225

Biochimica et Biophysics @ ElsevierlNorth-Holland

Acta,

575 (1979)

Biomedical

225-233

Press

BBA 57460

EFFECT OF GROWTH RATE ON LIPID AND LIPOTEICHOIC ACID COMPOSITION IN STREPTOCOCCUS FAECZUM

D. CARSON

=,*, R.A.

PIERINGER

b and L. DANEO-MOORE

a Department

a** *

University

of Microbiology and Immunology and b Department School of Medicine, Philadelphia, PA 19140 (U.S.A.)

(Received

June 27th,

of Biochemistry,

Temple

1979)

Key words: Lipid composition;

Lipoteichoic

acid; Growth

rate; (Streptococcus

faecium)

The lipid composition of Streptococcus faecium (S. faecalis ATCC 9790) was analyzed at various growth rates. Diphosphatidylglycerol and the non-ionic lipid fraction containing diacylglycerols and neutral glycolipids appeared to accumulate relative to cellular mass as the culture mass doubling time increased from 30 to 80 min. Within the same range of doubling times the non-ionic lipid fraction appeared to become substantially enriched with diacylglycerols. All lipid species and cellular lipoteichoic acid accumulated relative to the cellular mass at doubling times exceeding 80 min, although diacylglycerol accumulation exceeded that of all other compounds studied.

Introduction The role lipids play in providing cellular permeability barriers by virtue of their hydrophobic character is well-described [l]. This function alone, however, does not explain the existence of the wide variety of lipid types in bacteria. Certain ionic species have been proposed to act in stabilizing membranes against pH changes in the external mileu [2-41 or in mediating ion transport [5,6]. Furthermore, many enzymes have been shown to exhibit specific lipid requirements for activity [ 71. Also, certain lipids and lipoteichoic acid have been shown [S-10] to inhibit autolytic activities proposed to be of * Present address: Department of Physiological Chemistry. John Hopkins University School of Medicine, Baltimore, MD. U.S.A. ** To whom correspondence should be addressed.

226

primary importance in normal bacterial wall growth and division processes [ 11, 121. This study reports the effects of nutritional growth rate restriction on the lipid and Iipo~icIloic acid content of Streptococcus faecium. The relationships which the reported lipid alterations may bear to ‘stringent’ control [ 131 and to other biological phenomena are discussed. Materials

and Methods

wraith of cells. S. fuec~um (S. faecalis ATCC 9790) was grown at 37°C in a chemically defined medium 1141 from which glutamine was omitted and glutamate concentration was varied to alter growth rate [ 151. Culture turbidity was followed on a Coleman spectrophotometer model 14 at 675 nm. The absorbance was corrected for deviations from linearity [ 161 and this corrected value was used to estimate culture mass [15]. Cultures were maintained in a balanced exponential growth condition by repeated dilutions for a minimum of S-10 mass doublings prior to an experiment. Extraction and quantitation of lipids and iipoteichoic acid. Cultures were grown in the presence of [“Clor [2-3H]glycerol (0.5 Ci/l, 2.8 mg/ml; New England Nuclear Corp., Boston, MA) for a minimum of 8-10 generations prior to sampling, All samples were taken from cultures in the mid-exponenti~ phase of growth as judged turbidimetric~ly. Lipids were extracted with CHClJ CH30H (2 : 1, v/v) as described previously [17,18], with reagent volumes scaled up to accommodate 5-ml samples. Deacylation, chromatographic and electrophoretic separation of lipids were performed as described by Pieringer and Ambron [ 191. Chromatographic separations of non-ionic species were performed on samples eluted from the origin of electrophoreto~~s with CH30H/H20 (1 : 1, v/v). The deacylated non-ionic lipids were separated on Whatman No. 1 paper developed overnight with n-butanol/pyridine/water (6 : 4 : 3, v/v/v). Lanes from either electrophoretograms or chromatograms were cut into sequential 0.5 cm strips and placed in scintillation vials containing 0.5 ml doubly distilled water and 5 ml Formula 947 (New England Nuclear Corp., Boston, MA), Cellular lipoteichoic acid content was estimated essentially as described previously [ 171 using the hot phenol/water extraction procedure of Wicken and Knox 1201, except that 0.01 M MgClz was used in the aqueous phase [21]. Total cellular isotope incorporation was estimated in 0.5-ml culture samples either by precipitation in 4.5 ml 10% trichloroacetic acid as described previously 1171 or by filtration of cells on 0.4-gum pore size filters (25 mm diameter; Nucleopore Corp., Pleasanton, CA) followed by two 2-ml washes with ice-cold 0.08% (w/v) glycerol. Filters were then placed in scintillation vials, treated with 0.5 ml 90% NCS (Amersham/Searle Corp., Des Plaines, IL) for 2 h at 55’C, cooled, and 5 ml of a toluene-based scintillation fluid was added to each vial. All radioactive samples were counted in a Mark I liquid scintillation counter (Ame~h~/Se~le Corp., Des Plaines, IL) and dpm were calculated using an external standard. The counting efficiencies were approx. 55% for i4C and 20% for 3H. Specific actiuity determination. Deacylated lipid extracts from 5ml cultures prelabeled with [“Clglycerol as described above were hydrolyzed in 3 ml 2 N

227

HCl in sealed tubes for 48 h at 125’C. The samples were then lyophilized, resuspended in 0.1 ml CH30H/B20 (1 : 1, v/v) streaked for chromatography on Whatman No. 1 paper side with a [ “Clglycerol standard, and developed overnight with n-butanol/pyridine/water (6 : 4 : 3, v/v/v) [ 191. Strip scans of the chromatograms were performed and areas corresponding in mobility to the glycerol standard were eluted and adjusted to a final volume of 1 ml with doubly distilled water. Glycerol content was measured enzymically in 50200~~1 aliquots of each eluate [22]. 14C con%nt was measured by placing 50-d aliquots of each eluate in scintillation vials containing 450 ,LJ doubly distilled water and 5 ml Formula 947, and by counting as described above. These specific activity determinations were used to calculate the cellular content of each lipid species at the approximate growth rate. Such data are herein presented as pmol glycerol (found in the indicated lipid type)/pg cellular dry weight. Results Characterization of labeling conditions Pieringer and Ambron [19] have shown that exogenously added glycerol isotope is incorporated solely into the glycerol moiety of the lipids of S. faecium. Nonetheless, cells may harbor intracellular ‘pools’ of free isotope which would complicate quantitative analyses. Preparations of intact cells are expected to retain these pools, while procedures such as acid precipitation should release pools but not cell structure-associated material into soluble form. The difference in the values obtained by the two procedures should represent an upper limit estimate of the cellular glycerol in unbound form, i.e. glycerol pools. To test for the presence of glycerol pools, duplicate samples of cultures continuously labeled with either [‘HI- or [‘4C]glycerol were taken during mid-exponential growth, and either precipitated in ice-cold 10% trichloroacetic acid, or collected on filters and washed extensively with a glycerol solution to remove any exogenous. label. The ratio of these values (Fig. 1) indicated that cultures contained no more than 3-4s of their cellular glycerol in free form, and that this value did not change with growth rate.

, 200

I

I

I

I

I

100

60

50

40

30

Td lmnl

Fig. 1. Es&mate of maximal free glycerol pool size at various growth rates. Total Rl~cerol isotope incorporation in dpm into 0.5 ml of continuously labeled culture was estimated either by trichloroacetic acid precipitation (TCA ppt) or filtration of whole cells. The ratio of these values is plotted against the instantaneous growth rate constant of the culture (a = In Z/doubling time (Td) in h). -. the arithmetic mean ratio (0.977 t 0.037).

228

In order to relate glycerol ~n~o~oration to glycerol content at various growth rates the specific activity of the glycerol incorporated was determined at several growth rates. The specific activity of the label changed little for doubling times between 30 and 110 min, but decreased substantially at growth rates slower than 110 min. Specifically, the values obtained in three determinations (and their standard deviations) were 617 ?; 56, 568 f 13, 300 jI 22 and 288 rt 32 dpm/nmol glycerol at doubling times of 30, 109, 188 and 345 min, respectively . Distribution of ~~4C~g~~ce~o~ with growth rate As the growth rate of cultures declined, the ratio of total celhdar glycerol to cell dry weight increased (Fig. 2). The increase was substanti~, amounting to approximately a three-fold increase in glycerol content as the doubling times increased from 30 to 80 min. The increase was ten-fold between doubling times of 30 and 200 min. The distribution of label between lipoteichoic acid and lipid appeared to remain essentially constant (Fig. 3). Lipoteichoic acid and lipid accounted for 22.0 + 7.3% and 73.4 rf 6.4%, respectively, of the total cellular glycerol label over the range of growth rates studied (doubling times 30-150 min). The estimate obtained for cellular lipoteichoic acid is in good agreement with estimates of acylated (95-100%) plus deacylated (O-S%) cellular lipoteichoic acid obtained by agarose gel electrophoresis [21]. Distribution of ~‘~C]g~~~e~o~prong i~dividuu~lipids at different ~owth rates Lipid species separated electrophoretic~ly following deacylation [ 191 generated the profile shown in Fig. 4. This procedure allows for 80-100% recovery of original tracer input, and separates the extract into five fractions, i.e. a non-ionic fraction originating from diacylglycerol and its mono- and diglucosyl derivatives, and four anionic fractions originating from phosphatidyl

I

1

I

I

200

100

I

/

I

60 50 40 Td (mint

I

i

30

Fig. 2. Cellular glycerol content at various growth rates. Total cellular glycerol contenting cellular dry weight is plotted versus the instantaneous growth rate constant of the culture (ff). The empirical equation presented here is: nmol glycerol/fig cellular dry weight = t/(&86ru - 0.66).

II

n

.1 .I i I

0.5

I

I

1 .o

1.4

.I

I

I

c2(hr-‘1

I

,

I

h

I

I

200

100

60

50

40

30

0

5

10

15

20

cm migrated from 0rlq1r1

Td tmln)

Fig. 3. Distribution of glycerol label at various growth rates. Lipid and lipoteichoic acid (LTA) contents as C3Hl- or [ 1J~lglyceroi were estimated and related to the total cellular isotope content of continuously labeled cultures on a percent basis. O-----0, lipid (line drawn represents mean value of 73.4 f 6.4%); l-, LTA (line drawn represents mean value of 22.0 +_7.6%). Fig. 4. Profile of a typical electrophoretogmm. The profile is from a typical deacylated lipid extract of a culture doubling its mass every 30-33 min. Peaks originate from non-ionic llpids including diacylglycerols and their mono- and diglucosyl derivatives (I). phosphatidyldi~ucosyldiacyl~ycerol (II), phosphatidylglycerol and aminoac~l phosphatidylglycerol (III), phosphatidic acid (IV) and diphosphatidylglycerol (V).

diglu~osyldiacylgly~erol, phosphatidylglycerol and its amino acyl derivatives, phosphatidic acid and diphosphatidylglycerol. The deacylated derivatives of the non-ionic lipids gave on chromatography the profile shown in Fig. 5. The three peaks were identified as diglucosylglycerol from diglucosyldia~ylglycerol, I

I

I

I

I

I

20

25

.m

l

I

0

0

5

10

15

distance migrated from origin icmi

Fig. 5. Profile of a typical chromatogram of deacylated mono- and diglucosyl (NI) lipid derivatives. The profile presented is from an extract of a culture doubting in mass every 75 min. Peaks originate from diglucosyldiacylglyceride (I). monoglucosyldiacylglycerol (II) and diacylglycerol (III).

230 TABLE

I

LIPID COMPOSITION

OF S. FAECALIS

ATCC

9790

CULTURES

GROWN

AT A DOUBLING

TIME OF

30 min a, for charged lipids, values are the means of twelve are the means of three determinations. b, recalculated

determinations f 1 S.D. For non-ionic from Pieringer and Ambron [ 191.

pm01 glycerol/fig

15.8 0.6 41.3 2.6 11.0

Diglycosyldiacylglycerol Monoglucosyldiacylglycerol Diacylgzlycerol

cellular dry weight b

a Diphosphatidylglycerol Phosphatidic acid Phosphatidylglycerol Phosphatidyldiglyc~roldiacylglycerol Non-ionic fraction

lipids, values

7.4 1.2 3.4

f + * * f

4.4 0.6 6.5 1.7 3.2

11.2 0.7 42.9 1.5 13.6 10.5 1.3 2.2

monoglucosylglycerol from monoglucosyldiacylglycerol, and glycerol from diacylglycerol [ 191. The lipid composition of exponential phase cultures growing with a doubling time of 30 min is given in Table I. The data on lipid composition obtained from

100 -

?.

=50-• I?

. \ l

\-.-L.*

S O-* t @z 250 cl Le 200 - \ ?i 1501 3 \ = 100 **he. u :..-‘--# q50. ’ 0.;: :::: 6 150-

0

t

! 100

-

: diPG

0.8

0.4

400

PG

1.2

(Y (hr-‘1

4

:::

Td

60 50 (mini

-

40

30

Fig. 6. Lipid and lipoteichoic acid composition at various growth rates. The [14C]g.lycerol content of each lipid class was determined and converted to pm01 glycerol based on glycerol specific activity determinations. For the purpose of this graph, lipoteichoic acid (LTA) was considered as a lipid containing 29 glycerol residues molecule 1201. The data are as pm01 glycerol/~g cellular dry weight versus oi. Abbreviations. of lipids are as in Fig. 4.

231

TABLE

II

NON-IONIC Doubling

LIPID time

FRACTION pm01

COMPOSITION glycerol/@

cellular

AT

VARIOUS

dry

weight

GROWTH

RATES

(min) Dilglucosyl-

MonoglucosyI-

diacylglcyerol

diacYlglcyero1

Diacylglycerol

30

7.4

1.2

3.4

53

8.7

1.3

4.4

15

14.4

3.0

53.6

80

28.2

7.1

40.2

115

41.1

5.4

43.5

207

68.2

1.0

27.9

201

48.0

1.0

52.8

220

44.6

8.4

56.4

twelve individual determinations are compared to those reported by Pieringer and Ambron [19] who used silica gel paper chromatography for the initial separation of lipids, followed by deacylation and either paper electrophoresis or paper chromatography for further separation and identification. The major lipids in these cultures appear to be phosphatidylglycerol (41.3 pmol glycerol/pug dry wt.), followed by diphosphatidylglycerol (15.8 pmol glycerol/ E.rgdry wt.) and the non-ionic lipid fraction (11 pmol glycerol/pug dry wt.) with diglucosyldiacylglycerol as its major component (7.4 pmol glycerol/pg dry wt.). Phosphatidyldiglucosyldiacylglycerol and phosphatidic acid were present in amounts of 2.6 and 0.6 pmol glycerol/pg dry wt., respectively. Fig. 6 shows the results obtained when similar analyses were performed in extracts of cultures grown at various growth rates. The data are expressed as pmol glycerol/pg cellular dry weight based on an extrapolation of the specific activity of the label. The results indicate that the phosphatidylglycerol, phosphatidyldiglucosyldiacylglycerol, phosphatidic acid and lipoteichoic acid fractions maintained a fairly constant relationship to the cellular mass over a range of doubling times between 30 and 80 min. Over this same range of growth rates, the diphosphatidylglycerol and the non-ionic lipid fractions appear to accumulate as the doubling time increased. All lipids and lipoteichoic acid accumulated at doubling times exceeding 80 min. When the non-ionic lipid fraction was separated further by chromatography, it appeared that diacylglycerol constituted an increasing proportion of this fraction as the growth rate declined (Table II). Discussion Consistent

with earlier

work on Bacillus megaterium [23] and Escherichia that the total lipid content of S. faecium increases with decreasing growth rate of the culture. Our determinations are based on quantitative analysis of individual lipid species using a specific [19] isotope tracer, supported by specific activity measurements at various growth rates. Experiments performed to estimate glycerol pool sizes indicated that these were small (at most 3-4s of total) and constant over the range of growth rates studied.

coli [24], we have found

232

All lipid species were found to increase relative to the cellular mass when the doubling time was increased from 80 to 210 min. At doubling times increasing from 30 to 80 min, diphosphatidylglyeerol and the non-ionic lipid fraction appeared to accumulate substantially, whereas other lipid species and lipoteichoic acid maintained a relatively constant relationship to the cellular mass. When the non-ionic lipid fraction was further fractionated, it appeared that between doubling times of 30 and 80 min diacylglycerol accumulated to a greater extent (i.e. about ten-fold) than any other lipid species examined. We have also observed a large (ten fold) accumulation of diacylglycerol in amino acid-starved cultures (unpublished results) consistent with ‘stringent’ control of phospholipid synthesis [25--271 in both [13,28] situations. Earlier studies [25] of amino acid-starved bacteria have also indicated similar accumulations of non-phospho~s~ontaining lipids, suggesting or inferring a generalized bacterial response. Diacylglycerol accumulations in these situations are consistent with a common mechanism for the regulation of diacylglycerol levels during the ‘stringent’ response and at slow growth rates. Under these suboptimal growth conditions, alterations in the membrane lipid composition, i.e. acitivities not diacylglycerol enrichment, may serve to inhibit enzymic currently advantageous to the survival of the organism, e.g. autolysins. In some cases, these effects may not necessarily be the result of a depletion of certain lipids required for normal enzyme function (see Introduction for examples). These lipids may be depleted rapidly by converting them into diacylglycerols. There are several biological advantages to accumulating diacyl~ycerols under ~owth-limiting conditions. In general, dia~ylglycerols may serve as fuel reserves, as in depot fats, for times of more favorable growth conditions. They may be rapidly synthesized from phospholipids while their glycerophosphate byproducts are, at least temporarily, conserved as lipoteichoic acid in the case of Gram-positive organisms [29--321 or as other cellular compounds [33,34] in Gram-negative organisms. Additionally, diacylglycerols may be readily reconverted to phospholipid through the action of diacylglycerol kinase [35, 361. It is equally important that all these conversions can take place at the cell membrane allowing for a rapid, concerted response to changing environmental conditions.

This research was supported by Public Health Service grants AI 05044 and AI 05730 from the National Institute of Allergy and Infectious Diseases. D.C. was supported by predoctoral training grant GM 00983 from the National Institute of General Medical Sciences. Thanks are given to Professor G.D. Shockman for a critical reading of this manuscript, References 1

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