Antonie van Leeuwenhoek 40 (1974) 103-120

103

Interrelations between glycogen, poly-~-hydroxybutyric acid and lipids during accumulation and subsequent utilization in a Pseudomonas L. P. T. M. ZEVENHUIZEN AND ANNEKE G. EBBINK Laboratory of Microbiology, Agricultural University, Wageningen, the Netherlands

ZEVENHUIZEN,L. P. T. M. and EBBINK, A. G. 1974. Interrelations between glycogen, polyq3-hydroxybutyric acid and lipids during accumulation and subsequent utilization in a Pseudomonas. Antonie van Leeuwenhoek 40: 103-120. A pleomorphic pseudomonad, V-19, was isolated from activated sludge on the basis of its floc-forming capacity in 0.1 ~ casitone - 0.035 ~ yeast extract (CYE) - 0.2~ glycerol medium. In the late exponential phase of growth morphological changes and flocculation phenomena took place accompanied by a massive deposition of reserve granules in the cell. Chemical and electron-microscopical examination revealed 3 types of storage products: glycogen, poly-~-hydroxybutyric acid (PHB) and ether-extractable lipids. These products were isolated and chemically characterized. In CYE medium supplied with 0.5 ~ glucose or glycerol as the carbon source mainly ether-soluble lipids and glycogen were synthesized. On continued incubation these materials were slowly utilized, which enabled the cells to survive for long periods of time. Growth in inorganic salts medium (0.1 ~ ammonium sulfate; 1 7oo carbon source) yielded cells containing different accumulated products, depending on the carbon source used. Glycerol-grown cells contained mainly glycogen, but also ether-soluble lipid, and no PHB. Glucose was largely converted into gluconic acid and excreted into the medium before being deposited in the form of PHB as the primary product of assimilation. Subsequently, PHB was metabolized thereby being partly transformed into glycogen and ether-soluble lipid. Addition of ammonium sulfate to nitrogen-starved cells caused a ready mobilization of the accumulated products, resulting in a net synthesis of reservefree cell material and an increase in the number of cells.

INTRODUCTION From activated sludge a bacterium was isolated which, in CYE - 0.2~ glycerol medium, gave flocs consisting of egg-shaped cells in an otherwise fully clear, cell-free culture liquid (Deinema and Zevenhuizen, 1971). Upon treatment

104

L. P. T. M. ZEVENHUIZENAND A. G. EBBINK

of the sediment with cellulase, the flocs could not be solubilized, although large amounts of reducing sugar (glucose; 20 ~ of the dry weight) were released. It was shown that these sugars had been liberated from intracellular glycogen by the action of an ~-glucosidase impurity of the cellulase preparation. These phenomena prompted us to study the accumulation of storage compounds in relation to cell morphology, cell aggregation and cell longevity. This was done by electron-microscopical examination of ultrathin sections, and by analysing the cells for carbohydrate and lipid contents at various stages of growth in different media.

METHODS

Cultural methods. The following media were used: Casitone - yeast extract medium with the composition: Bacto-casitone (Difco), 0 . 1 ~ ; Bacto yeast extract, 0.035 ~ in tap water (CYE basal medium), and supplied with glycerol or glucose in various concentrations as indicated. When glucose was used as the carbon source 0.4 ~ calcium carbonate was added. Inorganic salts medium composed of (NH4)2SO4, 0.1 ~o; K2HPO4, 0.1 ~ ; MgCI2, 0.02 ~ ; CaCO3, 0.4 and trace elements: FeCla'6H20, 2.5; HaBOa, 0.01; ZnSO4"7H20, 0.01; CoC12" 6H20, 0.01 ; CuSO4" 5H20, 0.01 ; MnCl2, 1 ; Na2MoO 4. 2H20, 0.01 mg/litre of distilled water (SA basal medium). The experiments were carried out in 50 ml of liquid culture, in 100 ml Erlenmeyer flasks, on a rotary shaker at 25 C. Bacteria were maintained on slants of CYE - 0.2 ~ glycerol - agar. Cell counts were made by the plating technique on tryptone (Oxoid), 0.25 ~ - yeast extract, 0 . 1 5 ~ - agar, 1.2~o. Analysis of cultures. Hydrochloric acid was added to dissolve excess of calcium carbonate. Cultures were centrifuged and the cells washed with water. Glucose in the supernatant was analysed by the method of Somogyi-Nelson (Somogyi, 1952). Acid production was estimated by titration of the supernatant with 0.1 N sodium hydroxide. The titration value was corrected for the titration value of the blank medium treated in the same way. The washed cells were resuspended in I0 ml of water and analysed for dry weight and reserve deposits. Total carbohydrate was determined with the anthrone - sulfuric acid reagent using glucose as a standard (Trevelyan and Harrison, 1952). Insoluble glycogen was determined by adding 1 ml of 2 N sodium hydroxide to 1 ml of cell suspension and heating the mixture in a loosely stoppered tube at 100 C for 2 hr. The insoluble residue was centrifuged, washed with water, resuspended in 2 ml of water and analysed with the anthrone- sulfuric acid reagent. For lipid analysis 1 ml of 2 N hydrochloric acid was added to 1 ml of cell suspension. Mixtures

GLYCOGEN,

PHB AND LIPIDS IN A PSEUDOMONAS

105

were digested in loosely stoppered tubes at 100 C for 2 hr, cooled and extracted by shaking 2 times with 2.5 ml of chloroform. Samples, containing 10-100 ~.g of lipids, were transferred into test tubes and the chloroform was evaporated in a boiling water bath. Then 5 ml of sulfuric acid (96 ~o) were added, and the samples were heated at 100 C for 10 rain. Ultraviolet spectra were recorded between 200 and 350 nm. The amount of ether-soluble lipid was derived from the extinction at 295 nm (E295 = 0.62 per sample of 100 ~g); poly-~3-hydroxybutyric acid was calculated from the extinction at 235 nm after correction for interference by the lipid component (E 235 = 0.35 per 10 ~g PHB). Structural analysis of glycogen. Chemical end group determinations of glycogen were performed by methylation and periodate oxidation (Zevenhuizen, 1973). For enzymic analysis of glycogen the enzyme Cytophaga isoamylase (from lytic enzyme complex L-I, BDH), which completely debranches glycogen, was used. The reducing end groups set free were determined according to the method of Somogyi-Nelson, expressed as maltose equivalents, and average chain lengths @T) were determined (Gunja-Smith, Marshall and Smith, 1971). The extent of ~3-amylolysis of glycogen was determined by exhaustive treatment of the polysaccharide with ~-amylase (from barley, Fluka). Lipid analysis. Lipids were saponified with methanolic potassium hydroxide, and the resulting fatty acids were esterified with 10 ~ BCI3 in methanol (Brian and Gardner, 1967). Equivalent chain lengths (ECL) of fatty acid methyl esters were calculated by comparison of the retention times with those of known fatty acid methyl esters in the range 12 to 26 (Jamieson, 1970). Unsaturated fatty acids were characterized more fully by dissolving the sample in methanol, followed by hydrogenation with palladium (5 ~ ) on carbon as a catalyst for 4 hr at room temperature. The gas chromatogram of the original and the hydrogenated sample were then compared. Separation techniques. Fatty acid methyl esters were separated in a gas chromatograph, equipped with a flame ionization detector at a gas flow rate of 20 ml nitrogen per min. Columns: 2000 • 4 mm stainless steel, containing 10 9/0 (w/w) of diethylene glycol succinate (DEGS) on Chromosorb G-HP (80-100 mesh) and operating at 180 C; 2000 • 4 mm glass column, containing 39/00 Apiezon L (w/w) on Chromosorb N-AW-DMCS (80-100 mesh), operating at 200 C. Sugars were separated as their alditol acetates on a 3 % (w/w) OV-225 column on Chromosorb W-HP (100-120 mesh) at 200 C. Electron-microscopical examination. Cells were collected from late exponential-phase cultures by centrifugation at 5000 • g for 10 min. Pelleted cells were fixed in 1 ~o osmium tetroxide in Kellenberger buffer and embedded in 3 9/ooagar. Small blocks were stained with uranyl acetate, dehydrated in an alcohol series and embedded in Epon 812. Sections were poststained with lead citrate.

L. P. T. M. ZEVENHUIZENAND A. G. EBBINK

106

RESULTS

Cultural characteristics of Pseudomonas V-19. V-19 cells are gram-negative, strictly aerobic, catalase-positive and oxidase-positive. In the young stage the cells are motile rods, measuring 1 by 3 to 4 ~m. Later on they gradually change into oval cells of 1 by 1 to 2 ~zm,while at the same time cells tend to aggregate. Flagella-staining of motile cells by Gray's method revealed one or more polar flagella at one end. Gelatin was not liquefied, arginine was metabolized, while nitrite was produced from nitrate by this organism. Base composition of DNA was 59.9 ~ 0.7 ~o GC. Good growth occurred with D-glucose, D-galactose, D-mannose, D-fructose, L-arabinose, D-gluconate, DL-[3-hydroxybutyrate, pyruvate, oL-lactate, glycerol and acetate. No growth was obtained with D-maltose, saccharose, D-lactose, o-cellobiose, starch, D-xylose, L-rhamnose, D-sorbitol and citrate. When carbon sources were in relative excess, large amounts of glycogen and lipids were deposited inside the cells. Acid was produced from hexoses, especially from glucose. Not only complex nitrogenous substrates, like peptone and yeast extract, but also ammonium sulfate in a fully synthetic salts medium could be used as a nitrogen source. In none of the above-mentioned media fluorescence was observed, with the exception of SA-sodium acetate medium in which the bacteria exhibited a very strong yellow-green fluorescence. Acid production from glucose. In CYE medium with glucose as the carbon source V-19 cells excreted large amounts of acid into the medium. The pH of the unbuffered culture dropped to 3.7 and total cell death occurred. Cells were then long, nonmotile rods. In the presence of 0.4 ~ calcium carbonate the pH was kept at a neutral value and growth proceeded undisturbed. With 1 ~ of

9500 mtiGlucose &O0 300 [ ~ " ~ Gluconicocid

.tY

Dry weight

IO0

0

1

2

3

4

5

Doys

6

Fig. 1. Growth and acid production of V-19 during incubation in C Y E - 1 % glucose medium.

GLYCOGEN~

PHB AND LIPIDS IN A PSEUDOMONAS

107

glucose more than half of it was converted into acid (calculated as gluconic acid; Fig. 1) which on continued incubation was further metabolized. The organic acids were isolated by passing the supernatant over a Dowex-2 anionexchange column to yield a brown oil. The acid was identified by esterification with 4 ~ methanolic-HC1, followed by reduction of the ester with sodium borohydride in water, and acetylation with pyridin-acetic anhydride. Gas-liquid chromatography on a OV-225 column at 200 C gave mainly one peak corresponding with D-glucito1 acetate, from which it was concluded that the original acid was D-gluconic acid.

Cell morphology, cell aggregation and accumulation of storage products at various growth stages. Cell flocs from a 24 hr old culture in CYE - 0.2 ~ glycerol medium, consisting of egg-shaped cells (Fig. 2a), were inoculated into 50 ml of fresh medium and incubated at 25 C. Three hours after inoculation young, growing cells began to detach from the flocs, going into the culture fluid as short, yet nonmotile rods, mostly in pairs and measuring 1 by 2 to 3 [xm (Fig. 2b); 6 hr after inoculation flocs had totally disappeared and a homogeneous suspension of very motile rods measuring 1 by 3 to 4 [xm was present (Fig. 2c). Up to this moment no accumulation of storage products had taken place (Fig. 3). At 12 hr cells again fragmented into small, oval cells (Fig. 2d). During the period of transition of rods into egg-shaped cells in the late exponential growth phase, glycogen and lipids were laid down in the cells (Fig. 3). At the same time the culture began to flocculate, which may suggest a correlation between both properties. To study the position of the stored products, cells grown in CYE - 1 ~o glycerol medium were sectioned and examined in the electron microscope (Fig. 4). Two types of intracellular deposits are present: several large, central lipid globules, and around these a layer of many smaller particles presumably consisting of glycogen. Isolation and identification of glycogen. In order to isolate glycogen, cells were heated with sodium hydroxide (see Methods). The alkali-insoluble residues still showed the original cell shape under the light microscope (Fig. 5a). However, cell remnants were smaller and had lower contrast because the lightrefractive lipid inclusions had disappeared. The resulting alkali-stable glucan particles were completely soluble in 30 ~ sodium hydroxide at 100 C. The resulting clear, opalescent solution precipitated upon the addition of ethanol. Acid hydrolysis of the precipitated material gave more than 9 0 ~ reducing sugar identified as glucose; {3-amylase gave 30~o reducing sugar as maltose. Moreover, I.R.-analysis of V-19 glucan gave bands at 930, 850 and 750 cm-1, a pattern characteristic for ~-1,4-glucosidic linkages (Barker, Bourne and Whiffen, 1956). The glucan consumed 1 mole of periodate and gave 0.14 mole of formic acid per mole anhydroglucose unit; methylation gave 12~ 2,3,4,6-tetra-O-

108

L. P. T. M . ZEVENHUIZEN AND A. G. EBBINK

Fig. 2. Growth cycle of Pseudomonas, strain V-19, in C Y E 4 ) . 2 ~ glycerol medium at 25 C. Photomicrographs of cells correspond to the growth phases of Fig. 3. a. Inoculum of egg-shaped cells from a 24 hr old preculture. To show cell form more clearly, flocs were disrupted by pressing and rubbing the cover slip over the microscope slide; b. 3 hr, c. 6 hr and d. 12 hr after inoculation; 1625 x.

GLYCOGEN, PHB AND LIPIDS IN A PSEUDOMONAS

mg

f e ~ e ~ O r y

t,O /?

"/

|1'

Egg-ilke cells Clumping

109

weight

~

o

30

/ 20

/~ Rods ,/

3

/ /x

9 0

Totol reserves ...... o------._.._.Totel lipids

./ ~'e~'~'-"'--~p...___.._.____~.Totacorbohydrote

6

12

24

t

36

Hours

Fig. 3. Deposition of carbohydrates and lipids in V-19 cells during growth in CYE--0.2~ glycerol medium at 25 C. Growth phases correspond to the photomicrographs of Fig. 2.

Fig. 4. Uttrathin section of V-19 cells, grown in CYE-1 ~/oglycerol medium at 25 C; 33 000 •

110

L. P. T. M. ZEVENHUIZENAND A. G. EBBINK

Fig. 5. Alkaline and acid treatment of V-19. Cells were precultivated in CYE-0.5 % glycerol medium at 25 C. a. Cell residues after treatment with 1 N NaOH at 100 C for 1 hr; 1250 • b. Lipid granules after treatment with 1 N HCI at 100 C for 1 hr; 1250 • methyl-D-glucose, 76 ~ 2,3,6-tri-O-methyl-D-glucose and 12 ~ 2,3-di-O-methylD-glucose; action of Cytophaga isoamylase produced 1 2 ~ end groups. F r o m these data mean chain lengths (C--L)of 7.1, 8.3 and 8.3 respectively were derived. Isolation and fractionation of lipid components. The strongly light-refractive lipid inclusions of V-19 cells were isolated by treatment of the cell suspension with an equal volume of 2 U hydrochloric acid at 100 C for 2 hr. By this treatment lipid globules free of other cell constituents were obtained. They were visible under the light microscope as bodies strongly adhering to each other (Fig. 5b). Lipids were extracted by shaking the acid digest with chloroform and centrifugation. Only a small amount of insoluble residue between the two layers remained. Total lipid, after heating in strong sulfuric acid, had an absorption spectrum with two maxima at 235 nm and at 295 nm respectively (Fig. 6). The chloroform extract was evaporated to a small volume. On addition of ether, two fractions were obtained: a. an ether-insoluble fraction precipitating as a white solid and showing the characteristic absorption spectrum of crotonic acid in sulfuric acid (maximum at 235 nm; cf. Slepecky and Law, 1960). Moreover, the I.R.-spectrum was identical with the I.R.-spectra of poly-~-hydroxybutyric acid obtained from other bacterial sources (Lundgren et al., 1965); b. an ethersoluble fraction which on evaporation of the solvent gave a highly viscous oil with an absorption spectrum in sulfuric acid, showing a maximum at 295 nm

GLYCOGEN,

PHB

111

A N D LIPIDS IN A P S E U D O M O N A S

Fig. 6. U.V.-absorption spectra of total lipid and lipid fractions from V-19 cells cultivated in CYE-glycerol medium. At the top : total chloroform-extractable lipids; below: ether-insoluble PHB, 17.6 [zg/5 ml sulfuric acid; ether-soluble lipid, 138 ~xg/5 ml sulfuric acid.

o_|E

Total f i p i d s ( C H C I 3 - e x t r a c t of V19)

.b

i"~O

o< L\ O. ]

\

I

II

07

"

/lll~l~ll\

9 Lipid ( 2 9 5 n m ~

a

01 I

0.4

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i

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I

\

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0

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i

220

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:\ 260

I

\

'-

I

I

I

I

280

300

320

3':0

I

360

Nm

(Fig. 6). Long-chain unsaturated, hydroxy and cyclic fatty acids give a nonspecific absorption band at 290-300 nm in concentrated sulfuric acid, while normal, saturated fatty acids do not absorb above 200 nm (Zevenhuizen, 1974). Because V-19 lipid gave an identical spectrum with about the same intensity at the same ),. . . . it was concluded that fatty acid components of V-19 lipid are largely hydroxylated, cyclic and/or unsaturated. Saponification of the ethersoluble lipid yielded a mixture of fatty acids which were separated as their methyl esters by gas-liquid chromatography. On a DEGS column at 180 C more than 12 peaks were obtained with ECL values ranging from 12 to 26. On mild hydrogenation of the bacterial fatty acid methyl ester mixture with hydrogen/Pd at room temperature, a number of peaks (unsaturated fatty acids) disappeared. The absorption at 295 nm decreased but did not disappear completely. By changing the polar DEGS column for the apolar Apz-L column a large number of peaks shifted towards lower ECL vatues (range 12 to 22), indicating the polar character of these components, probably by the presence of hydroxyl-substituents (Jamieson, 1970). A number of the peaks were collected from the gaschromatographic column and identified by mass spectrometry. Among them were 13-hydroxy fatty acids: 3-OH 10:0 and 3-OH 12:1, and the cyclic fatty acid C-17: 0.

112

L. P. T. M. ZEVENHUIZEN AND A. G. EBBINK

Distribution of different types of storage products in V-19, cultivated on different carbon sources. W i t h m o s t c a r b o n sources listed in T a b l e 1, when given in excess to the m e d i u m , V-19 yielded cells with a high c a r b o h y d r a t e a n d lipid content. Cell yield without reserve materials in all cases was c o n s t a n t (50-60 mg) a n d only d e p e n d e d on the a m o u n t o f the growth-limiting nutrient (50 m g a m m o n i u m sulfate = 10 m g N). In sugar m e d i a in which acid was p r o d u c e d the p H was k e p t c o n s t a n t by a d d e d calcium c a r b o n a t e . Organic acids were given in the f o r m o f the calcium salt in o r d e r to m a i n t a i n a neutral p H . Different c a r b o n sources in the m e d i u m gave rise to a varied c o m p o s i t i o n o f intracellular storage p r o d u c t s . G l u c o s e a n d organic acids (gluconic acid, ~3h y d r o x y b u t y r i c acid) which are intermediates on the route to P H B , a n d citric acid cycle intermediates (succinate, malate) gave p r i n c i p a l l y d e p o s i t i o n o f P H B . Other sugars acted quite differently: m a n n o s e did not give a c c u m u l a t i o n at all; fructose gave cells with a very high lipid content without P H B . G l y c o g e n was present in considerable a m o u n t s in cells grown on each c a r b o n source. Because o f the transient c h a r a c t e r o f reserve materials a n d the possibility o f t r a n s f o r m a t i o n o f one type o f storage c o m p o u n d into a n o t h e r type, experiments were carried out with t w o selected c a r b o n sources, viz. glucose a n d glycerol. In these experiments the different types o f stored p r o d u c t s were studied in the course o f their a c c u m u l a t i o n a n d subsequent utilization.

Table 1. Growth yields and storage products of V-19 grown in SA-medium supplied with different carbon sources, at 25 C for 2 days. All values are expressed in mg per 50 ml of culture. Carbon sources were added in amounts corresponding to 500 mg of the ash-free, anhydric compound. Carbon source

Dry weight

Carbohydrate 1

PHB 2

Lipid2

D-Glucose D-Galactose D-Mannose D-Fructose L-Arabinose Ca-D-gluconate Na-DL-~3-hydroxybutyrate Ca-succinate Ca-Dt-malate Glycerol Ca-DL-lactate Na-acetate

150 128 62 141 115 118 128 102 84 100 125 78

20.0 30.6 3.5 32.6 31.4 13.3 12.1 9.5 7.4 29.8 14.9 2.9

60.0 11.3 0.4 38.2 42.8 38.6 10.7 2.9 24.0 2.9

18.0 39.4 5.4 54.5 25.6 11.8 30.8 11.3 7.2 14.3 13.0 15.9

i Expressed as glucose; 2 Calculated from the U.V.-spectrum in sulfuric acid at 235 nm (PHB), and at 295 nm (ethersoluble lipid) respectively.

GLYCOGEN, PHB AND LIPID5 IN A PSEUDOMONAS

113

Storage products of V-19 during growth and starvation CYE 0.5% glycerol and CYE - 0.5% glucose media. In these media V-19 synthesized large amounts of glycogen and ether-soluble lipid at the end of the logarithmic phase (Figs. 7 and 8). PHB was hardly present in glycerol-grown cells. In glucose-grown cells the moderate quantities of PHB synthesized were again rapidly utilized by the cells on further incubation. Lipid and glycogen were metabolized at much lower rates and were still present at the end of an incubation period of 21 days. As in these experiments growth of V-19 cells was dispersed without flocs, viable cell counts could be made by the plating technique. They amounted to 0.35 • 101~ cells/ml of culture, corresponding with -

mg 70 84

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Fig. 7. Accumulation and utilization of storage compounds by strain V-I 9, cultivated in CYE -0.5 ~ glycerol medium at 25 C. mg 50

30

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1

2

Total r~

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Fig. 8. Accumulation and utilization of storage compounds by strain V-19, cultivated in CYE-O.5 ~o glucose medium.

114

L. P. T. M. ZEVENHUIZENAND m. G. EBBtNK (NH/,)2 SO/, added

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10 o ~ q : E u ~ . _ o O ~ o - - L i pid 0 L-~ ' "-'----'---'---''--'----'----' 1 2 3 t, S 6 7

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8 Days

9

Fig. 9. Storage compounds during growth and starvation of strain V-19, cultivated in SA-1 glycerol medium. - - - - (NH4)2SO4 added. Total reserves calculated from experimental data. 0.5 mg dry cells without reserve material, and remained constant over the entire 3 week incubation period. When on prolonged incubation ceils were deprived of nearly all their endogenous substrates, they began to die at a noticable rate until about half the original numbers were left after 63 days. S A - 1 ~ glycerol and S A - 1 ~ glucose media. With glycerol as carbon source cells were obtained with low PHB and moderate lipid content but with carbohydrate rising to a level of 30 ~ of the dry weight (Fig. 9). Total reserve-free cell material, amounting to 56 mg, remained essentially constant during the entire starvation period. Addition of ]00 nag of ammonium sulfate to the culture at the 2nd day resulted in a prompt reduction of glycogen and lipid contents by 22 mg at the 9th day, when compared with ceils which had not been given nitrogen. On comparing dry cell weights, without reserves, of cells with, and without added a m m o n i u m sulfate (65 and 56 mg resp.) it can be concluded that part of the mobilized reserves had been converted into essential cell material. In SA - 1 ~ glucose medium (Fig. 10) large amounts of gluconic acid were excreted into the medium. At 12 hr, when glucose was consumed, gluconic acid reached its maximum value, corresponding to 50 ~o conversion of glucose. It was rapidly metabolized further during logarithmic growth thereby giving a sharp rise in PHB content of the cells, until dry weight reached a maximum of 160 mg per 50 ml of culture at 24 hr. F r o m that time PHB gradually decreased but glycogen and lipid still rose to higher values, necessarily at the expense of PHB. During subsequent starvation the amount of essential cell constituents = dry weight - total reserves remained at a constant level of 60 ~ 5 rag. Addition of 100 mg of ammonium sulfate at the third day caused an immediate mobilization

PHB

GLYCOGEN~

A N D L[PIDS IN A P S E U D O M O N A S

0 v' ' ', "'uc~ ~ ~ oc,d 150

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115

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.

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.

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4

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5

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Fig. 10. Storage compounds during growth and starvation of strain V-J9, cultivated in S A - ] ~ glucose medmm. - . . . . (NH4)2SO4 added. Total reserves calculated from experimental data.

of glycogen; PHB was also attacked but lipid was metabolized slower. At the 8th day, total amount of reserves was decreased by 33 mg when compared with cells which had not been given nitrogen. Nevertheless total dry weight did not appreciable alter as a result of the addition of the limiting nutrient, indicating a net synthesis of essenlial cell materials at the expense of intracellular reserves. Increase of reserve-free cell material at the 8th day was 29 mg, ~o that the major part of the mobilized reserves must have been converted into cell constituents. Ammonium sutfate additiorI also had a bearing on viaNe cetI counts. The number of viaMe cells first increased to a maximum value at the 4th day, but as the cells were then exhausted they began to die at a faster rate than cells without added nitrogen, which remained nearly 100 ~ viable during the experiment. At the end of a 4 week incubation period only a few ~ of the nitrogen-treated cells were then still al/ve.

116

L. P. T. M. ZEVENHUIZENAND A. G. EBBINK DISCUSSION

Pseudomonas V-19, developing both in complex and in minimal, synthetic media displays a definite growth cycle of straight, regular rods in the early logarithmic phase and of oval, egg-shaped cells in the stationary phase of growth. As the morphological changes occur in nitrogen- as well as in carbonlimited media, they are only related to the phase of growth and do not depend on the accumulation of reserve materials. When V-19 was freshly isolated from activated sludge the bacteria exhibited excellent floc-forming growth in CYE - 0.2 ~ glycerol medium. Flocculation was particularly pronounced in media in which excess of carbon was present and was only moderate in media in which the carbon source was omitted. Besides, cell fiocs could be deflocculated by exhaustion of the endogenous reserves on prolonged incubation of the culture. In the course of this investigation the ability of V-19 to form flocs under the experimental conditions decreased and finally disappeared. A theory based on the role of intracellular lipids on bacterial flocculation has been put forward by Crabtree et al. (1966). In this theory the assumption was made that, during the process of flocculation, accumulated PHB is released by the cells to form polymeric bridges between cells. Shadow-cast preparations of well flocculated V-19 cells indeed revealed exocellular material between the cells in the form of parallel bundles of straight threads (Deinema and Zevenhuizen, 1971). This material appeared to be non-cellulosic and alkalisoluble. According to this theory the total amount of accumulated lipids is not decisive foi the floc-forming properties. Only cells which release particular lipid components are determinative for the adhering properties. With our strain V-19 this "aberrant" cell form disappeared by a process of (self?) selection during storage for prolonged periods of time on agar slants. Among the endogenous substrates of bacteria, glycogen and lipids including PHB are considered as carbon and energy stores (Dawes and Ribbons, 1964). Glycogen is present in most bacteria, and in a large number of cells is accompanied by PHB and/or lipid inclusions (Table 2). Most reports on bacterial reserves deal with only one type of product namely either glycogen or PHB. In some organisms only one type was believed to be present; in other organisms in which two or more types have been recognized they have been studied separately. In only a few reports two types of endogenous reserves have been studied simultaneously in the same organism, e.g. glycogen and PHB in Rhodospirillum rubrum (Stanier et al., 1959) and glycogen and lipid in Mycobacterium spp. (Antoine and Tepper, 1969a, b). The presence of ether-soluble lipids in the lipid inclusions is reported for

GLYCOGEN,

PHB

117

AND LIPIDS IN A PSEUDOMONAS

Table 2. Occurrence o f m a j o r e n d o g e n o u s reserves in a n u m b e r o f bacteria. 50 ~ survival times listed were calculated by Ensign (1970) f r o m literature d a t a ; in each case, the longest starvation half-live, which was f o u n d in the literature, is represented. Organism

E n d o g e n o u s reserve PHB

Agrobacterium tumefaciens Sphaerotilus natans Sphaerotilus discophortls Streptococcus mitis Streptococcus lactis Escherichia coli Aerobacter aerogenes Azotobacter agilis Rhodospirillum rubrum Bacillus megaterium Bacillus cereus Arthrobacter spp. Mycobacterium spp. Pseudomonas spp. Pseudomonas V-19

Lipid

50 ~ survival Glycogen x1 x2

x2 x

x x x x x x 11 x 12 x l2

x 19,20 x

133 133

127 137

12 h 4 22 h s 30 h 6 36 h s 45 h 9 5 0 h lo

X 11

x 13

10

x x

7-9 3'~4 7-918

80-100 d x5'I6

X 17

X

x

8

60 d 21

1 M a d s e n , 1963 ; 2 M u l d e r et al., 1962; 3 Zevenhuizen, 1966b; 4 Stokes a n d Parson, 1968; 5 v a n H o u t e a n d Jansen, 1970; 6 T h o m a s a n d Batt, 1968; 7 Sigal, Cattaneo a n d Segel, 1964; s D a w e s a n d Ribbons, 1965; 9 Strange, W a d e a n d Ness, 1963; Jo Sobek, C h a r b a a n d Foust, 1966; t~ Stanier et al., 1959; ~2 Williamson a n d Wilkinson, 1958; 13 Barry et al., 1953; 1,~ G h o s h a n d Preiss, 1965; 1~ Zevenhuizen, 1966a; t6 Ensign, 1970; 17 A n t o i n e a n d Tepper, 1969a; 18 A n t o i n e a n d Tepper, 1969b; 19 KaIlio a n d Harrington, 1960; 2o Levine a n d Wolochow, 1960; 21 present investigation.

instance in Rhodospirillum rubrum (Stanier et al., 1959), Pseudomonas methanica (Kallio and Harrington, 1960) and Bacillus spp. (Williamson and Wilkinson, 1958). However, these materials were not examined with regard to their possible function as endogenous carbon somces. Lipids are present in small, constant amounts in all bacteria. They usually do not function as carbon and energy reserves and have to be considered as structural components of membranes (phospholipids) and of cell walls (lipopolysaccharides) (Kates, 1964; Lennarz, 1966). Only in Mycobacterium, lipid was found to function as an endogenous substrate although glycogen, which was present at the same time, appeared to be the preferred substrate (Antoine and Tepper, 1969a). In V-19 cells, glycogen, PHB and lipid may be considered as endogenous reserves, owing to the following properties: (a) synthesis of these products begins during logarithmic growth but accumulation takes place at the end of the log phase and in the stationary phase when growth is limited by a necessary nutrient (nitrogen) and carbon is available in excess, (b) cells which have accumulated utilize these reserves on continued incubation in a medium without nutrients by

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oxidation to carbon dioxide and water (endogenous respiration). By these means energy and carbon substrates are provided for their survival, (c) endogenous carbon substrates can be utilized for the synthesis of essential cell constituents. Large differences exist in survival rates of vegetative cells of bacteria which are aerated in buffer without nutrients at physiological temperatures, ranging from a few hours to several weeks (Table 2). Suspensions of Aerobacter aerogenes containing approximately 2 0 ~ of carbohydrate (mainly glycogen) in their cells respired most of it during the initial 25 hr of incubation (Dawes and Ribbons, 1964). The question was then raised, how glycogen can contribute towards maintenance and survival if during starvation these reserves are depleted at such a high rate. Cells of Escherichia coli grown in nitrogen-deficient medium survived longer when their carbohydrate (glycogen) content was high, but in all cases carbohydrate was depleted to about 50 ~ of the initial value within 4 hr (Strange, 1968). Viability of Streptococcus mitis, rich in glycogen, decreased to about 40 ~o during a 16 hr storage period; viability of strains without reserves decreased 10 000 fold after the same storage period (van Houte and Jansen, 1970). From these findings it was concluded that the possession of glycogen by S. mitis favors survival. On the other hand bacteria exist which survive much longer under the rigorous conditions of starvation. Suspensions of Arthrobacter cells in buffer were more than 75 ~ viable after an incubation period of several weeks (Zevenhuizen, 1966a; Ensign, 1970). The rate at which reserve materials are broken down is an important factor in the survival of the organism. In particular, a relation can be found between the rate of glycogen breakdown and the structure of the glycogen concerned. Cells which break down glycogen rapidly, like Escherichia coli and Aerobacter aerogenes, have glycogens structurally similar to those glycogens which are normally found in many cells of animal or microbial origin and have C---L-- 12-15. Arthrobacter and Mycobacterium have glycogens with a high degree of branching with cL = 7-9, and are in this respect comparable to phosphorylase limit dextrins. These dextrins are not susceptible to phosphorylase action and have first to be debranched by a debranching (~-l,6-glucosidase) enzyme. Therefore it was concluded that in these cells glycogen breakdown is limited by the (low)activity of the debranching enzyme, and the synthesis of it thus leads to accumulation of glycogen in this particular form (Zevenhuizen, 1966b). Pseudomonas V-19 is another example of a cell with a deviating glycogen structure with r163 = 8. Under starvation conditions its glycogen is broken down at a low rate. Its lipids too, are also only slowly utilized. The substratesparing effect of these products may be considered important factors for the

GLYCOGEN. P H B AND LIPIDS IN A PSEUDOMONAS

119

l o n g - t e r m survival o f V-19 cells, when c o m p a r e d with that o f cells devoid o f such substrates. PHB, which a p p e a r s to be m u c h m o r e easily attacked, is utilized n o t only by c o m b u s t i o n for the generation o f energy but also functions as an i n t e r m e d i a t e for the synthesis o f fatty acids a n d glycogen. In the presence o f excess o f c a r b o n (glucose), overflow is firstly directed t o w a r d s the synthesis o f P H B a n d next to m o r e stable c a r b o n deposits like fatty acids a n d glycogen. It is generally assumed that the enzymes for their b r e a k d o w n are present b u t that they are inhibited by the intermediate p r o d u c t s o f lipid a n d c a r b o h y d r a t e catabolism. A d d i t i o n o f the growth-limiting nutrient (nitrogen) causes withd r a w a l o f these intermediates a n d thus restores the activities o f the b r e a k d o w n enzymes, thus leading to an i m m e d i a t e m o b i l i z a t i o n o f the stored p r o d u c t s a n d to a net synthesis o f n i t r o g e n o u s p r o d u c t s as building stones o f new cell material. The a u t h o r s wish to t h a n k the technicians o f the Technical a n d Physical Engineering Research Service at W a g e n i n g e n for help in p r e p a r i n g ultrathin sections a n d in t a k i n g the electron m i c r o g r a p h s . T h a n k s are also due to Mr. W. H. J. C r o m b a c h for r e c o r d i n g t h e r m a l d e n a t u r a t i o n curves o f isolated D N A .

Received 4 April 1973

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