 DAWES AND RIBBONS acetate are added to the extracts. These data suggest a pathway for catabolism of PHB as shown in Fig. 2. f3-Hydroxybutyrate and aceto- acetate were also detected as products of PHB degradation in B. megaterium when cells were starved under N2. These acids accounted for over 80% of the polymer depleted under these condi- tions. Some properties of a D-(-)-O-hydroxy- butyrate dehydrogenase from B. megaterium have been described (24). GLUCOSE - N H4* salt reaction is optimal over a pH range of 6.8 to 8.5. The dehydrogenase is insensitive to sulfhydryl group reagents; ethylenediaminetetraacetic acid (EDTA) inhibition may be reversed by Mg2 if incubation is not prolonged. The specific activity of the D-(-)-3-hydroxy- butyrate dehydrogenase is dependent upon the cultural conditions at the time of harvest; low specific activities are observed during periods of PHB assimilation, but specific activities greater GLUCOSE_-TQYPTONE TRYPTONE 0-35 0-3 151 >1~~~~~~~~~~~~~~~~~~~~~~~~~0 0O25L E ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~10 F 3. Cdarbohydofcardtt 015 ~~~~~~~~~~~~~~~~8 ~0.1N/35 ~0 0 I010S I10 18 6 2 Time (/77/n) FIG. 3. Comparison of carbohydrate utilization and ammonia production by endogenously respiring cells of Escherichia coli harvested from stationary-phase glucose-ammonium salts, glucose-Tryptone, and Tryptone cultures (Ribbons and Dawes, 65). Reproduced by kind permission of The New York Academy of Sciences. Shuster and Doudoroff (74) found the D(-)3- hydroxybutyrate dehydrogenase from R. rubrum to be cold-sensitive. A 250-fold purification of the enzyme was obtained in 30% yield, and this was unstable in dilute solutions or inactivated by freezing. The enzyme is freely reversible, and the reaction products are acetoacetate and reduced NAD (NADH2). Although NAD phosphate (NADP) would not substitute for NAD, a-oxo- valerate showed 6% of the activity obtained with acetoacetate. Furthermore, a-oxovalerate compet- itively inhibited acetoacetate reduction, because of the lower affinity of acetoacetate. The rate of by a factor of 2 are observed with older cells depleted of polymer. Succinate-grown cells con- tain no PHB, and only low specific activities of the enzyme are found. Glycogenlike Reserves The majority of microorganisms synthesize polysaccharides of one form or another which may be of intracellular or extracellular origin. Only the former type come within the scope of the present survey since, by definition, extra- cellular polysaccharides [reviewed by Wilkinson in 1958 (92)] cannot serve as endogenous sub- 132 BACTERIOL. R.EV. EN21ENDOGENOUS METABOLISM OF BACTERIA strates. It is frequently possible to differentiate intracellular polysaccharide into "reserve" and "structural" carbohydrate, the criterion being whether or not the material is utilized under con- ditions of starvation. The term "structural" in this context may be incorrect in many instances, since it does not follow ipso facto that material not metabolized is necessarily part of the archi- tecture of the bacterial cell. The role of reserve carbohydrates in yeast was also reviewed recently (15). We have investigated the role of glycogen as an endogenous substrate in Escherichia coli and have obtained considerable evidence for its rapid utilization during periods of complete starvation under either aerobic or anaerobic conditions (16, 17, 65). The possession of glycogen by the cells prevents a net degradation of nitrogenous ma- TrABLE 2. Oxygen consumption and carbohydrate utilization by endogenously respiring Escherichia coli 02 re- 02 re- Time 02 quired for Difference quired for RQconsumed glycogen ribose corn- combustion bustion min jmoles jimoles Mmoles Pmoles 60 11.38 11.01 0.37 0.73 1.01 120 18.89 16.01 2.88 1.85 1.03 184 23.2 17.48 5.72 2.03 1.01 270 26.6 18.47 8.13 2.87 1.01 terials with liberation of ammonia; this was demonstrated with cells harvested from media allowing massive, moderate, and no deposition of glycogen. Subsequent starvation of washed suspensions of these cells showed that ammonia is released only after the glycogen has been oxi- dized, and in the case of Tryptone-grown cells, which do not contain glycogen, oxidation of nitrogenous materials commences immediately upon starvation (Fig. 3). The regulatory mecha- nisms of this phenomenon have not yet been studied. Other data indicate that, during oxida- tion of endogenous glycogen, very little else is oxidized, since the consumption of oxygen and evolution of CO2 correspond initially to that required for the complete combustion of glycogen to CO2 and water (Table 2). Longer periods of starvation reveal that oxygen consumption is in excess of glycogendepletion, and a part of this can be accounted for by oxidation of ribose, which is produced in the degradation of the RNA that occurs under these circumstances. The exact role of the glycogen under these starvation conditions is difficult to appreciate, since depletion of most of it is complete within 2 to 3 hr. If the possession of glycogen by E. coli favors survival by providing a store of energy, then one might expect a slower rate of utilization of this storage product. However, the exact con- ditions of starvation will influence considerably the fate of the polysaccharide, and those used in our experiments may favor an uncoupling of oxidative phosphorylation or other rate-limiting process. Even under a nitrogen atmosphere, part of the glycogen is rapidly degraded, with the evo- lution of hydrogen and carbon dioxide. It may be that in E. coli the glycogen functions as a re- serve of carbon skeletons and not of energy. Evi- dence in support of this concept is the transfer of glycogen carbon atoms to other cell constituents (36). The deposition and fate of glycogen in Aero- bacter aerogenes was studied by Strange, Dark, and Ness (81). Cells grown on Tryptone-glucose media contain 15 to 20%7, carbohydrate, and most of this is glycogen. It is utilized after approxi- mately 25 hr of incubation of the washed suspen- sions in buffer. Ammonia is released from glyco- gen-containing cells, but at a lower rate than from cells that have not stored glycogen. The complete suppression of ammonia release by glycogen as recorded with E. coli (65) was not observed, although the glycogen is oxidized much less quickly in A. aerogenes. Ultraviolet-absorb- ing materials are also released from glycogen- containing cells of A. aerogenes much more slowly than from cells harvested from tryptic meat broth or defined media (carbon-limiting). Glycogenlike polysaccharides have been de- scribed as reserves in a variety of bacterial species; some examples are given in Table 3. The amount stored was reported to be as high as 41 to 75% of the dry weight in Arthrobacter species by Mulder et al. (58). When washed suspensions of exponential-phase cells of Arthrobacter are starved for 4 days at 30 C, only 40% of their carbohydrate is utilized. At this stage, the oxygen consumption of the suspensions is almost zero. Thus, not all of the original carbohydrate is a substrate for endogenous respiration. R. rubrum, it may be recalled, is an example of a microorganism that is able to store more than V o ,. 28, 1964 133 DAWES AND RIBBONS one polymer as a reserve material, and the poly- mer that is deposited within the cells is deter- mined solely by the chemical nature of the carbon substrate supplied. Thus, acetate and butyrate are substrates for PHB storage, whereas carbon dioxide, succinate, propionate, and malate are photoassimilated principally to a glycogenlike polysaccharide (78). The photoassimilation of specifically labeled C'4-succinate by starved cells of R. rubrum was studied in detail by Stanier et al. (78). The polysaccharide was the most strongly labeled fraction when either 1-C14_ or 2-C'4-succinate was photometabolized; 75% of the assimilated suc- cinate flowed into the polysaccharide and only 14% into PHB. Carboxyl-labeled succinate labels the hexose residues at C-3 and C4, and TABLE 3. Occurrence of glycogen in bacteria* Species Reference Escherichia coli .......... 35, 36 Aerobacter aerogenes .. 81 Rhodospirillum rubrum ... 78 Arthrobacter sp...................... 58 Agrobacterium tumefaciens .. 48 Bacillus cereus...................... 58 B. megaterium...................... 3 Mycobacterium phlei ............. ... 25 M. tuberculosis...................... 11 * This is not a complete list of the bacterial species that store glycogen. the methylene carbon atoms of succinate enter C-1, C-2, C-5, and C-6 of the glucose residues. The specific activities of the incorporated ma- terial showed that the carboxyl-labeled succinate is diluted by a factor greater than two, whereas the methylene carbon atoms are diluted only slightly. The main mechanism of hexose syn- thesis, therefore, seems to involve decarboxyla- tion of the succinate chain (probably as oxalo- acetate), the product of which is channelled by reversal of the reactions of glycolysis to hexose phosphate. The chemical nature of the carbon nutrient determines the storage product formed; those compounds that are converted to acetate without intermediate formation of pyruvate (or phos- phoenolpyruvate) yield PHB, and those yielding the 3-carbon compound produce glycogen. Dur- ing photometabolism in R. rubrum, glycogen serves as a source of carbon and reducing power for further CO2 assimilation; it may also provide some energy, although this has not been tested. It appears to represent a "hot-house" shunt product (as does PHB in R. rubrum) as suggested by Foster (22), since massive deposits are formed rapidly by assimilation of exogenous carbon sources at rates greater than overall cellular syn- thesis. The fate or synthesis of the glycogen has not been studied during aerobic metabolism of this organism; here, as with PHB, the glycogen might be expected to serve additionally as an energy source. Hot water or hot 75% ethanol extracts a poly- glucose compound of low molecular weight from. glucose-peptone grown Sarcina lutea (5, 9, 65). We have shown that this polyglucose is only synthesized during growth on peptone media supplemented with glucose, and that it can serve as a substrate for endogenous respiration. The oxi- dation of this carbohydrate occurs during the de- pletion of the amino acid pool; i.e., the provision of a readily metabolizable carbohydrate as an endogenous reserve does not spare the nitrogen reserves, since free ammonia is released during the starvation period. Further, the oxygen con- sumption is in excess of that required by the polyglucose oxidized. The metabolic pathways of glycogen biosyn- thesis and degradation in bacteria have received very little attention. Rogers (66) suggested that bacterial enzymes degrading glycogen are un- known, although many systems that decompose starch, amylose, and amylopectin are described. Glycogen metabolism is, however, well docu- mented in animals and plants. [For reviews see Stetten and Stetten (79) and Whelan (90).] Current ideas of glycogen metabolism suggest that biosynthesis and dissimilation occur by separate routes. The uridine diphosphoglucose (UDPG) pathway is involved in synthesis and the phosphorylase pathway in degradation. The initial reactions of glycogen breakdown were delineated by Parnas and co-workers as early as 1935, when they showed that inorganic phos- phate is consumed and a hexose monophosphate, later identified as glucose 1-phosphate, is accumu- lated. The reaction catalyzed by glycogen phos- phorylase is: Pi + glucosyl-(al1,4') primer glucose 1-phosphate + primer In mammalian systems, complex interrelation- 134 BACTERIOL. REV.- ENDOGENOUS METABOLISM OF BACTERIA ships exist between inactive and active forms of phosphorylase, and the enzymes obtained from different tissues of the same species are not iden- tical (79). The bacterial phosphorylases are, how- ever, not well studied. The UDPG pathway of glycogen synthesis has been described in bacteria by Madsen (48, 49), and in yeast by Algranati and Cabib (1). Glyco- gen acts as a primer with the glycogen synthetase enzymes of Agrobacterium tumefaciens and yeast, but, unlike the mammalian enzymes, glucose 6-phosphate does not stimulate the reaction: UDPG + polysaccharide primer uridinediphosphate (UDP) + glucosyl (1,4') primer A cyclic scheme of glycogen degradation and synthesis has been proposed for mammalian UDP GLYCOGEN UDPG glycogen VI synthetase P UDPG Phosphorylase PP UDPG p \rophosphorylaseUTP Glucose-1- phosphate 1 Phosphoglucomutase GLUCOSE E.AT . Glucose 6-phosphatelRexoki~nase FIG. 4. Biosynthesis and degradation of glycogen. systems (Fig. 4). A similar system probably oper- ates in bacteria, since the necessary enzymes have been demonstrated and partially separated in A. tumefaciens (48), and UDPG has been shown to be a competitive inhibitor of phosphorylase in the same organism, suggesting that the concentration of UDPG may regulate glycogen synthesis not only directly, but also by inhibiting the degrada- tive enzymes (49). Madsen (50) has provided further evidence to support the postulate that the control of glyco- gen metabolism is effected by UDPG. The UDPG pathway to glycogen is essentially irreversible, and the equilibrium of the phosphorylase reac- tion is slightly in favor of glycogen synthesis, yet the concentration of glycogen in A. tume- faciens varies considerably. Madsen analyzed A. tumefaciens during its growth in batch culture for glycogen and UDPG content. Both increase initially during a short lag phase and then de- crease at the beginning of exponential growth. In the stationary phase (growth limited by nitro- gen), the concentrations of both compounds rise again, the UDPG slightly before the glycogen. A linear relationship between glycogen concentra- tion and UDPG concentration was demonstrated. Replenishment of the nitrogen supply in the stationary phase in another experiment caused resumption of growth, depletion of UDPG, and cessation of glycogen synthesis. During aeration of washed suspensions of A. tumefaciens in buf- fered salt solution, the glycogen was utilized. The UDPG concentration during this period re- mained low and constant. Glycogen is a highly branched polysaccharide, and little or no reference has been made to the synthesis of (al ,6') links; the UDPG synthetase will only synthesize (al,4') bonds although a branched primer is required. A branching en- zyme similar to Q-enzyme has been described in yeast which will synthesize glycogen from amylose by transglucosylation (al ,4' to al,6'). Amino Acid Pools The first indication that the free amino acid pool could serve as a source of substrates for endogenous respiration was provided by Dawes and Holms (14). Aeration of washed suspensions of stationary-phase, peptone-grown S. lutea re- duced the endogenous Qo2 values to negligible levels, during which time the free amino acid pool was depleted to one-half its original level and ammonia was released into the supernatant fluid. Hydrolysis and analysis of the hot water-soluble pool also showed that some peptide material was being used as substrates of endogenous respira- tion. The total oxygen consumption during the period of endogenous respiration corresponded to 7.5 and 3.4 jsmoles of oxygen per Emole of utilized amino acid in the unhydrolyzed and hydrolyzed pools, respectively. The latter value is a reasona- ble average figure for the oxidation of a mix- ture of the amino acids that are utilized during the starvation. Glycine, threonine, leucine, tryptophan, and a-aminobutyric acid were completely utilized, and much of the serine, glutamate, and alanine of the pool was oxidized. Very little loss of amino acids to the suspending media occurred (about 2% of the initial concentration of the hydrolyzed pool). Glutamate plays a most important role in the endogenous respiration of S. lutea; it accounts for 20% of the total pool amino acids in freshly VOL. 28, 1964 135 DAWES AND RIBBONS harvested peptone-grown cells, and this declines to 1% after a 5-hr aeration period. Although glucose-peptone grown S. lutea also stores a polyglucose compound as a reserve mate- rial, the free amino acid pool is depleted just as rapidly during starvation of these cells (9, 65). The utilization of free amino acid pools as en- dogenous substrates has since been shown to occur in Nocardia rugosa (2) and Staphylococcus aureus (64). During starvation of washed mycelial sus- pensions of N. rugosa, both the endogenous Qo, and Qo, (glucose) fall by approximately the same value. There is a loss of weight from the mycelia that can be accounted for almost completely by loss of protein; a little acid-soluble carbohydrate, mainly hexose, is also utilized. There was no sig- nificant utilization of lipid during starvation for 16 hr. The pH value changed from 7.5 to 8.2 during the incubation, and free ammonia was formed at the expense of the mycelial protein. Oxo-acids did not accumulate but were oxidized. Chromatography of the free amino acid pools showed that fresh mycelia contained large amounts of glutamate, alanine, aspartate, leu- cine, and valine, whereas only smaller amounts of leucine, alanine, and glutamate remained after starvation. The RNA and DNA contents of the cell are not utilized endogenously. It was con- cluded that the main substrates of endogenous respiration are the amino acids that are present in the pool, and also obtained from the hydrolysis of cell protein; an acid-soluble carbohydrate is also utilized. The presence of an exogenous source of glucose inhibits the endogenous consumption of protein in N. rugosa. Ramsey (64) showed that washed suspensions of S. aureus respire endogenously with an RQ of 0.83 to 0.86. The carbohydrate content of the cells remained constant (at the very low value of 1.18% of dry weight) during starvation, and ammonia was released into the supernatant fluid. The 02-NH3 ratio was 6.2, and glutamate was utilized from the free amino acid pool. This indi- cated that substances other than glutamate were being utilized (02-NH3 ratio for glutamate is 4.5). The endogenous respiration of incompletely C'4-labeled cells confirmed this view, since apart from the utilization of glutamate in the pool there was also some loss of C14 from the hot tri- chloroacetic acid-insoluble fraction of the cells; further, the oxygen consumption was much in excess of that required for oxidation of glu- tamate alone (glutamate can account for only 10% of the 02 consumed). More completely labeled cells were obtained by growth on U-C14- glucose and C'4 algal hydrolysate, and the change in cell components during respiration was fol- lowed. Only about one-third of the radioactivity lost from the cells is recovered as C'402. The rest can be traced to the suspending buffer, and rather more than one-third to deposition in the hot trichloroacetic acid fraction. The bulk of the radioactivity was lost from the hot trichloroacetic acid-insoluble fraction. Apart from the utilization of glutamate from the free amino acid pool, evi- dence was presented to show that aspartate was a probable substrate and, to a lesser extent, alanine. Cells, allowed to assimilate C'4-glycine into the pool, washed, and subsequently starved, liberated C1402 only slowly. Comparisons of the endogenous metabolism of coagulase-positive (+) and -negative (-) S. aureus have revealed marked differences (D. Ivler, personal communication). During starvation of organisms grown on Brain Heart Infusion, the RQ of + cells showed little change (from 1.08 to 0.95), whereas that of - cells fell from 1.10 to approximately 0.5 in 60 min. The Qo2 of + cells was higher than that of - cells, but both values decreased rapidly with starvation. High rates of endogenous respiration of + cells suppressed the rate of oxidation of added glucose, but no such effect was apparent with - cells. Measurement of 02-NH3 ratios during starvation of washed suspensions showed that values fell from 4.7 to 3.0 with + cells and from 13.4 to 10 with - cells. Of considerable note was the fact that while the free amino acid pool content of both types of cell diminished, the bulk of the loss could be accounted for as free amino acids in the super- natant fluid (as much as 90% recovery with - cells); nonetheless, ammonia appeared in thesupernatant at a greater rate and to a greater extent with + cells, indicating the net degrada- tion of nitrogenous materials. The total carbo- hydrate content of both + and - cells did not alter during starvation. RNA Metabolism Mlore typical storage compounds such as glycogen or PHB are characterized by their deposition during conditions of carbon source excess or nitrogen limitation, and by their de- pletion to almost negligible amounts during 136 BACTERIOL. REV. ENDOGENOUS METABOLISM OF BACTERIA starvation. However, as Herbert (34) empha- sized, the amounts of such basal materials of the bacterial cell as RNA, DNA, and protein are subject to wide variation, and this can be con- trolled by environmental conditions. Strange, Dark, and Ness (81) showed that RNA is metabolized endogenously during starva- tion of washed stationary-phase suspensions of A. aerogenes, and net degradation occurs. The extent of RNA catabolism varied, and this depended on the source of the cells; 40% of the RNA of cells harvested from defined media was utilized in 70 hr, during which time about 70% of the population remained viable. [See also E. coli (7)]. Cells harvested from glucose-Tryptone media contain much less RNA (about 11% of the dry weight), and little is utilized. These cells contain glycogen that is almost completely respired within 25 hr. Tryptic meat broth grown A. aerogenes, on the other hand, catabolize nearly half their RNA, from about 13% to 7% of the dry weight, within about 50 hr. The products of RNA metabolism that have been detected in the suspending fluid include ammonia, inorganic phosphate, and the free bases hypoxanthine, uracil, and guanine (62, 81, 83), and small amounts of adenine (83); hypoxanthine was the major component of the bases in the supernatant (83). Nucleotides and nucleosides did not ac- cumulate to any significant extent, and most of the pentose was apparently oxidized. The ultra- violet-absorbing materials were readily released into the supernatants, and acid-soluble inter- mediates did not accumulate within the cells (83). The amount of ultraviolet-absorbing material released corresponded well with the amount of RNA lost from the cell, and this loss occurred almost entirely from the RII sedimentation fraction (83), as had been demonstrated in the case of loss of RNA from E. coli ribosomes (85). RII is the cell fraction sedimented at 78,000 X g for 7.5 hr. The endogenous utilization of RNA is not peculiar to A. aerogenes, but appears to be very widespread, and has been demonstrated in E. coli (17, 18), S. lutea (9), and P. aeruginosa (27). The utilization of the ribose portion of the RNA by endogenously respiring E. coli was briefly mentioned in the section on glycogenlike reserves, and in Table 2. It is not yet clear whether the purine and pyrimidine bases are completely oxidized, although this is unlikely as ultraviolet-absorbing compounds accumulate in the supernatants. The degradation of RNA in E. coli was studied by Wade (85), who concluded that two pathways exist. The M route (Mg2+- dependent) results in the formation of nucleo- side 5'-phosphates characteristic of phosphodi- esterases. The rate of formation of these nucleo- tides is further stimulated by inorganic phos- phate, suggesting that polynucleotide phos- phorylase was also depolymerizing RNA (86). Autodegradation of ribosomes in the presence of inorganic P32-orthophosphate gave only labeled nucleoside diphosphate. The nucleoside mono- phosphates appeared to be formed by an inde- pendent route (86). The second route, the V route, results in the degradation of RNA into nucleoside 2', 3'-cyclic phosphates in the presence of sufficient EDTA to remove the M\g2+. The cyclic phosphates are further hydrolyzed to nucleoside 3'-phosphates. The V route then employs ribonuclease-type enzymes. The enzymes of both routes are located in the ribosomal frac- tion. The observation of Kiguchi and Uemura (84a) that citrate and phosphate enhance the release of RNA degradation products from yeast cells is, perhaps, relevant. These authors believe that magnesium is removed from the cell membrane by chelation, since added Mg2+ countered the effect of these agents. Starving washed suspensions of P. aeruginosa release much ultraviolet-absorbing material into the supernatant with an Emax at 260 mIu. Nucleo- tides, nucleosides, and free bases were detected. The effect of Mg2+ ions on ribosomal particles is well known (8), and Gronlund and Campbell (27) have used this as evidence for the utilization of RNA as a substrate for endogenous respiration. Oxygen consumption is depressed in the presence of Mg2+ ions, which allow the formation of 70S ribosomes. When P. aeruginosa was grown in the presence of C14-uracil, the radioactivity was con- tained primarily in the nucleic acid fraction, and this yielded C'402 during subsequent starvation. The C0402 was derived solely from the RNA, and came largely from the ribosome fraction. The enzymatic degradation of ribosomes was demon- strated and inhibited by EDTA; phosphate markedly increased ribosome degradation, sug- gesting a role for polynucleotide phosphorylase. The fate of the ribose portion of the RNA was not determined, although ribose (free and com- bined?) was detected in supernatant fluids. 137VOL. 28, 1964 DAWES AND RIBBONS By calculations based on the values of C1402 released from uniformly C'4-labeled cells, 2-C04- uracil-labeled cells, and U-C'4-proline-labeled cells, Gronlund and Campbell deduced that the C'402 liberated from 2-C'4-uracil-labeled and from U-C14-proline-labeled cells is equivalent to the total amount of C1402 liberated by uniformly labeled cells. From this, they infer that RNA and protein are the only endogenous substrates oxidized. Their calculations are based, however, on the assumption that the fate of the 2-C of uracil is representative of all RNA carbon atoms, since, for example, the ribose was presumably unlabeled by this technique and its fate was not determined. The fate of the proline C atoms is also assumed to reflect the destiny of all the other carbon atoms of protein. Protein The net utilization of protein as an endogenous substrate was first demonstrated by Strange et al. (81) with starving suspensions of A. aerogenes, and Gronlund and Campbell (26) indicated that ammonia release by endogenously respiring cells was a general phenomenon. Thus, washed sus- pensions of E. coli, P. aeruginosa, P. fluorescens, Achromobacter sp., B. subtilis, and S. faecalis all liberate ammonia during endogenous respiration (15, 26, 87). To this list may be added A. aerog- enes (81) [although Gronlund and Campbell (26) did not detect ammonia production with their strain], B. cereus (12, 47), S. lutea (14), S. aureus (64), and N. rugosa (2). Even so, the original work of Strange et al. is the most decisive demon- stration of protein degradation, since they used simple chemical analysis. Other workers have concluded from radiochemical evidence that protein is a substrate of endogenous respiration; in some bacteria, it appears to be the main substrate. With P. aeruginosa, Gronlund and Campbell (27) labeled the cells with U-C14-proline and demonstrated the endogenous evolution of C1402 from the hot trichloroacetic acid-insoluble fraction. It is interesting to note that the alcohol- soluble protein of P. aeruginosa accounts for 20% of the total protein (60) and yet this is not utilized during starvation. The hot trichloroacetic acid- insoluble residue yields less C1402 when cells are starved in the presence of Mg2+; under these con- ditions, there is also a slight decrease in the alco- hol-soluble protein. Pine (60) demonstrated that the alcohol-soluble proteins of E. coli do not dis- appear during starvation; the solubility proper- ties of this fraction alter, however, and cautiousinterpretations with respect to the fluctuations of this fraction are required. Strange et al. (83) showed that protein is lost from all ultracentrifugal fractions during starva- tion of A. aerogenes; i.e., ribosomal and soluble proteins are utilized. The products of protein catabolism appear to be ammonia and carbon dioxide, as only traces of amino acids accumulate in the suspending fluid. Attention must be directed to the data ob- tained by Strange et al. (81) for the release of am- monia from cells grown in different media. If, on the basis of their figures, a calculation is made to compare the nitrogen accounted for as NH3 with the nitrogen of the degraded protein and RNA, considerable discrepancies are apparent. Thus 78.8, 25.2, and 30.2% nitrogen are unaccounted for as NH3 in cells harvested from glucose-Tryp- tone, Tryptone meat broth, and defined media, respectively, after 25 hr of starvation. The fate of this nitrogen has not been ascertained, and some cellular redistribution might be envisaged. Other Potential Substrates Our knowledge of bacterial lipids is very limited (34), especially concerning their role as endogenous reserves of energy. It appears that conventional lipids are stored by E. coli under favorable conditions; e.g., provision of acetate stimulates lipid deposition (13). The utilization of lipid and phospholipid materials during starva- tion has been discussed (15). The massive deposi- tion of lipids by the yeasts Rhodotorula gracilis, Lipomyces starkeyi, R. graminis, and R. glutinis was recently described by Mulder and co-workers (58). DNA is generally considered to be a stable cell constituent whose function is storage of informa- tion, and generally no utilization of DNA occurs during starvation. However, Strange, Wade, and Ness (83) showed that the DNA of starving A. aerogenes increased by 17%, and they ruled out the possibility of cell division occurring. A DNA increase at the expense of RNA was also demon- strated with phosphate-deficient E. coli (37). On the other hand, utilization of DNA after 19 hr of starvation was noted in A. aerogenes (30). ENERGY OF MAINTENANCE Cellular processes, whether mechanical or chemical, require energy for their performance, 138 BACTERIOL. REV. ENDOGENOUS METABOLISM OF BACTERIA and unless a supply of energy is readily available these essential processes will cease and the cell will die. The provision of energy by exogenous nutrients is well established, e.g., the use of glu- cose as an energy (but not carbon) source by Streptococcus faecalis (4). Under conditions of starvation, mobilization of endogenous sub- strates must furnish the energy necessary to allow the various cellular activities to continue. The energy required for these processes of cell sur- vival has been called the "energy of maintenance." Since the performance of work is required for all these activities, which include resynthesis, osmotic regulation, and heat loss to the external environment, presumably they will eventually cease owing to lack of energy, and the cell will no longer be able to maintain its status quo. To maintain the intact living cell, structures such as the cell wall, flagella, cell membrane, and cell particles must be kept in good repair. It is pos- sible that some of these structures may be dis- pensed with for the sake of survival; indeed, flagella have been removed from bacterial cells without affecting viability (45), while suitably prepared protoplasts possess metabolic activities that are identical with those of whole cells (71). The possession of a rigid cell wall provides dis- tinct advantages, since protoplasts remain intact only in solutions of high osmotic pressure. The cell wall is probably an essential feature of cell survival under natural conditions. Protoplasts are able to grow (increase in size) but apparently are unable to undergo cell division unless rever- sion to bacillary form (in gram-negative organ- isms only) occurs (55a). Mandelstam (52) reported that starved sus- pensions of E. coli break down and resynthesize their macromolecular components (RNA and protein) at a rate of 5% per hr. On the other hand, growing cells appear not to exhibit any appreci- able turnover of protein. The continued resynthe- sis of macromolecular components during starva- tion requires energy, which may be supplied by the components that are undergoing transforma- tion. This situation pinpoints a feature not gen- erally appreciated, namely, that the maintenance requirement is not necessarily a constant feature independent of growth rate; i.e., at the extremes of unrestricted growth and absence of growth, protein turnover (and, therefore, the energy re- quired for this process) varies from 0 to 5% per hr. Consequently, unrestricted growth should have a lower maintenance requirement. Microorganisms are able to survive for con- siderable periods during starvation and conse- quently must maintain soluble constituents, often against considerable concentration gradients. The regulation of the cytoplasmic osmotic pres- sure and pH value appear to be highly selective processes, since considerable quantities of some cell substances, e.g., RNA from bacteria and yeast, and also smaller molecular moieties, such as amino acids and bases of RNA, ribose, and in- organic phosphate, are able to diffuse into the suspending fluids without loss of viability (7, 17, 27, 68, 81). For bacteria to remain motile, a source of energy is required; tactic responses were dis- cussed at some length by Weibull (89). In the absence of exogenous substrates, energy must be supplied from endogenous sources. Phototrophic organisms present a somewhat different case in that their source of energy is light. If light is the sole source of energy available to green and purple sulfur bacteria, then incubation of washed suspensions in the dark should exclude mecha- nisms of ATP synthesis, and it would be of in- terest to know the survival characteristics of these species in the dark. This situation is similar to the survival of strict aerobes under anaero- biosis. Although we are principally concerned with the concept of maintenance energy in starving cells, it is obvious that some consideration of growing cells should be included; additional demands for energy may be manifest here. The actual proc- esses of cell division may require larger amounts of energy than the other phases of the growth cycle of individual cells which, in the main, would be supplied by exogenous sources. Lowered cell yields at low growth rates may, in fact, be caused by the diversion of energy from synthesis of cell substances to the physical and energetic task of maintaining a cell that spends a long period over division. Alternatively, cells actually dividing may dissociate energy-yielding reactions from synthesis without affecting the rate of utilization of energy source. The chemical and mechanical activities of the microbial cell, therefore, lead one to postulate that some energy is utilized to maintain the cell in a functional and viable condition, and that during starvation this energy must be derived from VOL. 28, 1964 139 DAWES AND RIBBONS endogenous sources. We think few scientists would now deny the energy-of-maintenance re- quirement, although there are several widely quoted experiments that have been cited as evidence against such a concept, often because it could not be detected. This is especially apparent in the growth-yield experiments of Monod (57) and Bauchop and Elsden (4). Microbial growth yields are (within certain limits) directly propor- tional to the concentration of limited nutrient. Even when the carbon and energy source limits the yield of cells, the relationship holds for very low concentrations; extrapolation of the experi- mental points indicates that no intercept occurs,i.e., at zero concentration of nutrient no growth occurs. If some portion of the energy source were utilized for functions other than growth, then one should observe that the addition of very small amounts of an energy source would not permit growth. This then would assume that growth is a secondary feature of energy utiliza- tion and that energy is preferentially channelled to maintenance purposes. The concentrations of the energy source used in these experiments were such that, although they limited the maximal population attainable, they did not limit the rate of growth. Monod also limited the rate of growth of E. coli by limiting aeration, and although this doubled the time taken to achieve maximal density in one culture the cell yield was un- changed. He concluded that since the rate of growth did not influence the cell crop any energy-of-maintenance values were nil. It seems to us that many of the experiments designed to test the use of a portion of the exoge- nous energy source for maintenance rather than growth are complicated by the fact that the energy source is also a source of carbon for growth. More definitive evidence might be ob- tained with microorganisms which do not in- corporate their energy source into cell substance; there are numerous systems available for such experiments-phototrophs, autotrophs, and the nutritionally fastidious anaerobes that ferment carbohydrates almost solely as a source of energy. Furthermore, experiments designed to show that some portion of the energy (and carbon) source is not utilized for growth, and many do demon- strate this, are not entirely convincing arguments for the maintenance concept. These experiments do not show that the energy that is diverted from growth is utilized specifically for mainte- nance. It seems feasible that at slower growth rates the cell yield might be decreased because the cell physiology with respect to regulatory mecha- nisms has altered in response to the changed en- vironment, and that a portion of the energy source is uncoupled at the enzymatic sites of phosphor) lation; i.e., the decreased cell yield is merely a reflection of decreased efficiency of conversion f the energy source into high-energy phosphate. It is perhaps too obvious that the reactions of energy-yielding metabolism are not always coupled to the growth of the organism. The more extreme cases of this are most often observed in batch cultures that have reached a stationary population but still consume considerable quan- tities of substrate. This same feature is seen dur- ing growth limitation (rate or yield) of some nutrient other than energy source, and is most dramatically demonstrated by washed suspen- sions of nonproliferating cells metabolizing added carbon or energy substrates. Where the energy source is also the carbon source, usage of carbon skeletons occurs without net growth. In some cases, the carbon skeletons are removed from the medium and stored within the cells (assimilation) ready for utilization under duress. However, uncoupling of assimilation has also been observed, in that products of metabo- lism often appear in supernatants, and presum- ably these are lost to the cell. Further, it has been suggested that the products of metabolism under conditions of carbon and energy excess are shunt products, and that assimilation into reserves merely reflects this glut. Limitations of the rate of microbial growth by nutrients other than the energy source do not control the extent of oxida- tion of the energy source; e.g., Rosenberger and Elsden (67) showed that in tryptophan-limited growth S. faecalis produced large amounts of lactate. A discussion of the theoretical principles of continuous culture (as controlled by nutrient limitation), and a comparison of these with results obtained experimentally, led Herbert (33) to postulate that A. aerogenes displays a constant rate of endogenous metabolism during exponen- tial growth. The curve derived experimentally relating steady-state bacterial concentration to dilution rate (growth rate) does not coincide with the theoretically predicted curve. At low dilution rates, the cell yield is less than that expected when the carbon and energy source is the growth- 140 BACTERIOL. REV. ENDOGENOUS METABOLISM OF BACTERIA limiting nutrient. Cultures whose growth is limited by nutrients other than the carbon and energy source do not show this phenomenon at low growth rates, so that the decreased cell yields observed under these conditions are not simply a property of the growth rate. The lowered cell yield recorded during slow growth on a limiting carbon and energy source was explained by sug- gesting that, in addition to cell synthesis from the carbon substrate, there is also a constant oxi- dation of cell substance to C02, i.e., some turn- over is occurring during growth. (The experimen- tal evidence for this is considered later.) The equation representing the exponential growth of a bacterial culture can be modified from dx ds -= sx = -Y-dt dt to dx du= _- k)xdt cohere x = cell concentration; s = substrate utilized; t = time; A = growth rate; k = a con- stant representing the endogenous metabolism; and Y = yield coefficient. Thus, by lowering the dilution or growth rate, k becomes proportionally larger in relation to A and, therefore, the cell density falls; the cell yield is lower because pro- portionally more cell material is oxidized relative to the amount of limiting nutrient that is assimi- lated. In toto, the net result is an uncoupling of growth from oxidation of carbon substrate, since proportionally more nutrient is oxidized than is assimilated per cell. This idea was expanded by Marr et al. (53), who designed experiments to determine the value of k (these workers substitute a for k) which they call the specific maintenance. Mathematically, the lowered cell yield at low dilution rates can be expressed as: dx (u-kx Yds -Z = (~a-k)x =-Yddt dt Rearranging, Y ds x dt Now Y. x, ds/dt, and At (or D, the dilution rate) can all be determined experimentally, and, there- fore, k may be evaluated. Substitution of k into the continuous culture equations enables plots of steady-state cell con- centration versus dilution rate to be made that correspond exactly with those obtained experi- mentally (33). Evidence to interpret k as repre- senting a constant endogenous metabolism that occurs during growth is demonstrated by com- paring the rates of respiration of A. aerogenes in continuous culture at different growth rates. Extrapolation to zero growth rate of Qo2 and Qco2 values determined at different growth rates in media containing limiting glycerol gave values on the ordinate that were identical to the Qo2 (endogenous) and Qco2 (endogenous) of these cells. It is suggested that the respiration consists of (i) substrate oxidation that is proportional to the growth rate and (ii) a constant rate of oxida- tion of endogenous material, that occurs at all growth rates. Carbon balances (details of which were not recorded) were also claimed to indicate that proportionally more cell carbon than sub- strate carbon is oxidized. The lowered cell yields at low dilution rates (carbon and energy source limiting) have been observed for other bacteria and for Torula utilis (33). Marr et al. (53) noted that E. coli is unable to maintain cell density at low dilution rates, and they calculated the specific maintenance as 0.025 hr-1. The carbon balances of substrate utilization at different growth rates received attention from Marr et al. (53) with batch cultures. U-C14-glucose was (i) added to a batch culture, giving exponen- tial growth; (ii) fed rapidly to a culture so that only a small fraction would be used for mainte- nance, giving linear growth; and (iii) fed slowly so that a large fraction was used for maintenance,giving curvilinear growth. It had previously been shown that the cell crops obtained increased in the order: slowly fed cultures < from batch cultures < from rapidly fed cultures. The radiochemical results confirmed this observation and also showed that more of the glucose is oxidized to C1402 in slowly fed than in batch, which in turn is greater than in rapidly fed cultures. The reverse order was found for C14 assimilated by the cells. It is not entirely clear why the batch cultures should lie between the fast and slow feeding of energy source, but some ex- planations may be offered. During exponential growth in batch culture, the rate of growth is not limited by glucose concentration and it appears that glucose is utilized less efficiently; e.g., it is not oxidized to CO2 immediately, possibly owing to a limitation of oxygen concentration. Conse- 141VOL. 28, 1964 DAWES AND RIBBONS quently, growth may cease and the stationary- phase cells oxidize the accumulated intermediates to CO2. Lower growth yields in batch than in continuous or nutrient-limited cultures were also observed by Pirt (61). The energy required for turnover of macro- molecules appears to account for a larger propor- tion of the energy source that is not utilized for growth in E. coli (53), but these workers could not demonstrate that accumulation of methyl- thiogalactoside was responsible for any signifi- cant amount of energy expenditure, although Kepes (40) noted that addition of this compound to E. coli suspensions resulted in a doubling of the rate of endogenous metabolism. The earlier ideas concerning the concept of energy of maintenance were excellently sum- marized by Mallette (51) and McGrew and Mallette (55). They indicated that lack of sensi- tive techniques was among the reasons for the difficulty of demonstration of the energy of main- tenance. To overcome this problem, they used a high cell density in relation to a low concentration of carbon and energy source to study the mainte- nance requirement of E. coli. Low concentrations of glucose were fed to suspensions of E. coli in an otherwise complete growth medium, and the tur- bidity changes were recorded. When very small additions of glucose were made, the extinction did not alter appreciably from control cultures after a standard time. Higher concentrations of glucose permitted growth to occur. Thus, they demon- strated that a threshold concentration of glucose is required before growth can occur. Cell suspen- sions starved with respect to the carbon and energy source showed rapid decreases in turbidity for 5 days, at which time only 15% of the cells were viable, and then remained constant while the viability continued to fall. A small addition of glucose at 6-hr intervals, sufficient to maintain the turbidity at a constant value, also suppressed the rate of loss of viability; e.g., after 5 days only 20% of the cells had died. Glucose additions that permitted a very slow growth (20% increase in extinction over 10 days) did not prevent death of the cells. (After 5 days, approximately 10% had died.) Thus, it would seem that small amounts of glucose can provide energy to maintain the cell without allowing growth to occur. The loss of viability that occurs during the slow growth may be due to the interval method (6 hr) of feeding, as pointed out by Marr et al. (53); i.e., some breakdown of cellular material occurs before the next glucose supplement, and this is insufficient to allow reclamation of the lost cell materials. A criticism which may be leveled at this type of ex- periment is that growth may be occurring al- though it is not revealed by turbidity measure- ments. The number of cells dying may be such that the turbidity undergoes no net change as growth occurs. The influence of higher cell con- centrations on these phenomena is not known, and regrowth (28) may assume special impor- tance; perhaps open systems should be considered in experimental design. STARVATION AND SURVIVAL The early literature concerning the effects of starvation upon the survival of microorganisms was critically reviewed by Postgate and Hunter (62). They also draw attention to the pitfalls and difficulties that may be encountered during the estimation of viabilities. The cleanliness of labo- ratory ware and purity of chemicals is considered to be critical, as trace impurities may either per- mit growth of otherwise starving organisms (23) or kill the cells. The growth of bacteria at the expense of their companions has been termed cryptic growth (70), cannibalism (28), and re- growth (81). This growth is a function of cell density and can considerably influence the sur- vival behavior of starving suspensions, since a "population turnover" may occur. Apart from the phenomenon of regrowth, the initial cell density of starving suspensions also affects their death rate. Harrison (28) first showed the relationship between cell density and death rate of starving suspensions of A. aerogenes, and an optimal density for survival was demon- strated. He concluded that an interaction be- tween individual cells favors survival, and the work of Postgate and Hunter (62) removes any doubt about the possibility of regrowth occurring. The latter authors made a very thorough study of many factors that influence the survival of starving suspensions of A. aerogenes. They elimi- nated ambiguity that would arise from cryptic growth, growth on impurities, and toxicity of suspending fluids, by a suitable choice of cell density [20 ,ug (dry weight) of cells per ml] and suspending fluid [saline-tris(hydroxymethyl)- aminomethane buffer-EDTA solution]. High illu- mination, high temperatures, high pH values, and high potassium ion concentrations increased the 142 BACTERIOL. REV. ENDOGENOUS METABOLISM OF BACTERIA death rates of starving suspensions. For example, A. aerogenes survives better at 20 C than at 30, 40, or 10 C. Anaerobiosis accelerated death, and this was attributed to the acid conditions produced. We also have observed that anaerobically starved E. coli die faster than the aerobically starved sus- pensions (18); however, the pH value during anaerobiosis fell only from 7.2 to 6.8 with our more strongly buffered suspensions (unpublished data). Various nutrients, or the previous history of suspensions of A. aerogenes, markedly affected the death rates. Ca2+, Mg2+, and to a lesser extent Fe2+, when added to the saline-buffer, prolonged the life of the cells. The slower the rate of growth of the bacteria, the greater was their death rate upon fasting. This applied to organisms whose growth rate was limited by C, N, P, and S, but with Mg2+-limited growth the reverse was true and the cells that had most rapidly proliferated died fastest when starved. The effect of nutrient additives upon death rates is, however, more com- plex and depends upon the previous history of the cells. Thus, Postgate and Hunter (63) observed a general phenomenon of substrate-accelerated death, in which the addition of the growth-limit- ing nutrient to starving suspensions increased the death rate. Glycerol-limited cells of A. aerogenes showed glycerol-accelerated death (metabolites of glycerol, e.g., pyruvate, also accelerate death); NH4+-limited cells are killed by NH4+ additions but not by glycerol, which is slightly protective. Phosphate-limited cells behaved similarly, as did other carbon source-limited cells. Sulfate-limited cells were not killed quickly by sulfate, but in- stead showed glycerol-accelerated death. Mg2+- limited cells were another exception, in that addi- tion of Mg2+ actually prolonged the life of these cells, as it does of other cells, a feature quite inde- pendent of the nutrient that limited their growth. Harrison and Lawrence (30) also noted that the effect of nutrient additions to starving suspen- sions is influenced
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