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DEWES & RIBBONS, 1964

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role for it was
not convincingly demonstrated until some 30
years later. PHB was first isolated by chloroform
extraction of an aerobic bacillus by Lemoigne
(42), following his earlier discovery that (3-
hydroxybutyrate was a metabolic product of the
organism. Since that time, PHB has been demon-
strated in a wide variety of bacterial species
(Table 1), and in some instances has been im-
plicated as an assimilatory product, from meas-
urements of gaseous exchange during photo-
metabolism of fatty acids and gross elemental
composition, by Gaffron (cited in 78). The quan-
tities of PHB within the bacterial cell vary enor-
mously; contents of up to 50% of the dry weight
have been recorded. It is a reserve that is peculiar
to microorganisms, and its functions, formation,
and synthesis have been studied extensively in
recent years, mainly by Doudoroff and Stanier,
Gibbons, Schlegel, Wilkinson, and their col-
laborators.
Sudanophilic granules present in bacteria were
considered by Lemoigne, Delaporte, and Croson
(43) to be composed of PHB; the data of Weibull
(88) subsequently supported this proposition.
However, it was not until the work of William-
VOL. 28, 1964 127
DAWES AND RIBBONS
son and Wilkinson (95) that the intracellular
lipid granules of Bacillus cereus and B. mega-
terium were demonstrated unequivocally to be
composed mainly of PHB (about 90%), although
neither this nor the remaining 10%, lipid is re-
sponsible for the sudanophilic properties of the
granules that are observed in situ. Macrae and
Wilkinson (46, 47) also studied the effect of
various cultural conditions on the synthesis of
PHB in B. megaterium. When the glucose con-
TABLE 1. Occurrence of poly-,3-hydroxybutyrate in
bacteria*
Species Reference
Bacillus megaterium............... 43, 77, 95
B. cereus.......................... 43, 95
B. mycoides....................... 43
B. anthracis....................... 43
Azotobacter chroococcum ........... 43
A. agilis.......................... 21
A. vinelandii...................... 21
Rhizobium sp...................... 21
Vibrio sp.......................... 31, 32
Chromobacterium violaceum........ 21
Pseudomonas solanacearum........ 32
P. antimycetica ................... 32
P. methanica...................... 39
P. pseudomallei................... 44
P. saccharophila .................. 19
Pseudomonas AMi ................ 59
Micrococcus halodenitrificans ...... 75, 76
Sphaerotilus natans ............... 58, 69
Hydrogenomonas sp................ 73
Rhodospirillum rubrum............ 19, 78
Rhodopseudomonas spheroides.... . 10
Chromatiurn okenii................ 72
Spirillum itersonnii............... 54
S. anulus......................... 54
S. serpens......................... 32, 54
* This is not a complete list of the bacterial
species that synthesize PHB.
centration in the growth medium was raised,
more of the polymer was synthesized; exhaustion
of the nitrogen source in the presence of excess
carbon and energy source permitted deposition
of about four times the amount of PHB as was
formed when glucose limited growth. Glucose,
pyruvate, and f-hydroxybutyrate were suitable
substrates for PHB production by washed sus-
pensions, but acetate, although itself unable to
effect synthesis (compare with Rhodospirillum
rubrum), enhanced PHB formation when supple-
menting other substrates. Anaerobic conditions
prevented PHB synthesis, as did dinitrophenol.
Concentrations of oxygen greater than 5% also
inhibited the assimilatory process. B. cereus, but
not B. megaterium, could effect PHB synthesis
under hydrogen, although no net uptake of
hydrogen was detected; PHB is not formed under
nitrogen.
When washed suspensions of these two bacilli
were shaken under air or under nitrogen, in the
absence of an exogenous carbon and energy
source, stored PHB was metabolized. The
anaerobic degradation of reserves was slower;
e.g., in B. megaterium, 61 and 17% degradation
of PHB occurred under air and nitrogen, respec-
tively, within 8 hr. Metabolic products detected
included f-hydroxybutyrate, acetoacetate, and
acetate, although aerobically CO2 and water
were the major products and only small amounts
of acetoacetate accumulated. Some correlation
was evident between the PHB content of cells
and their endogenous respiration; e.g., cells with
PHB-total N ratios of 0.83 and 3.27 had, respec-
tively, endogenous Qo2 values of 169 and 536.
Macrae and Wilkinson (46) also claimed that
N-deficient cells with a high content of PHB are
better able to withstand death and autolysis
than those with a low PHB content. Autolysis
was estimated by the total N content which, in
4 hr, fell by 12% in PHB-poor cells compared
with 5% in PHB-rich cells; the PHB content
of the latter cells decreased to the greatest ex-
tent. The method of estimation of autolysis is
not entirely satisfactory, because it has been
shown in numerous cases that nitrogenous mate-
rials are oxidized endogenously, releasing am-
monia, and that substantial amounts of nitrog-
enous compounds may diffuse from the cell
without loss of viability. Specifically in the case
of B. cereus, Clifton and Sobek showed that am-
monia is produced endogenously under some
conditions (12), and clearly B. megaterium
utilizes an endogenous substrate other than,
although concurrently with, PHB, since the
oxygen consumption is in excess of that required
for complete combustion of PHB; this other
substrate is not polysaccharide (47). It is pos-
sible, however, that autolysis may have occurred
to a similar extent in both PHB-rich and PHB-
poor cells, but the greater amount of reserve
material in the PHB-rich cells permitted utiliza-
tion of the liberated nitrogenous material and
128 BACTERIOL. REV.
ENDOGENOUS METABOLISM OF BACTERIA
some limited cellular synthesis occurred. Against
this possibility may be set the authors' observa-
tion that growth did not occur when PHB-rich
cells were held in a medium lacking a carbon
and energy source; growth was measured by
total N so that in these bacilli PHB may serve
as a reserve of energy but not as a source of
carbon skeletons for synthesis.
Doudoroff and Stanier (19) have made some
interesting observations concerning the role of
PHB in oxidative assimilation by Pseudomonas
saccharophila and in photoassimilation by
R. rubrum. They found with most substrates that
a major portion of the assimilated carbon (60 to
90%) initially accumulates within the cells as
PHB; when the exogenous substrate is removed,
a rapid intracellular degradation of the polymer
occurs, suggesting a physiological role as a re-
serve material. When cells are subjected to
standard fractionation procedures, the chemical
properties of PHB result in its appearance in the
hot trichloroacetic acid insoluble fraction (pro-
tein fraction). This fact had led Wiame and
Doudoroff (91) earlier to conclude that C14 is
incorporated into nitrogenous materials during
oxidative assimilation.
Incubation of starved washed suspensions of
R. rubrum with C'4-acetate allowed deposition
of 70% of the assimilated C'4 into PHB, with no
significant dilution. With C'4-butyrate, some
dilution occurred, and the polymer contained
only 58% of the assimilated C14. The fate of the
polymer in the light over a period of 12 hr was
studied under a variety of conditions, e.g., in the
presence and absence of a source of organic sub-
strates but in the presence of a N source and
CO2. The absence of an exogenous organic carbon
source led to the disappearance of more than 90%
of the polymer, but much of the C14 of this
material was redistributed into other cellular
components. The authors did not study the fate
of PHB in both nitrogen and carbon starvation,
or in the dark, so its behavior under these con-
ditions is not yet known. The rate of degrada-
tion of PHB and its conversion to other cellular
components was