JOURNAL OF BACrIOLOGY, Apr. 1972, p. 281-290Copyright i 1972 American Society for Microbiology

Vol. 110, No. 1Printed in U.S.A.

Regulation of the Bacterial Cell Wall: Analysisof a Mutant of Bacillus subtilis Defective in

Biosynthesis of Teichoic AcidR. J. BOYLAN, N. H. MENDELSON, D. BROOKS,' AND F. E. YOUNG

Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, NewYork 14642, and Department of Microbiology, University of Arizona, Tucson, Arizona 85721

Received for publication 27 October 1971

Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at thenonpermissive temperature resulted in a deficiency of teichoic acid in the cellwall. A decrease in teichoic acid synthesis coupled with the rapid turnover ofthis polymer led to a progressive loss until less than 20% of the level found inwild-type rods remained in spheres. Extracts of cells grown at 45 C containedamounts of the enzymes involved in the biosynthesis and glucosylation of tei-choic acids that were equal to or greater than those found in normal rods. Cellwalls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-L-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformationdemonstrated that the lesion responsible for these effects (tag-i) is tightlylinked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis.

During the past decade, efforts of many in-vestigators have resulted in the elucidation ofthe primary structure and biosynthesis of thecell wall of microorganisms (9, 16). The earlystudies of Salton and Marshall (18), whichwere extended by Young, Spizizen, and Craw-ford (28) and Warth (Ph.D. thesis, Univ. ofWisconsin, Madison, 1969), demonstrated therelative chemical simplicity of the cell wall ofBacillus subtilis. In view of the well-definedmechanisms of genetic exchange (transforma-tion and transduction) and the ability of thecells to carry out a complex morphogeneticevent (sporulation), B. subtilis was chosen as amodel system for studying the regulation ofsynthesis of the cell wall. Previous investiga-tions demonstrated that the genes regulatingthe glucosylation of teichoic acid, gtaA, gtaB,and gtaC (24, 26), and the structure of flagella(10) reside in the mid-portion of the chromo-some in a region which constitutes less than2% of the genome (26). In the present paper,structural, biochemical, and genetic studiesestablish that a temperature-sensitive Rod-mutant which forms spheres under nonpermis-

' Present address: Department of Medicine, University ofCambridge, Cambridge CB21QT, England.

sive conditions (1) is defective in the biosyn-thesis of teichoic acid and that the mutationlies in the same region of the chromosome asother genes regulating surface properties. Thedata suggest that loss of teichoic acid drasti-cally alters the structure of the cell surface,resulting in pheiotropic effects which influencecellular division and cellular morphology.

MATERIALS AND METHODSStrains of B. subtilis. All of the strains used

were derivatives of B. subtilis 168. Markers were in-troduced by congression (8, 15) either by B. Reilly(BR strains) or in our laboratory (Table 1). By con-gression, high levels of transforming deoxyribonu-cleic acid (DNA) were used to introduce markerswhich could not be selected directly into recipientstrains. Because saturating levels of DNA were used,nonlinked markers were introduced on differentDNA molecules. For example, an A- marker couldbe introduced into a B- strain by selecting B+ trans-formants and examining them for the presence ofthe A- marker.

Media. Spizizen's minimal medium supple-mented with 22 mM glucose, 5 mM MgSO4, 0.02%acid-hydrolyzed casein (Nutritional BiochemicalsCorp.), and 50 ;ig of the appropriate auxotrophicrequirements per ml was used as the basic growthmedium (GM medium) in this study. In experiments

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TABLE 1. Summary of strains

Strain Genotype designation Origin Parent strain Source

RUB1000 trpC2 tag-la Congression5 BRl9RUB1001 hisAl tag-i Congression BR19RUB1002 trpC2 gtaA 12a Congression BR19BR19 trpC2hisAl SB1 B. Reilly168 trpC2 thy168ts-200B trpC2 thy tag-I NTGC 168BY12 trpC2gtaAl2 168BR290 trpC2gtaB290 168 B. ReillyBY51 trpC2 gtaC51 168G-5 hisA I argC4 gtaA12 Congression 168 G. GrantBR85 trpC2argC4 Congression 168 B. ReillyBH21 trpC2hisAI cysB3 168

a The symbol tag denotes teichoic acid glycerol as contrasted to tar for teichoic acid ribitol; gta genes regu-late the glucosylation of teichoic acid (26).

b Markers introduced by transformation (15).c Nitrosoguanidine treatment of parent strain.

designed to label the teichoic acid with 32p, the cellswere grown in GM medium buffered with 0.05 M tris(hydroxymethyl)aminomethane (Tris), pH 7.2, con-taining only 1 mm phosphate (GM-P medium; 25).The GM medium was modified for transformationstudies. GM1 medium contained 100 ml of Spizi-zen's minimal medium supplemented with 22 mMglucose, 0.02% acid-hydrolyzed casein, 0.1% yeastextract, and 50 gg of amino acids per ml to supple-ment the auxotrophic requirements of the strains(Erickson and Copland, unpublished data). GM2medium was similar to GM1 medium with the ex-ception that CaCl2 and MgCl2 were added to bringthe final concentrations to 5 and 2.5 mM, respec-tively. The tryptose blood agar base (TBAB, Difco),antibiotic medium no. 3 (PAB, Difco), and minimalglucose agar (MGA) were prepared and utilized asdescribed previously (26).

Transduction. The procedures for preparing ly-sates for transduction and growth of the recipientstrains were modified from the method of Young etal. (26). When selecting either phage-resistant (Phar)or teichoic acid-producing (Tag+) recombinants, theplates were incubated for 3 hr at 30 C prior tooverlay with phage t25 or shift to 45 C, respectively.All experiments included a phage control and a cellcontrol. Transductants were picked with steriletoothpicks and subcultured on the medium used forselection before being examined for unselected mar-kers. In one experiment in which Tag+ recombinantswere selected, the clones were restreaked, and iso-lated colonies were examined for unselected mar-kers. No difference was found between these andclones examined in the usual way; therefore, trans-ductants were not cloned prior to analysis for unse-lected traits. Cotransduction of tag-i (teichoic acidglycerol) with prototrophic or Phar recombinants wasdetermined by replica-plating the subcultured cloneson TBAB at 30 and 45 C. Tag+ recombinants weregrown at 30 C before they were examined for auxo-trophic markers or phage resistance. Donor and re-cipient strains were included as controls.

Transformation. The procedure for isolation ofDNA was similar to that described previously (27).Development of competence and transformationwere carried out according to a method devised byR. Erickson and J. Copeland (unpublished data).Essentially, 10 ml of GM1 medium was inoculatedwith a small amount of the recipient strain in theevening (105 cells/ml) and left overnight withoutshaking at room temperature. In the morning, theculture was transferred, sterilely, to a flask and incu-bated with shaking (250 rev/min) at 37 C. The tur-bidity of the culture was followed in a Klett-Sum-merson colorimeter (filter no. 66) at hourly intervalsfor the first 2 hr, and at 15-min intervals thereafter.One Klett unit equals approximately 2 x 106cells/ml. Turbidity was plotted as a function of log2until growth departed from a linear relationship (T.).At 90 min after To the cells were diluted 1:10 intoGM2 medium and incubated for 60 min. Cells wereincubated with DNA (final concentrations rangingfrom 0.01 to 10.0 gg/ml) for 30 min at 37 C with aer-ation. Deoxyribonuclease (20 Ag/ml, WorthingtonBiochemical Corp.) was added to each tube, and thecells were diluted in minimal medium. Samples (0.1ml) were spread on appropriate plates. Transformedcolonies obtained from the lowest dilution of trans-forming DNA giving a minimum of 200 colonies perplate were examined for incorporation of unselectedmarkers in the same manner as transduced colonies.Preparation of cell walls. Cell walls were pre-

pared from strain RUB1000 (Table 1) grown in GMmedium for the times and temperatures indicated inthe tables. The cells were centrifuged at 10,000rev/min and washed once with minimal medium;the pellets were stored at -20 C. The harvestingsand centrifugations were carried out at 4 C to pre-vent autolysis.

Cells were ruptured in a Braun homogenizer (28)or in a Hughes press with the pressure maintainedover 1,000 lb. Cell walls were isolated, washed, andlyophilized as described previously (28).Determination of physical, enzymatic, and

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chemical properties of cell walls. The adsorptionof phage 4'25 to cell walls isolated from strainsgrown at 30 or 45 C was determined by incubatingcell walls (100 ,ug/ml) with 4'25 (17) for various timesat 30 C. Samples (0.1 ml) were removed, diluted 100-fold, and centrifuged at 8,000 rev/min; the superna-tant fraction was assayed for plaque-forming units(24).The activity of the cell wall-associated autolysin

N-acyl-muramyl-L-alanine amidase was determinedby turbidimetric analysis in (NH4)2CO3 (pH 8.9,0.025 M) at 30 C (23).The amino acid, amino sugar, phosphorus, and

glucose determinations were performed as describedpreviously for cell wall hydrolysates (24, 28). All datawere corrected for degradation of amino sugarsduring hydrolysis in 4 N HCl at 105 C for 11 hr (28).The protein concentration of cytoplasmic mem-

branes was determined by the biuret reaction in thepresence of sodium deoxycholate to solubilize themembranes (11).Assay of enzymes involved in biosynthesis of

teichoic acid. Cells were grown to the end of loga-rithmic phase in GM medium, harvested, washedonce in GM medium, and stored at -20 C untilneeded. All assays were done on the same prepara-

tion to preclude variation from culture to culture.Uridine diphosphate (UDP)-glucose: polyglycerol-teichoic acid glucosyl transferase (TAG transferase)was assayed by use of protoplast membranes isolatedfrom frozen pellets after treatment with lysozyme,deoxyribonuclease, and ribonuclease (2).

Cytidine diphosphate (CDP)-glycerol: phospho-glycerol transferase was assayed by use of thesame membrane preparation as for TAG transferase.A typical reaction mixture contained: CDP-[14C]-glycerol, 20 nmoles (approximately 60,000counts/min); MgCl2 *6H2O, 6 gmoles; Tris-hydro-chloride, pH 7.5, 10 umoles; enzyme [suspended in0.05 M N-tris(hydroxylmethyl)methyl-a-aminoethanesulfuric acid (TES)-NaOH buffer, pH 7, at a proteinconcentration of about 10 mg/ml], 0.05 ml; andglass-distilled water to a final volume of 0.11 ml.The mixture was incubated for 15 min at 37 C, andthe amount of polymer formed was measured by thefilter technique of Brooks et al. (2).

CDP-glycerol pyrophosphorylase was isolated bythe method of Shaw (19) through the first ammo-

nium sulfate fractionation (step 2). A typical reac-tion mixture contained: Tris-hydrochloride, pH 8.0,5 gmoles; MgCl2 6H2O, 1 gmole; disodium ethyl-enediaminetetraacetate (EDTA), 0.05 Amole; cyti-dine triphosphate (CTP; neutralized), 4 gmoles; in-organic pyrophosphatase, 5 units; DL-a-glycerolphosphate, 2.6 Mmoles; L-a-[14C]-glycerol phosphate,1 yCi (specific activity 20 mCi/mmole); enzyme (dis-solved in 5 mmoles of Tris-hydrochloride, pH 7.5, ata concentration of 10 mg of protein/ml), 0.1 ml; andglass-distilled water to give a final volume of 0.28ml. The mixtures were incubated at 37 C for 10 min,and the reactions were stopped by heating for 10min in a boiling-water bath. The complete reactionmixtures were applied to Whatman 3MM paper andsubjected to descending chromatography in ethanol-

1 M ammonium acetate (2:1, v/v), pH 3.6 (2). Theradioactivity was located by cutting the chromato-grams and counting the pieces in a Beckman LS-230scintillation counter. The amount of CDP-glycerolformed was calculated as a percentage of the ra-dioactivity recovered from a reaction mixture inwhich the enzyme had been inactivated by boilingprior to the incubation period.Turnover of teichoic acid. To label the teichoic

acid with 32p, strain RUB1000 was incubated for 15hr at 30 C in GM-P medium and then diluted 100-fold into GM-P medium containing 2.5 ACi of 32p(International Chemical and Nuclear Co.) per ml.After growth to late log phase at 30 C with aeration,31P (1 mmole/ml) was added to the culture. The cellswere grown for an additional 30 min at 30 C, filteredthrough a type HA filter (0.45 Aim; Millipore Corp.),washed three times with prewarmed GM medium,and resuspended in GM-P medium at 30 or 45 C at30 Klett units. At transfer, greater than 93% of the32p was bound to the cells. Samples (1.0 ml) wereremoved from these cultures at various times and fil-tered through type HA filters. After determination ofthe radioactivity of 0.1-ml samples of the filtrate bythe method of Haviland and Bieber (12) in aBeckman LS-230 scintillation counter, the re-mainder was added to a column (50 by 3 cm) of 4%Sepharose (Pharmacia Fine Chemicals, Inc.). Theteichoic acid was separated from nucleic acids byelution with potassium phosphate buffer (pH 8.0, 10mM) with a flow rate of 30 ml/hr (25). The radioac-tivity and the optical density at 220 and 260 nm ofthe fractions were determined. As shown in Fig. 1,only two peaks were obtained. Peak A is typical ofthe elution profile of teichoic acid (25). The 260 nmadsorption of the fractions yielding peak B suggests

-

4

30- 040 50 60 70 80 90

FRACTION NUMBER

FIG. 1. Chromatography of B. subtilis RUB1000medium on 4% agarose. B. subtilis RUB1000 waslabeled with 32p, in GM-P medium at 30 C, washed,and then grown in GM-P medium at 45 C for 2 hr asdescribed in Materials and Methods. The cells werefiltered, and a sample of the filtrate was chromato-graphed on a column (3 by 50 cm) of 4% agarose andeluted with 10 mM phosphate buffer (pH 8.0) at aflow rate of 30 ml per hr. Symbols: 0, radioactivity;0, absorbancy at 220 nm; U, absorbancy at 260 nm.

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that they contain nucleotides. The radioactive frac-tions yielding peak B were pooled, lyophilized,treated with deoxyribonuclease and ribonuclease (50,gg/ml), and rechromatographed on the same Seph-arose column. This procedure did not alter theelution, suggesting that these fractions containednucleotides which are eluted near the internalvolume of the column. The proportion of radioac-tivity in macromolecular teichoic acid in the super-natant fractions taken at times other than 60 or 120min was determined by multiplying the total ra-dioactivity in the supernatant fractions by the av-erage percentage of radioactivity that was in peak Aafter 60 and 120 min of incubation at either 30 or 45C.

Materials UDP-14C,-glucose (uniformly labeled,237 mCi/mmole) and L-a-14C-glycerol phosphate (20and 26.5 mCi/mmole) were purchased from NewEngland Nuclear Corp. CDP-glycerol was preparedby the following procedure. CDP-glycerol pyrophos-phorylase was extracted from B. subtilis 168 by themethod of Shaw (19). The reaction mixture con-tained: Tris-hydrochloride, pH 8.0, 150 ptmoles;MgCl2 - 6H2O, 20 jsmoles; disodium EDTA, 1.6Amoles; CTP (neutralized), 39 Mmoles; DL-a-glycerolphosphate, 13 umoles; inorganic pyrophosphatase, 60units; L-a-14C-glycerol phosphate (26.5 mCi/mmole),50 uCi; enzyme (dissolved in 5 mM Tris-hydrochlo-ride, pH 7.5, at a concentration of 10 mg ofprotein/ml), 1.0 ml; and glass-distilled water to givea final volume of 1.35 ml. The mixture was incu-bated at 37 C for 60 min and then subjected to de-scending chromatography on sheets of Whatman3MM paper in ethanol-i M ammonium acetate, pH3.6 (2: 1, v/v; 2). The area corresponding to the CDP-glycerol was located with a paper chromatogramstrip scanner (Nuclear-Chicago Corp.) and cut out ofthe strip, and the paper was washed with ethanol toremove the ammonium acetate. The CDP-glycerolwas eluted with water and rechromatographed in thesame solvent. This procedure was repeated until theCDP-glycerol ran as one homogeneous spot co-chro-matographing with a CDP-glycerol standard. Thisprocedure yields approximately 10 umoles of CDP-'4C-glycerol with a specific activity of 3.3 x 106counts per min per Mmole.

UDP-glucose, inorganic pyrophosphatase, and a-glycerol phosphate were purchased from SigmaChemical Co. CTP and TES were purchased fromCalbiochem. Lysozyme, deoxyribonuclease, and ribo-nuclease were purchased from Worthington Bio-chemical Corp.

RESULTSChemical composition of cell walls of

strain RUB1000 at 30 and 45 C. B. subtilis168 (carrying tag-i and hisAl) was'grown at 30C. After an increase from 8 to 25 Klett units,half of the population was shifted to 45 C.Within 2 hr, the morphology of the culture hadchanged from rods to irregular spheres. Thecultures were harvested and the cell walls wereisolated, as described in Materials and

Methods. There was a fourfold reduction in theconcentration of phosphorus/mg of cell wall incells shifted to 45 C. This remaining phos-phorus could be due to residual teichoic acidfrom growth at 30 C. Because spores do notcontain teichoic acid (5, 18), Rod- spores weregerminated at 30 and 45 C. The cultures wereharvested at 137 Klett units, and the cell wallswere analyzed, as described in Materials andMethods. As shown in Table 2, the decreasesin the components of teichoic acid were 5-fold,12-fold, and 26-fold for glucose, phosphorus,and galactosamine, respectively. Since pre-vious studies have demonstrated a close corre-lation in the glycerol to phosphorus ratio inteichoic acid (Young, unpublished data), glyc-erol was not determined directly.

Concomitant with the decrease in teichoicacid at 45 C, there was an increase in the pro-portion of peptidoglycan in the cell wall. Whenthe total amount of alanine in the cell wall wascorrected for the amount of alkali-labile D-ala-nine, the molar ratio of alanine to glutamicacid decreased from 2.3 to 1.5 (24). Thus, themajor components of the peptidoglycan in thecell wall were in similar proportions at the twotemperatures despite the decrease in teichoicacid and the twofold increase in the relativeproportion of peptidoglycan. The residualamounts of phosphorus and glucose suggestthat teichoic acid biosynthesis was not com-pletely inhibited at 45 C. Cell walls were alsoisolated from strain RUB1000 2 hr after a shiftfrom 45 to 30 C. As shown in Table 2, therewas an increase in the components of teichoicacid and a decrease in peptidoglycan. Twostriking differences were observed, however.First, the molar ratio of alanine to glutamicacid was 1:1 instead of the expected 1.5:1.Second, the amount of galactosamine was two-fold lower than would be predicted from theamount of phosphorus in the cell wall.Enzymatic studies. The preponderant auto-

lytic activity in the cell walls of sporogenicstrains of B. subtilis 168 is N-acyl-muramyl-L-alanine amidase (4, 24). To investigate whetherthe loss of teichoic acid was associated with aconcomitant loss of autolysin, cell walls wereisolated from cultures grown at 30 and 45 C,and were assayed for bound autolysin, as de-scribed in Materials and Methods. Cell wallsfrom strain RUB1000 grown at 45 C did notautolyze (Fig. 2). The addition of crude autol-ysin from autolyzed cell walls from strainRUB1000 grown at 30 C resulted in lysis of the45 C cell walls, suggesting that the amount ofbound enzyme rather than modification ofsubstrate was the major difference between the

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TABLE 2. Analysis of cell walls of Bacillus subtilis RUB1000

30C 45C 45CC 30CMajor component

Amta Rb Amt R Amt R

Phosphorus ........................ 1,330 3.5 115 0.2 1,130 2.3Glucose ........................... 672 1.8 136 0.2 582 1.2Glucosamine ....................... 362 1.0 610 0.9 452 0.9Muramic acid ...................... 430 1.0 660 1.0 460 0.9Diaminopimelic acid ................ 380 1.0 641 0.9 460 0.9Glutamic acid ...................... 380 1.0 680 1.0 500 1.0Alanine ........................... 890 2.3 1,020 1.6 520 1.0Galactosamine ..................... 181 0.4 7 <0.1 70 0.1

a Nanomoles per milligram of cell wall."Molar ratio based on glutamic. acid equal to one. Spores of B. subtilis RUB1000 were germinated and

grown in GM medium at 30 or 45 C. Cells were harvested at 5 hr for cell wall analyses. A sample of the cellsgrown at 45 C was transferred to 30 C and grown for 2 hr for the 45 C , 30 C cell wall analysis.

e 0.9-0.8-

0.7

k Q5-

~0A4

Q3-

0.1-

0 30 60 90 120TIME (MIN.)

FIG. 2. Autolysis of cell walls of B. subtilisRUB1000. Cell walls of B. subtilis RUB1000 were

isolated from cells either grown continuously at 30 Cor grown at 30 C for 15 hr and then diluted andgrown at 45 C for 2 hr as described in Materials andMethods. The walls were suspended in 0.025 M

ammonium carbonate (pH 8.9) at a concentration of1.5 mg per ml and were incubated in a water bath at30 C. The walls of cells grown at 45 C were dilutedfurther to obtain an initial absorbancy at 600 nm of1.0 for a more accurate reading. Control tubes con-tained 30 C or 45 C walls boiled for 5 min in dis-tilled water. Symbols: 0, 45 C control; *, 45 Cwalls; 0, 30 C control; 0, 30 C walls.

two preparations of cell wall.The biosynthesis and glucosylation of tei-

choic acid require three enzymes. To deter-mine whether a deficiency in any of these en-

zymes could be responsible for the loss of tei-choic acid under the nonpermissive condition,cells were grown at 30 and 45 C, and crude

extracts were examined for the presence ofCDP:glycerol pyrophosphorylase, CDP:gly-cerol polyglycerol transferase, and TAG trans-ferase. Table 3 shows that all are presentin cell-free extracts of strain RUB1000 grownfrom spores at 30 and 45 C. Thus, the defi-ciency of teichoic acid in spheres cannot bedue to inactivation of these enzymes at 45 C.In fact, there is an increase in the specific ac-tivity of CDP:glycerol pyrophosphorylase andTAG transferase at 45 C.Adsorption of phage 4)25. Glucosylation of

teichoic acid is essential for adsorption ofmany of the phages that infect B. subtilis 168.Because secondary binding sites could beunmasked by the loss of polyglycerol phos-phates, cell walls isolated from strainRUB1000 at 30 and 45 C were used for adsorp-tion of 425. Cell walls obtained from cellsgrown at 45 C did not adsorb virus, whereaswalls obtained from cells grown at 30 C ad-sorbed 4)25 at apparently normal rates (Fig. 3).Turnover of teichoic acid. The teichoic

acid in the cell wall of the parent (BR19) andthe mutant (RUB1000) was labeled with 32p at30 C, as described in Materials and Methods.After a shift to 45 C, the parent released mac-romolecular teichoic acid at a linear rate for2.5 hr (Fig. 4). The parent incubated at 30 C orthe mutant grown at either 30 or 45 C showeda lag in the release of teichoic acid. After ap-proximately 1 hr, each of these populationsbegan to release teichoic acid more rapidly,and the rate approximated that of the parentincubated at 45 C. At both temperatures, lessteichoic acid was released by the mutant thanby the parent at corresponding times after theshift. The 2.5-hr samples indicated that allpopulations except the mutant grown at 45 Cwere still releasing teichoic acid. This observa-

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TABLE 3. Activity of enzymes involved in thesynthesis and glucosylation of teichoic acid

Specific activity"

Temp (C)a CDP glycerol CDP glycerol TAGpyrophos- polyglycerol transferasephorylase transferase

30 18.0 1.8 3.745 31.0 1.6 12.5

aTemperature at which the cells from which theenzymes were isolated were grown.

b Expressed as nanomoles of substrate convertedper minute per milligram of protein.

TIME (MIN.)

FIG. 3. Adsorption of phage $25 to cell walls ofB. subtilis RUB1000. Phage $25 (0.1 ml, 107 plaque-forming units per ml) and 0.1 ml of cell walls (025mg per ml) were added to 0.8 ml of PAB and incu-bated at 30 C with bubbling aeration. At intervals,0.1-ml samples were removed and centrifuged, andthe supernatant fluids were assayed for unadsorbedphage. Symbols: 0, 45 C walls; 0, 30 C walls.

tion may explain the higher residual teichoicacid in shift experiments at 45 C as comparedwith germination of mutant spores at the non-

permissive temperatures.Genetic studies. The generalized trans-

ducing phage PBS1 contains large fragmentsof bacterial DNA ranging up to 8% of the cellgenome (8). Therefore, initial localization ofthe tag-i mutation was accomplished byPBS1-mediated transduction of standard re-

cipient strains. Of the markers tested, linkagewas limited to hisAl. As in previous mappingstudies, in this region there was no linkage to

either of the outside markers cysB3 or argC4(Table 4).To determine whether only one locus was

responsible for the phenotype expressed in168ts-200B, tag-i was introduced into strainBR19 by congression (15). In this way, we con-

structed two strains (RUB1000 and RUB1001),

0

75--

50-

IK

2 3TIME (HRS.)

FIG. 4. Rate of turnover of teichoic acid in B. sub-tilis BR19 and B. subtilis RUBIOOO. B. subtilis BR19and B. subtilis RUB1000 were grown in the presenceof 32)P) at 30 C, chased with 32p,, and then treatedand grown at 30 or 45 C as described in Materialsand Methods. Filtrates of the 1- and 2-hr samples ofeach culture were chromatographed as described inFig. 1. The proportion of radioactivity in teichoicacid in the filtrates taken at other times was deter-mined by multiplying the total radioactivity of thefiltrate by the percentage of radioactivity that was inpeak A at 1 and 2 hr of incubation. Symbols: *,RUB1000 grown at 30 C; 0, BR19 grown at 30 C; 0,

RUB1000 grown at 45 C; 0, BR19 grown at 45 C.

TABLE 4. Cotransduction of tag-i with selectedauxotrophic markersa

Percentage of Rod- recombinants inRecipient transduction from auxotrophy to

strain prototrophy

cysB hisAl argC4

BH21 .......0 (0/208) 22 (63/285)BR19 35 (133/376)G-5 42 (87/208)BR85 0 (0/285)

a A PBS1 lysate from strain 168ts-200B was usedto transduce the strains listed above to independ-ence of their auxotrophic marker(s). The recombi-nants were then examined for incorporation of thetag-i gene.

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carrying either the trpC2 or the hisAl muta-tion and the tag-1 mutation. To confirm thelocation of the tag locus and to compare thebehavior of the tag-i lesion in strains 168ts-200B and RUB1000, lysates of PBS1 propa-gated on these strains were used for a three-point cross with strain G-5 (carrying hisAl,gtaA12) as the recipient. Transductants wereselected for His+ and examined for the incor-poration of the unselected marker traits Pharand Tag- (Table 5). The tag-1 and gtaA12markers were cotransduced with the hisA locuswith a frequency of 42% and 38% in strains168ts-200B and RUB1000, respectively. In bothcrosses, there was one transductant that wasphage-sensitive but temperature-insensitive,which indicated that the tag-i lesion was lo-cated to the right of the gtaA 12 lesion in B.subtilis.

Strain RUB1002 (carrying trpC2 andgtaA 12) was derived by transformation ofstrain BR19 with DNA isolated from strainBY12. A PBS1 lysate propagated on strainRUB1002 was used to transduce strainRUB1001 to either His+ or Tag+ (Table 6).Selection for phage resistance was not satisfac-tory because of the high number of phage-re-sistant spontaneous revertants (26). The tightlinkage between the gtaA12 and tag-i markerscan be seen in either His+ or Tag+ transduc-tants. For instance, in Table 6a, of 157 His+transductants that had also incorporated thegtaA12 lesion, 147 (94%) were also Tag+. WhenTag+ transductants were selected (Table 6b),gtaA12 was cotransduced at a frequency of98%. The relative frequency of recombinants inlines 1 and 4 of Tables 6a and 6b demonstratethat the gtaA locus is to the left of tag on thechromosome of B. subtilis.DNA-mediated transformation was used for

fine-structure analysis. Strain RUB1000 was

TABLE 5. Comparison of location of tag-i gene in168ts-200B and RUB1000

Selectonfo His+ No. of recombinantsSelection for His+ with PBS1 lysate from

hisAl gtaA12 tag-I 200B RUB1000

la 1 1 87 781 0 1 1

1 0 0 120 1291 0 1 0 0

a The designations 1 and 0 refer to donor and re-cipient genotypes, respectively (as described in 26).PBS1 lysates grown on either strain 168ts-200B orstrain RUB1000 were used to transduce strain G-5(hisAl argC4 gtaA12) to His+. The recombinantswere examined for gtaA12 and tag-i genotypes.

transformed from Rod- to Rod+ with DNApreparations isolated from three strains, BY12,BR290, and BY51 (Table 1), carrying differentgta loci. The transformants were examined forsensitivity to phage M25. The DNA isolatedfrom strain BY12 (carring gtaA12) was alsoused to select Phar transformants which weresubsequently examined for incorporation ofTag+. To minimize the production of pseudo-doubles in the transformants, limiting concen-trations of DNA (ca. 0.1 ,ug/ml) were used. Theresults of these crosses (Table 7) clearly dem-onstrate that tag-i is more closely linked togtaA12 than it is to the other two gta markers.The gtaA12, gtaB290, and gtaC51 loci werecotransformed with tag-i with frequencies of50%, 10%, and 1%, respectively. Phar (gtaA12)

TABLE 6. Transduction of RUBIOOI (hisAl tag-i)with PBSI lysate from RUB1002 (gtaA12 trp-2),

three-factor cross(a) Selection for His+

hisAlgtaA12 tag-1 No. of recombi-hisAlI gtaAi12 tag-I nants

la 1 1 1471 1 0 101 0 0 1781 0 1 0

(b) Selection for Tag+

tag-I gtaA12 hisAl No. of recombi-nants

1 1 1 3121 1 0 2481 0 0 111 0 1 2

a The designation 1 and 0 refer to donor and recip-ient genotypes, respectively.

TABLE 7. Transformation of RUB1000 to Tag+ orPhar with DNA isolated from three different phage-

resistant strains

Se- Unse- Se- Unse-Donor DNAa lected lected lected lectedmarker marker marker marker

Tag+b Phar c Pharb Tag+ c

BY12 (gtaA12) ... 409 202 100 78BR290 (gtaB290) 300 30 NDd NDBY51 (gtaC51) ... 400 3 ND ND

a Transforming DNA isolated from Tag+ strainscarrying one of the three Phar loci.

Number of colonies examined for selected mar-kers Tag+ or Pha'.cNumber of colonies containing the unselected

markers in clones selected for Tag+ or Phar.d Not determined.

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transformants examined for incorporation oftag-i also demonstrated a high degree oflinkage between the two markers (78%). Theseexperiments coupled with the earlier studies(10, 26) establish the most probable order forthe markers in this region, as shown in Fig. 5.

DISCUSSIONPrevious morphological studies of B. subtilis

have indicated that the cell wall of this orga-nism consists of a trilaminar structure withinner and outer discrete electron-dense areasseparated by a thicker homogeneous zone (6).At 45 C, the middle layer of the cell wall of themutant (RUB1000) is markedly thickened.There is a concomitant loss of the inner andouter lamella (6). The inner and outer lamellareappear after a shift to the permissive tem-perature. The following biochemical and ge-netic analysis of strain RUB1000 indicates thatthe morphological alterations under nonper-missive conditions are related to a loss of tei-choic acid: (i) a shift from the permissive (30C) to the nonpermissive conditions (45 C) isaccompanied by a marked reduction of tei-choic acid in the cell wall; (ii) germination ofmutant spores at the nonpermissive tempera-ture results in the formation of distortedspheres which contain less than 9% of the wild-type level of teichoic acid; (iii) the molar ratiosof the peptidoglycan in the wild type andmutant are similar at 30 and 45 C; and (iv)transfer of the locus from the original strain toother standard recipients by PBS1-mediatedtransduction or DNA-mediated transformationis accompanied by an association between theRod- phenotype and loss of teichoic acid fromthe cell wall. Furthermore, this locus is linkedtightly to the genes regulating the glucosyla-tion of teichoic acid. To date, the genes regu-lating the structure of the cell wall and itsappendages reside in a region that constitutesless than 2% of the genome. Cotransformationexperiments are in progress with DNA treatedwith nitrous acid to determine whether othercell wall loci reside in this region.The deficiency in teichoic acid at nonper-

missive temperatures cannot be explained bydeficiencies in the enzymes known to be in-

uvr i orgC4

hisAl tj9B A spoCl gtoC ie| metA

(IaAAtog (loC argOtfloB

FIG. 5. Partial map of the chromosome of Bacillussubtilis 168 showing the location of the tag gene.

volved in the biosynthesis of teichoic acid.Shaw et al. (20) also were unable to correlateenzymatic defects with the inability to synthe-size ribitol teichoic acid in a pleiotropic tei-choic acid-deficient mutant of Staphylococcusaureus 52A5. It will be important to determinewhether these represent fundamental modifi-cations in the organization of enzymes in thecytoplasmic membrane or whether there is amore trivial explanation, such as a change inthe acceptor that precludes the transfer of nas-cent chains of wall polymers. Nevertheless,transfer to restrictive conditions results in aloss of teichoic acid from the cell wall. Thefailure of biosynthesis to keep pace with therapid tumover of the cell wall, as demon-strated in labeling experiments, is probablyresponsible for this loss. For instance, after alag of 60 min in the mutant, the rate of releaseof teichoic acid at 45 C is similar to that in theparent at 45 C. Chromatography of the super-natant fractions from the wild type and mu-tant at 30 and 45 C demonstrates that 50% ofthe released radioactivity is in macromolecularteichoic acid and is not due to extensive degra-dation by enzymes such as teichoicase (Wise etal., Bacteriol. Proc., p. 49, 1971). However, thedifferences between the peaks in region B ofthe 32p and the absorbancy curves (Fig. 1) sug-gest that there may be some smaller fragmentsof teichoic acid in the supernatant fraction.The abrupt cessation of synthesis of teichoicacid after 2 hr observed in the mutant but notin the wild type suggests that biosynthesis ofteichoic acid may be necessary for continualtumover. In a closely related strain, B. subtilisW23, which contains ribitol rather than glyc-erol teichoic acid (14), there was a 50% turn-over of cell wall per generation. This observa-tion is substantiated by electron micrographs,which show fragmentation of the cell wall of B.subtilis 168 during growth (6). Studies in prog-ress should establish whether the N-acyl-mur-amyl-L-alanine amidase (23), a hexaminidase(4), or other enzymes are involved in this tum-over process.

Shift from the nonpermissive to permissiveconditions of growth results in the reinitiationof synthesis of teichoic acid; however, the ga-lactosamine which is associated with the tei-choic acid fraction does not increase as rapidlyas the other components of teichoic acid. Anal-ysis of the peptidoglycan during reversion in-dicates that significant remodeling of the cellwall occurs. After correction for the amount ofD-alanine in teichoic acid, the molar ratios ofthe major components in the peptide of thecell wall at 30 and 45 C are 1.5:1:1 for ala-

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nine, glutamic acid, and diaminopimelic acid,respectively, whereas during reversion themolar ratios of these compounds were 1:1: 0.9,respectively. Structural studies correlated withanalyses of and turnover of peptidoglycanshould distinguish between excision of preex-isting cross-bridges and merely the synthesisof non-cross-linked peptides.Although the rigid structure of the cell wall

has been attributed to peptidoglycan in B.subtilis, these observations suggest that defi-ciency of teichoic acid may result in profoundmorphological alterations in the cell wall. Hep-tinstall, Archibald, and Baddiley (13) sug-gested that teichoic acid may play a centralrole in maintaining the ionic environment forthe cytoplasmic membrane. In addition, thestudies of Tomasz (21, 22) have shown that themere substitution of ethanolamine for cholinein the teichoic acid of pneumococci can modifyautolysis of the cells and inhibit cellular sepa-ration. Other modifications, such as the chem-ical removal of the D-alanine residues on tei-choic acid in Streptococcus zymogenes, renderthe wall susceptible to lysis by its endogenousautolysin(s) (7). A further relationship betweenautolysins and teichoic acid is revealed in thisstudy. Cell walls of strain RUB1000 do notautolyze spontaneously at 45 C. However, theaddition of autolysin from lysates of cell wallsgrown at 30 C results in autolysis, indicatingthat modification of the substrate is not re-sponsible for this inhibition of autolysis. Onthe other hand, if cells of strain RUB1000 aresuspended in 1.2 M NaCl, they lyse more rap-idly than wild-type cells (Chatterjee andYoung, unpublished data). A similar phenom-enon was observed with the teichoic acid-defective strain of Staphylococcus aureus 52A5(Gilpin et al., Bacteriol. Proc., p. 49, 1971).Furthermore, previous studies have shown thatthe N-acyl-muramyl-L-alanine amidase istightly associated with teichoic acid (3). There-fore, the teichoic acid may selectively bindautolytic enzymes to the cell wall and modu-late their action. Thus, the distorted mor-phology of these cells could result from theexcess layers of peptidoglycan which accumu-late when teichoic acid and its associated au-tolysins are decreased. In view of these find-ings, it will be important to determine whetherteichoic acid plays a role in regulation of pep-tidoglycan synthesis or supramolecular organi-zation.

ACKNOWLEDGMENTSThis study was supported by grant GB24379 from the

National Science Foundation (F.E.Y.), Public Health

Service grant AI-10141 from the National Institute of Al-lergy and Infectious Diseases, (F.E.Y.) and grant GB17022from the National Science Foundation (N.H.M.). A portionof this investigation was supported by a President's Fellow-ship of the American Society for Microbiology (RJ.B.) and aNATO Postdoctoral Fellowship (D.B.).

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2. Brooks, D., L. L. Mays, Y. Hatefi, and F. E. Young.1971. Glucosylation of teichoic acid: solubilizationand partial characterization of the uridine diphospho-glucose:polyglycerolteichoic acid glucosyl transferasefrom membranes of Bacillus subtilis. J. Bacteriol. 107:223-229.

3. Brown, W. C., D. K. Fraser, and F. E. Young. 1970.Problems in purification of a Bacillus subtilis auto-lytic enzyme caused by association with teichoic acid.Biochim. Biophys. Acta 198:308-315.

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22. Tomasz, A. 1968. Biological consequences of the replace-ment of choline by ethanolamine in the cell wall ofPneumococcus: chain formation, loss of transforma-bility, and loss of autolysis. Proc. Nat. Acad. Sci.U.S.A. 59:86-93.

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24. Young, F. E. 1967. Requirement of glucosylated teichoicacid for adsorption of phage in Bacillus subtilis 168.Proc. Nat. Acad. Sci. U.S.A. 58:2377-2384.

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