JOURNAL OF BACTERIOLOGY, Oct. 1980, p. 238-246 Vol. 144, No. 10021-9193/80/10/0238/09$02.00/0

Effects of Carbon Source and Growth Rate on Cell WallComposition of Bacillus subtilis subsp. nigerFRED J. KRUYSSEN,t WIM R. DE BOER,4 AND JAN T. M. WOUTERS*

Laboratorium voor Microbiologie, Universiteit van Amsterdam, 1018 WS Amsterdam, The Netherlands

A study was made to determine whether factors other than the availability ofphosphorus were involved in the regulation ofsynthesis ofteichoic and teichuronicacids in Bacillus subtilis subsp. niger WM. First, the nature of the carbon sourcewas varied while the dilution rate was maintained at about 0.3 h-'. Irrespectiveof whether the carbon source was glucose, glycerol, galactose, or malate, teichoicacid was the main anionic wall polymer whenever phosphorus was present inexcess of the growth requirement, and teichuronic acid predominated in the wallsof phosphate-limited cells. The effect of growth rate was studied by varying thedilution rate. However, only under phosphate limitation did the wall compositionchange with the growth rate: walls prepared from cells grown at dilution ratesabove 0.5 h-1 contained teichoic as well as teichuronic acid, despite the culturestill being phosphate limited. The wall content of the cells did not vary with thenature of the growth limitation, but a correlation was observed between thegrowth rate and wall content. No indications were obtained that the compositionof the peptidoglycan of B. subtilis subsp. nigerWM was phenotypically variable.

The cell wall of Bacillus subtilis and relatedbacilli such as B. licheniformis and B. stearo-thermophilus consists mainly of peptidoglycanplus one or more anionic polymers, teichoic andteichuronic acids (for reviews, see 2, 9). Altera-tions in the relative proportions of these anionicpolymers have been, until now, one of the fewwell-documented examples of phenotypic varia-bility in the composition of bacterial cell walls.The effect of the metabolism of phosphate oncell wall composition is clear: teichoic acid, thephosphorus-containing anionic polymer, is pres-ent under all growth conditions, except when thephosphate concentration in the growth environ-ment is low and is limiting the growth rate; thenteichuronic acid (a non-phosphorus-containingpolymer) is observed in the wall (8). With B.licheniformis, the synthesis of teichoic acid issimilarly regulated; however, teichuronic acidalso is present (though in small amounts) underconditions of phosphate excess (12, 17).Most research into the phenotypic variability

of bacterial cell walls has been performed withcultures of B. subtilis subsp. niger. This workhas resulted in an insight into the structure ofits anionic polymers (teichoic acid being a glu-cosylated glycerol phosphate polymer [6, 8] andteichuronic acid being a polymer consisting of

t Present address: Department of Biochemistry and Micro-biology, University of Victoria, Victoria, B.C., V8W 2Y2, Can-ada.

t Present address: Gist-Brocades N.V., Microbiology Re-search Laboratory, 2600 MA Delft, The Netherlands.

alternating glucuronic acid and N-acetylgalac-tosamine [8]) and in some understanding of theregulation of syntheses of both polymers in re-lation to their possible functioning in cationtransport (10). Moreover, it has led to a highlyspeculative regulation scheme based upon reg-ulation of activity of the enzyme(s) of the bio-synthetic pathway of one of the anionic poly-mers by the concentration of an intermediate(s)of the other (9).We wished to investigate the influence of fac-

tors other than the concentration of Pi on thesynthesis of the anionic polymers and decidedalso to use B. subtilis subsp. niger as the testorganism. In this article we report on the effectsof varying the growth rate and the nature of thecarbon source on the wall composition. All re-sults were obtained with steady-state culturesgrowing in a chemostat.The vast majority of the experiments bearing

on the subject that are described in the literatureentail the use of glucose-grown organisms. Wehave used here glycerol, galactose, and malate,as well as glucose. Figure 1 shows the pathwaysleading from these compounds to the variouswall polymers in B. subtilis subsp. niger. Reac-tion 1 in Fig. 1, in which glycerol phosphate istransposed into CDPglycerol, is specific for thepathway ofbiosynthesis ofteichoic acid, whereasreaction 2, the oxidation of UDPglucose toUDPglucuronic acid, and reaction 3, the epimer-ization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, are specific for the tei-

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CELL WALL COMPOSITION OF B. SUBTILIS 239

churonic acid biosynthetic pathway in this or-ganism. The activity (or synthesis) of these cy-toplasmic, pathway-specific enzymes is generallyconsidered to be a target of the mechanismsregulating the biosynthesis of the respective pol-ymers (1, 12, 17, 23).The metabolic distances between the carbon

source and the polymeric wall compound ulti-mately formed differ widely when the varioussubstrates are compared, as is evident from Fig.1. This implies that the actual activity of the"target" enzymes (determined by their specifickinetic properties and the levels of genetic andallosteric controls) may vary considerably, de-pending on the carbon source used, and conse-quently the relative rate of biosynthesis of bothpolymers also may vary.Growth rate is another factor possibly in-

volved in the control of biosynthesis of anionicpolymers. Two types ofqualitative change in thecell wall of B. subtilis with growth rate havebeen reported. Both refer to a change in teichoicacid content of the wall. First, Ellwood (8) ob-served an increase in teichoic acid from 50 to75%, at the expense of peptidoglycan, under

magnesium limitation at dilution rates between0.1 and 0.3 h-1. Second, Wright and Heckels (33)found, with B. subtilis W23, that under phos-phate limitation teichoic acid was synthesized ata dilution rate of 0.3 h-1, in contrast to the datathey obtained with cells grown at a lower dilu-tion rate. At the higher growth rate teichoic acidappeared to replace the teichuronic acid origi-nally present in the walls. We have verified thesefindings, and analyzed the walls isolated fromorganisms which had been grown at widely dif-ferent growth rates.

(The results reported here are taken from thePh.D. dissertation by F. K. [University of Am-sterdam, Amsterdam, The Netherlands 1979].)

MATERIALS AND METHODSStrain. B. subtilis subsp. nigerWM (white mutant)

is a spontaneously occurring reversible variant strainfrom B. subtilis subsp. niger (ATCC 3972). Wild-typecultures invariably give rise to some white colonieswhen plated on peptone agar plus 1% glucose. These"white mutants" were used throughout the experi-ments.Growth conditions. Continuous cultures were

maintained in either a custom-built Porton-type

---., ...-TAGalactose - Goloctose- I-P )gUDPGal O <

Glucose -Glucose-6- P- Glucose-I- P-->-UDPGlc -y' UDPGIcUA

3 ?_TUAFructose- 6-P .- UDPGlcNAc -UDPGoINAc

Fructose-I1,6- dip

Glycerol DHF /-PG

Glycerol-3-PII/ UDPMurNAcPEP pentopeptide

CDPGly

e Mo late

TAFIG. 1. Metabolic routes in B. subtilis, leading from various carbon sources to the ultimate precursors of

the wall polymers peptidoglycan (PG), teichoic acid (TA), and teichuronic acid (TUA). Polymerizing,membrane-bound reactions are represented by bold arrows; the broken arrows indicate that more enzymereactions are involved. The enzymes mentioned in the text are designated by the following numbers: (1)CDPglycerol pyrophosphorylase (EC 2.7.7.39); (2) UDPglucose dehydrogenase (EC 1.1.1.22); (3) UDPacetyl-glucosamine 4-epimerase (EC 5.1.3.7); (4) glycerol-3-phosphate dehydrogenase (NAD-independent; EC1.1.99.5); (5) glycerol dehydrogenase. Abbreviations: DHA(P), dihydroxyacetone (phosphate); GAP, glyceral-dehyde 3-phosphate; PEP,phosphoenolpyruvate; CDPGly, CDP glycerol; UDPGlc, UDPglucose; UDPGlcUA,UDPglucuronic acid; UDPGkcNAc, UDP-N-acetylglucosamine; UDPGalNAc, UDP-N-acetylgalactosamine;UDPMurNAcpentapeptide, UDP-N-acetylmuramic acid, carrying a side chain with the composition L-Ala.D-Glu.DAP.D-Ala.D-Ala.

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240 KRUYSSEN, DE BOER, AND WOUTERS

chemostat or in an L.H.-chemostat (L.H. EngineeringCo., Ltd., Stoke Poges, England) with working volumesof 0.5 or 0.6 to 1.0 liter, respectively. The temperaturewas regulated at 370C and the pH was controlled at7.0 (by the addition of 2 N NaOH); polypropyleneglycol (Fluka AG, Basel, Switzerland) was added atregular time intervals as an antifoaming agent (finalconcentration, about 0.1%, vol/vol). The medium com-position was based upon the recipe of Evans et al. (11).When glucose or glycerol (used in equal amounts ona weight basis) was the sole carbon source, 75% of theconcentrations reported-by Evans et al. (11) wereroutinely used. When malate was used, the concentra-tions of the mineral compounds were lowered to one-half thoge specified above, D,L-malate being added toa concentration of either 7 or 21 g/liter, for carbon-limiting and carbon-excess conditions, respectively.The complete media were sterilized for 60 min at1200C (after adjustment with NaOH to a pH of about6 when glycerol or malate was the carbon source).Glucose was autoclaved separately for 20 min at 1100Cbefore it was added to the bulk sterilized medium.When growth was to be initiated in a glucose-basedmedium, the mineral medium in the vessel was inoc-ulated with 50 ml of a nutrient broth culture. Themedium pump was started immediately and set at adilution rate of 0.1 to 0.2 h-'. When glycerol or malatewas the prospective carbon source, a starter culturewas transferred serially into media containing reducedamounts of nutrient broth, but supplemented with thecarbon source in question. Once growth had started,daily routine checks were carried out to control thepurity of the culture (light microscope, Gram staining,plating on solid media), the dilution rate, and thesteady state, which was assumed to have been enteredafter five generations at a certain dilution rate (by Ew0and dry weight measurements; see below). Occasion-ally, the nature of the limitation was verified by mea-suring s (that is, the concentration of the growth-limiting nutrient in the extracellular fluid) and somenonlimiting nutrients (e.g., magnesium, phosphate, orcarbon source; see below). The experiments involvingvariation of the dilution rate were conducted as fol-lows. Some 3 or 4 days after inoculation and growth ata dilution rate of about 0.2 h-', the first sample waswithdrawn. From then on, the dilution rate was raisedin small steps (increments of c0.05 h-'), until a growthrate was attained at which the next sample was due.

Sampling. Samples (<50 ml) were withdrawn di-rectly from the culture vessel by using a sample port.When larger quantities of cells were needed to preparecell walls, the culture overflow tubing was led througha glass vessel placed in a glycerol bath which wasthermostated at 1000C. This was arranged in such away that the average residence time of the cells ex-posed at this temperature could be set at about 20min, independent of the dilution rate, before theyflowed over into an ice-cooled reservoir. By means ofthis procedure, cell lysis during harvest was preventedto a large extent. After the cells had been spun down,they were washed once with water and stored eitheras a wet pellet at -20°C or in a freeze-dried form.Preparation of cell walls. When performed on a

large scale, the procedure used for preparing cell wallswas identical to that described by De Boer et al. (6),

J. BACTERIOL.

except for the method of breaking of the cells. Here,the cells were divided into portions of 1.5 g, boiled in10 ml of water for 10 min, and transferred to 25-mlglass bottles containing 10 g of Ballotini beads (diam-eter, 0.1 mm). The cells were broken by shaking for 10min in a Braun MSK homogenizer operated at thehighest speed and fitted with a C02 cooling device.

For a small-scale preparation (dry weight of cells,about 50 mg), the procedure was essentially the sameas that described above. Some modifications wereneeded, however, to allow the scaling down. Water-washed cells were suspended in 9 ml of ice-cold dis-tilled water and boiled for 10 min. They were thentransferred to a glass bottle (ca. 15 ml) containing 1.5g of Ballotini beads (diameter, 0.1 mm) and 1 drop ofpolypropylene glycol to prevent excessive foaming.The ratio of cells to beads appeared to be critical:when a lower cell concentration was used, much morebreakage of the beads occurred, hampering the furtherpurification steps. The bottle (originally with an alu-minum screw cap, later with a Teflon cover) wasplaced in a specially designed adaptor in a BraunMSKhomogenizer, and the cells were shaken for 10 min atthe highest speed while being cooled by C02. About100% efficiency of breaking was obtained under theseconditions, as observed microscopically. The beadswere removed by sucking the suspension through asintered-glass filter (G3). Filter and vial were rinsedwith ice-cold buffer (50 mM Tris-hydrochloride-10mM MgCl2, pH 7.5). The washes and the originalsuspension were pooled and centrifuged for 20 min at17,000 x g. The pellet was suspended in 5 ml of bufferand transferred to a 10-ml centrifuge tube, in whichthe wall preparation was held during all the subse-quent purification steps. After the addition of 0.2 mlof DNase (1 mg/ml; Boehringer, Mannheim, WestGermany) and 0.3 ml of RNase (1 mg/ml; also fromBoehringer), the suspension was incubated for 30 minat 370C; then 0.5 ml of trypsin (6 mg/ml; Merck AG,Darmstadt, West Germany) was added, and the incu-bation was continued for another 30 min, with inter-mittent shaking. The suspension was made 2% withrespect to sodium dodecyl sulfate and was incubatedat 600C for 30 min; then the preparation was centri-fuged at 17,000 x g for 20 min. The resulting pelletwas suspended in distilled water, by use of a Teflonpestle that fitted the centrifuge tube, and washedanother two times with water, prior to final resuspen-sion in 5 ml of distilled water. The "purified walls"were stored at -20°C. Up to 16 samples could beprocessed this way in 1 day.

Hydrolysis of cell walls. Cell walls prepared ineither of the two ways described above were hydro-lyzed prior to analysis of various compounds (seebelow). For this hydrolysis, 5 to 10 mg of walls (ifnecessary, after a freeze-drying step) were transferredto a glass-stoppered tube (10 ml), and 2 ml of 6 N HCl(Suprapure) was added. The tube was locked tightlywith Teflon tape and a stainless-steel spring bolt andwas incubated for 6 h at 1000C in a thermostatedglycerol bath. After hydrolysis, the samples were ly-ophilized repeatedly over NaOH, and finally the ma-terial was dissolved in 2 ml of distilled water andstored as "cell wall hydrolysate." Amino sugars aresubject to destruction during the hydrolysis procedure.

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CELL WALL COMPOSITION OF B. SUBTILIS 241

To assess the extent of this process, we included theproper standards in every run. Variable degrees ofdestruction, both per compound and per experiment,were found (ranging between 20 and 40% loss). Cor-rection for these losses was made only when the resultsof the determinations were to be used to calculate theconcentration of the polymers present in the cell wall

prior to hydrolysis.Analytical procedures. Cell dry weight was de-

termined on duplicate 10-ml samples. These were cen-trifuged at 12,000 x g for 10 min, washed once withwater, and dried at 95°C for 18 h.The amount of Pi in culture fluids and media was

determined as described by Chen et al. (4). Magnesiumcontent was determined with a Perkin-Elmer model303 atomic absorption spectrophotometer. Glucoseand galactose were assessed with the commerciallyavailable specific reagent kits Glox and Galax (ABKabi, Stockholm, Sweden). Glycerol and malate were

determined enzymatically by standard proceduresbased on glycerol kinase and glycerol phosphate de-hydrogenase (32) and on malate dehydrogenase (15),respectively.

Total phosphorus in cell walls was determined as

described by Chen et al. (4); the oxidation procedurewas adapted to process small amounts of material.Therefore, samples containing maximally 150 mol ofphosphorus (in 25 to 500 pl) were dried in small glasstubes (10 by 75 mm) at 90°C for 18 h. Oxidation was

performed with 50 A1 of HC104 (70%) on a sand bathuntil the initially blackened sample was colorless. Du-plicate samples were oxidized; after the samples hadcooled to room temperature, reagents as prescribedwere added directly to the tubes. The residual per-

chloric acid did not interfere with the development ofthe color, and the results obtained were identical tothose obtained with the larger-scale oxidations byChen et al. (4), who used concentrated H2SO4 andHC104. Total carbohydrates in cell walls were deter-mined by using the anthrone reagent as described byeither Ashwell (3) or Jermyn (19). The latter methodallows the assessment of 10 times smaller amounts ofmaterial (up to 25 jug). Uronic acid was measured withthe hannine reagent (31). Protein was determined bythe method of Lowry et al. (20) with bovine serumalbumin used as a standard.

Glycerol content of cell wall hydrolysates was de-termined as described above after overnight incuba-tion of the hydrolysate with Escherichia coli alkalinephosphatase (Boehringer) at pH 9.0. Galactosaminewas determined with the aid of the Galax reagent (seeabove). Amino acids and amino sugars were deter-mined on an amino acid analyzer (Locarte, London,England). Samples were prepared by freeze-drying aportion of the wall hydrolysate (about 200 ,ug of walls)and dissolving the residue in an appropriate citratebuffer.

Calculation of cell wall yield and composition.The teichoic acid figures are routinely based on phos-phate determinations. Whenever tested, glycerol andglucose proved to be present in about equimolaramounts. Their ratio to phosphate was between 0.8and 1.0 (see also 6). Teichuronic acid values are basedon uronic acid and galactosamine detenninations (thelatter after correction for hydrolytic loss). The amount

of peptidoglycan was derived from the diaminopimelicacid content of the wall hydrolysates. To transformthe analytical results (all in nanomoles per milligramof dry weight) to percentages (wt/wt), we used thefollowing molecular-weight values of the repeatingunits of the respective polymers, based on literaturedata on their chemical composition: 900 for peptido-glycan (27), 320 for teichoic acid (6), and 400 forteichuronic acid (8, 33). Since in samples prepared bythe small-scale procedure the cell wall recovery couldnot be measured gravimetrically, as a consequence ofremnants of the breaking procedure (glass and alumi-num powder), the wall contents of the cells werecalculated as the weight sum of the three wall poly-mers. Most often, the measured dry weight of thelarge-scale cell wall preparations could be attributedonly to the three above-mentioned polymers. Small-scale preparations usually gave slightly higher recov-eries of these polymers on a cell-weight basis than wascalculated from the large-scale preparations on thesame samples. The wall hydrolysates appeared to con-tain invariably less than 3% protein, based on aminoacid analysis (though Lowry assays prior to hydrolysisoften suggested the presence of up to 12% protein inthe samples).

RESULTSEffect of glycerol and malate on cell wall

composition. Table 1 reveals the phenotypicvariability of the cell wall of Bacillus subtilissubsp. niger WM. Clearly, teichoic acid synthe-sis was strongly suppressed under conditions ofPi limitation. Teichuronic acid was then themain anionic polymer in the wall, and the tei-choic acid content amounted to between 3 and7%. This was true for all carbon sources tested.

TABLE 1. Influence of the carbon source upon thelimitation-dependent cell wall composition of B.

subtilis subsp. niger WM'Percent'

Limitation Carbon sourcePG TA TUA

Carbon Glucose 37 60 3Glycerol 37 57 6Malate 38 59 3

Magnesium Glucose 45 51 3Glycerol 51 47 3

Phosphate Glucose 43 4 54Glycerol 43 3 56Malate 46 4 50

a Cultures were grown at dilution rates between 0.2and 0.3 h-', as described in Materials and Methods.Cell walls were prepared by either of the two proce-dures mentioned in the same section; they were foundinvariably to make up between 15 and 20% of thecellular dry weight.

'Relative proportions of the polymers peptidogly-can (PG), teichoic acid (TA), and teichuronic acid(TUA) in the cell wall.

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242 KRUYSSEN, DE BOER, AND WOUTERS

Under Pi-excess conditions teichoic acid pre-dominated in the wall, teichuronic acid incor-poration being low.

It appeared (data not shown) that the extentto which the incorporation of teichuronic acidwas suppressed varied with each experimentwith the glucose-grown cultures. While generallyless than 5% of the wall was made up of teichu-ronic acid under these conditions, in some ex-periments up to 15% teichuronic acid was de-tected. Neither culture dry weight nor phospho-rus content of the cells in these experimentsdeviated from the values found in cultures dis-playing a low teichuronic acid content. Thus, itmay be concluded that anomalies in Pi con-sumption are not involved in this phenomenon.

Effect of galactose on wall composition.The influence which galactose exerts on cell wallsynthesis in bacilli was investigated by Forsberget al. (12). These authors showed that in a wild-type strain of B. licheniformis (NCTC 6346)teichuronic acid synthesis was stimulated bygalactose under conditions of Pi excess in a

growth medium based on glycerol. We sought toverify this effect with B. subtilis. Therefore,galactose was added to a (Pi-sufficient) glycerol-limited culture by replacing the glycerol mediumwith one which contained, beside 5 g of glycerolper liter, 3 g of galactose per liter. Analysesperformed on the cell-free culture fluid after 23h (ca. nine generations), when the culture wasconsidered to have entered a new steady state,indicated that the cells were still glycerol lim-ited, whereas about 60% of the galactose addedwas being consumed. Under these circumstancesno changes were observed in either dry weightof cells or cell wall composition (Table 2).Effect of growth rate on cell wall com-

position: Pi-excess cultures. The actualgrowth rate in chemostat cultures in steady stateis equal to the dilution rate imposed. The max-imal growth rate (glmax) in the chemically defmed

TABLE 2. Response of cell wall composition to theaddition ofgalactosea

Growth in Percent'presence ofgalactose (h) PG TA TUA

0 40 56 45.5 41 55 4

23 32 64 5a To a glycerol-limited culture (dilution rate, 0.26

h-1; 5 g of glycerol per liter), galactose was added to a

final concentration of 3 g/liter. At the times indicated,cells were harvested and subjected to the small-scaleprocedure to prepare cell walls. The recovery of cell

walls did not vary in the three samples (16% on a

cellular dry weight basis).b See Table 1.

J. BACTERIOL.

medium of the composition used here appearedto vary with the nature of the carbon source. Avalue of 0.75 h-' was found in experiments em-

ploying cultures growing on glucose. When glyc-erol was used as the sole carbon source, ,u,ax wasfound to be 0.65 h-1, whereas malate allowed amaximal growth rate of 0.45 h-'.When we compared the wall composition of

cell samples from a magnesium-limited culturegrown at various dilution rates, we did not detectvariations in the content of each of the main

polymers (Table 3). Identical results were ob-tained with carbon-limited cultures, irrespectiveof the carbon source used (data not shown).

Effect of growth rate on wall composi-tion: Pi-limited cultures. Figure 2a shows thedry weight of a Pi-limited culture of B. subtilissubsp. niger WM, growing on glucose, as a func-tion ofthe growth rate. The value ofthe maximalgrowth rate, obtained by extrapolation, wasfound to be ca. 0.75 h-1, as was reported before(see above).In contrast to the aforementioned constancy

in wall composition, changes that were depend-ent on the dilution rate were observed in Pi-limited cultures, as is exemplified by Fig. 2b.Obviously, the teichoic acid content of the wallsincreased, corresponding to a decrease in teichu-ronic acid content, whereas the peptidoglycancontent of the walls was constant throughoutthe experiment.

It was crucially important to know whetherthe appearance of teichoic acid at the higherdilution rates related to relief of the phosphatelimitation. Since the concentration of unusedsubstrate (s) is considered as a criterion of thelimitation, the increase of s, in the various ex-periments, with the dilution rate was measuredand is shown in Fig. 2a. The conclusion seems

TABLE 3. Effect of the dilution rate (D) on cell wallcomposition of B. subtilis subsp. niger WMgrown

under magnesium limitationaPercentb

D (h-')CW PG TA TUA

0.18 20 46 49 <50.32 14 49 43 <80.43 16 42 53 <50A9 11 38 60 20.63 10 42 56 30.67 8 42 56 20.77 10 47 50 3

a Cells were grown in a chemostat, and walLs wereprepared by the small-scale procedure described inMaterials and Methods.

b CW, wall recovery (on a cellular dry weight basis),calculated as the sum of the three wall polymers. PG,TA, and TUA: see Table 1.

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CELL WALL COMPOSITION OF B. SUBTILIS 243

DRYWEIGHTI (g/1)

0.2 03 04 05 06 0 7 0S

° b

0

ii,* s o. I

//-

03 04 05 0.6 0.7 0.6

DILUTION RATE (h-')

FIG. 2. Dry weight, gp,, and cell wall composition in relation to the dilution rate underphosphate limitation.B. subtilis subsp. niger WM was grown until steady state was reached at the dilution rates indicated (inputwas ca. 1.5 mM Pi; glucose as the carbon source). Cell walls were prepared by the small-scale proceduredescribed in Materials and Methods. Symbols: *, dry weight; 0, s,, residual concentration of the limitingsubstrate in the cell-free culture fluid; E, peptidoglycan; 0, teichoic acid; V, teichuronic acid.

justified that the cells harvested at dilution ratesbetween 0.5 and 0.7 h-1 had been growing underconditions ofphosphate limitation but neverthe-less displayed a significant amount of teichoicacid in their walls.Phenotypic variations in the wall con-

tent. We were not able to detect limitation-specific variations in the wall content of thecells: invariably 15 to 20% of the dry weight ofthe cells was recovered as wall polymers (see thefootnotes of Tables 1 and 2), either as theweighed sum ofpeptidoglycan plus anionic poly-mers in the case of large-scale preparations or as

the calculated sum of the polymers in small-scale preparations (for calculations, see Mate-rials and Methods).From experiments described in the literature,

it emerged that the higher the rate at which an

organism is growing, the larger its size. This hasbeen demonstrated for gram-negative species(25, 30) and for bacilli (16, 22, 24, 29), though

some controversy exists with respect to the sit-uation in B. subtilis (28). In accord with thisexperimentally observed rule is the inverse re-

lationship between growth rate and wall contentwhich we observed occasionally (see, for in-stance, Table 3) and which was reported also byEllwood (8) for B. subtilis subsp. niger (at dilu-tion rates below 0.4 h-'). Often however, espe-cially in experiments using phosphate-limnitedcultures, we were not able to detect such strikingvariations in the wall content (data not shown).Phenotypic variations in peptidoglycan

composition. It is generally accepted in theliterature that the composition of peptidoglycan,with some well-known exceptions, is phenotypi-cally invariable (for a review, see 26). Two re-

ports in which chemostat-cultured bacilli were

analyzed did, however, indicate variations de-pending on the growth conditions, measured as

variations in the mutual ratios between the re-

spective peptidoglycan constituents (9, 12).

sp (mM)

30

20

I o

0

60

501-

401-

w

g-34

0-301

201.

to

02

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244 KRUYSSEN, DE BOER, AND WOUTERS

Here, we report the composition of the pepti-doglycan prepared from B. subtilis subsp. nigerWM cells which had been cultivated under thesets of conditions specified previously. Table 4shows the results as the molar ratio towarddiaminopimelic acid of the various constituentsin the wall hydrolysates. In contrast to the find-ings of Ellwood and Tempest (9), who reportedlarge variations in these parameters in B. sub-tilis W23 (e.g., alanine/diaminopimelic acid var-ied up to 28%, dependent on the limitation), wefound only minor variations which, consideringthe cumbersome procedures involved in produc-ing these data, are too small to be consideredsignificant.An exception to the invariable composition of

peptidoglycan proposed above may be the ratiobetween glucosamine and diaminopimelic acid,which apparently is significantly higher in pep-tidoglycan from carbon- and magnesium-limitedcells than in that from phosphate-limited ones.This difference, however, may be due to thepresence of a linking unit between teichoic acidand peptidoglycan, which has been shown, inmany organisms, to contain glucosamine (5).

DISCUSSIONIn the experiments described here, we have

considered the cell wall to be made of peptido-glycan and anionic polymers only. As was ex-plained in Materials and Methods, we had foundno indications for the presence of other poly-meric material in our. preparations, thoughamounts of protein, as found in B. subtilis 168by Doyle et al. (7), cannot be excluded. Clearly,though, wall components occurring in suchquantities (c1%) are not relevant in the studiescarried out here.

The results in this paper accord with theobservations on B. subtilis subsp. niger andother B. subtilis strains (9) regarding the effectof phosphate upon the quality of the anionicpolymer in the cell wall. It is clear, however, thatfor as yet unknown reasons the prevention ofteichuronic acid synthesis in phosphate-rich me-dia and at a low dilution rate (O0.3 h-') was notalways complete. This was, however, observedonly when glucose was used as the carbonsource. We are not at this point able to providea reasonable explanation for this phenomenon.By varying the carbon source we sought to

determine whether intermediary metabolismhad any influence on the phosphate-dependentregulation system of cell wall assembly.The metabolism ofglycerol is thought to occur

via an NADH-independent glycerol phosphatedehydrogenase (reaction 4 in Fig. 1), as wasproposed by Mindich (21), and not via glyceroldehydrogenase (reaction 5 in Fig. 1). The formerenzyme was indeed present in cells of B. subtilissubsp. niger WM in large quantities under allgrowth conditions, at least when glycerol waspresent in the medium (F. J. Kruyssen, Ph.D.thesis, University of Amsterdam, Amsterdam,The Netherlands, 1979). One might imaginethat, when glycerol is used as a substrate, thelevel of the intracellular pool of glycerol phos-phate will be high, even under conditions ofphosphate limitation, and thus allow teichoicacid synthesis to proceed alongside, or insteadof, teichuronic acid biosynthesis. Our data, how-ever, do not lend any support to this hypothesis:under conditions of phosphate limitation theincorporation of teichoic acid was suppressed tothe same extent in glycerol-grown cells as in cellsgrowing on glucose.

TABLE 4. Limitation-dependent variations in peptidoglycan composition, measured as the molar ratiosbetween the constituents and diaminopimelic acida

No. of Peptidoglycan component:Limitation Carbon source cul- Glutamic Muramic Glucosa-

tures Alanine acid acid mineCarbon Glucose, glycerol, malateb 8 1.52 (0.12) 1.01 (0.14) 0.83 (0.14) 0.91 (0.13)

Magnesium Glucose 9 1.43 (0.06) 0.94 (0.05) 0.84 (0.14) 0.93 (0.08)Glycerol 5 1.55 (0.05) 1.01 (0.12) 0.78 (0.09) 1.02 (0.07)

Phosphate Glucose 9 1.47 (0.05) 1.01 (0.07) 0.85 (0.11) 0.86 (0.03)Glycerol 6 1.53 (0.07) 0.98 (0.10) 0.80 (0.18) 0.83 (0.09)

a For every culture (dilution rate, ca. 0.2 h-') the average values of the molar ratios in the peptidoglycan weredetermined. To obtain the statistical parameters per lmitation, we averaged these values for the individualcultures (standard deviation between parameters is given in parentheses). The amino sugar figures are notcorrected for hydrolytic loss.

b All cultures were taken together, irrespective of the carbon source used, since the paucity of data did notpermit separate statistical processing.

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CELL WALL COMPOSITION OF B. SUBTILIS 245

Also, during growth on galactose and malate(when the balance between the activities of gly-colysis and gluconeogenesis is completelyshifted), the regulation of cell wall compositionwas observed not to be modified. It is not rele-vant to consider this question in greater detailhere; however, one is bound to conclude thatcell wall metabolism is, to a large extent, inde-pendent of the pathways of carbon-substratemetabolism. In the experiments describedherein, no influence of carbon metabolism onthe rate and quality of the process of wall syn-thesis could be detected. In comparison to thisindependence, the direct influence that phos-phate exerts on cell wall synthesis is the moreremarkable.Growth rate was observed to affect the wall

composition only in phosphate-limited cultures.The finding of Ellwood (8) that a high dilutionrate favored the synthesis of teichoic acid at theexpense of peptidoglycan in magnesium-limitedcultures could not be substantiated.The onset of teichoic acid biosynthesis which

we noticed in phosphate-limited cultures wasremarkable, since it was established that it oc-curred at dilution rates at which the existence ofa phosphate limitation was unequivocal (basedupon measurements of §p). In contrast, Wrightand Heckels (33) failed to draw this conclusionfrom a comparable experiment; using B. subtilisW23, they found a replacement of teichuronicacid by teichoic acid at a dilution rate of 0.3 h-',accompanied, however, by the presence of (fur-ther undetermined) phosphorus in the culturefluid.An obvious explanation for the effect we ob-

served is that there are growth rate-dependentchanges in the synthesis or the activity of theenzymes involved in the biosynthesis of precur-sors of the respective anionic polymers. How-ever, this explanation must be discarded since ithas been found recently (W. R. De Boer et al.,in preparation) that teichuronic acid could bedetected in the cell-free culture fluid, whichcould not be accounted for by wall turnover.This secretion of anionic polymer was most pro-nounced under conditions of high dilution rateand a limitation of phosphate, when teichuronicacid was found exclusively in the culture fluid(and not in the wall). This implies a controlmechanism acting at the level of cell wall assem-bly, which is responsible for the fact that thevarious anionic polymers are incorporated intothe wall in a ratio different from that in whichthey are synthesized.Research on the molecular mechanisms of

reactions involved in wall assembly is still at anearly stage (13). No data are yet available that

provide evidence as to how the incorporation ofone anionic polymer, instead of the other, maybe brought about. Thus, whether different en-zyme complexes exist, each catalyzing the cou-pling of a different anionic polymer to peptido-glycan, or whether there is only one, which canbe altered phenotypically in affinity or specific-ity towards the various polymeric substrates isunknown. Modifications in the activity of en-zymes involved in the terminal stages of wallassembly do occur, as was demonstrated byGlaser and Loewy (14) for B. subtilis and alsoby Ishiguro and Ramey for E. coli (18). Verylikely, compounds to enter such interactions arePs-derived intracellular metabolites. It should bementioned, however, that preliminary experi-ments carried out in this laboratory did not giveany indication of correlations between the poollevels of relevant intermediates and the cell wallcomposition (comparing carbon- and phosphate-limited cultures of B. subtilis subsp. niger WM,growing at different dilution rates; De Boer,unpublished data).

ACKNOWLEDGMENTSWe are grateful to Pieter Buijsman, Margreet de Nie, and

Diederick Meyer for expert technical assistance. Thanks arealso due to D. W. Tempest for a critical reading of themanuscript.

The work was supported by the Foundation for Fundamen-tal Biological Research (B.I.O.N.), which is subsidized by theNetherlands Organization for the Advancement of Pure Re-search (Z.W.O.).

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