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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 Substrates ......................................
Endogenous 144
SUMMARY AND FUTURE OUTLOOK ............................................................... 145
LITERATURE CITED ............................................................................. 146

"One great use of a Review, indeed, is to have used the term "endogenous" loosely in
make men wise in ten pages, who have no relation to the activities of cell extracts (see, for
appetite for a hundred pages; to condense example, 84).
nourishment, to work with pulp and essence, It must also be recognized that the reactions
and to guard the stomach from idle burden characteristic of endogenous metabolism may
and unmeaning bulk."
Sidney Smith continue in the presence of exogenous substrates,
Edinburgh Review, 1825 and, since metabolism of the latter compounds
is usually effected after they have been taken
into the cell, in the final analysis the distinction
INTRODUCTION between the metabolism of endogenous and
Current interest in the endogenous metab- exogenous substrates may become largely a
olism of microorganisms has been reflected by matter of semantics, although problems of com-
two recent symposia (Focal Topic A7, Intern. partmentation within the cell should be appre-
Congr. Microbiol., 7th, Montreal, 1962; Ann. ciated (22a). This is particularly the case where
N.Y. Acad. Sci., vol. 102, art. 3, p. 515-793, the oxidation of an exogenous substrate by a
1963) and by our own previous survey (15). The washed suspension of microorganisms leads to the
present article is intended to be a more discursive assimilation of material which subsequently can
review of the present situation in this area of be utilized as an endogenous substrate. Analysis
study and to deal in greater detail with those of the status of the endogenous metabolism in
aspects which were not previously elaborated. the presence of added substrates presents many
Endogenous metabolism may be defined as experimental problems, but these have already
the total metabolic reactions that occur within been adequately reviewed (6, 15).
the living cell when it is held in the absence of The possible significance of endogenous metab-
compounds or elements which may serve as olism in relation to the survival of microor-
specific exogenous substrates. Some products of ganisms is a topic presently under study in
endogenous metabolism may be released into several laboratories. One view which may be
the surrounding medium and are often utilized held is that endogenous metabolism occurs
by the cells, sometimes resulting in regrowth, a simply because the organism cannot help it, and
phenomenon which is discussed later. It is im- it therefore bears no relationship, direct or other-
portant to stress the viability and integrity of wise, to the period of survival (22). The other
the cell in this context, because some authors extreme outlook is that the survival character-
126
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 127
istics are related directly to the endogenous SUBSTRATES FOR ENDOGENOUS
metabolic activities of the cell. The evidence METABOLISM
available at the time of writing suggests that,
for those organisms which have been studied, A consideration of possible substrates for
the truth lies somewhere between these extremes, endogenous metabolism naturally focuses atten-
as will be discussed later. tion on energy-storage compounds. Wilkinson
The existence of an energy of maintenance for (93) described three main classes of compounds
microorganisms has attracted considerable ex- that could possibly act as energy-storage com-
perimental attention of late. The idea that a pounds. These are polysaccharides, lipids (includ-
definite amount of energy must be expended to ing PHB), and polyphosphate; all occur in
enable a microbial cell to maintain its integrity widely varying amounts dependent upon the
without growth or death occurring seems a particular species and the environmental condi-
reasonable concept, and appears now to be sup- tions. However, it is now apparent that there
ported by some evidence. The question of are many other substrates for endogenous
whether a cell can exist in a condition such that metabolism; these include ribonucleic acid
it is committed neither to growth nor to death is, (RNA) and protein (both are also subject to
however, still a debatable issue. The experi- turnover) and free amino acid and peptide pools.
mental problems posed by the death and regrowth Possible substrates not yet implicated in endoge-
of bacterial cultures make difficult a conclusive nous metabolism include deoxyribonucleic acid
answer to this query at present. (DNA), cell-wall polymers, and cell-membrane
Aspects of endogenous metabolism concerning materials.
macromolecular turnover and cell differentiation
into spores were focal points of the symposium Role of PHB
published by the New York Academy of Sciences, The elucidation of the role of PHB in bacterial
and will not be elaborated here. A symposium on endogenous metabolism is a fascinating story,
microbial reaction to environment has also for, although the polymer was originally dis-
directed attention to the considerable variation covered in 1927, a physiological role for it was
in composition and properties that can be pro- not convincingly demonstrated until some 30
duced by alteration of experimental conditions, years later. PHB was first isolated by chloroform
and additionally to the influence of the previous extraction of an aerobic bacillus by Lemoigne
history of the cells. Although the effect of some (42), following his earlier discovery that (3-
environmental factors on endogenous metab- hydroxybutyrate was a metabolic product of the
olism has been investigated (65), there are many organism. Since that time, PHB has been demon-
other aspects of this same problem which have strated in a wide variety of bacterial species
not been explored. Indeed, there are so many (Table 1), and in some instances has been im-
possible external influences on endogenous plicated as an assimilatory product, from meas-
metabolism that considerable caution should be urements of gaseous exchange during photo-
exercised in making generalized statements con- metabolism of fatty acids and gross elemental
cerning the endogenous metabolic behavior of composition, by Gaffron (cited in 78). The quan-
microorganisms. tities of PHB within the bacterial cell vary enor-
The present review will pay particular atten- mously; contents of up to 50 % of the dry weight
tion to the role of poly-f3-hydroxybutyrate have been recorded. It is a reserve that is peculiar
(PHB) as a substrate for endogenous metab- to microorganisms, and its functions, formation,
olism, because, as a storage compound unique to and synthesis have been studied extensively in
bacteria, it holds a position of considerable recent years, mainly by Doudoroff and Stanier,
interest. Several groups of investigators have Gibbons, Schlegel, Wilkinson, and their col-
demonstrated the presence of the polymer and laborators.
Sudanophilic granules present in bacteria were
have studied its metabolism in various bacterial considered by Lemoigne, Delaporte, and Croson
species, but there has so far been no attempt to (43) to be composed of PHB; the data of Weibull
correlate their findings in relation to endogenous (88) subsequently supported this proposition.
metabolism. However, it was not until the work of William-
128 DAWES AND RIBBONS BACTERIOL. REV.

son and Wilkinson (95) that the intracellular menting other substrates. Anaerobic conditions
lipid granules of Bacillus cereus and B. mega- prevented PHB synthesis, as did dinitrophenol.
terium were demonstrated unequivocally to be Concentrations of oxygen greater than 5% also
composed mainly of PHB (about 90%), although inhibited the assimilatory process. B. cereus, but
neither this nor the remaining 10%, lipid is re- not B. megaterium, could effect PHB synthesis
sponsible for the sudanophilic properties of the under hydrogen, although no net uptake of
granules that are observed in situ. Macrae and hydrogen was detected; PHB is not formed under
Wilkinson (46, 47) also studied the effect of nitrogen.
various cultural conditions on the synthesis of When washed suspensions of these two bacilli
PHB in B. megaterium. When the glucose con- were shaken under air or under nitrogen, in the
absence of an exogenous carbon and energy
TABLE 1. Occurrence of poly-,3-hydroxybutyrate in
bacteria* source, stored PHB was metabolized. The
anaerobic degradation of reserves was slower;
Species Reference e.g., in B. megaterium, 61 and 17% degradation
of PHB occurred under air and nitrogen, respec-
Bacillus megaterium ............... 43, 77, 95 tively, within 8 hr. Metabolic products detected
B. cereus .......................... 43, 95 included f-hydroxybutyrate, acetoacetate, and
B. mycoides ....................... 43 acetate, although aerobically CO2 and water
B. anthracis ....................... 43 were the major products and only small amounts
Azotobacter chroococcum ........... 43
A. agilis .......................... 21 of acetoacetate accumulated. Some correlation
A. vinelandii ...................... 21 was evident between the PHB content of cells
Rhizobium sp ...................... 21 and their endogenous respiration; e.g., cells with
Vibrio sp .......................... 31, 32 PHB-total N ratios of 0.83 and 3.27 had, respec-
Chromobacterium violaceum ........ 21 tively, endogenous Qo2 values of 169 and 536.
Pseudomonas solanacearum ........ 32 Macrae and Wilkinson (46) also claimed that
P. antimycetica ................... 32 N-deficient cells with a high content of PHB are
P. methanica ...................... 39 better able to withstand death and autolysis
P. pseudomallei ................... 44 than those with a low PHB content. Autolysis
P. saccharophila .................. 19 was estimated by the total N content which, in
Pseudomonas AMi ................ 59
Micrococcus halodenitrificans ...... 75, 76 4 hr, fell by 12% in PHB-poor cells compared
Sphaerotilus natans ............... 58, 69 with 5% in PHB-rich cells; the PHB content
Hydrogenomonas sp ................ 73 of the latter cells decreased to the greatest ex-
Rhodospirillum rubrum ............19, 78 tent. The method of estimation of autolysis is
Rhodopseudomonas spheroides.... . 10 not entirely satisfactory, because it has been
Chromatiurn okenii ................ 72 shown in numerous cases that nitrogenous mate-
Spirillum itersonnii ............... 54 rials are oxidized endogenously, releasing am-
S. anulus ......................... 54 monia, and that substantial amounts of nitrog-
S. serpens ......................... 32, 54 enous compounds may diffuse from the cell
* This is not a complete list of the bacterial without loss of viability. Specifically in the case
species that synthesize PHB. of B. cereus, Clifton and Sobek showed that am-
monia is produced endogenously under some
centration in the growth medium was raised, conditions (12), and clearly B. megaterium
more of the polymer was synthesized; exhaustion utilizes an endogenous substrate other than,
of the nitrogen source in the presence of excess although concurrently with, PHB, since the
carbon and energy source permitted deposition oxygen consumption is in excess of that required
of about four times the amount of PHB as was for complete combustion of PHB; this other
formed when glucose limited growth. Glucose, substrate is not polysaccharide (47). It is pos-
pyruvate, and f-hydroxybutyrate were suitable sible, however, that autolysis may have occurred
substrates for PHB production by washed sus- to a similar extent in both PHB-rich and PHB-
pensions, but acetate, although itself unable to poor cells, but the greater amount of reserve
effect synthesis (compare with Rhodospirillum material in the PHB-rich cells permitted utiliza-
rubrum), enhanced PHB formation when supple- tion of the liberated nitrogenous material and
VO0L.- 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 129
some limited cellular synthesis occurred. Against absence of an exogenous carbon source. The
this possibility may be set the authors' observa- reason for this became apparent when it was
tion that growth did not occur when PHB-rich shown that succinate is photoassimilated princi-
cells were held in a medium lacking a carbon pally to a glycogen-like polysaccharide, and the
and energy source; growth was measured by dry weight of the cells increased by 40%.
total N so that in these bacilli PHB may serve Similar studies with P. saccharophila revealed
as a reserve of energy but not as a source of that washed suspensions of glucose-grown cells
carbon skeletons for synthesis. incorporated 66% of the carbon assimilated from
Doudoroff and Stanier (19) have made some U-C14-glucose into PHB, and without appre-
interesting observations concerning the role of ciable dilution. An even greater amount (about
PHB in oxidative assimilation by Pseudomonas 80%) of the assimilated carbon from acetate or
saccharophila and in photoassimilation by butyrate appeared in the polymer. Although ex-
R. rubrum. They found with most substrates that perimental details were not given, the authors
a major portion of the assimilated carbon (60 to claimed that tracer experiments indicated the
90%) initially accumulates within the cells as role of PHB as a substrate for endogenous metab-
PHB; when the exogenous substrate is removed, olism in the absence of an exogenous carbon
a rapid intracellular degradation of the polymer source; its metabolism was reported to be much
occurs, suggesting a physiological role as a re- slower than in R. rubrum, and transfer of polymer
serve material. When cells are subjected to carbon to other cell constituents could not be
standard fractionation procedures, the chemical demonstrated.
properties of PHB result in its appearance in the The use of PHB as an endogenous store of
hot trichloroacetic acid insoluble fraction (pro- carbon skeletons for synthesis in R. rubrum was
tein fraction). This fact had led Wiame and studied further by Stanier et al. (78), who showed
Doudoroff (91) earlier to conclude that C14 is that for conversion of stored PHB to other cell
incorporated into nitrogenous materials during materials CO2 is essential. This was demon-
oxidative assimilation. strated with cells which had assimilated acetate
Incubation of starved washed suspensions of and were then incubated in the presence of (i)
R. rubrum with C'4-acetate allowed deposition NH4Cl and He, (ii) NH4Cl and He-CO2 mixture,
of 70% of the assimilated C'4 into PHB, with no and (iii) He-CO2 mixture in the absence of a
significant dilution. With C'4-butyrate, some nitrogen source. After 16 hr, very small changes
dilution occurred, and the polymer contained in total dry weight had occurred and, in the
only 58% of the assimilated C14. The fate of the absence of C02, the PHB content of the cells
polymer in the light over a period of 12 hr was fell slightly but with no increase in the carbo-
studied under a variety of conditions, e.g., in the hydrate or nitrogen content. In the presence of
presence and absence of a source of organic sub- CO2 without a nitrogen source, the PHB content
strates but in the presence of a N source and fell by about 50% and the carbohydrate content
CO2. The absence of an exogenous organic carbon showed a corresponding gain, but there was no
source led to the disappearance of more than 90 % change in nitrogen content. WNhen both nitrogen
of the polymer, but much of the C14 of this and CO2 were furnished, the PHB disappeared
material was redistributed into other cellular almost completely with concomitant increases in
components. The authors did not study the fate both carbohydrate and nitrogen content, includ-
of PHB in both nitrogen and carbon starvation, ing protein. None of the experiments was de-
or in the dark, so its behavior under these con- signed to test the possibility that PHB may
ditions is not yet known. The rate of degrada- serve as an energy store, as it does, perhaps, in
tion of PHB and its conversion to other cellular B. megaterium and presumably also in P. sac-
components was decreased when an exogenous charophila. It is considered that PHB serves as a
source of butyrate was supplied. In marked con- store of carbon and reducing power for further
trast, succinate, which is also metabolized under CO2 assimilation, which is essential for PHB
these conditions, was quite unable to prevent utilization. The anaerobic utilization of PHB in
polymer breakdown and transfer of its C skele- the absence of CO2 and a nitrogen source would
tons to other cell constituents; these processes appear to be limited, but neither its formation
occurred to about the same extent as in the nor degradation aerobically in the dark has been
130 DAWES AND RIBBONS BACTERIOL. REV.

studied, and here one might expect PHB to conditions, some lysis of cells occurred and the
serve as a source of energy. endogenous Qo2 remained at 40 throughout this
The presence of PHB as an endogenous reserve period. Some product of metabolism or lysis
in Hydrogenomonas is documented by the work was inhibiting further endogenous respiration,
of Schlegel, Gottschalk, and von Bartha (73). since the inhibition could be relieved by washing
When chemolithotrophic growth, in an atmos- the cells. Changes in the cell constituents of
phere of H2-02-CO2 (60:30:10), is limited by PHB-poor cells (containing 10% PHB) showed
nitrogen exhaustion (NH4Cl) in the medium, cells that 3 hr of starvation produced no changes in
in the stationary phase continue to increase in size total N, either soluble material or carbohydrate,
although no division occurs. The increase in dry but almost half the PHB had been consumed.
weight is accounted for entirely by PHB forma- The endogenous Qo2 value decreased with poly-
tion. Washed suspensions synthesize PHB from mer depletion. A similar but extended study
CO2 by oxidation of hydrogen, and the stoichi- was made with PHB-rich cells; after 96 hr of
ometry corresponds to: starvation had elapsed, the PHB content had
25H2 + 802 + 4CO2 -- (C4H602) + 22H20 diminished from 50 to 10% of the dry weight, and
the endogenous Qo2 remained constant. Further
The rate of endogenous respiration of PHB- starvation rapidly reduced the endogenous Qo2.
poor cells (about 10% of the dry weight) was From these and other data, it seems clear that
considerably less than that of PHB-rich cells the rate of PHB oxidation is slower during
(about 50% of the dry weight). It was little starvation experiments than in the respirometer:
affected by the addition of a nitrogen source if, as appears to be the case, PHB is the sole
(NH4Cl) which had a marked stimulatory effect substrate for respiration, cells containing 50 %
on the respiration of the latter cells. Determina- of their dry weight could respire for only 15 hr
tions of cellular carbon and total nitrogen during with a Qo2 of 40.
endogenous respiration, and during hydrogen Biosynthesis of PHB. In view of the unique
oxidation in the absence of C02, were carried occurrence of PHB as a storage polymer (or
out with PHB-rich cells, both with and without metabolic shunt product) in bacteria, it is some-
a nitrogen source. In air, only about 8% of the what surprising that, at the time of writing,
PHB was consumed in 12 hr by endogenous there is very little information regarding the
metabolism, but with added NH4Cl the total metabolic routes and enzymes concerned with
cellular nitrogen increased significantly and the its biosynthesis. The only paper of significance
PHB decreased by some 73%. Under an at- is that of Merrick and Doudoroff (56). They
mosphere of C02-free H2-02, the increase in cell demonstrated that polymer particles (obtained
nitrogen was much greater, although not much by differential centrifugation of lysozyme-treated
more PHB was utilized (76 %). These results bacteria) of B. megaterium KM were able to
suggest that in Hydrogenomonas stored PHB may incorporate C'4-f-hydroxybutyryl-coenzyme A
serve as a carbon and energy source and can (CoA) into PHB. This was independent of the
support protein synthesis in the presence of a presence of the soluble fraction of the cell. Ap-
suitable source of nitrogen. When hydrogen is proximately 40% of the total radioactivity be-
provided as an additional energy source, the came incorporated into the polymer, which cor-
efficiency of protein synthesis is increased. responded to the amount of thioester decomposed.
Conditions for the accumulation of PHB in the A mixture of labeled g-hydroxybutyrate and un-
halophile, Micrococcus halodenitrificans, were labeled CoA did not allow incorporation of C14
determined by Sierra and Gibbons (75). These into the polymer.
authors further studied the role of PHB and its Similar experiments by these authors were
relationship to endogenous respiration and sur- extended to the particulate fraction of R. rubrum
vival of the organism (76). The RQ of the en- which contained all the activity for the incor-
dogenous respiration of M. halodenitrificans was poration of f-hydroxybutyryl-CoA into PHB.
0.87 i 0.05. (That required for complete com- However, these particles also contained a very
bustion of PHB is 0.88.) Aeration of washed active depolymerase which could degrade the
suspensions at 25 C reduced the PHB content PHB. With crude extracts, supplemented with
slowly (from 55 to 29%G in 127 hr); under these CoA, adenosine triphosphate (ATP) and reduced
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 131
nicotinamide dinucleotides, a small incorporation CO2 by l3-hydroxybutyrate was recorded; this
of C'4-acetate into polymer occurred. Activa- depolymerization is inhibited by the esterase
tion of f3-hydroxybutyrate could not be demon- inhibitor, diethyl-p-nitrophenyl phosphate (para-
strated. oxon). Further, the rate of release of f3-hydroxy-
Although activation of ,3-hydroxybutyrate butyrate was identical with the rate at which
could not be demonstrated in extracts of R. ru- oxygen is consumed for the complete combustion
brum, whole cells of Hydrogenomonas incorporate of PHB. For example, 0.76 umole of ,3-hydroxy-
l3-hydroxybutyrate into PHB (73). Crotonic butyrate was liberated per hr by 2 mg of cells
acid can also be used as a substrate; in either containing 50% of their dry weight as PHB;
case, the presence of hydrogen is not mandatory. this requires an oxygen consumption of 3.4
Presumably, the oxidation of a portion of these Mmoles per hr for complete combustion. This is a
substrates provides the energy necessary for figure realized by the recorded Qo2 values of 39
polymerization. The utilization of crotonic acid [(39 X 2)/22.4 Mmoles of 02 per 2 mg of cells per
is particularly interesting since there may exist a
Poly - hydroxybutyrate
2n ATP y 2n CH3CO2H
2n ADP 2n CH3CO-SCoA IN PHB esterase
(depolymerase)
Acetyl GoA
V HSCoA
n CH3COCH2CO- SCoA D- (-)-A - I ydroxybutyrate
Acetoacetyl CoA
_______ V2n[H] r NAD
4,- I4ydroxybutyrate
- - -

CH3CHOHCHC0,H-I n CH3CHOH CH2CO' SCoA


4- Hydroxybutyrate 4-Hydroxybutyryl GoA NADH2 dehydrogenase
,, .4HSCoA
CH3CH-CHCO2H----CHCH-CH-COSCIoA1 Acetoacetate
Crotonate Crotonyl CoA (CH3- H- CHaCO-)n
Hydrogenomonas
I 9
Poly-A-hydroxybutyrate
ATP, HSCoA, Mg2+
FIG. 1. Biosynthesis of poly-f3-hydroxybutyrate. Acetyl GoA
possible pathway of 13-hydroxybutyrate synthesis Oxaloacetate -
5 Citrate
omitting acetoacetate.
Tricarboxylic,
CH3-CH = CHC02H + H20 Acid
-* CH3 CHOHCH2 CO2H Cycle
Reactions leading to PHB are schematically
shown in Fig. 1. FIG. 2. Catabolism of poly-f3-hydroxybutyrate.
Catabolism of PHB. As with the biosynthesis
of PHB, comparatively little is known of its hr = 3.48]. On this basis, the depolymerization
degradation. There are, however, indications appears to be rate-limiting. Sierra and Gibbons
that the initial stages of depolymerization are later showed that depolymerase activity is
extremely complex and appear to be bound up markedly dependent upon Nat and Lit ions (76,
with the structural integrity of the polymer 76a). Cells washed and resuspended in KCl or
particles (56, 94). Thus, Merrick and Doudoroff water do not oxidize their PHB reserves unless
(56) demonstrated that extracts of R. rubrum Na+ or Li+ is added.
contain enzymes that degrade (i) native PHB Enzymes involved in the degradation of /3-hy-
from B. megaterium or (ii) boiled PHB particles droxybutyrate were also demonstrated by Sierra
from R. rubrum, but do not degrade the purified and Gibbons (76). DL-f3-Hydroxybutyrate is
polymer. oxidized by crude extracts to acetoacetate by
Sierra and Gibbons (76) demonstrated PHB a nicotinamide adenine dinucleotide (NAD)-spe-
esterase (depolymerase) activity in Ml. halode- cific enzyme. Only the D(-)-f-hydroxybutyrate
nitrificans by the anaerobic release of CO2 from is utilized, and the acetoacetate is not further
bicarbonate buffer. A stoichiometric release of metabolized unless ATP, Mg2, CoA, and oxalo-
132 DAWES AND RIBBONS BACTERIOL. R.EV.

acetate are added to the extracts. These data reaction is optimal over a pH range of 6.8 to 8.5.
suggest a pathway for catabolism of PHB as The dehydrogenase is insensitive to sulfhydryl
shown in Fig. 2. f3-Hydroxybutyrate and aceto- group reagents; ethylenediaminetetraacetic acid
acetate were also detected as products of PHB (EDTA) inhibition may be reversed by Mg2 if
degradation in B. megaterium when cells were incubation is not prolonged.
starved under N2. These acids accounted for over The specific activity of the D-(-)-3-hydroxy-
80% of the polymer depleted under these condi- butyrate dehydrogenase is dependent upon the
tions. Some properties of a D-(-)-O-hydroxy- cultural conditions at the time of harvest; low
butyrate dehydrogenase from B. megaterium have specific activities are observed during periods of
been described (24). PHB assimilation, but specific activities greater
GLUCOSE - N H4* salt GLUCOSE_-TQYPTONE TRYPTONE
0-35

0-3 1 15
>1~
~~
~~~~~~~~~~~~~~~~~~~~~~0

0O25L

E ~~~~~~~~~~~~~~~
F 3. of cardtt
Cdarbohyd

015
~~~~~~~~~~~~
~0.1N/35
~0

0 I010
S 10 18 6
I
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- by a factor of 2 are observed with older cells
hydroxybutyrate dehydrogenase from R. rubrum depleted of polymer. Succinate-grown cells con-
to be cold-sensitive. A 250-fold purification of the tain no PHB, and only low specific activities of
enzyme was obtained in 30% yield, and this was the enzyme are found.
unstable in dilute solutions or inactivated by
freezing. The enzyme is freely reversible, and the Glycogenlike Reserves
reaction products are acetoacetate and reduced The majority of microorganisms synthesize
NAD (NADH2). Although NAD phosphate polysaccharides of one form or another which
(NADP) would not substitute for NAD, a-oxo- may be of intracellular or extracellular origin.
valerate showed 6% of the activity obtained with Only the former type come within the scope of
acetoacetate. Furthermore, a-oxovalerate compet- the present survey since, by definition, extra-
itively inhibited acetoacetate reduction, because cellular polysaccharides [reviewed by Wilkinson
of the lower affinity of acetoacetate. The rate of in 1958 (92)] cannot serve as endogenous sub-
V o ,. 28,2 1
1964 ENDOGENOUS METABOLISM OF BACTERIA
EN 133
strates. It is frequently possible to differentiate produced in the degradation of the RNA that
intracellular polysaccharide into "reserve" and occurs under these circumstances.
"structural" carbohydrate, the criterion being The exact role of the glycogen under these
whether or not the material is utilized under con- starvation conditions is difficult to appreciate,
ditions of starvation. The term "structural" in since depletion of most of it is complete within
this context may be incorrect in many instances, 2 to 3 hr. If the possession of glycogen by E. coli
since it does not follow ipso facto that material favors survival by providing a store of energy,
not metabolized is necessarily part of the archi- then one might expect a slower rate of utilization
tecture of the bacterial cell. The role of reserve of this storage product. However, the exact con-
carbohydrates in yeast was also reviewed recently ditions of starvation will influence considerably
(15). the fate of the polysaccharide, and those used
We have investigated the role of glycogen as in our experiments may favor an uncoupling of
an endogenous substrate in Escherichia coli and oxidative phosphorylation or other rate-limiting
have obtained considerable evidence for its rapid process. Even under a nitrogen atmosphere, part
utilization during periods of complete starvation of the glycogen is rapidly degraded, with the evo-
under either aerobic or anaerobic conditions (16, lution of hydrogen and carbon dioxide. It may
17, 65). The possession of glycogen by the cells be that in E. coli the glycogen functions as a re-
prevents a net degradation of nitrogenous ma- serve of carbon skeletons and not of energy. Evi-
dence in support of this concept is the transfer of
TrABLE 2. Oxygen consumption and carbohydrate glycogen carbon atoms to other cell constituents
utilization by endogenously respiring (36).
Escherichia coli The deposition and fate of glycogen in Aero-
02 re- 02 re-
bacter aerogenes was studied by Strange, Dark,
02 quired for quiredcorn-
Time consumed glycogen Difference ribose for RQ and Ness (81). Cells grown on Tryptone-glucose
combustion bustion media contain 15 to 20%7, carbohydrate, and most
of this is glycogen. It is utilized after approxi-
min jmoles jimoles Mmoles Pmoles
mately 25 hr of incubation of the washed suspen-
60 11.38 11.01 0.37 0.73 1.01 sions in buffer. Ammonia is released from glyco-
120 18.89 16.01 2.88 1.85 1.03
184 23.2 17.48 5.72 2.03 1.01
gen-containing cells, but at a lower rate than
270 26.6 18.47 8.13 2.87 1.01 from cells that have not stored glycogen. The
complete suppression of ammonia release by
glycogen as recorded with E. coli (65) was not
terials with liberation of ammonia; this was observed, although the glycogen is oxidized much
demonstrated with cells harvested from media less quickly in A. aerogenes. Ultraviolet-absorb-
allowing massive, moderate, and no deposition ing materials are also released from glycogen-
of glycogen. Subsequent starvation of washed containing cells of A. aerogenes much more slowly
suspensions of these cells showed that ammonia than from cells harvested from tryptic meat broth
is released only after the glycogen has been oxi- or defined media (carbon-limiting).
dized, and in the case of Tryptone-grown cells, Glycogenlike polysaccharides have been de-
which do not contain glycogen, oxidation of scribed as reserves in a variety of bacterial
nitrogenous materials commences immediately species; some examples are given in Table 3. The
upon starvation (Fig. 3). The regulatory mecha- amount stored was reported to be as high as 41
nisms of this phenomenon have not yet been to 75% of the dry weight in Arthrobacter species
studied. Other data indicate that, during oxida- by Mulder et al. (58). When washed suspensions
tion of endogenous glycogen, very little else is of exponential-phase cells of Arthrobacter are
oxidized, since the consumption of oxygen and starved for 4 days at 30 C, only 40% of their
evolution of CO2 correspond initially to that carbohydrate is utilized. At this stage, the oxygen
required for the complete combustion of glycogen consumption of the suspensions is almost zero.
to CO2 and water (Table 2). Longer periods of Thus, not all of the original carbohydrate is a
starvation reveal that oxygen consumption is in substrate for endogenous respiration.
excess of glycogen depletion, and a part of this can R. rubrum, it may be recalled, is an example of
be accounted for by oxidation of ribose, which is a microorganism that is able to store more than
134 DAWES AND RIBBONS BACTERIOL. REV.-

one polymer as a reserve material, and the poly- serves as a source of carbon and reducing power
mer that is deposited within the cells is deter- for further CO2 assimilation; it may also provide
mined solely by the chemical nature of the carbon some energy, although this has not been tested.
substrate supplied. Thus, acetate and butyrate It appears to represent a "hot-house" shunt
are substrates for PHB storage, whereas carbon product (as does PHB in R. rubrum) as suggested
dioxide, succinate, propionate, and malate are by Foster (22), since massive deposits are formed
photoassimilated principally to a glycogenlike rapidly by assimilation of exogenous carbon
polysaccharide (78). sources at rates greater than overall cellular syn-
The photoassimilation of specifically labeled thesis. The fate or synthesis of the glycogen has
C'4-succinate by starved cells of R. rubrum was not been studied during aerobic metabolism of
studied in detail by Stanier et al. (78). The this organism; here, as with PHB, the glycogen
polysaccharide was the most strongly labeled might be expected to serve additionally as an
fraction when either 1-C14_ or 2-C'4-succinate was energy source.
photometabolized; 75% of the assimilated suc- Hot water or hot 75% ethanol extracts a poly-
cinate flowed into the polysaccharide and only glucose compound of low molecular weight from.
14% into PHB. Carboxyl-labeled succinate glucose-peptone grown Sarcina lutea (5, 9, 65).
labels the hexose residues at C-3 and C4, and We have shown that this polyglucose is only
synthesized during growth on peptone media
TABLE 3. Occurrence of glycogen in bacteria* supplemented with glucose, and that it can serve
as a substrate for endogenous respiration. The oxi-
Species Reference dation of this carbohydrate occurs during the de-
pletion of the amino acid pool; i.e., the provision
Escherichia coli . ......... 35, 36 of a readily metabolizable carbohydrate as an
Aerobacter aerogenes . . 81
endogenous reserve does not spare the nitrogen
Rhodospirillum rubrum ... 78
reserves, since free ammonia is released during
Arthrobacter sp . ..................... 58
Agrobacterium tumefaciens . . 48
the starvation period. Further, the oxygen con-
Bacillus cereus . ..................... 58 sumption is in excess of that required by the
B. megaterium ...................... 3 polyglucose oxidized.
Mycobacterium phlei ............. ... 25 The metabolic pathways of glycogen biosyn-
M. tuberculosis . ..................... 11 thesis and degradation in bacteria have received
* This is not a complete list of the bacterial
very little attention. Rogers (66) suggested that
bacterial enzymes degrading glycogen are un-
species that store glycogen. known, although many systems that decompose
starch, amylose, and amylopectin are described.
the methylene carbon atoms of succinate enter Glycogen metabolism is, however, well docu-
C-1, C-2, C-5, and C-6 of the glucose residues. mented in animals and plants. [For reviews see
The specific activities of the incorporated ma- Stetten and Stetten (79) and Whelan (90).]
terial showed that the carboxyl-labeled succinate Current ideas of glycogen metabolism suggest
is diluted by a factor greater than two, whereas that biosynthesis and dissimilation occur by
the methylene carbon atoms are diluted only separate routes. The uridine diphosphoglucose
slightly. The main mechanism of hexose syn- (UDPG) pathway is involved in synthesis and
thesis, therefore, seems to involve decarboxyla- the phosphorylase pathway in degradation. The
tion of the succinate chain (probably as oxalo- initial reactions of glycogen breakdown were
acetate), the product of which is channelled by delineated by Parnas and co-workers as early as
reversal of the reactions of glycolysis to hexose 1935, when they showed that inorganic phos-
phosphate. phate is consumed and a hexose monophosphate,
The chemical nature of the carbon nutrient later identified as glucose 1-phosphate, is accumu-
determines the storage product formed; those lated. The reaction catalyzed by glycogen phos-
compounds that are converted to acetate without phorylase is:
intermediate formation of pyruvate (or phos-
phoenolpyruvate) yield PHB, and those yielding Pi + glucosyl-(al1,4') primer
the 3-carbon compound produce glycogen. Dur- glucose 1-phosphate + primer
ing photometabolism in R. rubrum, glycogen In mammalian systems, complex interrelation-
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 135

ships exist between inactive and active forms of In the stationary phase (growth limited by nitro-
phosphorylase, and the enzymes obtained from gen), the concentrations of both compounds rise
different tissues of the same species are not iden- again, the UDPG slightly before the glycogen. A
tical (79). The bacterial phosphorylases are, how- linear relationship between glycogen concentra-
ever, not well studied. tion and UDPG concentration was demonstrated.
The UDPG pathway of glycogen synthesis has Replenishment of the nitrogen supply in the
been described in bacteria by Madsen (48, 49), stationary phase in another experiment caused
and in yeast by Algranati and Cabib (1). Glyco- resumption of growth, depletion of UDPG, and
gen acts as a primer with the glycogen synthetase cessation of glycogen synthesis. During aeration
enzymes of Agrobacterium tumefaciens and yeast, of washed suspensions of A. tumefaciens in buf-
but, unlike the mammalian enzymes, glucose fered salt solution, the glycogen was utilized.
6-phosphate does not stimulate the reaction: The UDPG concentration during this period re-
mained low and constant.
UDPG + polysaccharide primer Glycogen is a highly branched polysaccharide,
uridinediphosphate (UDP) and little or no reference has been made to the
+ glucosyl (1,4') primer synthesis of (al ,6') links; the UDPG synthetase
A cyclic scheme of glycogen degradation and will only synthesize (al,4') bonds although a
synthesis has been proposed for mammalian branched primer is required. A branching en-
zyme similar to Q-enzyme has been described in
yeast which will synthesize glycogen from amylose
UDP GLYCOGEN
UDPG glycogen VI
by transglucosylation (al ,4' to al,6').
synthetase
P UDPG Phosphorylase Amino Acid Pools
PP
UDPG The first indication that the free amino acid
p \rophosphorylase
UTP
pool could serve as a source of substrates for
Glucose-1- phosphate endogenous respiration was provided by Dawes
1 Phosphoglucomutase and Holms (14). Aeration of washed suspensions
E.AT .
GLUCOSE lRexoki~nase Glucose 6-phosphate of stationary-phase, peptone-grown S. lutea re-
duced the endogenous Qo2 values to negligible
FIG. 4. Biosynthesis and degradation of glycogen. levels, during which time the free amino acid pool
was depleted to one-half its original level and
systems (Fig. 4). A similar system probably oper- ammonia was released into the supernatant fluid.
ates in bacteria, since the necessary enzymes have Hydrolysis and analysis of the hot water-soluble
been demonstrated and partially separated in A. pool also showed that some peptide material was
tumefaciens (48), and UDPG has been shown to being used as substrates of endogenous respira-
be a competitive inhibitor of phosphorylase in the tion. The total oxygen consumption during the
same organism, suggesting that the concentration period of endogenous respiration corresponded to
of UDPG may regulate glycogen synthesis not 7.5 and 3.4 jsmoles of oxygen per Emole of utilized
only directly, but also by inhibiting the degrada- amino acid in the unhydrolyzed and hydrolyzed
tive enzymes (49). pools, respectively. The latter value is a reasona-
Madsen (50) has provided further evidence to ble average figure for the oxidation of a mix-
support the postulate that the control of glyco- ture of the amino acids that are utilized during
gen metabolism is effected by UDPG. The UDPG the starvation.
pathway to glycogen is essentially irreversible, Glycine, threonine, leucine, tryptophan, and
and the equilibrium of the phosphorylase reac- a-aminobutyric acid were completely utilized,
tion is slightly in favor of glycogen synthesis, and much of the serine, glutamate, and alanine of
yet the concentration of glycogen in A. tume- the pool was oxidized. Very little loss of amino
faciens varies considerably. Madsen analyzed A. acids to the suspending media occurred (about
tumefaciens during its growth in batch culture for 2% of the initial concentration of the hydrolyzed
glycogen and UDPG content. Both increase pool). Glutamate plays a most important role in
initially during a short lag phase and then de- the endogenous respiration of S. lutea; it accounts
crease at the beginning of exponential growth. for 20% of the total pool amino acids in freshly
136 DAWES AND RIBBONS BACTERIOL. REV.

harvested peptone-grown cells, and this declines tamate alone (glutamate can account for only
to 1% after a 5-hr aeration period. 10% of the 02 consumed). More completely
Although glucose-peptone grown S. lutea also labeled cells were obtained by growth on U-C14-
stores a polyglucose compound as a reserve mate- glucose and C'4 algal hydrolysate, and the change
rial, the free amino acid pool is depleted just as in cell components during respiration was fol-
rapidly during starvation of these cells (9, 65). lowed. Only about one-third of the radioactivity
The utilization of free amino acid pools as en- lost from the cells is recovered as C'402. The rest
dogenous substrates has since been shown to occur can be traced to the suspending buffer, and rather
in Nocardia rugosa (2) and Staphylococcus aureus more than one-third to deposition in the hot
(64). During starvation of washed mycelial sus- trichloroacetic acid fraction. The bulk of the
pensions of N. rugosa, both the endogenous Qo, radioactivity was lost from the hot trichloroacetic
and Qo, (glucose) fall by approximately the same acid-insoluble fraction. Apart from the utilization
value. There is a loss of weight from the mycelia of glutamate from the free amino acid pool, evi-
that can be accounted for almost completely by dence was presented to show that aspartate was
loss of protein; a little acid-soluble carbohydrate, a probable substrate and, to a lesser extent,
mainly hexose, is also utilized. There was no sig- alanine. Cells, allowed to assimilate C'4-glycine
nificant utilization of lipid during starvation for into the pool, washed, and subsequently starved,
16 hr. The pH value changed from 7.5 to 8.2 liberated C1402 only slowly.
during the incubation, and free ammonia was Comparisons of the endogenous metabolism of
formed at the expense of the mycelial protein. coagulase-positive (+) and -negative (-) S.
Oxo-acids did not accumulate but were oxidized. aureus have revealed marked differences (D.
Chromatography of the free amino acid pools Ivler, personal communication). During starvation
showed that fresh mycelia contained large of organisms grown on Brain Heart Infusion, the
amounts of glutamate, alanine, aspartate, leu- RQ of + cells showed little change (from 1.08
cine, and valine, whereas only smaller amounts to 0.95), whereas that of - cells fell from 1.10 to
of leucine, alanine, and glutamate remained after approximately 0.5 in 60 min. The Qo2 of + cells
starvation. The RNA and DNA contents of the was higher than that of - cells, but both values
cell are not utilized endogenously. It was con- decreased rapidly with starvation. High rates of
cluded that the main substrates of endogenous endogenous respiration of + cells suppressed the
respiration are the amino acids that are present rate of oxidation of added glucose, but no such
in the pool, and also obtained from the hydrolysis effect was apparent with - cells. Measurement
of cell protein; an acid-soluble carbohydrate is of 02-NH3 ratios during starvation of washed
also utilized. The presence of an exogenous source suspensions showed that values fell from 4.7 to
of glucose inhibits the endogenous consumption 3.0 with + cells and from 13.4 to 10 with - cells.
of protein in N. rugosa. Of considerable note was the fact that while the
Ramsey (64) showed that washed suspensions free amino acid pool content of both types of
of S. aureus respire endogenously with an RQ of cell diminished, the bulk of the loss could be
0.83 to 0.86. The carbohydrate content of the accounted for as free amino acids in the super-
cells remained constant (at the very low value of natant fluid (as much as 90% recovery with -
1.18% of dry weight) during starvation, and cells); nonetheless, ammonia appeared in the
ammonia was released into the supernatant fluid. supernatant at a greater rate and to a greater
The 02-NH3 ratio was 6.2, and glutamate was extent with + cells, indicating the net degrada-
utilized from the free amino acid pool. This indi- tion of nitrogenous materials. The total carbo-
cated that substances other than glutamate were hydrate content of both + and - cells did not
being utilized (02-NH3 ratio for glutamate is alter during starvation.
4.5). The endogenous respiration of incompletely
C'4-labeled cells confirmed this view, since apart RNA Metabolism
from the utilization of glutamate in the pool Mlore typical storage compounds such as
there was also some loss of C14 from the hot tri- glycogen or PHB are characterized by their
chloroacetic acid-insoluble fraction of the cells; deposition during conditions of carbon source
further, the oxygen consumption was much in excess or nitrogen limitation, and by their de-
excess of that required for oxidation of glu- pletion to almost negligible amounts during
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 137

starvation. However, as Herbert (34) empha- ultraviolet-absorbing compounds accumulate in


sized, the amounts of such basal materials of the the supernatants. The degradation of RNA in
bacterial cell as RNA, DNA, and protein are E. coli was studied by Wade (85), who concluded
subject to wide variation, and this can be con- that two pathways exist. The M route (Mg2+-
trolled by environmental conditions. dependent) results in the formation of nucleo-
Strange, Dark, and Ness (81) showed that side 5'-phosphates characteristic of phosphodi-
RNA is metabolized endogenously during starva- esterases. The rate of formation of these nucleo-
tion of washed stationary-phase suspensions of tides is further stimulated by inorganic phos-
A. aerogenes, and net degradation occurs. The phate, suggesting that polynucleotide phos-
extent of RNA catabolism varied, and this phorylase was also depolymerizing RNA (86).
depended on the source of the cells; 40% of the Autodegradation of ribosomes in the presence of
RNA of cells harvested from defined media was inorganic P32-orthophosphate gave only labeled
utilized in 70 hr, during which time about 70% nucleoside diphosphate. The nucleoside mono-
of the population remained viable. [See also E. phosphates appeared to be formed by an inde-
coli (7)]. Cells harvested from glucose-Tryptone pendent route (86). The second route, the V
media contain much less RNA (about 11% of route, results in the degradation of RNA into
the dry weight), and little is utilized. These cells nucleoside 2', 3'-cyclic phosphates in the presence
contain glycogen that is almost completely of sufficient EDTA to remove the M\g2+. The
respired within 25 hr. Tryptic meat broth grown cyclic phosphates are further hydrolyzed to
A. aerogenes, on the other hand, catabolize nearly nucleoside 3'-phosphates. The V route then
half their RNA, from about 13% to 7% of the employs ribonuclease-type enzymes. The enzymes
dry weight, within about 50 hr. The products of of both routes are located in the ribosomal frac-
RNA metabolism that have been detected in the tion. The observation of Kiguchi and Uemura
suspending fluid include ammonia, inorganic (84a) that citrate and phosphate enhance the
phosphate, and the free bases hypoxanthine, release of RNA degradation products from yeast
uracil, and guanine (62, 81, 83), and small cells is, perhaps, relevant. These authors believe
amounts of adenine (83); hypoxanthine was the that magnesium is removed from the cell
major component of the bases in the supernatant membrane by chelation, since added Mg2+
(83). Nucleotides and nucleosides did not ac- countered the effect of these agents.
cumulate to any significant extent, and most of Starving washed suspensions of P. aeruginosa
the pentose was apparently oxidized. The ultra- release much ultraviolet-absorbing material into
violet-absorbing materials were readily released the supernatant with an Emax at 260 mIu. Nucleo-
into the supernatants, and acid-soluble inter- tides, nucleosides, and free bases were detected.
mediates did not accumulate within the cells (83). The effect of Mg2+ ions on ribosomal particles is
The amount of ultraviolet-absorbing material well known (8), and Gronlund and Campbell (27)
released corresponded well with the amount of have used this as evidence for the utilization of
RNA lost from the cell, and this loss occurred RNA as a substrate for endogenous respiration.
almost entirely from the RII sedimentation Oxygen consumption is depressed in the presence
fraction (83), as had been demonstrated in the of Mg2+ ions, which allow the formation of 70S
case of loss of RNA from E. coli ribosomes (85). ribosomes. When P. aeruginosa was grown in the
RII is the cell fraction sedimented at 78,000 X g presence of C14-uracil, the radioactivity was con-
for 7.5 hr. tained primarily in the nucleic acid fraction, and
The endogenous utilization of RNA is not this yielded C'402 during subsequent starvation.
peculiar to A. aerogenes, but appears to be very The C0402 was derived solely from the RNA, and
widespread, and has been demonstrated in E. came largely from the ribosome fraction. The
coli (17, 18), S. lutea (9), and P. aeruginosa (27). enzymatic degradation of ribosomes was demon-
The utilization of the ribose portion of the strated and inhibited by EDTA; phosphate
RNA by endogenously respiring E. coli was markedly increased ribosome degradation, sug-
briefly mentioned in the section on glycogenlike gesting a role for polynucleotide phosphorylase.
reserves, and in Table 2. It is not yet clear The fate of the ribose portion of the RNA was
whether the purine and pyrimidine bases are not determined, although ribose (free and com-
completely oxidized, although this is unlikely as bined?) was detected in supernatant fluids.
138 DAWES AND RIBBONS BACTERIOL. REV.

By calculations based on the values of C1402 appear during starvation; the solubility proper-
released from uniformly C'4-labeled cells, 2-C04- ties of this fraction alter, however, and cautious
uracil-labeled cells, and U-C'4-proline-labeled interpretations with respect to the fluctuations of
cells, Gronlund and Campbell deduced that the this fraction are required.
C'402 liberated from 2-C'4-uracil-labeled and Strange et al. (83) showed that protein is lost
from U-C14-proline-labeled cells is equivalent to from all ultracentrifugal fractions during starva-
the total amount of C1402 liberated by uniformly tion of A. aerogenes; i.e., ribosomal and soluble
labeled cells. From this, they infer that RNA and proteins are utilized. The products of protein
protein are the only endogenous substrates catabolism appear to be ammonia and carbon
oxidized. Their calculations are based, however, dioxide, as only traces of amino acids accumulate
on the assumption that the fate of the 2-C of in the suspending fluid.
uracil is representative of all RNA carbon atoms, Attention must be directed to the data ob-
since, for example, the ribose was presumably tained by Strange et al. (81) for the release of am-
unlabeled by this technique and its fate was not monia from cells grown in different media. If, on
determined. The fate of the proline C atoms is the basis of their figures, a calculation is made to
also assumed to reflect the destiny of all the other compare the nitrogen accounted for as NH3 with
carbon atoms of protein. the nitrogen of the degraded protein and RNA,
considerable discrepancies are apparent. Thus
Protein 78.8, 25.2, and 30.2% nitrogen are unaccounted
The net utilization of protein as an endogenous for as NH3 in cells harvested from glucose-Tryp-
substrate was first demonstrated by Strange et tone, Tryptone meat broth, and defined media,
al. (81) with starving suspensions of A. aerogenes, respectively, after 25 hr of starvation. The fate of
and Gronlund and Campbell (26) indicated that this nitrogen has not been ascertained, and some
ammonia release by endogenously respiring cells cellular redistribution might be envisaged.
was a general phenomenon. Thus, washed sus- Other Potential Substrates
pensions of E. coli, P. aeruginosa, P. fluorescens, Our knowledge of bacterial lipids is very
Achromobacter sp., B. subtilis, and S. faecalis all limited (34), especially concerning their role as
liberate ammonia during endogenous respiration endogenous reserves of energy. It appears that
(15, 26, 87). To this list may be added A. aerog- conventional lipids are stored by E. coli under
enes (81) [although Gronlund and Campbell (26) favorable conditions; e.g., provision of acetate
did not detect ammonia production with their stimulates lipid deposition (13). The utilization of
strain], B. cereus (12, 47), S. lutea (14), S. aureus lipid and phospholipid materials during starva-
(64), and N. rugosa (2). Even so, the original tion has been discussed (15). The massive deposi-
work of Strange et al. is the most decisive demon- tion of lipids by the yeasts Rhodotorula gracilis,
stration of protein degradation, since they used Lipomyces starkeyi, R. graminis, and R. glutinis
simple chemical analysis. Other workers have was recently described by Mulder and co-workers
concluded from radiochemical evidence that (58).
protein is a substrate of endogenous respiration; DNA is generally considered to be a stable cell
in some bacteria, it appears to be the main constituent whose function is storage of informa-
substrate. With P. aeruginosa, Gronlund and tion, and generally no utilization of DNA occurs
Campbell (27) labeled the cells with U-C14-proline during starvation. However, Strange, Wade, and
and demonstrated the endogenous evolution of Ness (83) showed that the DNA of starving A.
C1402 from the hot trichloroacetic acid-insoluble aerogenes increased by 17%, and they ruled out
fraction. It is interesting to note that the alcohol- the possibility of cell division occurring. A DNA
soluble protein of P. aeruginosa accounts for 20% increase at the expense of RNA was also demon-
of the total protein (60) and yet this is not utilized strated with phosphate-deficient E. coli (37). On
during starvation. The hot trichloroacetic acid- the other hand, utilization of DNA after 19 hr of
insoluble residue yields less C1402 when cells are starvation was noted in A. aerogenes (30).
starved in the presence of Mg2+; under these con-
ditions, there is also a slight decrease in the alco- ENERGY OF MAINTENANCE
hol-soluble protein. Pine (60) demonstrated that Cellular processes, whether mechanical or
the alcohol-soluble proteins of E. coli do not dis- chemical, require energy for their performance,
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 139

and unless a supply of energy is readily available hr. Consequently, unrestricted growth should
these essential processes will cease and the cell have a lower maintenance requirement.
will die. The provision of energy by exogenous Microorganisms are able to survive for con-
nutrients is well established, e.g., the use of glu- siderable periods during starvation and conse-
cose as an energy (but not carbon) source by quently must maintain soluble constituents, often
Streptococcus faecalis (4). Under conditions of against considerable concentration gradients.
starvation, mobilization of endogenous sub- The regulation of the cytoplasmic osmotic pres-
strates must furnish the energy necessary to allow sure and pH value appear to be highly selective
the various cellular activities to continue. The processes, since considerable quantities of some
energy required for these processes of cell sur- cell substances, e.g., RNA from bacteria and
vival has been called the "energy of maintenance." yeast, and also smaller molecular moieties, such
Since the performance of work is required for as amino acids and bases of RNA, ribose, and in-
all these activities, which include resynthesis, organic phosphate, are able to diffuse into the
osmotic regulation, and heat loss to the external suspending fluids without loss of viability (7, 17,
environment, presumably they will eventually 27, 68, 81).
cease owing to lack of energy, and the cell will For bacteria to remain motile, a source of
no longer be able to maintain its status quo. To energy is required; tactic responses were dis-
maintain the intact living cell, structures such as cussed at some length by Weibull (89). In the
the cell wall, flagella, cell membrane, and cell absence of exogenous substrates, energy must be
particles must be kept in good repair. It is pos- supplied from endogenous sources. Phototrophic
sible that some of these structures may be dis- organisms present a somewhat different case in
pensed with for the sake of survival; indeed, that their source of energy is light. If light is the
flagella have been removed from bacterial cells sole source of energy available to green and
without affecting viability (45), while suitably purple sulfur bacteria, then incubation of washed
prepared protoplasts possess metabolic activities suspensions in the dark should exclude mecha-
that are identical with those of whole cells (71). nisms of ATP synthesis, and it would be of in-
The possession of a rigid cell wall provides dis- terest to know the survival characteristics of
tinct advantages, since protoplasts remain intact these species in the dark. This situation is similar
only in solutions of high osmotic pressure. The to the survival of strict aerobes under anaero-
cell wall is probably an essential feature of cell biosis.
survival under natural conditions. Protoplasts Although we are principally concerned with the
are able to grow (increase in size) but apparently concept of maintenance energy in starving cells,
are unable to undergo cell division unless rever- it is obvious that some consideration of growing
sion to bacillary form (in gram-negative organ- cells should be included; additional demands for
isms only) occurs (55a). energy may be manifest here. The actual proc-
Mandelstam (52) reported that starved sus- esses of cell division may require larger amounts of
pensions of E. coli break down and resynthesize energy than the other phases of the growth cycle
their macromolecular components (RNA and of individual cells which, in the main, would be
protein) at a rate of 5% per hr. On the other hand, supplied by exogenous sources. Lowered cell
growing cells appear not to exhibit any appreci- yields at low growth rates may, in fact, be caused
able turnover of protein. The continued resynthe- by the diversion of energy from synthesis of cell
sis of macromolecular components during starva- substances to the physical and energetic task of
tion requires energy, which may be supplied by maintaining a cell that spends a long period over
the components that are undergoing transforma- division. Alternatively, cells actually dividing
tion. This situation pinpoints a feature not gen- may dissociate energy-yielding reactions from
synthesis without affecting the rate of utilization
erally appreciated, namely, that the maintenance of energy source.
requirement is not necessarily a constant feature The chemical and mechanical activities of the
independent of growth rate; i.e., at the extremes microbial cell, therefore, lead one to postulate
of unrestricted growth and absence of growth, that some energy is utilized to maintain the cell in
protein turnover (and, therefore, the energy re- a functional and viable condition, and that during
quired for this process) varies from 0 to 5% per starvation this energy must be derived from
140 DAWES AND RIBBONS BACTERIOL. REV.

endogenous sources. We think few scientists It seems feasible that at slower growth rates
would now deny the energy-of-maintenance re- the cell yield might be decreased because the cell
quirement, although there are several widely physiology with respect to regulatory mecha-
quoted experiments that have been cited as nisms has altered in response to the changed en-
evidence against such a concept, often because it vironment, and that a portion of the energy
could not be detected. This is especially apparent source is uncoupled at the enzymatic sites of
in the growth-yield experiments of Monod (57) phosphor) lation; i.e., the decreased cell yield is
and Bauchop and Elsden (4). Microbial growth merely a reflection of decreased efficiency of
yields are (within certain limits) directly propor- conversion f the energy source into high-energy
tional to the concentration of limited nutrient. phosphate.
Even when the carbon and energy source limits It is perhaps too obvious that the reactions of
the yield of cells, the relationship holds for very energy-yielding metabolism are not always
low concentrations; extrapolation of the experi- coupled to the growth of the organism. The more
mental points indicates that no intercept occurs, extreme cases of this are most often observed in
i.e., at zero concentration of nutrient no growth batch cultures that have reached a stationary
occurs. If some portion of the energy source were population but still consume considerable quan-
utilized for functions other than growth, then tities of substrate. This same feature is seen dur-
one should observe that the addition of very ing growth limitation (rate or yield) of some
small amounts of an energy source would not nutrient other than energy source, and is most
permit growth. This then would assume that dramatically demonstrated by washed suspen-
growth is a secondary feature of energy utiliza- sions of nonproliferating cells metabolizing added
tion and that energy is preferentially channelled carbon or energy substrates.
to maintenance purposes. The concentrations of Where the energy source is also the carbon
the energy source used in these experiments were source, usage of carbon skeletons occurs without
such that, although they limited the maximal net growth. In some cases, the carbon skeletons
population attainable, they did not limit the rate are removed from the medium and stored within
of growth. Monod also limited the rate of growth the cells (assimilation) ready for utilization under
of E. coli by limiting aeration, and although this duress. However, uncoupling of assimilation has
doubled the time taken to achieve maximal also been observed, in that products of metabo-
density in one culture the cell yield was un- lism often appear in supernatants, and presum-
changed. He concluded that since the rate of ably these are lost to the cell. Further, it has been
growth did not influence the cell crop any suggested that the products of metabolism under
energy-of-maintenance values were nil. conditions of carbon and energy excess are shunt
It seems to us that many of the experiments products, and that assimilation into reserves
designed to test the use of a portion of the exoge- merely reflects this glut. Limitations of the rate of
nous energy source for maintenance rather than microbial growth by nutrients other than the
growth are complicated by the fact that the energy source do not control the extent of oxida-
energy source is also a source of carbon for tion of the energy source; e.g., Rosenberger and
growth. More definitive evidence might be ob- Elsden (67) showed that in tryptophan-limited
tained with microorganisms which do not in- growth S. faecalis produced large amounts of
corporate their energy source into cell substance; lactate.
there are numerous systems available for such A discussion of the theoretical principles of
experiments-phototrophs, autotrophs, and the continuous culture (as controlled by nutrient
nutritionally fastidious anaerobes that ferment limitation), and a comparison of these with results
carbohydrates almost solely as a source of energy. obtained experimentally, led Herbert (33) to
Furthermore, experiments designed to show that postulate that A. aerogenes displays a constant
some portion of the energy (and carbon) source rate of endogenous metabolism during exponen-
is not utilized for growth, and many do demon- tial growth. The curve derived experimentally
strate this, are not entirely convincing arguments relating steady-state bacterial concentration to
for the maintenance concept. These experiments dilution rate (growth rate) does not coincide with
do not show that the energy that is diverted the theoretically predicted curve. At low dilution
from growth is utilized specifically for mainte- rates, the cell yield is less than that expected
nance. when the carbon and energy source is the growth-
V OL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 141
limiting nutrient. Cultures whose growth is centration versus dilution rate to be made that
limited by nutrients other than the carbon and correspond exactly with those obtained experi-
energy source do not show this phenomenon at mentally (33). Evidence to interpret k as repre-
low growth rates, so that the decreased cell yields senting a constant endogenous metabolism that
observed under these conditions are not simply a occurs during growth is demonstrated by com-
property of the growth rate. The lowered cell paring the rates of respiration of A. aerogenes in
yield recorded during slow growth on a limiting continuous culture at different growth rates.
carbon and energy source was explained by sug- Extrapolation to zero growth rate of Qo2 and
gesting that, in addition to cell synthesis from Qco2 values determined at different growth rates
the carbon substrate, there is also a constant oxi- in media containing limiting glycerol gave values
dation of cell substance to C02, i.e., some turn- on the ordinate that were identical to the Qo2
over is occurring during growth. (The experimen- (endogenous) and Qco2 (endogenous) of these
tal evidence for this is considered later.) The cells. It is suggested that the respiration consists
equation representing the exponential growth of a of (i) substrate oxidation that is proportional to
bacterial culture can be modified from the growth rate and (ii) a constant rate of oxida-
tion of endogenous material, that occurs at all
dx ds growth rates. Carbon balances (details of which
-= sx = -Y-
dt dt were not recorded) were also claimed to indicate
to that proportionally more cell carbon than sub-
dx strate carbon is oxidized.
du
dt = _- k)x The lowered cell yields at low dilution rates
(carbon and energy source limiting) have been
cohere x = cell concentration; s = substrate observed for other bacteria and for Torula utilis
utilized; t = time; A = growth rate; k = a con- (33). Marr et al. (53) noted that E. coli is unable
stant representing the endogenous metabolism; to maintain cell density at low dilution rates, and
and Y = yield coefficient. Thus, by lowering the they calculated the specific maintenance as 0.025
dilution or growth rate, k becomes proportionally hr-1.
larger in relation to A and, therefore, the cell The carbon balances of substrate utilization at
density falls; the cell yield is lower because pro- different growth rates received attention from
portionally more cell material is oxidized relative Marr et al. (53) with batch cultures. U-C14-glucose
to the amount of limiting nutrient that is assimi- was (i) added to a batch culture, giving exponen-
lated. In toto, the net result is an uncoupling of tial growth; (ii) fed rapidly to a culture so that
growth from oxidation of carbon substrate, since only a small fraction would be used for mainte-
proportionally more nutrient is oxidized than is nance, giving linear growth; and (iii) fed slowly
assimilated per cell. This idea was expanded by so that a large fraction was used for maintenance,
Marr et al. (53), who designed experiments to giving curvilinear growth. It had previously
determine the value of k (these workers substitute been shown that the cell crops obtained
a for k) which they call the specific maintenance. increased in the order: slowly fed cultures <
Mathematically, the lowered cell yield at low from batch cultures < from rapidly fed
dilution rates can be expressed as: cultures. The radiochemical results confirmed this
observation and also showed that more of the
dx
-Z
dt
= (u-kx Yds
(~a-k)x =-Yddt 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
Rearranging, assimilated by the cells. It is not entirely clear
Y ds why the batch cultures should lie between the fast
x dt and slow feeding of energy source, but some ex-
planations may be offered. During exponential
Now Y. x, ds/dt, and At (or D, the dilution rate) growth in batch culture, the rate of growth is not
can all be determined experimentally, and, there- limited by glucose concentration and it appears
fore, k may be evaluated. that glucose is utilized less efficiently; e.g., it is
Substitution of k into the continuous culture not oxidized to CO2 immediately, possibly owing
equations enables plots of steady-state cell con- to a limitation of oxygen concentration. Conse-
142 DAWES AND RIBBONS BACTERIOL. REV.

quently, growth may cease and the stationary- breakdown of cellular material occurs before the
phase cells oxidize the accumulated intermediates next glucose supplement, and this is insufficient
to CO2. Lower growth yields in batch than in to allow reclamation of the lost cell materials. A
continuous or nutrient-limited cultures were also criticism which may be leveled at this type of ex-
observed by Pirt (61). periment is that growth may be occurring al-
The energy required for turnover of macro- though it is not revealed by turbidity measure-
molecules appears to account for a larger propor- ments. The number of cells dying may be such
tion of the energy source that is not utilized for that the turbidity undergoes no net change as
growth in E. coli (53), but these workers could growth occurs. The influence of higher cell con-
not demonstrate that accumulation of methyl- centrations on these phenomena is not known,
thiogalactoside was responsible for any signifi- and regrowth (28) may assume special impor-
cant amount of energy expenditure, although tance; perhaps open systems should be considered
Kepes (40) noted that addition of this compound in experimental design.
to E. coli suspensions resulted in a doubling of the
rate of endogenous metabolism. STARVATION AND SURVIVAL
The earlier ideas concerning the concept of The early literature concerning the effects of
energy of maintenance were excellently sum- starvation upon the survival of microorganisms
marized by Mallette (51) and McGrew and was critically reviewed by Postgate and Hunter
Mallette (55). They indicated that lack of sensi- (62). They also draw attention to the pitfalls and
tive techniques was among the reasons for the difficulties that may be encountered during the
difficulty of demonstration of the energy of main- estimation of viabilities. The cleanliness of labo-
tenance. To overcome this problem, they used a ratory ware and purity of chemicals is considered
high cell density in relation to a low concentration to be critical, as trace impurities may either per-
of carbon and energy source to study the mainte- mit growth of otherwise starving organisms (23)
nance requirement of E. coli. Low concentrations or kill the cells. The growth of bacteria at the
of glucose were fed to suspensions of E. coli in an expense of their companions has been termed
otherwise complete growth medium, and the tur- cryptic growth (70), cannibalism (28), and re-
bidity changes were recorded. When very small growth (81). This growth is a function of cell
additions of glucose were made, the extinction did density and can considerably influence the sur-
not alter appreciably from control cultures after vival behavior of starving suspensions, since a
a standard time. Higher concentrations of glucose "population turnover" may occur.
permitted growth to occur. Thus, they demon- Apart from the phenomenon of regrowth, the
strated that a threshold concentration of glucose initial cell density of starving suspensions also
is required before growth can occur. Cell suspen- affects their death rate. Harrison (28) first showed
sions starved with respect to the carbon and the relationship between cell density and death
energy source showed rapid decreases in turbidity rate of starving suspensions of A. aerogenes, and
for 5 days, at which time only 15% of the cells an optimal density for survival was demon-
were viable, and then remained constant while strated. He concluded that an interaction be-
the viability continued to fall. A small addition tween individual cells favors survival, and the
of glucose at 6-hr intervals, sufficient to maintain work of Postgate and Hunter (62) removes any
the turbidity at a constant value, also suppressed doubt about the possibility of regrowth occurring.
the rate of loss of viability; e.g., after 5 days only The latter authors made a very thorough study
20 % of the cells had died. Glucose additions that of many factors that influence the survival of
permitted a very slow growth (20% increase in starving suspensions of A. aerogenes. They elimi-
extinction over 10 days) did not prevent death of nated ambiguity that would arise from cryptic
the cells. (After 5 days, approximately 10% had growth, growth on impurities, and toxicity of
died.) Thus, it would seem that small amounts of suspending fluids, by a suitable choice of cell
glucose can provide energy to maintain the cell density [20 ,ug (dry weight) of cells per ml] and
without allowing growth to occur. The loss of suspending fluid [saline-tris(hydroxymethyl)-
viability that occurs during the slow growth may aminomethane buffer-EDTA solution]. High illu-
be due to the interval method (6 hr) of feeding, mination, high temperatures, high pH values, and
as pointed out by Marr et al. (53); i.e., some high potassium ion concentrations increased the
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 143

death rates of starving suspensions. For example, A. aerogenes.] Unfortunately, it is not clear that
A. aerogenes survives better at 20 C than at 30, regrowth does not occur here. The concentrations
40, or 10 C. Anaerobiosis accelerated death, and this of glucose added as an energy source were very
was attributed to the acid conditions produced. much less than those used by Postgate and
We also have observed that anaerobically starved Hunter (62) to demonstrate substrate-accelerated
E. coli die faster than the aerobically starved sus- death.
pensions (18); however, the pH value during
anaerobiosis fell only from 7.2 to 6.8 with our Death
more strongly buffered suspensions (unpublished The survival characteristics of a suspension of
data). organisms can be defined only by experimental
Various nutrients, or the previous history of design; for example, cells that have not divided
suspensions of A. aerogenes, markedly affected within 3 hr are generally considered dead when
the death rates. Ca2+, Mg2+, and to a lesser extent estimated by slide culture, but longer incubation
Fe2+, when added to the saline-buffer, prolonged (24 hr) by more traditional methods sometimes
the life of the cells. The slower the rate of growth gives higher counts, especially with aged cells.
of the bacteria, the greater was their death rate Again, the washing procedure and recovery
upon fasting. This applied to organisms whose media will determine to a large extent the viable
growth rate was limited by C, N, P, and S, but counts or the percentage viability obtained. As
with Mg2+-limited growth the reverse was true we have pointed out (15), a situation is possible
and the cells that had most rapidly proliferated where the loss of a single enzyme from the cell
died fastest when starved. The effect of nutrient could lead to its "death" in certain media; this is
additives upon death rates is, however, more com- clearly seen with techniques that select nutri-
plex and depends upon the previous history of the tional mutants via minimal media. The actual
cells. Thus, Postgate and Hunter (63) observed causes of death by starvation may be quite di-
a general phenomenon of substrate-accelerated verse and include factors such as the loss of the
death, in which the addition of the growth-limit- capacity for nucleic acid and protein synthesis.
ing nutrient to starving suspensions increased the Postgate and Hunter (62) were able to demon-
death rate. Glycerol-limited cells of A. aerogenes strate that death of their starving suspensions
showed glycerol-accelerated death (metabolites was not due to alterations or degradation of the
of glycerol, e.g., pyruvate, also accelerate death); osmotic barrier since, among other factors, starv-
NH4+-limited cells are killed by NH4+ additions ing populations did not become permeable to
but not by glycerol, which is slightly protective. fluorescent dyes at the same rate as their death
Phosphate-limited cells behaved similarly, as did rate. The Qo2 (glycerol) and glycerol dehydroge-
other carbon source-limited cells. Sulfate-limited nase activity decline in parallel with the loss of
cells were not killed quickly by sulfate, but in- viability; it may be that the enzymes catabolizing
stead showed glycerol-accelerated death. Mg2+- the energy source are the critical factors in life
limited cells were another exception, in that addi- and death in this case.
tion of Mg2+ actually prolonged the life of these If it were possible to maintain a bacterial popu-
cells, as it does of other cells, a feature quite inde- lation by the addition of a small amount of an
pendent of the nutrient that limited their growth. energy source, then continuous cultivation of
Harrison and Lawrence (30) also noted that the &ells at decreasing dilution rates should give a
effect of nutrient additions to starving suspen- situation where the dilution rate is significantly
sions is influenced by the cell density used. Thus, larger than the growth rate, and eventually reach
log-phase A. aerogenes (106 to 107 cells per ml) a point when the growth rate is zero and the dilu-
may become sterile within a few hours in distilled tion rate has some finite value. It may not be easy
water, but phosphate delays death. Higher cell to demonstrate technically, as other factors such
densities (108 to 109 cells per ml) survive as well as bacterial density may interfere; e.g., Jannasch
in water as they do in phosphate buffer. has shown the influence of cells upon each other
McGrew and Mallette (55), however, pre- (38). In practice, it has not been possible to ob-
sented results which show that E. coli may be tain such a steady state, i.e., slow exponential
maintained by very small quantities of a carbon washout, because many cells do not survive at
and energy source. [See also Harrison (28) with low growth rates and, consequently, the effluent
144 DAWES AND RIBBONS BACTERIOL. REV.

contains a proportion of dead cells; the number of protein and polysaccharide, occurred during the
dead cells appearing becomes larger as the genera- starvation of carbon-limited A. aerogenes (81).
tion time increases. This is a property not simply Little protein degradation occurred during the
confined to carbon- and energy-limited growth first few hours of starvation, and the free amino
but occurs also with S-, Mg-, and N-limited acid pool content remained constant. The poly-
growth. Postgate and Hunter (62) are of the saccharide that was degraded was completely
opinion that these bacteria are able only to oxidized within 2 hr under these conditions (com-
multiply or to die! pare with E. coli), and the endogenous Qo2 value
declined rapidly during this period. It appears
Relationship Between Survival and that RNA is the most expendable reserve; fur-
Endogenous Substrates ther, fast-growing cells contain more RNA and
It has been suggested that the oxidation of also die more slowly. Fast-growing cells are also
endogenous materials by microorganisms is of larger than slow-growing cells (33), and thus each
importance to the cell for its survival during individual will contain proportionally more
starvation. Therefore, it might be concluded that expendable material; this may account for their
cells which contain large amounts of storage slower death rate. However, this apparent rela-
polymers have an advantage over those cells tionship of the death rate and initial RNA con-
which do not. Strange, Dark, and Ness (81) have, tent is not supported by other data. Thus,
in fact, shown that A. aerogenes survives best Strange et al. (83) showed that carbon-limiting
when harvested from glucose-Tryptone media and defined media yielded stationary-phase cells that
these cells have assimilated much glycogen. Sta- contain 18% of their weight as RNA, and these
tionary-phase cells obtained from defined manni- cells do not survive as well as those harvested
tol-limiting media contain no glycogen, whereas from tryptic meat broth and which contain 11%
those grown in the same medium but with nitro- RNA. This was especially apparent during star-
vation under N2 at 20 to 22 C. The other interest-
gen limiting growth deposit glycogen. Again, the ing observation is that of Harrison and Lawrence
latter cells survived better than those that had
not deposited glycogen. It would seem that the (30), who were able to isolate starvation-resistant
possession of glycogen, in A. aerogenes at least, mutants from starved suspensions of A. aerogenes.
favors survival. The provision of an exogenous These mutants are slower growing and, therefore,
source of glucose causes death, and 50% of the
contain less RNA than the wild type; they utilize
cells died in 4 hr, during which time starved cells their RNA, as well as other materials, during
remained almost completely viable. However, it starvation, since up to 20% of the weight of the
is difficult to demonstrate unequivocally that the cell is lost before there is any loss in viability. The
mutant, however, actually degrades more of its
possession of glycogen by these cells is in fact re- RNA than the wild type. Thus, we may conclude
sponsible for their longevity. On the other hand, that it is not simply the quantity of substrate for
Dawes and Ribbons (18) could not conclude that endogenous metabolism that favors survival but
possession of glycogen is an aid to survival in E. rather the capacity of the cell to utilize the sub-
coli, although their experiments are much less strate to advantage.
extensive. The only survival studies of which we are
The glycogen of starving suspensions of E. coli aware that correlate survival with PHB content
was oxidized at a very rapid rate, as much as 20 % are those of Sierra and Gibbons (76). PHB-rich
of the dry weight being completely oxidized in 3 cells of M. halodenitrificans are able to respire
hr; yet, little or no loss of viability occurred for endogenously at a constant rate for long periods
12 hr. Similarly Burleigh, Dawes, and Ribbons (96 hr) while their PHB reserves are being oxi-
(9) were unable to demonstrate a relation between dized. Then, at a critical concentration of about
the free amino acid pool of S. lutea or its Qo2
(endogenous) and the capacity of the cells for 10%, when first-order kinetics are initiated, the
survival. Starved cells of S. lutea remain viable Qo2 falls and the cells begin to die. When PHB-
for considerable periods (30 hr), after the amino poor cells (10% PHB) are starved, the Qo2
acid pool has been depleted. (endogenous), viability, and PHB content all de-
Degradation of RNA, and to a lesser extent cline.
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA 145

SUMMARY AND FUTURE OUTLOOK components of the cell-protein, RNA, and


Studies of endogenous metabolism and survival DNA-might be expected to occur, e.g., in cases
lead to several interesting considerations. First, of an amino acid deficiency. Thus, depending on
in connection with the substrates of endogenous the nature of the medium used to grow A. aerog-
respiration, it is apparent that various cell con- enes, different polymers in the main are utilized
stituents are depleted during starvation. The ex- for endogenous respiration. Starvation of glucose-
tent of the degradation of individual substrates Tryptone grown A. aerogenes results in a rapid
appears to depend on their initial concentrations
loss of glycogen; little RNA and protein are oxi-
in the cell, which, in turn, are determined by the dized initially. RNA-rich bacteria, obtained by
nutritional status of the organism. The pattern of growth on defined media, utilize their RNA to a
endogenous metabolism observed is thus a reflec- greater extent than their protein. (These were
tion of the nutritional condition of the cell. It carbon-limited cells and did not store glycogen.)
seems not without significance that a similar con-
Cells from tryptic meat broth, however, contain
clusion was reached by Postgate and Hunter much less RNA, and protein makes the largest
with respect to survival. Certain materials within contribution of intermediates for combustion.
the bacterial cell are believed to be expendable, The phenomenon of substrate-accelerated
e.g., glycogen and PHB, because cells may sur-
death has widespread implications, for it may
vive without them. Other polymers such as pro- mean that many experiments with "whole cells"
tein, DNA, and RNA are thought to be essential or washed suspensions of bacteria are lethal to the
for continued existence. Nonetheless, with the organism. Although great care may be taken to
possible exceptions of the information center ensure that cells remain viable during harvesting
(DNA) and the framework (cell-wall and mem- and washing procedures, the introduction of an
brane polymers), it has been shown for several or- oxidizable carbon compound to those carbon-
ganisms that most materials are depleted during limited cells can quickly kill them. The provision
stresses of starvation. There appear to be, at of a substrate gives a condition of plenty, and in
least, expendable fractions of the "essential" life some cases leads to high rates of metabolism. This
constituents. For example, ribosomal RNA is contrasts strongly with those conditions which
utilized during starvation; extrapolation of the are associated with longevity and which often
ribosomal RNA content of Salmonella typhimu- lower the rates of metabolism, as for example
rium to zero rate of growth shows that these cells with bacterial spores. Even more far reaching are
contain virtually no ribosomal RNA (20). In some the consequences of industrial and sewage proc-
instances, the expendable reserves are utilized esses in which the use of whole cells may, in fact,
preferentially, as for instance in E. coli where mean the inclusion of a large proportion of dead
glycogen spares the nitrogen-containing com- cells.
ponents of the cell; the carbohydrate reserve of A. The direct measurement of the ATP content of
aerogenes also seems to suppress partially the de-
cells has been made during starvation (82).
composition of RNA and protein. Conversely, the Strange, Wade, and Dark, however, could find no
polyglucose of S. lutea does not suppress the en- relationship between ATP content and viability,
dogenous respiration of the free amino acid pool. since the ATP content of A. aerogenes fell to a
However, another view has been put forward; very low value about 30 hr before any loss of
namely, the reserves that are utilized initially are viability occurred. They do provide evidence
those present in the cell in excess. Cells which are which suggests a direct relationship between the
arrested in growth due to the limitation of one ability of the cell to form ATP and its viability.
essential nutrient may continue to synthesize The provision of ATP by endogenous respiration
certain cellular components from nutrients that was demonstrated earlier by Strange (80), who
are still available in the medium. Such com-
concluded that the loss of the ability of starved
ponents may then be considered to be in "excess," cells to synthesize f3-galactosidase was not due to
i.e., at a concentration higher than that found in a shortage of energy. The intermediates of enzyme
the exponentially growing cells. This situation is synthesis were in short supply, since an inducer
one of considerable interest in organisms which do and a suitable nitrogen source allowed enzyme
not contain significant amounts of glycogen or induction to occur. We suggested earlier (15) that
PHB, for then imbalance between the essential the general functions of endogenous metabolism
146 DAWES AND RIBBONS BACTERIOL. REV.

were to provide (i) energy and (ii) carbon skele- 5. BINNIE, B., E. A. DAWES, AND W. H. HOLMS.
tons, and thus it seems in this case at least that 1960. Metabolism of Sarcina lutea. IV. Pat-
the carbon and nitrogen skeletons were not avail- terns of oxidative assimilation. Biochim.
able for synthesis although available for ATP pro- Biophys. Acta 40:237-251.
duction. Some examples are known where a trans- 6. BLUMENTHAL, H. J. 1963. Endogenous metabo-
lism of filamentous fungi. Ann. N.Y. Acad.
fer of carbon skeletons to other cell constituents Sci. 102:688-706.
occurs. However, we cannot agree with Gronlund 7. BOREK, E., A. RYAN, AND J. ROCKENBACH.
and Campbell (27) that the ribonucleoprotein 1955. Nucleic acid metabolism in relation to
serves as a source of amino acids and nucleotides the lysogenic phenomenon. J. Bacteriol.
for the synthesis of a greater quantity of enzymes 69:460-467.
concerned with endogenous respiration, although 8. BOWEN, T. J., S. DAGLEY, AND J. SYKES. 1959.
ribonucleoprotein may be able to maintain the A ribonucleoprotein component of Esch-
concentration of enzymes already present. To erichia coli. Biochem. J. 72:419-425.
the best of our knowledge, there are no cases 9. BURLEIGH, I. G., E. A. DAWES, AND D. W.
RIBBONS. 1963. Endogenous metabolism and
where the rate of endogenous respiration increases survival of Sarcina lutea. Biochem. J.
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The final aspect concerns the suggestion of 10. CARR, N. G., AND J. LASCELLES. 1961. Some
Lamanna and Mallette (41) that evolution enzymic reactions concerned in the metabo-
should have selected microbes capable of with- lism of acetoacetic-coenzyme A in Athio-
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ACKNOWLEDGMENTS Metabolism of Sarcina lutea. III. Endoge-
nous metabolism. Biochim. Biophys. Acta
We are greatly indebted to I. G. Burleigh for 30:278-293.
helpful discussion and constructive criticism, and 15. DAWES, E. A., AND D. W. RIBBONS. 1962. The
also to The Royal Society for a grant-in-aid. endogenous metabolism of micro-organisms.
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