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
make men wise in ten pages, who have no
appetite for a hundred pages; to condense
nourishment, to work with pulp and essence,
and to guard the stomach from idle burden
and unmeaning bulk."
Sidney Smith
Edinburgh Review, 1825
INTRODUCTION
Current interest in the endogenous metab-
olism of microorganisms has been reflected by
two recent symposia (Focal Topic A7, Intern.
Congr. Microbiol., 7th, Montreal, 1962; Ann.
N.Y. Acad. Sci., vol. 102, art. 3, p. 515-793,
1963) and by our own previous survey (15). The
present article is intended to be a more discursive
review of the present situation in this area of
study and to deal in greater detail with those
aspects which were not previously elaborated.
Endogenous metabolism may be defined as
the total metabolic reactions that occur within
the living cell when it is held in the absence of
compounds or elements which may serve as
specific exogenous substrates. Some products of
endogenous metabolism may be released into
the surrounding medium and are often utilized
by the cells, sometimes resulting in regrowth, a
phenomenon which is discussed later. It is im-
portant to stress the viability and integrity of
the cell in this context, because some authors
126
have used the term "endogenous" loosely in
relation to the activities of cell extracts (see, for
example, 84).
It must also be recognized that the reactions
characteristic of endogenous metabolism may
continue in the presence of exogenous substrates,
and, since metabolism of the latter compounds
is usually effected after they have been taken
into the cell, in the final analysis the distinction
between the metabolism of endogenous and
exogenous substrates may become largely a
matter of semantics, although problems of com-
partmentation within the cell should be appre-
ciated (22a). This is particularly the case where
the oxidation of an exogenous substrate by a
washed suspension of microorganisms leads to the
assimilation of material which subsequently can
be utilized as an endogenous substrate. Analysis
of the status of the endogenous metabolism in
the presence of added substrates presents many
experimental problems, but these have already
been adequately reviewed (6, 15).
The possible significance of endogenous metab-
olism in relation to the survival of microor-
ganisms is a topic presently under study in
several laboratories. One view which may be
held is that endogenous metabolism occurs
simply because the organism cannot help it, and
it therefore bears no relationship, direct or other-
wise, to the period of survival (22). The other
extreme outlook is that the survival character-
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
son and Wilkinson (95) that the intracellular
lipid granules of Bacillus cereus and B. mega-
terium were demonstrated unequivocally to be
composed mainly of PHB (about 90%), although
neither this nor the remaining 10%, lipid is re-
sponsible for the sudanophilic properties of the
granules that are observed in situ. Macrae and
Wilkinson (46, 47) also studied the effect of
various cultural conditions on the synthesis of
PHB in B. megaterium. When the glucose con-
TABLE 1. Occurrence of poly-,3-hydroxybutyrate in
bacteria*
Species Reference
Bacillus megaterium ............... 43, 77, 95
B. cereus .......................... 43, 95
B. mycoides ....................... 43
B. anthracis ....................... 43
Azotobacter chroococcum ........... 43
A. agilis .......................... 21
A. vinelandii ...................... 21
Rhizobium sp ...................... 21
Vibrio sp .......................... 31, 32
Chromobacterium violaceum ........ 21
Pseudomonas solanacearum ........ 32
P. antimycetica ................... 32
P. methanica ...................... 39
P. pseudomallei ................... 44
P. saccharophila .................. 19
Pseudomonas AMi ................ 59
Micrococcus halodenitrificans ...... 75, 76
Sphaerotilus natans ............... 58, 69
Hydrogenomonas sp ................ 73
Rhodospirillum rubrum ............19, 78
Rhodopseudomonas spheroides.... . 10
Chromatiurn okenii ................ 72
Spirillum itersonnii ............... 54
S. anulus ......................... 54
S. serpens ......................... 32, 54
* This is not a complete list of the bacterial
species that synthesize PHB.
centration in the growth medium was raised,
more of the polymer was synthesized; exhaustion
of the nitrogen source in the presence of excess
carbon and energy source permitted deposition
of about four times the amount of PHB as was
formed when glucose limited growth. Glucose,
pyruvate, and f-hydroxybutyrate were suitable
substrates for PHB production by washed sus-
pensions, but acetate, although itself unable to
effect synthesis (compare with Rhodospirillum
rubrum), enhanced PHB formation when supple-
BACTERIOL. REV.
menting other substrates. Anaerobic conditions
prevented PHB synthesis, as did dinitrophenol.
Concentrations of oxygen greater than 5% also
inhibited the assimilatory process. B. cereus, but
not B. megaterium, could effect PHB synthesis
under hydrogen, although no net uptake of
hydrogen was detected; PHB is not formed under
nitrogen.
When washed suspensions of these two bacilli
were shaken under air or under nitrogen, in the
absence of an exogenous carbon and energy
source, stored PHB was metabolized. The
anaerobic degradation of reserves was slower;
e.g., in B. megaterium, 61 and 17% degradation
of PHB occurred under air and nitrogen, respec-
tively, within 8 hr. Metabolic products detected
included f-hydroxybutyrate, acetoacetate, and
acetate, although aerobically CO2 and water
were the major products and only small amounts
of acetoacetate accumulated. Some correlation
was evident between the PHB content of cells
and their endogenous respiration; e.g., cells with
PHB-total N ratios of 0.83 and 3.27 had, respec-
tively, endogenous Qo2 values of 169 and 536.
Macrae and Wilkinson (46) also claimed that
N-deficient cells with a high content of PHB are
better able to withstand death and autolysis
than those with a low PHB content. Autolysis
was estimated by the total N content which, in
4 hr, fell by 12% in PHB-poor cells compared
with 5% in PHB-rich cells; the PHB content
of the latter cells decreased to the greatest ex-
tent. The method of estimation of autolysis is
not entirely satisfactory, because it has been
shown in numerous cases that nitrogenous mate-
rials are oxidized endogenously, releasing am-
monia, and that substantial amounts of nitrog-
enous compounds may diffuse from the cell
without loss of viability. Specifically in the case
of B. cereus, Clifton and Sobek showed that am-
monia is produced endogenously under some
conditions (12), and clearly B. megaterium
utilizes an endogenous substrate other than,
although concurrently with, PHB, since the
oxygen consumption is in excess of that required
for complete combustion of PHB; this other
substrate is not polysaccharide (47). It is pos-
sible, however, that autolysis may have occurred
to a similar extent in both PHB-rich and PHB-
poor cells, but the greater amount of reserve
material in the PHB-rich cells permitted utiliza-
tion of the liberated nitrogenous material and
VO0L.- 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
some limited cellular synthesis occurred. Against
this possibility may be set the authors' observa-
tion that growth did not occur when PHB-rich
cells were held in a medium lacking a carbon
and energy source; growth was measured by
total N so that in these bacilli PHB may serve
as a reserve of energy but not as a source of
carbon skeletons for synthesis.
Doudoroff and Stanier (19) have made some
interesting observations concerning the role of
PHB in oxidative assimilation by Pseudomonas
saccharophila and in photoassimilation by
R. rubrum. They found with most substrates that
a major portion of the assimilated carbon (60 to
90%) initially accumulates within the cells as
PHB; when the exogenous substrate is removed,
a rapid intracellular degradation of the polymer
occurs, suggesting a physiological role as a re-
serve material. When cells are subjected to
standard fractionation procedures, the chemical
properties of PHB result in its appearance in the
hot trichloroacetic acid insoluble fraction (pro-
tein fraction). This fact had led Wiame and
Doudoroff (91) earlier to conclude that C14 is
incorporated into nitrogenous materials during
oxidative assimilation.
Incubation of starved washed suspensions of
R. rubrum with C'4-acetate allowed deposition
of 70% of the assimilated C'4 into PHB, with no
significant dilution. With C'4-butyrate, some
dilution occurred, and the polymer contained
only 58% of the assimilated C14. The fate of the
polymer in the light over a period of 12 hr was
studied under a variety of conditions, e.g., in the
presence and absence of a source of organic sub-
strates but in the presence of a N source and
CO2. The absence of an exogenous organic carbon
source led to the disappearance of more than 90 %
of the polymer, but much of the C14 of this
material was redistributed into other cellular
components. The authors did not study the fate
of PHB in both nitrogen and carbon starvation,
or in the dark, so its behavior under these con-
ditions is not yet known. The rate of degrada-
tion of PHB and its conversion to other cellular
components was decreased when an exogenous
source of butyrate was supplied. In marked con-
trast, succinate, which is also metabolized under
these conditions, was quite unable to prevent
polymer breakdown and transfer of its C skele-
tons to other cell constituents; these processes
occurred to about the same extent as in the
129
absence of an exogenous carbon source. The
reason for this became apparent when it was
shown that succinate is photoassimilated princi-
pally to a glycogen-like polysaccharide, and the
dry weight of the cells increased by 40%.
Similar studies with P. saccharophila revealed
that washed suspensions of glucose-grown cells
incorporated 66% of the carbon assimilated from
U-C14-glucose into PHB, and without appre-
ciable dilution. An even greater amount (about
80%) of the assimilated carbon from acetate or
butyrate appeared in the polymer. Although ex-
perimental details were not given, the authors
claimed that tracer experiments indicated the
role of PHB as a substrate for endogenous metab-
olism in the absence of an exogenous carbon
source; its metabolism was reported to be much
slower than in R. rubrum, and transfer of polymer
carbon to other cell constituents could not be
demonstrated.
The use of PHB as an endogenous store of
carbon skeletons for synthesis in R. rubrum was
studied further by Stanier et al. (78), who showed
that for conversion of stored PHB to other cell
materials CO2 is essential. This was demon-
strated with cells which had assimilated acetate
and were then incubated in the presence of (i)
NH4Cl and He, (ii) NH4Cl and He-CO2 mixture,
and (iii) He-CO2 mixture in the absence of a
nitrogen source. After 16 hr, very small changes
in total dry weight had occurred and, in the
absence of C02, the PHB content of the cells
fell slightly but with no increase in the carbo-
hydrate or nitrogen content. In the presence of
CO2 without a nitrogen source, the PHB content
fell by about 50% and the carbohydrate content
showed a corresponding gain, but there was no
change in nitrogen content. WNhen both nitrogen
and CO2 were furnished, the PHB disappeared
almost completely with concomitant increases in
both carbohydrate and nitrogen content, includ-
ing protein. None of the experiments was de-
signed to test the possibility that PHB may
serve as an energy store, as it does, perhaps, in
B. megaterium and presumably also in P. sac-
charophila. It is considered that PHB serves as a
store of carbon and reducing power for further
CO2 assimilation, which is essential for PHB
utilization. The anaerobic utilization of PHB in
the absence of CO2 and a nitrogen source would
appear to be limited, but neither its formation
nor degradation aerobically in the dark has been
130 DAWES AND RIBBONS
studied, and here one might expect PHB to
serve as a source of energy.
The presence of PHB as an endogenous reserve
in Hydrogenomonas is documented by the work
of Schlegel, Gottschalk, and von Bartha (73).
When chemolithotrophic growth, in an atmos-
phere of H2-02-CO2 (60:30:10), is limited by
nitrogen exhaustion (NH4Cl) in the medium, cells
in the stationary phase continue to increase in size
although no division occurs. The increase in dry
weight is accounted for entirely by PHB forma-
tion. Washed suspensions synthesize PHB from
CO2 by oxidation of hydrogen, and the stoichi-
ometry corresponds to:
25H2 + 802 + 4CO2 -- (C4H602) + 22H20
The rate of endogenous respiration of PHB-
poor cells (about 10% of the dry weight) was
considerably less than that of PHB-rich cells
(about 50% of the dry weight). It was little
affected by the addition of a nitrogen source
(NH4Cl) which had a marked stimulatory effect
on the respiration of the latter cells. Determina-
tions of cellular carbon and total nitrogen during
endogenous respiration, and during hydrogen
oxidation in the absence of C02, were carried
out with PHB-rich cells, both with and without
a nitrogen source. In air, only about 8% of the
PHB was consumed in 12 hr by endogenous
metabolism, but with added NH4Cl the total
cellular nitrogen increased significantly and the
PHB decreased by some 73%. Under an at-
mosphere of C02-free H2-02, the increase in cell
nitrogen was much greater, although not much
more PHB was utilized (76 %). These results
suggest that in Hydrogenomonas stored PHB may
serve as a carbon and energy source and can
support protein synthesis in the presence of a
suitable source of nitrogen. When hydrogen is
provided as an additional energy source, the
efficiency of protein synthesis is increased.
Conditions for the accumulation of PHB in the
halophile, Micrococcus halodenitrificans, were
determined by Sierra and Gibbons (75). These
authors further studied the role of PHB and its
relationship to endogenous respiration and sur-
vival of the organism (76). The RQ of the en-
dogenous respiration of M. halodenitrificans was
0.87 i 0.05. (That required for complete com-
bustion of PHB is 0.88.) Aeration of washed
suspensions at 25 C reduced the PHB content
slowly (from 55 to 29%G in 127 hr); under these
BACTERIOL. REV.
conditions, some lysis of cells occurred and the
endogenous Qo2 remained at 40 throughout this
period. Some product of metabolism or lysis
was inhibiting further endogenous respiration,
since the inhibition could be relieved by washing
the cells. Changes in the cell constituents of
PHB-poor cells (containing 10% PHB) showed
that 3 hr of starvation produced no changes in
total N, either soluble material or carbohydrate,
but almost half the PHB had been consumed.
The endogenous Qo2 value decreased with poly-
mer depletion. A similar but extended study
was made with PHB-rich cells; after 96 hr of
starvation had elapsed, the PHB content had
diminished from 50 to 10% of the dry weight, and
the endogenous Qo2 remained constant. Further
starvation rapidly reduced the endogenous Qo2.
From these and other data, it seems clear that
the rate of PHB oxidation is slower during
starvation experiments than in the respirometer:
if, as appears to be the case, PHB is the sole
substrate for respiration, cells containing 50 %
of their dry weight could respire for only 15 hr
with a Qo2 of 40.
Biosynthesis of PHB. In view of the unique
occurrence of PHB as a storage polymer (or
metabolic shunt product) in bacteria, it is some-
what surprising that, at the time of writing,
there is very little information regarding the
metabolic routes and enzymes concerned with
its biosynthesis. The only paper of significance
is that of Merrick and Doudoroff (56). They
demonstrated that polymer particles (obtained
by differential centrifugation of lysozyme-treated
bacteria) of B. megaterium KM were able to
incorporate C'4-f-hydroxybutyryl-coenzyme A
(CoA) into PHB. This was independent of the
presence of the soluble fraction of the cell. Ap-
proximately 40% of the total radioactivity be-
came incorporated into the polymer, which cor-
responded to the amount of thioester decomposed.
A mixture of labeled g-hydroxybutyrate and un-
labeled CoA did not allow incorporation of C14
into the polymer.
Similar experiments by these authors were
extended to the particulate fraction of R. rubrum
which contained all the activity for the incor-
poration of f-hydroxybutyryl-CoA into PHB.
However, these particles also contained a very
active depolymerase which could degrade the
PHB. With crude extracts, supplemented with
CoA, adenosine triphosphate (ATP) and reduced
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
nicotinamide dinucleotides, a small incorporation
of C'4-acetate into polymer occurred. Activa-
tion of f3-hydroxybutyrate could not be demon-
strated.
Although activation of ,3-hydroxybutyrate
could not be demonstrated in extracts of R. ru-
brum, whole cells of Hydrogenomonas incorporate
l3-hydroxybutyrate into PHB (73). Crotonic
acid can also be used as a substrate; in either
case, the presence of hydrogen is not mandatory.
Presumably, the oxidation of a portion of these
substrates provides the energy necessary for
polymerization. The utilization of crotonic acid
is particularly interesting since there may exist a
2n ATP y 2n CH3CO2H
2n ADP 2n CH3CO-SCoA
Acetyl GoA
V HSCoA
n CH3COCH2CO- SCoA
Acetoacetyl CoA
_______ V2n[H]
- - -
CH3CHOHCHC0,H-I n CH3CHOH CH2CO' SCoA
4- Hydroxybutyrate 4-Hydroxybutyryl GoA
,, .4HSCoA
CH3CH-CHCO2H----CHCH-CH-COSCIoA1
Crotonate Crotonyl CoA (CH3- H- CHaCO-)n
Hydrogenomonas
I 9
Poly-A-hydroxybutyrate
FIG. 1. Biosynthesis of poly-f3-hydroxybutyrate.
possible pathway of 13-hydroxybutyrate synthesis
omitting acetoacetate.
CH3-CH = CHC02H + H20
-* CH3 CHOHCH2 CO2H
Reactions leading to PHB are schematically
shown in Fig. 1.
Catabolism of PHB. As with the biosynthesis
of PHB, comparatively little is known of its
degradation. There are, however, indications
that the initial stages of depolymerization are
extremely complex and appear to be bound up
with the structural integrity of the polymer
particles (56, 94). Thus, Merrick and Doudoroff
(56) demonstrated that extracts of R. rubrum
contain enzymes that degrade (i) native PHB
from B. megaterium or (ii) boiled PHB particles
from R. rubrum, but do not degrade the purified
polymer.
Sierra and Gibbons (76) demonstrated PHB
esterase (depolymerase) activity in Ml. halode-
nitrificans by the anaerobic release of CO2 from
bicarbonate buffer. A stoichiometric release of
131
CO2 by l3-hydroxybutyrate was recorded; this
depolymerization is inhibited by the esterase
inhibitor, diethyl-p-nitrophenyl phosphate (para-
oxon). Further, the rate of release of f3-hydroxy-
butyrate was identical with the rate at which
oxygen is consumed for the complete combustion
of PHB. For example, 0.76 umole of ,3-hydroxy-
butyrate was liberated per hr by 2 mg of cells
containing 50% of their dry weight as PHB;
this requires an oxygen consumption of 3.4
Mmoles per hr for complete combustion. This is a
figure realized by the recorded Qo2 values of 39
[(39 X 2)/22.4 Mmoles of 02 per 2 mg of cells per
Poly - hydroxybutyrate
IN PHB esterase
(depolymerase)
D- (-)-A - I ydroxybutyrate
r NAD
4,- I4ydroxybutyrate
NADH2 dehydrogenase
Acetoacetate
ATP, HSCoA, Mg2+
Acetyl GoA
Oxaloacetate -
5 Citrate
Tricarboxylic,
Acid
Cycle
FIG. 2. Catabolism of poly-f3-hydroxybutyrate.
hr = 3.48]. On this basis, the depolymerization
appears to be rate-limiting. Sierra and Gibbons
later showed that depolymerase activity is
markedly dependent upon Nat and Lit ions (76,
76a). Cells washed and resuspended in KCl or
water do not oxidize their PHB reserves unless
Na+ or Li+ is added.
Enzymes involved in the degradation of /3-hy-
droxybutyrate were also demonstrated by Sierra
and Gibbons (76). DL-f3-Hydroxybutyrate is
oxidized by crude extracts to acetoacetate by
a nicotinamide adenine dinucleotide (NAD)-spe-
cific enzyme. Only the D(-)-f-hydroxybutyrate
is utilized, and the acetoacetate is not further
metabolized unless ATP, Mg2, CoA, and oxalo-
132 DAWES AND RIBBONS BACTERIOL. R.EV.

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

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-
hydroxybutyrate dehydrogenase from R. rubrum
to be cold-sensitive. A 250-fold purification of the
enzyme was obtained in 30% yield, and this was
unstable in dilute solutions or inactivated by
freezing. The enzyme is freely reversible, and the
reaction products are acetoacetate and reduced
NAD (NADH2). Although NAD phosphate
(NADP) would not substitute for NAD, a-oxo-
valerate showed 6% of the activity obtained with
acetoacetate. Furthermore, a-oxovalerate compet-
itively inhibited acetoacetate reduction, because
of the lower affinity of acetoacetate. The rate of
by a factor of 2 are observed with older cells
depleted of polymer. Succinate-grown cells con-
tain no PHB, and only low specific activities of
the enzyme are found.
Glycogenlike Reserves
The majority of microorganisms synthesize
polysaccharides of one form or another which
may be of intracellular or extracellular origin.
Only the former type come within the scope of
the present survey since, by definition, extra-
cellular polysaccharides [reviewed by Wilkinson
in 1958 (92)] cannot serve as endogenous sub-
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
ships exist between inactive and active forms of
phosphorylase, and the enzymes obtained from
different tissues of the same species are not iden-
tical (79). The bacterial phosphorylases are, how-
ever, not well studied.
The UDPG pathway of glycogen synthesis has
been described in bacteria by Madsen (48, 49),
and in yeast by Algranati and Cabib (1). Glyco-
gen acts as a primer with the glycogen synthetase
enzymes of Agrobacterium tumefaciens and yeast,
but, unlike the mammalian enzymes, glucose
6-phosphate does not stimulate the reaction:
UDPG + polysaccharide primer
uridinediphosphate (UDP)
+ glucosyl (1,4') primer
A cyclic scheme of glycogen degradation and
synthesis has been proposed for mammalian
UDP GLYCOGEN
UDPG glycogen VI
synthetase
P UDPG Phosphorylase
PP
UDPG
p \rophosphorylase
UTP
Glucose-1- phosphate
1 Phosphoglucomutase
E.AT .
GLUCOSE lRexoki~nase Glucose 6-phosphate
FIG. 4. Biosynthesis and degradation of glycogen.
systems (Fig. 4). A similar system probably oper-
ates in bacteria, since the necessary enzymes have
been demonstrated and partially separated in A.
tumefaciens (48), and UDPG has been shown to
be a competitive inhibitor of phosphorylase in the
same organism, suggesting that the concentration
of UDPG may regulate glycogen synthesis not
only directly, but also by inhibiting the degrada-
tive enzymes (49).
Madsen (50) has provided further evidence to
support the postulate that the control of glyco-
gen metabolism is effected by UDPG. The UDPG
pathway to glycogen is essentially irreversible,
and the equilibrium of the phosphorylase reac-
tion is slightly in favor of glycogen synthesis,
yet the concentration of glycogen in A. tume-
faciens varies considerably. Madsen analyzed A.
tumefaciens during its growth in batch culture for
glycogen and UDPG content. Both increase
initially during a short lag phase and then de-
crease at the beginning of exponential growth.
135
In the stationary phase (growth limited by nitro-
gen), the concentrations of both compounds rise
again, the UDPG slightly before the glycogen. A
linear relationship between glycogen concentra-
tion and UDPG concentration was demonstrated.
Replenishment of the nitrogen supply in the
stationary phase in another experiment caused
resumption of growth, depletion of UDPG, and
cessation of glycogen synthesis. During aeration
of washed suspensions of A. tumefaciens in buf-
fered salt solution, the glycogen was utilized.
The UDPG concentration during this period re-
mained low and constant.
Glycogen is a highly branched polysaccharide,
and little or no reference has been made to the
synthesis of (al ,6') links; the UDPG synthetase
will only synthesize (al,4') bonds although a
branched primer is required. A branching en-
zyme similar to Q-enzyme has been described in
yeast which will synthesize glycogen from amylose
by transglucosylation (al ,4' to al,6').
Amino Acid Pools
The first indication that the free amino acid
pool could serve as a source of substrates for
endogenous respiration was provided by Dawes
and Holms (14). Aeration of washed suspensions
of stationary-phase, peptone-grown S. lutea re-
duced the endogenous Qo2 values to negligible
levels, during which time the free amino acid pool
was depleted to one-half its original level and
ammonia was released into the supernatant fluid.
Hydrolysis and analysis of the hot water-soluble
pool also showed that some peptide material was
being used as substrates of endogenous respira-
tion. The total oxygen consumption during the
period of endogenous respiration corresponded to
7.5 and 3.4 jsmoles of oxygen per Emole of utilized
amino acid in the unhydrolyzed and hydrolyzed
pools, respectively. The latter value is a reasona-
ble average figure for the oxidation of a mix-
ture of the amino acids that are utilized during
the starvation.
Glycine, threonine, leucine, tryptophan, and
a-aminobutyric acid were completely utilized,
and much of the serine, glutamate, and alanine of
the pool was oxidized. Very little loss of amino
acids to the suspending media occurred (about
2% of the initial concentration of the hydrolyzed
pool). Glutamate plays a most important role in
the endogenous respiration of S. lutea; it accounts
for 20% of the total pool amino acids in freshly
136 DAWES AND RIBBONS
harvested peptone-grown cells, and this declines
to 1% after a 5-hr aeration period.
Although glucose-peptone grown S. lutea also
stores a polyglucose compound as a reserve mate-
rial, the free amino acid pool is depleted just as
rapidly during starvation of these cells (9, 65).
The utilization of free amino acid pools as en-
dogenous substrates has since been shown to occur
in Nocardia rugosa (2) and Staphylococcus aureus
(64). During starvation of washed mycelial sus-
pensions of N. rugosa, both the endogenous Qo,
and Qo, (glucose) fall by approximately the same
value. There is a loss of weight from the mycelia
that can be accounted for almost completely by
loss of protein; a little acid-soluble carbohydrate,
mainly hexose, is also utilized. There was no sig-
nificant utilization of lipid during starvation for
16 hr. The pH value changed from 7.5 to 8.2
during the incubation, and free ammonia was
formed at the expense of the mycelial protein.
Oxo-acids did not accumulate but were oxidized.
Chromatography of the free amino acid pools
showed that fresh mycelia contained large
amounts of glutamate, alanine, aspartate, leu-
cine, and valine, whereas only smaller amounts
of leucine, alanine, and glutamate remained after
starvation. The RNA and DNA contents of the
cell are not utilized endogenously. It was con-
cluded that the main substrates of endogenous
respiration are the amino acids that are present
in the pool, and also obtained from the hydrolysis
of cell protein; an acid-soluble carbohydrate is
also utilized. The presence of an exogenous source
of glucose inhibits the endogenous consumption
of protein in N. rugosa.
Ramsey (64) showed that washed suspensions
of S. aureus respire endogenously with an RQ of
0.83 to 0.86. The carbohydrate content of the
cells remained constant (at the very low value of
1.18% of dry weight) during starvation, and
ammonia was released into the supernatant fluid.
The 02-NH3 ratio was 6.2, and glutamate was
utilized from the free amino acid pool. This indi-
cated that substances other than glutamate were
being utilized (02-NH3 ratio for glutamate is
4.5). The endogenous respiration of incompletely
C'4-labeled cells confirmed this view, since apart
from the utilization of glutamate in the pool
there was also some loss of C14 from the hot tri-
chloroacetic acid-insoluble fraction of the cells;
further, the oxygen consumption was much in
excess of that required for oxidation of glu-
BACTERIOL. REV.
tamate alone (glutamate can account for only
10% of the 02 consumed). More completely
labeled cells were obtained by growth on U-C14-
glucose and C'4 algal hydrolysate, and the change
in cell components during respiration was fol-
lowed. Only about one-third of the radioactivity
lost from the cells is recovered as C'402. The rest
can be traced to the suspending buffer, and rather
more than one-third to deposition in the hot
trichloroacetic acid fraction. The bulk of the
radioactivity was lost from the hot trichloroacetic
acid-insoluble fraction. Apart from the utilization
of glutamate from the free amino acid pool, evi-
dence was presented to show that aspartate was
a probable substrate and, to a lesser extent,
alanine. Cells, allowed to assimilate C'4-glycine
into the pool, washed, and subsequently starved,
liberated C1402 only slowly.
Comparisons of the endogenous metabolism of
coagulase-positive (+) and -negative (-) S.
aureus have revealed marked differences (D.
Ivler, personal communication). During starvation
of organisms grown on Brain Heart Infusion, the
RQ of + cells showed little change (from 1.08
to 0.95), whereas that of - cells fell from 1.10 to
approximately 0.5 in 60 min. The Qo2 of + cells
was higher than that of - cells, but both values
decreased rapidly with starvation. High rates of
endogenous respiration of + cells suppressed the
rate of oxidation of added glucose, but no such
effect was apparent with - cells. Measurement
of 02-NH3 ratios during starvation of washed
suspensions showed that values fell from 4.7 to
3.0 with + cells and from 13.4 to 10 with - cells.
Of considerable note was the fact that while the
free amino acid pool content of both types of
cell diminished, the bulk of the loss could be
accounted for as free amino acids in the super-
natant fluid (as much as 90% recovery with -
cells); nonetheless, ammonia appeared in the
supernatant at a greater rate and to a greater
extent with + cells, indicating the net degrada-
tion of nitrogenous materials. The total carbo-
hydrate content of both + and - cells did not
alter during starvation.
RNA Metabolism
Mlore typical storage compounds such as
glycogen or PHB are characterized by their
deposition during conditions of carbon source
excess or nitrogen limitation, and by their de-
pletion to almost negligible amounts during
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
starvation. However, as Herbert (34) empha-
sized, the amounts of such basal materials of the
bacterial cell as RNA, DNA, and protein are
subject to wide variation, and this can be con-
trolled by environmental conditions.
Strange, Dark, and Ness (81) showed that
RNA is metabolized endogenously during starva-
tion of washed stationary-phase suspensions of
A. aerogenes, and net degradation occurs. The
extent of RNA catabolism varied, and this
depended on the source of the cells; 40% of the
RNA of cells harvested from defined media was
utilized in 70 hr, during which time about 70%
of the population remained viable. [See also E.
coli (7)]. Cells harvested from glucose-Tryptone
media contain much less RNA (about 11% of
the dry weight), and little is utilized. These cells
contain glycogen that is almost completely
respired within 25 hr. Tryptic meat broth grown
A. aerogenes, on the other hand, catabolize nearly
half their RNA, from about 13% to 7% of the
dry weight, within about 50 hr. The products of
RNA metabolism that have been detected in the
suspending fluid include ammonia, inorganic
phosphate, and the free bases hypoxanthine,
uracil, and guanine (62, 81, 83), and small
amounts of adenine (83); hypoxanthine was the
major component of the bases in the supernatant
(83). Nucleotides and nucleosides did not ac-
cumulate to any significant extent, and most of
the pentose was apparently oxidized. The ultra-
violet-absorbing materials were readily released
into the supernatants, and acid-soluble inter-
mediates did not accumulate within the cells (83).
The amount of ultraviolet-absorbing material
released corresponded well with the amount of
RNA lost from the cell, and this loss occurred
almost entirely from the RII sedimentation
fraction (83), as had been demonstrated in the
case of loss of RNA from E. coli ribosomes (85).
RII is the cell fraction sedimented at 78,000 X g
for 7.5 hr.
The endogenous utilization of RNA is not
peculiar to A. aerogenes, but appears to be very
widespread, and has been demonstrated in E.
coli (17, 18), S. lutea (9), and P. aeruginosa (27).
The utilization of the ribose portion of the
RNA by endogenously respiring E. coli was
briefly mentioned in the section on glycogenlike
reserves, and in Table 2. It is not yet clear
whether the purine and pyrimidine bases are
completely oxidized, although this is unlikely as
137
ultraviolet-absorbing compounds accumulate in
the supernatants. The degradation of RNA in
E. coli was studied by Wade (85), who concluded
that two pathways exist. The M route (Mg2+-
dependent) results in the formation of nucleo-
side 5'-phosphates characteristic of phosphodi-
esterases. The rate of formation of these nucleo-
tides is further stimulated by inorganic phos-
phate, suggesting that polynucleotide phos-
phorylase was also depolymerizing RNA (86).
Autodegradation of ribosomes in the presence of
inorganic P32-orthophosphate gave only labeled
nucleoside diphosphate. The nucleoside mono-
phosphates appeared to be formed by an inde-
pendent route (86). The second route, the V
route, results in the degradation of RNA into
nucleoside 2', 3'-cyclic phosphates in the presence
of sufficient EDTA to remove the M\g2+. The
cyclic phosphates are further hydrolyzed to
nucleoside 3'-phosphates. The V route then
employs ribonuclease-type enzymes. The enzymes
of both routes are located in the ribosomal frac-
tion. The observation of Kiguchi and Uemura
(84a) that citrate and phosphate enhance the
release of RNA degradation products from yeast
cells is, perhaps, relevant. These authors believe
that magnesium is removed from the cell
membrane by chelation, since added Mg2+
countered the effect of these agents.
Starving washed suspensions of P. aeruginosa
release much ultraviolet-absorbing material into
the supernatant with an Emax at 260 mIu. Nucleo-
tides, nucleosides, and free bases were detected.
The effect of Mg2+ ions on ribosomal particles is
well known (8), and Gronlund and Campbell (27)
have used this as evidence for the utilization of
RNA as a substrate for endogenous respiration.
Oxygen consumption is depressed in the presence
of Mg2+ ions, which allow the formation of 70S
ribosomes. When P. aeruginosa was grown in the
presence of C14-uracil, the radioactivity was con-
tained primarily in the nucleic acid fraction, and
this yielded C'402 during subsequent starvation.
The C0402 was derived solely from the RNA, and
came largely from the ribosome fraction. The
enzymatic degradation of ribosomes was demon-
strated and inhibited by EDTA; phosphate
markedly increased ribosome degradation, sug-
gesting a role for polynucleotide phosphorylase.
The fate of the ribose portion of the RNA was
not determined, although ribose (free and com-
bined?) was detected in supernatant fluids.
138 DAWES AND RIBBONS
By calculations based on the values of C1402
released from uniformly C'4-labeled cells, 2-C04-
uracil-labeled cells, and U-C'4-proline-labeled
cells, Gronlund and Campbell deduced that the
C'402 liberated from 2-C'4-uracil-labeled and
from U-C14-proline-labeled cells is equivalent to
the total amount of C1402 liberated by uniformly
labeled cells. From this, they infer that RNA and
protein are the only endogenous substrates
oxidized. Their calculations are based, however,
on the assumption that the fate of the 2-C of
uracil is representative of all RNA carbon atoms,
since, for example, the ribose was presumably
unlabeled by this technique and its fate was not
determined. The fate of the proline C atoms is
also assumed to reflect the destiny of all the other
carbon atoms of protein.
Protein
The net utilization of protein as an endogenous
substrate was first demonstrated by Strange et
al. (81) with starving suspensions of A. aerogenes,
and Gronlund and Campbell (26) indicated that
ammonia release by endogenously respiring cells
was a general phenomenon. Thus, washed sus-
pensions of E. coli, P. aeruginosa, P. fluorescens,
Achromobacter sp., B. subtilis, and S. faecalis all
liberate ammonia during endogenous respiration
(15, 26, 87). To this list may be added A. aerog-
enes (81) [although Gronlund and Campbell (26)
did not detect ammonia production with their
strain], B. cereus (12, 47), S. lutea (14), S. aureus
(64), and N. rugosa (2). Even so, the original
work of Strange et al. is the most decisive demon-
stration of protein degradation, since they used
simple chemical analysis. Other workers have
concluded from radiochemical evidence that
protein is a substrate of endogenous respiration;
in some bacteria, it appears to be the main
substrate. With P. aeruginosa, Gronlund and
Campbell (27) labeled the cells with U-C14-proline
and demonstrated the endogenous evolution of
C1402 from the hot trichloroacetic acid-insoluble
fraction. It is interesting to note that the alcohol-
soluble protein of P. aeruginosa accounts for 20%
of the total protein (60) and yet this is not utilized
during starvation. The hot trichloroacetic acid-
insoluble residue yields less C1402 when cells are
starved in the presence of Mg2+; under these con-
ditions, there is also a slight decrease in the alco-
hol-soluble protein. Pine (60) demonstrated that
the alcohol-soluble proteins of E. coli do not dis-
BACTERIOL. REV.
appear during starvation; the solubility proper-
ties of this fraction alter, however, and cautious
interpretations with respect to the fluctuations of
this fraction are required.
Strange et al. (83) showed that protein is lost
from all ultracentrifugal fractions during starva-
tion of A. aerogenes; i.e., ribosomal and soluble
proteins are utilized. The products of protein
catabolism appear to be ammonia and carbon
dioxide, as only traces of amino acids accumulate
in the suspending fluid.
Attention must be directed to the data ob-
tained by Strange et al. (81) for the release of am-
monia from cells grown in different media. If, on
the basis of their figures, a calculation is made to
compare the nitrogen accounted for as NH3 with
the nitrogen of the degraded protein and RNA,
considerable discrepancies are apparent. Thus
78.8, 25.2, and 30.2% nitrogen are unaccounted
for as NH3 in cells harvested from glucose-Tryp-
tone, Tryptone meat broth, and defined media,
respectively, after 25 hr of starvation. The fate of
this nitrogen has not been ascertained, and some
cellular redistribution might be envisaged.
Other Potential Substrates
Our knowledge of bacterial lipids is very
limited (34), especially concerning their role as
endogenous reserves of energy. It appears that
conventional lipids are stored by E. coli under
favorable conditions; e.g., provision of acetate
stimulates lipid deposition (13). The utilization of
lipid and phospholipid materials during starva-
tion has been discussed (15). The massive deposi-
tion of lipids by the yeasts Rhodotorula gracilis,
Lipomyces starkeyi, R. graminis, and R. glutinis
was recently described by Mulder and co-workers
(58).
DNA is generally considered to be a stable cell
constituent whose function is storage of informa-
tion, and generally no utilization of DNA occurs
during starvation. However, Strange, Wade, and
Ness (83) showed that the DNA of starving A.
aerogenes increased by 17%, and they ruled out
the possibility of cell division occurring. A DNA
increase at the expense of RNA was also demon-
strated with phosphate-deficient E. coli (37). On
the other hand, utilization of DNA after 19 hr of
starvation was noted in A. aerogenes (30).
ENERGY OF MAINTENANCE
Cellular processes, whether mechanical or
chemical, require energy for their performance,
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
and unless a supply of energy is readily available
these essential processes will cease and the cell
will die. The provision of energy by exogenous
nutrients is well established, e.g., the use of glu-
cose as an energy (but not carbon) source by
Streptococcus faecalis (4). Under conditions of
starvation, mobilization of endogenous sub-
strates must furnish the energy necessary to allow
the various cellular activities to continue. The
energy required for these processes of cell sur-
vival has been called the "energy of maintenance."
Since the performance of work is required for
all these activities, which include resynthesis,
osmotic regulation, and heat loss to the external
environment, presumably they will eventually
cease owing to lack of energy, and the cell will
no longer be able to maintain its status quo. To
maintain the intact living cell, structures such as
the cell wall, flagella, cell membrane, and cell
particles must be kept in good repair. It is pos-
sible that some of these structures may be dis-
pensed with for the sake of survival; indeed,
flagella have been removed from bacterial cells
without affecting viability (45), while suitably
prepared protoplasts possess metabolic activities
that are identical with those of whole cells (71).
The possession of a rigid cell wall provides dis-
tinct advantages, since protoplasts remain intact
only in solutions of high osmotic pressure. The
cell wall is probably an essential feature of cell
survival under natural conditions. Protoplasts
are able to grow (increase in size) but apparently
are unable to undergo cell division unless rever-
sion to bacillary form (in gram-negative organ-
isms only) occurs (55a).
Mandelstam (52) reported that starved sus-
pensions of E. coli break down and resynthesize
their macromolecular components (RNA and
protein) at a rate of 5% per hr. On the other hand,
growing cells appear not to exhibit any appreci-
able turnover of protein. The continued resynthe-
sis of macromolecular components during starva-
tion requires energy, which may be supplied by
the components that are undergoing transforma-
tion. This situation pinpoints a feature not gen-
erally appreciated, namely, that the maintenance
requirement is not necessarily a constant feature
independent of growth rate; i.e., at the extremes
of unrestricted growth and absence of growth,
protein turnover (and, therefore, the energy re-
quired for this process) varies from 0 to 5% per
139
hr. Consequently, unrestricted growth should
have a lower maintenance requirement.
Microorganisms are able to survive for con-
siderable periods during starvation and conse-
quently must maintain soluble constituents, often
against considerable concentration gradients.
The regulation of the cytoplasmic osmotic pres-
sure and pH value appear to be highly selective
processes, since considerable quantities of some
cell substances, e.g., RNA from bacteria and
yeast, and also smaller molecular moieties, such
as amino acids and bases of RNA, ribose, and in-
organic phosphate, are able to diffuse into the
suspending fluids without loss of viability (7, 17,
27, 68, 81).
For bacteria to remain motile, a source of
energy is required; tactic responses were dis-
cussed at some length by Weibull (89). In the
absence of exogenous substrates, energy must be
supplied from endogenous sources. Phototrophic
organisms present a somewhat different case in
that their source of energy is light. If light is the
sole source of energy available to green and
purple sulfur bacteria, then incubation of washed
suspensions in the dark should exclude mecha-
nisms of ATP synthesis, and it would be of in-
terest to know the survival characteristics of
these species in the dark. This situation is similar
to the survival of strict aerobes under anaero-
biosis.
Although we are principally concerned with the
concept of maintenance energy in starving cells,
it is obvious that some consideration of growing
cells should be included; additional demands for
energy may be manifest here. The actual proc-
esses of cell division may require larger amounts of
energy than the other phases of the growth cycle
of individual cells which, in the main, would be
supplied by exogenous sources. Lowered cell
yields at low growth rates may, in fact, be caused
by the diversion of energy from synthesis of cell
substances to the physical and energetic task of
maintaining a cell that spends a long period over
division. Alternatively, cells actually dividing
may dissociate energy-yielding reactions from
synthesis without affecting the rate of utilization
of energy source.
The chemical and mechanical activities of the
microbial cell, therefore, lead one to postulate
that some energy is utilized to maintain the cell in
a functional and viable condition, and that during
starvation this energy must be derived from
140 DAWES AND RIBBONS
endogenous sources. We think few scientists
would now deny the energy-of-maintenance re-
quirement, although there are several widely
quoted experiments that have been cited as
evidence against such a concept, often because it
could not be detected. This is especially apparent
in the growth-yield experiments of Monod (57)
and Bauchop and Elsden (4). Microbial growth
yields are (within certain limits) directly propor-
tional to the concentration of limited nutrient.
Even when the carbon and energy source limits
the yield of cells, the relationship holds for very
low concentrations; extrapolation of the experi-
mental points indicates that no intercept occurs,
i.e., at zero concentration of nutrient no growth
occurs. If some portion of the energy source were
utilized for functions other than growth, then
one should observe that the addition of very
small amounts of an energy source would not
permit growth. This then would assume that
growth is a secondary feature of energy utiliza-
tion and that energy is preferentially channelled
to maintenance purposes. The concentrations of
the energy source used in these experiments were
such that, although they limited the maximal
population attainable, they did not limit the rate
of growth. Monod also limited the rate of growth
of E. coli by limiting aeration, and although this
doubled the time taken to achieve maximal
density in one culture the cell yield was un-
changed. He concluded that since the rate of
growth did not influence the cell crop any
energy-of-maintenance values were nil.
It seems to us that many of the experiments
designed to test the use of a portion of the exoge-
nous energy source for maintenance rather than
growth are complicated by the fact that the
energy source is also a source of carbon for
growth. More definitive evidence might be ob-
tained with microorganisms which do not in-
corporate their energy source into cell substance;
there are numerous systems available for such
experiments-phototrophs, autotrophs, and the
nutritionally fastidious anaerobes that ferment
carbohydrates almost solely as a source of energy.
Furthermore, experiments designed to show that
some portion of the energy (and carbon) source
is not utilized for growth, and many do demon-
strate this, are not entirely convincing arguments
for the maintenance concept. These experiments
do not show that the energy that is diverted
from growth is utilized specifically for mainte-
nance.
BACTERIOL. REV.
It seems feasible that at slower growth rates
the cell yield might be decreased because the cell
physiology with respect to regulatory mecha-
nisms has altered in response to the changed en-
vironment, and that a portion of the energy
source is uncoupled at the enzymatic sites of
phosphor) lation; i.e., the decreased cell yield is
merely a reflection of decreased efficiency of
conversion f the energy source into high-energy
phosphate.
It is perhaps too obvious that the reactions of
energy-yielding metabolism are not always
coupled to the growth of the organism. The more
extreme cases of this are most often observed in
batch cultures that have reached a stationary
population but still consume considerable quan-
tities of substrate. This same feature is seen dur-
ing growth limitation (rate or yield) of some
nutrient other than energy source, and is most
dramatically demonstrated by washed suspen-
sions of nonproliferating cells metabolizing added
carbon or energy substrates.
Where the energy source is also the carbon
source, usage of carbon skeletons occurs without
net growth. In some cases, the carbon skeletons
are removed from the medium and stored within
the cells (assimilation) ready for utilization under
duress. However, uncoupling of assimilation has
also been observed, in that products of metabo-
lism often appear in supernatants, and presum-
ably these are lost to the cell. Further, it has been
suggested that the products of metabolism under
conditions of carbon and energy excess are shunt
products, and that assimilation into reserves
merely reflects this glut. Limitations of the rate of
microbial growth by nutrients other than the
energy source do not control the extent of oxida-
tion of the energy source; e.g., Rosenberger and
Elsden (67) showed that in tryptophan-limited
growth S. faecalis produced large amounts of
lactate.
A discussion of the theoretical principles of
continuous culture (as controlled by nutrient
limitation), and a comparison of these with results
obtained experimentally, led Herbert (33) to
postulate that A. aerogenes displays a constant
rate of endogenous metabolism during exponen-
tial growth. The curve derived experimentally
relating steady-state bacterial concentration to
dilution rate (growth rate) does not coincide with
the theoretically predicted curve. At low dilution
rates, the cell yield is less than that expected
when the carbon and energy source is the growth-
V OL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
limiting nutrient. Cultures whose growth is
limited by nutrients other than the carbon and
energy source do not show this phenomenon at
low growth rates, so that the decreased cell yields
observed under these conditions are not simply a
property of the growth rate. The lowered cell
yield recorded during slow growth on a limiting
carbon and energy source was explained by sug-
gesting that, in addition to cell synthesis from
the carbon substrate, there is also a constant oxi-
dation of cell substance to C02, i.e., some turn-
over is occurring during growth. (The experimen-
tal evidence for this is considered later.) The
equation representing the exponential growth of a
bacterial culture can be modified from
dx ds
-= sx = -Y-
dt dt
to
dx
du
dt = _- k)x
cohere x = cell concentration; s = substrate
utilized; t = time; A = growth rate; k = a con-
stant representing the endogenous metabolism;
and Y = yield coefficient. Thus, by lowering the
dilution or growth rate, k becomes proportionally
larger in relation to A and, therefore, the cell
density falls; the cell yield is lower because pro-
portionally more cell material is oxidized relative
to the amount of limiting nutrient that is assimi-
lated. In toto, the net result is an uncoupling of
growth from oxidation of carbon substrate, since
proportionally more nutrient is oxidized than is
assimilated per cell. This idea was expanded by
Marr et al. (53), who designed experiments to
determine the value of k (these workers substitute
a for k) which they call the specific maintenance.
Mathematically, the lowered cell yield at low
dilution rates can be expressed as:
dx
-Z
dt
= (u-kx Yds
(~a-k)x =-Yddt
Rearranging,
Y ds
x dt
Now Y. x, ds/dt, and At (or D, the dilution rate)
can all be determined experimentally, and, there-
fore, k may be evaluated.
Substitution of k into the continuous culture
equations enables plots of steady-state cell con-
141
centration versus dilution rate to be made that
correspond exactly with those obtained experi-
mentally (33). Evidence to interpret k as repre-
senting a constant endogenous metabolism that
occurs during growth is demonstrated by com-
paring the rates of respiration of A. aerogenes in
continuous culture at different growth rates.
Extrapolation to zero growth rate of Qo2 and
Qco2 values determined at different growth rates
in media containing limiting glycerol gave values
on the ordinate that were identical to the Qo2
(endogenous) and Qco2 (endogenous) of these
cells. It is suggested that the respiration consists
of (i) substrate oxidation that is proportional to
the growth rate and (ii) a constant rate of oxida-
tion of endogenous material, that occurs at all
growth rates. Carbon balances (details of which
were not recorded) were also claimed to indicate
that proportionally more cell carbon than sub-
strate carbon is oxidized.
The lowered cell yields at low dilution rates
(carbon and energy source limiting) have been
observed for other bacteria and for Torula utilis
(33). Marr et al. (53) noted that E. coli is unable
to maintain cell density at low dilution rates, and
they calculated the specific maintenance as 0.025
hr-1.
The carbon balances of substrate utilization at
different growth rates received attention from
Marr et al. (53) with batch cultures. U-C14-glucose
was (i) added to a batch culture, giving exponen-
tial growth; (ii) fed rapidly to a culture so that
only a small fraction would be used for mainte-
nance, giving linear growth; and (iii) fed slowly
so that a large fraction was used for maintenance,
giving curvilinear growth. It had previously
been shown that the cell crops obtained
increased in the order: slowly fed cultures <
from batch cultures < from rapidly fed
cultures. The radiochemical results confirmed this
observation and also showed that more of the
glucose is oxidized to C1402 in slowly fed than in
batch, which in turn is greater than in rapidly fed
cultures. The reverse order was found for C14
assimilated by the cells. It is not entirely clear
why the batch cultures should lie between the fast
and slow feeding of energy source, but some ex-
planations may be offered. During exponential
growth in batch culture, the rate of growth is not
limited by glucose concentration and it appears
that glucose is utilized less efficiently; e.g., it is
not oxidized to CO2 immediately, possibly owing
to a limitation of oxygen concentration. Conse-
142 DAWES AND RIBBONS
quently, growth may cease and the stationary-
phase cells oxidize the accumulated intermediates
to CO2. Lower growth yields in batch than in
continuous or nutrient-limited cultures were also
observed by Pirt (61).
The energy required for turnover of macro-
molecules appears to account for a larger propor-
tion of the energy source that is not utilized for
growth in E. coli (53), but these workers could
not demonstrate that accumulation of methyl-
thiogalactoside was responsible for any signifi-
cant amount of energy expenditure, although
Kepes (40) noted that addition of this compound
to E. coli suspensions resulted in a doubling of the
rate of endogenous metabolism.
The earlier ideas concerning the concept of
energy of maintenance were excellently sum-
marized by Mallette (51) and McGrew and
Mallette (55). They indicated that lack of sensi-
tive techniques was among the reasons for the
difficulty of demonstration of the energy of main-
tenance. To overcome this problem, they used a
high cell density in relation to a low concentration
of carbon and energy source to study the mainte-
nance requirement of E. coli. Low concentrations
of glucose were fed to suspensions of E. coli in an
otherwise complete growth medium, and the tur-
bidity changes were recorded. When very small
additions of glucose were made, the extinction did
not alter appreciably from control cultures after
a standard time. Higher concentrations of glucose
permitted growth to occur. Thus, they demon-
strated that a threshold concentration of glucose
is required before growth can occur. Cell suspen-
sions starved with respect to the carbon and
energy source showed rapid decreases in turbidity
for 5 days, at which time only 15% of the cells
were viable, and then remained constant while
the viability continued to fall. A small addition
of glucose at 6-hr intervals, sufficient to maintain
the turbidity at a constant value, also suppressed
the rate of loss of viability; e.g., after 5 days only
20 % of the cells had died. Glucose additions that
permitted a very slow growth (20% increase in
extinction over 10 days) did not prevent death of
the cells. (After 5 days, approximately 10% had
died.) Thus, it would seem that small amounts of
glucose can provide energy to maintain the cell
without allowing growth to occur. The loss of
viability that occurs during the slow growth may
be due to the interval method (6 hr) of feeding,
as pointed out by Marr et al. (53); i.e., some
BACTERIOL. REV.
breakdown of cellular material occurs before the
next glucose supplement, and this is insufficient
to allow reclamation of the lost cell materials. A
criticism which may be leveled at this type of ex-
periment is that growth may be occurring al-
though it is not revealed by turbidity measure-
ments. The number of cells dying may be such
that the turbidity undergoes no net change as
growth occurs. The influence of higher cell con-
centrations on these phenomena is not known,
and regrowth (28) may assume special impor-
tance; perhaps open systems should be considered
in experimental design.
STARVATION AND SURVIVAL
The early literature concerning the effects of
starvation upon the survival of microorganisms
was critically reviewed by Postgate and Hunter
(62). They also draw attention to the pitfalls and
difficulties that may be encountered during the
estimation of viabilities. The cleanliness of labo-
ratory ware and purity of chemicals is considered
to be critical, as trace impurities may either per-
mit growth of otherwise starving organisms (23)
or kill the cells. The growth of bacteria at the
expense of their companions has been termed
cryptic growth (70), cannibalism (28), and re-
growth (81). This growth is a function of cell
density and can considerably influence the sur-
vival behavior of starving suspensions, since a
"population turnover" may occur.
Apart from the phenomenon of regrowth, the
initial cell density of starving suspensions also
affects their death rate. Harrison (28) first showed
the relationship between cell density and death
rate of starving suspensions of A. aerogenes, and
an optimal density for survival was demon-
strated. He concluded that an interaction be-
tween individual cells favors survival, and the
work of Postgate and Hunter (62) removes any
doubt about the possibility of regrowth occurring.
The latter authors made a very thorough study
of many factors that influence the survival of
starving suspensions of A. aerogenes. They elimi-
nated ambiguity that would arise from cryptic
growth, growth on impurities, and toxicity of
suspending fluids, by a suitable choice of cell
density [20 ,ug (dry weight) of cells per ml] and
suspending fluid [saline-tris(hydroxymethyl)-
aminomethane buffer-EDTA solution]. High illu-
mination, high temperatures, high pH values, and
high potassium ion concentrations increased the
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
death rates of starving suspensions. For example,
A. aerogenes survives better at 20 C than at 30,
40, or 10 C. Anaerobiosis accelerated death, and this
was attributed to the acid conditions produced.
We also have observed that anaerobically starved
E. coli die faster than the aerobically starved sus-
pensions (18); however, the pH value during
anaerobiosis fell only from 7.2 to 6.8 with our
more strongly buffered suspensions (unpublished
data).
Various nutrients, or the previous history of
suspensions of A. aerogenes, markedly affected
the death rates. Ca2+, Mg2+, and to a lesser extent
Fe2+, when added to the saline-buffer, prolonged
the life of the cells. The slower the rate of growth
of the bacteria, the greater was their death rate
upon fasting. This applied to organisms whose
growth rate was limited by C, N, P, and S, but
with Mg2+-limited growth the reverse was true
and the cells that had most rapidly proliferated
died fastest when starved. The effect of nutrient
additives upon death rates is, however, more com-
plex and depends upon the previous history of the
cells. Thus, Postgate and Hunter (63) observed
a general phenomenon of substrate-accelerated
death, in which the addition of the growth-limit-
ing nutrient to starving suspensions increased the
death rate. Glycerol-limited cells of A. aerogenes
showed glycerol-accelerated death (metabolites
of glycerol, e.g., pyruvate, also accelerate death);
NH4+-limited cells are killed by NH4+ additions
but not by glycerol, which is slightly protective.
Phosphate-limited cells behaved similarly, as did
other carbon source-limited cells. Sulfate-limited
cells were not killed quickly by sulfate, but in-
stead showed glycerol-accelerated death. Mg2+-
limited cells were another exception, in that addi-
tion of Mg2+ actually prolonged the life of these
cells, as it does of other cells, a feature quite inde-
pendent of the nutrient that limited their growth.
Harrison and Lawrence (30) also noted that the
effect of nutrient additions to starving suspen-
sions is influenced by the cell density used. Thus,
log-phase A. aerogenes (106 to 107 cells per ml)
may become sterile within a few hours in distilled
water, but phosphate delays death. Higher cell
densities (108 to 109 cells per ml) survive as well
in water as they do in phosphate buffer.
McGrew and Mallette (55), however, pre-
sented results which show that E. coli may be
maintained by very small quantities of a carbon
and energy source. [See also Harrison (28) with
143
A. aerogenes.] Unfortunately, it is not clear that
regrowth does not occur here. The concentrations
of glucose added as an energy source were very
much less than those used by Postgate and
Hunter (62) to demonstrate substrate-accelerated
death.
Death
The survival characteristics of a suspension of
organisms can be defined only by experimental
design; for example, cells that have not divided
within 3 hr are generally considered dead when
estimated by slide culture, but longer incubation
(24 hr) by more traditional methods sometimes
gives higher counts, especially with aged cells.
Again, the washing procedure and recovery
media will determine to a large extent the viable
counts or the percentage viability obtained. As
we have pointed out (15), a situation is possible
where the loss of a single enzyme from the cell
could lead to its "death" in certain media; this is
clearly seen with techniques that select nutri-
tional mutants via minimal media. The actual
causes of death by starvation may be quite di-
verse and include factors such as the loss of the
capacity for nucleic acid and protein synthesis.
Postgate and Hunter (62) were able to demon-
strate that death of their starving suspensions
was not due to alterations or degradation of the
osmotic barrier since, among other factors, starv-
ing populations did not become permeable to
fluorescent dyes at the same rate as their death
rate. The Qo2 (glycerol) and glycerol dehydroge-
nase activity decline in parallel with the loss of
viability; it may be that the enzymes catabolizing
the energy source are the critical factors in life
and death in this case.
If it were possible to maintain a bacterial popu-
lation by the addition of a small amount of an
energy source, then continuous cultivation of
&ells at decreasing dilution rates should give a
situation where the dilution rate is significantly
larger than the growth rate, and eventually reach
a point when the growth rate is zero and the dilu-
tion rate has some finite value. It may not be easy
to demonstrate technically, as other factors such
as bacterial density may interfere; e.g., Jannasch
has shown the influence of cells upon each other
(38). In practice, it has not been possible to ob-
tain such a steady state, i.e., slow exponential
washout, because many cells do not survive at
low growth rates and, consequently, the effluent
144 DAWES AND RIBBONS
contains a proportion of dead cells; the number of
dead cells appearing becomes larger as the genera-
tion time increases. This is a property not simply
confined to carbon- and energy-limited growth
but occurs also with S-, Mg-, and N-limited
growth. Postgate and Hunter (62) are of the
opinion that these bacteria are able only to
multiply or to die!
Relationship Between Survival and
Endogenous Substrates
It has been suggested that the oxidation of
endogenous materials by microorganisms is of
importance to the cell for its survival during
starvation. Therefore, it might be concluded that
cells which contain large amounts of storage
polymers have an advantage over those cells
which do not. Strange, Dark, and Ness (81) have,
in fact, shown that A. aerogenes survives best
when harvested from glucose-Tryptone media and
these cells have assimilated much glycogen. Sta-
tionary-phase cells obtained from defined manni-
tol-limiting media contain no glycogen, whereas
those grown in the same medium but with nitro-
gen limiting growth deposit glycogen. Again, the
latter cells survived better than those that had
not deposited glycogen. It would seem that the
possession of glycogen, in A. aerogenes at least,
favors survival. The provision of an exogenous
source of glucose causes death, and 50% of the
cells died in 4 hr, during which time starved cells
remained almost completely viable. However, it
is difficult to demonstrate unequivocally that the
possession of glycogen by these cells is in fact re-
sponsible for their longevity. On the other hand,
Dawes and Ribbons (18) could not conclude that
possession of glycogen is an aid to survival in E.
coli, although their experiments are much less
extensive.
The glycogen of starving suspensions of E. coli
was oxidized at a very rapid rate, as much as 20 %
of the dry weight being completely oxidized in 3
hr; yet, little or no loss of viability occurred for
12 hr. Similarly Burleigh, Dawes, and Ribbons
(9) were unable to demonstrate a relation between
the free amino acid pool of S. lutea or its Qo2
(endogenous) and the capacity of the cells for
survival. Starved cells of S. lutea remain viable
for considerable periods (30 hr), after the amino
acid pool has been depleted.
Degradation of RNA, and to a lesser extent
BACTERIOL. REV.
protein and polysaccharide, occurred during the
starvation of carbon-limited A. aerogenes (81).
Little protein degradation occurred during the
first few hours of starvation, and the free amino
acid pool content remained constant. The poly-
saccharide that was degraded was completely
oxidized within 2 hr under these conditions (com-
pare with E. coli), and the endogenous Qo2 value
declined rapidly during this period. It appears
that RNA is the most expendable reserve; fur-
ther, fast-growing cells contain more RNA and
also die more slowly. Fast-growing cells are also
larger than slow-growing cells (33), and thus each
individual will contain proportionally more
expendable material; this may account for their
slower death rate. However, this apparent rela-
tionship of the death rate and initial RNA con-
tent is not supported by other data. Thus,
Strange et al. (83) showed that carbon-limiting
defined media yielded stationary-phase cells that
contain 18% of their weight as RNA, and these
cells do not survive as well as those harvested
from tryptic meat broth and which contain 11%
RNA. This was especially apparent during star-
vation under N2 at 20 to 22 C. The other interest-
ing observation is that of Harrison and Lawrence
(30), who were able to isolate starvation-resistant
mutants from starved suspensions of A. aerogenes.
These mutants are slower growing and, therefore,
contain less RNA than the wild type; they utilize
their RNA, as well as other materials, during
starvation, since up to 20% of the weight of the
cell is lost before there is any loss in viability. The
mutant, however, actually degrades more of its
RNA than the wild type. Thus, we may conclude
that it is not simply the quantity of substrate for
endogenous metabolism that favors survival but
rather the capacity of the cell to utilize the sub-
strate to advantage.
The only survival studies of which we are
aware that correlate survival with PHB content
are those of Sierra and Gibbons (76). PHB-rich
cells of M. halodenitrificans are able to respire
endogenously at a constant rate for long periods
(96 hr) while their PHB reserves are being oxi-
dized. Then, at a critical concentration of about
10%, when first-order kinetics are initiated, the
Qo2 falls and the cells begin to die. When PHB-
poor cells (10% PHB) are starved, the Qo2
(endogenous), viability, and PHB content all de-
cline.
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
SUMMARY AND FUTURE OUTLOOK
Studies of endogenous metabolism and survival
lead to several interesting considerations. First,
in connection with the substrates of endogenous
respiration, it is apparent that various cell con-
stituents are depleted during starvation. The ex-
tent of the degradation of individual substrates
appears to depend on their initial concentrations
in the cell, which, in turn, are determined by the
nutritional status of the organism. The pattern of
endogenous metabolism observed is thus a reflec-
tion of the nutritional condition of the cell. It
seems not without significance that a similar con-
clusion was reached by Postgate and Hunter
with respect to survival. Certain materials within
the bacterial cell are believed to be expendable,
e.g., glycogen and PHB, because cells may sur-
vive without them. Other polymers such as pro-
tein, DNA, and RNA are thought to be essential
for continued existence. Nonetheless, with the
possible exceptions of the information center
(DNA) and the framework (cell-wall and mem-
brane polymers), it has been shown for several or-
ganisms that most materials are depleted during
stresses of starvation. There appear to be, at
least, expendable fractions of the "essential" life
constituents. For example, ribosomal RNA is
utilized during starvation; extrapolation of the
ribosomal RNA content of Salmonella typhimu-
rium to zero rate of growth shows that these cells
contain virtually no ribosomal RNA (20). In some
instances, the expendable reserves are utilized
preferentially, as for instance in E. coli where
glycogen spares the nitrogen-containing com-
ponents of the cell; the carbohydrate reserve of A.
aerogenes also seems to suppress partially the de-
composition of RNA and protein. Conversely, the
polyglucose of S. lutea does not suppress the en-
dogenous respiration of the free amino acid pool.
However, another view has been put forward;
namely, the reserves that are utilized initially are
those present in the cell in excess. Cells which are
arrested in growth due to the limitation of one
essential nutrient may continue to synthesize
certain cellular components from nutrients that
are still available in the medium. Such com-
ponents may then be considered to be in "excess,"
i.e., at a concentration higher than that found in
the exponentially growing cells. This situation is
one of considerable interest in organisms which do
not contain significant amounts of glycogen or
PHB, for then imbalance between the essential
145
components of the cell-protein, RNA, and
DNA-might be expected to occur, e.g., in cases
of an amino acid deficiency. Thus, depending on
the nature of the medium used to grow A. aerog-
enes, different polymers in the main are utilized
for endogenous respiration. Starvation of glucose-
Tryptone grown A. aerogenes results in a rapid
loss of glycogen; little RNA and protein are oxi-
dized initially. RNA-rich bacteria, obtained by
growth on defined media, utilize their RNA to a
greater extent than their protein. (These were
carbon-limited cells and did not store glycogen.)
Cells from tryptic meat broth, however, contain
much less RNA, and protein makes the largest
contribution of intermediates for combustion.
The phenomenon of substrate-accelerated
death has widespread implications, for it may
mean that many experiments with "whole cells"
or washed suspensions of bacteria are lethal to the
organism. Although great care may be taken to
ensure that cells remain viable during harvesting
and washing procedures, the introduction of an
oxidizable carbon compound to those carbon-
limited cells can quickly kill them. The provision
of a substrate gives a condition of plenty, and in
some cases leads to high rates of metabolism. This
contrasts strongly with those conditions which
are associated with longevity and which often
lower the rates of metabolism, as for example
with bacterial spores. Even more far reaching are
the consequences of industrial and sewage proc-
esses in which the use of whole cells may, in fact,
mean the inclusion of a large proportion of dead
cells.
The direct measurement of the ATP content of
cells has been made during starvation (82).
Strange, Wade, and Dark, however, could find no
relationship between ATP content and viability,
since the ATP content of A. aerogenes fell to a
very low value about 30 hr before any loss of
viability occurred. They do provide evidence
which suggests a direct relationship between the
ability of the cell to form ATP and its viability.
The provision of ATP by endogenous respiration
was demonstrated earlier by Strange (80), who
concluded that the loss of the ability of starved
cells to synthesize f3-galactosidase was not due to
a shortage of energy. The intermediates of enzyme
synthesis were in short supply, since an inducer
and a suitable nitrogen source allowed enzyme
induction to occur. We suggested earlier (15) that
the general functions of endogenous metabolism
146 DAWES AND RIBBONS
were to provide (i) energy and (ii) carbon skele-
tons, and thus it seems in this case at least that
the carbon and nitrogen skeletons were not avail-
able for synthesis although available for ATP pro-
duction. Some examples are known where a trans-
fer of carbon skeletons to other cell constituents
occurs. However, we cannot agree with Gronlund
and Campbell (27) that the ribonucleoprotein
serves as a source of amino acids and nucleotides
for the synthesis of a greater quantity of enzymes
concerned with endogenous respiration, although
ribonucleoprotein may be able to maintain the
concentration of enzymes already present. To
the best of our knowledge, there are no cases
where the rate of endogenous respiration increases
with the duration of starvation.
The final aspect concerns the suggestion of
Lamanna and Mallette (41) that evolution
should have selected microbes capable of with-
standing starvation stresses. This view has been
amplified by the isolation of starvation-resistant
mutants from starved suspensions of A. aerogenes
by Harrison and Lawrence (29, 30). It is note-
worthy that these slower-growing mutants,
cells presumably with a slower metabolic rate,
have the greater capacity for longevity. This
characteristic they share with spores, in which an
extreme capacity for survival is allied to a barely
detectable metabolic rate. Harrison and Lawrence
conclude the discussion of their paper with the
fable of the hare and the tortoise!
ACKNOWLEDGMENTS
We are greatly indebted to I. G. Burleigh for
helpful discussion and constructive criticism, and
also to The Royal Society for a grant-in-aid.
LITERATURE CITED
1. ALGRANATI, I. D., AND E. CABIB. 1960. The
synthesis of glycogen in yeast. Biochim.
Biophys. Acta 43:141-142.
2. BARDI, U., AND G. BORETTI. 1958. Osservazioni
sul metabolismo endogeno di un Proto-
actinomyces: Nocardia rugosa. Giorn. Micro-
biol. 6:91-102.
3. BARRY, C., R. GAVARD, G. MILHAUD, AND
J. AUBERT. 1953. Etude du glycogene extrait
de Bacillus megatherium. Ann. Inst. Pasteur
34:605-610.
4. BAUCHOP, T., AND S. R. ELSDEN. 1960. The
growth of microorganisms in relation to
their energy supply. J. Gen. Microbiol.
23:457-469.
BACTERIOL. REV.
5. BINNIE, B., E. A. DAWES, AND W. H. HOLMS.
1960. Metabolism of Sarcina lutea. IV. Pat-
terns of oxidative assimilation. Biochim.
Biophys. Acta 40:237-251.
6. BLUMENTHAL, H. J. 1963. Endogenous metabo-
lism of filamentous fungi. Ann. N.Y. Acad.
Sci. 102:688-706.
7. BOREK, E., A. RYAN, AND J. ROCKENBACH.
1955. Nucleic acid metabolism in relation to
the lysogenic phenomenon. J. Bacteriol.
69:460-467.
8. BOWEN, T. J., S. DAGLEY, AND J. SYKES. 1959.
A ribonucleoprotein component of Esch-
erichia coli. Biochem. J. 72:419-425.
9. BURLEIGH, I. G., E. A. DAWES, AND D. W.
RIBBONS. 1963. Endogenous metabolism and
survival of Sarcina lutea. Biochem. J.
88:30-31P.
10. CARR, N. G., AND J. LASCELLES. 1961. Some
enzymic reactions concerned in the metabo-
lism of acetoacetic-coenzyme A in Athio-
rhodaceae. Biochem. J. 80:70-77.
11. CHARGAFF, E., AND D. H. MOORE. 1944. On
bacterial glycogen: the isolation from avian
tubercle bacilli of a polyglucosan of very
high particle weight. J. Biol. Chem. 155:493-
501.
12. CLIFTON, C. E., AND J. M. SOBEK. 1961. En-
dogenous respiration of Bacillus cereus. J.
Bacteriol. 82:252-256.
13. DAGLEY, S., AND A. R. JOHNSON. 1953. The
relation between lipid and polysaccharide
contents of Bact. coli. Biochim. Biophys.
Acta 11:158-160.
14. DAWES, E. A., AND W. H. HOLMS. 1958.
Metabolism of Sarcina lutea. III. Endoge-
nous metabolism. Biochim. Biophys. Acta
30:278-293.
15. DAWES, E. A., AND D. W. RIBBONS. 1962. The
endogenous metabolism of micro-organisms.
Ann. Rev. Microbiol. 16:241-264.
16. DAWES, E. A., AND D. W. RIBBONS. 1962. En-
dogenous metabolism of Escherichia coli.
Biochem J. 82:49P.
17. DAWES, E. A., AND D. W. RIBBONS. 1962. Effect
of environmental conditions on the endoge-
nous metabolism of Escherichia coli. Bio-
chem. J. 84:97P.
18. DAWES, E. A., AND D. W. RIBBONS. 1963. En-
dogenous metabolism and survival of Esch-
erichia coli in aqueous suspensions. J. Appl.
Bacteriol. 26:vi.
19. DOUDOROFF, M., AND R. Y. STANIER. 1959.
Role of poly-,B-hydroxybutyric acid in the
assimilation of organic carbon by bacteria.
Nature 123:1440-1442.
20. ECKER, R. E., AND M. SCHAECHTER. 1963.
VOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
Bacterial growth under conditions of limited
nutrition. Ann. N.Y. Acad. Sci. 102:549-
563.
21. FORSYTH, W. G. C., A. C. HAYWARD, AND J. B.
ROBERTS. 1958. The occurrence of poly-,3-
hydroxybutyrate in aerobic gram-negative
bacteria. Nature 182:800.
22. FOSTER, J. W. 1947. Some introspections of
mold metabolism. Bacteriol. Rev. 11:166-
191.
22a. FURANO, A. V., AND J. P. GREEN. 1963.
Differences in the disposition of endogenous
and exogenous substances by cells. Nature
199:380-381.
23. GARVIE, E. I. 1955. The growth of Escherichia
coli in buffer substrate and distilled water.
J. Bacteriol. 69 :393-398.
24. GAVARD, R., C. COMBRE, AND A. TUFFET. 1960.
etude de la D(-)-,-hydroxybutyrate deshy-
drogenase de Bacillus megatherium. Compt.
Rend. 251:1931-1933.
25. GERMAN, R. J., A. S. JONES, AND M. NADA-
RAJAH. 1961. Polysaccharides of Mycobac-
terium phlei. Nature 189:1008.
26. GRONLUND, A. F., AND J. J. R. CAMPBELL.
1961. Nitrogenous compounds as substrates
for endogenous respiration in microorgan-
isms. J. Bacteriol. 81:721-724.
27. GRONLUND, A. F., AND J. J. R. CAMPBELL.
1963. Nitrogenous substrates of endogenous
respiration in Pseudomonas aeruginosa. J.
Bacteriol. 86:58-66.
28. HARRISON, A. P., JR. 1960. The response of
Bacterium lactis-aerogenes, when held at
growth temperature in the absence of
nutrient: an analysis of survival curves.
Proc. Roy. Soc. (London) Ser. B 152:418-
428.
29. HARRISON, A. P., JR. 1961. Ageing and decline
of bacteria in phosphate buffer. Bacteriol.
Proc., p. 53.
30. HARRISON, A. P., JR., AND F. R. LAWRENCE.
1963. Phenotypic, genotypic, and chemical
changes in starving populations of Aero-
bacter aerogenes. J. Bacteriol. 85:742-750.
31. HAYWARD, A. C. 1959. Poly-j3-hydroxybutyrate
inclusions in the classifications of aerobic
Gram-negative bacteria. J. Gen. Microbiol.
21:2-3.
32. HAYWARD, A. C., W. G. C. FORSYTH, AND J. B.
ROBERTS. 1959. Synthesis and breakdown of
poly-3-hydroxybutryic acid by bacteria. J.
Gen. Microbiol. 20:510-518.
33. HERBERT, D. 1958. Recent progress in micro-
biology. Some principles of continuous
culture. Intern. Congr. Microbiol. 7th,
Stockholm, p. 381-396.
147
34. HERBERT, D. 1961. The chemical composition
of micro-organisms as a function of their
environment. Symp. Soc. Gen. Microbiol.
11:391-416.
35. HOLME, T. 1957. Continuous culture studies on
glycogen synthesis in Escherichia coli strain
B. Acta Chem. Scand. 2:763-775.
36. HOLME, T., AND H. PALMISTIERNA. 1956. On
the glycogen in Escherichia coli B; its syn-
thesis and breakdown and its specific label-
ling with 14C. Acta Chem. Scand. 10:1557-
1562.
37. HORUICHI, T. 1959. RNA degradation and
DNA and protein synthesis of E. coli in a
phosphate deficient medium. J. Biochem.
(Tokyo) 46:1467-1480.
38. JANNASCH, H. W. 1962. Bacterial growth at low
population densities. Nature 196:496-497.
39. KALLIO, R. E., AND A. A. HARRINGTON. 1960.
Sudanophilic granules and lipid of Pseu-
domonas methanica. J. Bacteriol. 80:321-
324.
40. KEPES, A. 1960. Etudes cinetiques sur la
galactoside-permease d'Escherichia coli.
Biochim. Biophys. Acta 40:70-84.
41. LAMANNA, C., AND M. F. MALLETTE. 1959.
Basic bacteriology, 2nd ed., p. 612-624.
The Williams & Wilkins Co., Baltimore.
42. LEMOIGNE, M. 1927. etudes sur lFautolyse
microbienne. Origine de lFacide f-hydroxy-
butyrique forme par autolyse. Ann. Inst.
Pasteur 41:148-165.
43. LEMOIGNE, M., B. DELAPORTE, AND S. CROSON.
1944. Contribution a l'6tude botanique et
biochimique des bacteriennes du geur Bacil-
lus. I. Valeur du test des lipides fl-hydroxy-
butyriques pour la characterization des
especes. Ann. Inst. Pasteur 70:224.
44. LEVINE, H. B., AND H. WOLOCHOW. 1960. Oc-
currence of poly-o-hydroxybutyrate in Pseu-
domonas pseudomallei. J. Bacteriol. 79:305-
306.
45. LURIA, S. E. 1960. The bacterial protoplasm:
composition and organization, p. 1-34. In
I. C. Gunsalus and R. Y. Stanier [ed.], The
bacteria, vol. 1. Academic Press, Inc., New
York.
46. MACRAE, R. M., AND J. F. WILKINSON. 1958.
Poly-,B-hydroxybutyrate metabolism in
washed suspension of Bacillus cereus and
Bacillus megaterium. J. Gen. Microbiol.
19:210-222.
47. MACRAE, R. M., AND J. F. WILKINSON. 1958.
The influence of cultural conditions on poly-
,B-hydroxybutyrate synthesis in Bacillus
megaterium. Proc. Roy. Soc. Edinburgh A
27:73-78.
148 DAWES AND RIBBONS
48. MADSEN, N. B. 1961. The occurrence and
enzymic synthesis of glycogen in extracts of
Agrobacterium tumefaciens. Biochim. Bio-
phys. Acta 50:194-195.
49. MADSEN, N. B. 1961. The inhibition of glycogen
phosphorylase by uridine diphosphate glu-
cose. Biochem. Biophys. Res. Commun.
6:310-312, 315.
50. MADSEN, N. B. 1963. The biological control of
glycogen metabolism in Agrobacterium tume-
faciens. Can. J. Biochem. Physiol. 41:561-
571.
51. MALLETTE, M. F. 1963. Validity of the concept
of energy of maintenance. Ann. N.Y. Acad.
Sci. 102:521-535.
52. MANDELSTAM, J. 1960. The intracellular turn-
over of protein and nucleic acids and its role
in biochemical differentiation. Bacteriol.
Rev. 24:289-308.
53. MARR, A. G., E. H. NILSON, AND D. J. CLARK.
1963. The maintenance requirement of Esch-
erichia coli. Ann. N.Y. Acad. Sci. 102:536-
548.
54. MARTINEZ, R. J. 1962. On the nature of the
granules of the genus Spirillum. Arch.
Mikrobiol. 44:334-343.
55. McGREW, S. B., AND M. F. MALLETTE. 1962.
Energy of maintenance in Escherichia coli.
J. Bat-teriol. 83:844-850.
56. MERRICK, J. M., AND M. DOUDOROFF. 1961.
Enzymatic synthesis of poly-,B-hydroxy-
butyric acid in bacteria. Nature 189:890-
892.
57. MONOD, J. 1942. Recherches sur la croissance
des cultures bacteriennes, p. 90-91. Her-
mann & Cie, Paris.
58. MULDER, E. G., M. H. DEINEMA, W. L. VAN
NEEN, AND L. P. T. M. ZEVENHUIZEN. 1962.
Polysaccharides, lipids and poly-,3-hydroxy-
butyrate in micro-organisms. Rec. Trav.
Chim. 81:797-809.
59. PEEL, D., AND J. R. QUAYLE. 1961. Microbial
growth on C1 compounds. I. Isolation and
characterization of Pseudomonas AM1. Bio-
chem. J. 81:465-469.
60. PINE, M. J. 1963. Alcohol-soluble protein of
microorganisms. J. Bacteriol. 85:301-305.
61. PIRT, S. J. 1957. The oxygen requirements of
growing cultures of Aerobacter species deter-
mined by means of the continuous culture
technique. J. Gen. Microbiol. 16:59-75.
62. POSTGATE, J. R., AND J. R. HUNTER. 1962. The
survival of starved bacteria. J. Gen. Micro-
biol. 29:233-263.
63. POSTGATE, J. R., AND J. R. HUNTER. 1963.
Acceleration of bacterial death by growth
substrates. Nature 198:273.
64. RAMSEY, H. H. 1962. Endogenous respiration
BACTERIOL. REV.
of Staphylococcus aureus. J. Bacteriol.
83:507-514.
65. RIBBONS, D. W., AND E. A. DAWES. 1963. En-
vironmental and growth conditions affecting
the endogenous metabolism of bacteria.
Ann. N.Y. Acad. Sci. 102:564-586.
66. ROGERS, H. J. 1961. The dissimilation of high
molecular weight substances, p. 257-318. In
I. C. Gunsalus and R. Y. Stanier [ed.], The
bacteria, vol. 2. Academic Press, Inc., New
York.
67. ROSENBERGER, R. F., AND S. R. ELSDEN. 1960.
The yields of Streptococcus faecalis grown in
continuous culture. J. Gen. Microbiol.
22:726-739.
68. ROTMAN, B. 1958. Regulation of enzymatic ac-
tivity in the intact cell: the ,B-D-galactosidase
of Escherichia coli. J. Bacteriol. 76:1-14.
69. ROUF, M. A., AND J. L. STOKES. 1962. Isola-
tion and identification of the sudanophilic
granules of Sphaerotilus natans. J. Bacteriol.
83:343-347.
70. RYAN, F. J. 1955. Spotaneous mutation in non-
dividing bacteria. Genetics 40:726-731.
71. SALTON, M. R. J. 1960. Surface layers of the
bacterial cell, p. 143-144. In I. C. Gunsalus
and R. Y. Stanier [ed.], The bacteria, vol. 1.
Academic Press, Inc., New York.
72. SCHLEGEL, H. G. 1962. Die Speicherstoffe von
Chromatium okanii. Arch. Mikrobiol. 42:110-
116.
73. SCHLEGEL, H. G., G. GOTTSCHALK, AND R. VON
BARTHA. 1961. Formation and utilization of
poly-,3-hydroxybutyric acid by Knallgas
bacteria (Hydrogenomonas). Nature 191:463-
465.
74. SHUSTER, C. W., AND M. DOUDOROFF. 1962. A
cold-sensitive D (-),3-hydroxybutyric acid
dehydrogenase from Rhodospirillum rubrum.
J. Biol. Chem. 237:603-607.
75. SIERRA, G., AND N. E. GIBBONS. 1962. Pro-
duction of poly-f3-hydroxybutyric acid gran-
ules in Micrococcus halodenitrificans. Can. J.
Microbiol. 8:249-253.
76. SIERRA, G., AND N. E. GIBBONS. 1962. Role and
oxidation pathway of poly-,3-hydroxy-
butyric acid in Micrococcus halodenitrificans.
Can. J. Microbiol. 8:255-269.
76a. SIERRA, G., AND N. E. GIBBONS. 1963. Sodium
requirement of poly-,B-hydroxybutyric acid
depolymerase of Micrococcus halodenitrifi-
cans. Can. J. Microbiol. 9:491-497.
77. SLEPECKY, R. A., AND J. H. LAW. 1961. Synthe-
sis and degradation of poly-,3-hydroxy-
butyric acid in connection with sporulation
of Bacillus megaterium. J. Bacteriol.
82 :37-42.
78. STANIER, R. Y., M. DOUDOROFF, R. KUNI-
VoOL. 28, 1964 ENDOGENOUS METABOLISM OF BACTERIA
SAWA, AND R. CONTOPOULOU. 1959. The role
of organic substrates in bacterial photo-
synthesis. Proc. Natl. Acad. Sci. U.S.
45:1246-1260.
79. STETTEN, DEW., JR., AND M. R. STETTEN.
1960. Glycogen metabolism. Physiol. Rev.
40:505-537.
80. STRANGE, R. E. 1961. Induced enzyme synthe-
sis in aqueous suspensions of starved station-
ary phase Aerobacter aerogenes. Nature
191:1272-1273.
81. STRANGE, R. E., F. A. DARK, AND A. G. NESS.
1961. The survival of stationary phase
Aerobacter aerogenes stored in aqueous sus-
pensions. J. Gen. Microbiol. 25:61-76.
82. STRANGE, R. E., H. E. WADE, AND F. A. DARK.
1963. Effect of starvation on adenosine tri-
phosphate concentration in Aerobacter
aerogenes. Nature 199:55-57.
83. STRANGE, R. E., H. E. WADE, AND A. G. NESS.
1963. The catabolism of protein and nucleic
acids in starved Aerobacter aerogenes. Bio-
chem. J. 86:197-203.
84. SUTTON, W. B. 1963. Relationship of the hexose
monophosphate shunt to the endogenous
metabolism of cell-free extracts of Myco-
bacterium phlei. J. Bacteriol. 85:476-484.
84a. HIGUCHI, M., AND T. VEMARA. 1959. Release
of nucleotides from yeast cells. Nature
184:1381.
85. WADE, H. E. 1961. The autodegradation of
ribonucleoprotein in Escherichia coli. Bio-
chem. J. 78:457-472.
149
86. WADE, H. E., AND S. LOVETT. 1961. Poly-
nucleotide phosphorylase in ribosomes from
Escherichia coli. Biochem. J. 81:319-328.
87. WARREN, R. A. J., A. F. ELLS, AND J. J. R.
CAMPBELL. 1960. Endogenous respiration of
Pseudomonas aeruginosa. J. Bacteriol. 79:
875-879.
88. WEIBULL, C. 1953. Characterization of the
protoplasmic constituents of Bacillus mega-
terium. J. Bacteriol. 66:696-702.
89. WEIBULL, C. 1960. Movement, p. 153-205. In I.
C. Gunsalus and R. Y. Stanier [ed.], The
bacteria, vol. 1. Academic Press, Inc., New
York.
90. WHELAN, W. J. 1961. Recent advances in the
biochemistry of glycogen and starch. Nature
190:954-957.
91. WIAME, J. M., AND M. DOUDOROFF. 1951.
Oxidative assimilation by Pseudomonas sac-
charophila with C14-labelled substrates. J.
Bacteriol. 62:187-193.
92. WILKINSON, J. F. 1958. The extracellular
polysaccharides of bacteria. Bacteriol. Rev.
22:46-73.
93. WILKINSON, J. F. 1959. The problem of energy-
storage compounds in bacteria. Exptl. Cell.
Res. Suppl. 7:111-130.
94. WILKINSON, J. F. 1963. Carbon and energy
storage in bacteria. J. Gen. Microbiol.
32:171-176.
95. WILLIAMSON, D. H., AND J. F. WILKINSON.
1958. The isolation and estimation of poly-
,B-hydroxybutyrate inclusions of Bacillus
species. J. Gen. Microbiol. 19:198-209.