JUlY 1991, p. 4341-4346 0021-9193/91/144341-06$02.00/0 Copyright C) 1991, American Society for Microbiology

JOURNAL OF BACTERIOLOGY,

Vol. 173, No. 14

Role and Expression of the Bacillus subtilis rodC Operon PATRICIA M. WAGNERt AND GEORGE C. STEWARTt* Department of Microbiology, The University of Kansas, Lawrence, Kansas 66045 Received 26 December 1990/Accepted 20 May 1991

The role of the rodC operon in Bacillus subtilis was investigated. The operon encodes two genes (rodD and rodC) necessary for the synthesis of the cell wail teichoic acid. Transcription of this operon is responsive to levels of phosphate and to concentrations of magnesium ions in the growth medium. This regulation of mRNA production corresponds to conditions that dictate the type of polymer that will be synthesized for the cell wall, i.e., teichoic or teichuronic acid. While the introduction of multiple copies of rodC was tolerated by the cells, multiple copies of rodD appeared to be lethal. The lethality of the rodD fragment was not exhibited if multiple copies of rodC were also present.

rodC mutants of Bacillus subtilis were first isolated and described by Rogers (19, 26). The initial characterizations of the rodCl mutant suggested this gene might be involved in cell division, since the mutant was temperature sensitive, forming normal-shaped rods at 30°C and cocci at 45°C (19, 26). However, recent mapping data of Karamata and colleagues indicated that the rodC operon is involved in teichoic acid synthesis and, more specifically, mapped to a region of the chromosome where the determinants for enzymes involved in the glucosylation of teichoic acids reside (19, 24). Teichoic acids are the phosphate-rich negatively charged polymers which constitute up to 50% of the gram-positive cell wall material. Not only do these polymers constitute bacteriophage receptors, but they also appear to play a role in the binding of magnesium ions and appear to be necessary for the structural stability of the cell wall (2-5, 9, 20, 26, 28, 30). However, under conditions of phosphate limitation, cells with teichoic acid in their walls will synthesize another negatively charged polymer, teichuronic acid, to replace the teichoic acid (2, 5, 9, 26, 28). Teichuronic acid, unlike teichoic acid, does not have a phosphate-rich backbone. The rodC operon has been cloned, and its nucleotide sequence has been determined (17, 18). Analysis of the nucleotide sequence of this operon revealed that it consists of two genes, rodD (gtaA) and rodC (Fig. 1). Furthermore, the sequence suggests that these two genes, which encode proteins of 78 kDa and 88 kDa, may be translationally coupled. The rodD gene is the first encoded by the operon, with the last base in the translational termination codon for rodD being the first base of the translational initiation codon of rodC. The message encoding these two proteins appears to be expressed from a rA-type promoter positioned directly upstream of the rodD gene. In this study, we have examined the relationship of the rodC operon to teichoic acid synthesis, the conditions under and the extent to which the operon is expressed, and the tolerance of B. subtilis for integration or duplication of the rodC region of the chromosome.

MATERIALS AND METHODS Bacterial strains. B. subtilis strains used were 168 trpC2 (1), 168 trp+ (1), and ROD113 (leuA8 rodCl) (19). Escherichia coli strains DH5a (F- recAI endAl gyrA96 thi-J supE44 hsdRJ7 (rK- MK+) relAl A(lacZYA-argF) U169 (4+80d lacZAM15) (Bethesda Research Laboratories) and LE392 (F- supE44 supF58 lacYl galT22 metBI trpR55 hsdR5J4 (rK- MK+) (as the source of plasmid DNA for B. subtilis transformation) (21) were also utilized. Plasmids utilized are presented in Table 1. Media and reagents. L broth or L agar (21) was used for the routine growth of E. coli, while tryptic soy broth (TSB; Scott Laboratories Inc.) was used for the routine growth of B. subtilis. Minimal medium for B. subtilis was made as previously described (11), with the following modifications: amino acids were added at a concentration of 25 p.g/ml, except L-tryptophan and L-leucine, which were added at a concentration of 50 ,ug/ml, to the appropriate cultures. The addition of amino acids replaced the addition of yeast extract and casein hydrolysate to the SP1 medium. Other modifications to the minimal medium were that phosphate-limiting medium used 1/10 the normal concentration of potassium phosphate, that high-salt-containing medium contained NaCl to a concentration of 0.6 M, and that high-osmolarity medium contained mannitol to a concentration of 0.11 M. Cells from an overnight culture were diluted 1:100 into the minimal medium (A500, 0.02 to 0.05) and grown to mid-logarithmic phase. Minicycle medium was 0.56 mM glucose-40 mM Tris-HCl (pH 7.5)-134 mM KCl-0.56 mM D-alanine, as established by Cloutier et al. (7). Transformation. Preparation of E. coli competent cells and plasmid transformation was by the method of Dagert and Ehrlich (8). B. subtilis competent-cell transformation was carried out essentially by the method of Erickson and

Copeland (11). Spore stock preparation. To generate spore stocks, the desired B. subtilis strain was streaked onto a TSA plate and incubated at room temperature for several days. The plate was scrubbed with sterile distilled water, and the suspension was placed in a sterile tube. The spore suspension was then heated at 85°C for 15 min to kill any remaining vegetative cells and stored in the refrigerator until needed. Before use, the suspension was reheated at 85°C for 15 min.

* Corresponding author. t Present address: Department of Microbiology, State University of New York at Buffalo, Buffalo, NY 14214. t Present address: Department of Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC

Isolation of RNA. RNA was isolated by the method of

Fujita et al. (14). A total of 100 ml of cells was grown to the desired absorbance and harvested. The pellet was resus-

29208. 4341

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E

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2179

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pAH101 FIG. 1. The physical map of the region of the clone pAH101 encoding rodD, rodC, and the putative promoter region. Restriction endonuclease sites: B, BamHI; Sc, ScaI; X, XbaI; P, PstI; K, KpnI; E, EcoRI. Pro designates the site of the promoter region, with the rightward arrow indicating the direction of transcription. P1 indicates the PstI site closest to the 5' end of the clone, P2 indicates the second PstI site, and P3 indicates the 3'-most PstI site. The opposing arrows (--) designate the position of the 15-bp inverted repeat. The subclones derived from pAH101 are indicated in Table 1.

pended in 6 ml of sucrose buffer (25% sucrose, 20 mM EDTA, 50 mM Tris-HCl [pH 7.5], with vanadylribonucleoside complex [Sigma Chemical Corp.] at a concentration of 10 mM and lysozyme at a concentration of 50 ,ug/ml) and incubated at 37°C for 10 min. After this time, proteinase K was added to a concentration of 10 ,ug/ml, followed by the addition of 10% sodium dodecyl sulfate (SDS) to a final concentration of 1%. This suspension was heated at 60°C for 15 min, and then 4 ml of cold sterile 5 M NaCl was added, and the lysate was incubated on ice for at least 15 min. The suspension was centrifuged, and the resulting supernatant was extracted with hot phenol. The resulting RNA was precipitated with 95% ethanol. The RNA pellet was then dried and suspended for treatment with RNase-free DNase I (Boehringer-Mannheim Biochemicals) in 0.1 M sodium acetate (pH 5.0)-S5 mM MgSO4 at 37°C for 45 min in the presence of 38 U of the RNase inhibitor RNAsin (Promega). The DNase was removed with phenol-chloroform extraction, and the resulting RNA was precipitated with 1/10 volume of 3 M sodium acetate (pH 5.0) and 3 volumes of 95% ethanol. Nick translation of probes. DNA was nick translated as described by Maniatis (21). Unincorporated nucleotides were separated from the labeled plasmid DNA on a Sephadex G-50 spin column. RNA dot blots. RNA was isolated and quantified by using TABLE 1. Plasmids utilized Plasmid

Insert (kb)

Vector (reference)

pAH120 pTW12 pTW873 pTW1250

4.0 PstI 2.7 BamHI-KpnI 4.0 PstI 2.7 BamHI-KpnI

pJH101 (12) pUC18 (29) pDH5060 (16, 23) pDH5060

rodC+ rodD+ rodC+ rodD+

pDH5060

rodD+ rodC+

pTW2056

5.3 BamHI-PstI3

Insert genotype

the A260. The RNA was prepared for loading onto the nitrocellulose membrane by adding 20% (vol/vol) 37% formaldehyde and 30% (vol/vol) 20x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) to the RNA sample and heating the sample at 65°C for 15 min. The samples were then loaded on the dot blot apparatus (Bethesda Research Laboratories) and filtered under a light vacuum. The nitrocellulose membrane was then removed, air dried, and baked in vacuo at 80°C for 60 to 90 min. Hybridization of RNA. Hybridization studies of RNA were carried out by using the conditions described by Smith and Summers (27). SEM sample preparation. Strains were grown overnight at 30°C to stationary phase in rich medium. The cells were then diluted 1:2 in the appropriate minimal medium and grown at either 30 or 42°C for 6 h. The cells were harvested, washed, and suspended in 0.75 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]). To this 0.75 ml, 0.25 ml of 10% glutaraldehyde was added and mixed. The cells were left standing in this solution at 4°C overnight and then rinsed sequentially with 30, 50, and 80% ethanol. These rinses were followed with two 95% ethanol washes and then two washes with absolute ethanol. The samples were critical point dried and coated with gold-palladium and viewed with a Philips 501 scanning electron microscope (SEM).

RESULTS Morphology of the rodCI mutant in response to environmental phosphate levels. If the rodC operon is indeed involved exclusively in teichoic acid synthesis, then growth of the rodCl mutant strain under phosphate-limiting conditions, a situation in which teichuronic acid would be produced instead of teichoic acid, should relieve the effect of the rodCI mutation. The rodCl mutant ROD113 was grown in minimal media under phosphate excess and phosphatelimiting conditions at 42°C for 6 h, and the cells were then prepared for SEM analysis. As can be seen in the micrographs in Fig. 2, mutant type coccus-shaped cells predominated in minimal medium containing excess phosphate at the elevated temperature of 42°C. However, the mutant phenotype was suppressed (i.e., normal rods predominated) in phosphate-limiting minimal medium at the same elevated temperature. At 30°C in phosphate excess and phosphatelimiting media, the normal rod shape was observed (not shown). The morphology of wild-type B. subtilis was unaffected by these alterations in medium composition or temperature. Thus, the limitation of phosphate in some way overrides the mutant phenotype normally expressed at 42°C in ROD113 cells. Concomitant with the decrease of phosphate in the medium is a decrease in the ionic strength of the medium. To determine whether the morphology of the mutant ROD113 cells in phosphate-limiting medium was simply due to the decrease in ionic strength, 0.6 M NaCl was added to the phosphate-limiting minimal medium to bring the ionic strength to a value equal to that of phosphate excess minimal medium. When the ROD113 cells were grown in this medium at 42°C, the mutant morphology was not suppressed (Table 2). Wild-type B. subtilis cells were unaffected by the change in medium. ROD113 cells were also grown in phosphatelimiting medium in which the osmotic strength was increased to a value equivalent to that of normal minimal medium (by the incorporation of mannitol, a sugar not utilized by B. subtilis, to 0.11 M). In this case, the cells grown at 42°C displayed the rod morphology. Thus, the shift in osmotic

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EXPRESSION OF rodC OF B. SUBTILIS

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FIG. 2. Electron micrographs of ROD113 grown in phosphate excess (A) and phosphate-limiting minimal media (B) at 42°C. Magnification, x 5,000.

strength of phosphate-limiting medium did

not

ies have demonstrated that the enzymes for the synthesis of teichoic acid are not present in B. subtilis cells under phosphate-limiting growth conditions (2, 9, 10, 13, 15, 28). Studies were thus done to determine whether the expression of the rodC operon was affected when cells were grown in

affect the

morphology of the ROD113 cells. ROD113 cells grown in phosphate excess in the presence of 0.11 M mannitol maintained the mutant coccus phenotype. Expression of the rodC operon. Previous biochemical stud-

TABLE 2. Morphology and media composition Normal phosphate at

Low phosphate at

Low phosphate + 0.6 M

Low phosphate + 0.11

temp ( C)

temp ( C)

NaCl at temp (°C)

M mannitol at temp (°C) 42 30

Cells

168 trpC2 ROD113

30

42

30

42

30

42

Rods Rods

Rods Cocci

Rods Rods

Rods Rods

Rods Rods

Rods Cocci

Rods Rods

Rods

Rods

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TABLE 3. Outgrowth potential

..

Relative survival'

Cells

Time (h)a

168 trpC2

0

1.0

3

0.77

6

0.65

0

0.03

3

0.66

6

0.95

4

ROD113

Time of incubation in TSB at 30°C following heat shock of spores. Relative survival = (CFU per milliliter at 42°C)/(CFU per milliliter at 30°C). a

b

4 .*

FIG. 3. RNA dot blot analyses. (A) RNA was isolated from mid-log-phase cells grown in phosphate excess minimal medium (NP) or phosphate-limiting minimal medium (LP). Amounts of RNA in micrograms loaded onto the filter were 1, 25; 2, 12.5; 3, 6.3; and 4, 3.1. The "R" samples were the NP RNAs treated with RNase A prior to being applied to the filter. The probe was nick-translated pTW12. (B) RNA isolated from B. subtilis grown in minimal medium (lane 1), phosphate-limited minimal medium (lane 2), minimal medium + 0.6 M NaCl (lane 3), and phosphate-limited minimal medium + 0.6 M NaCl (lane 4). RNA from each sample (30 ,ug) was loaded in its respective lane. The probe was nick-translated pTW12.

phosphate-limiting minimal medium. B. subtilis 168 wildtype cells were grown in phosphate excess minimal medium, phosphate-limiting minimal medium, phosphate excess minimal medium in the presence of 0.6 M NaCl, and phosphatelimiting minimal medium in the presence of 0.6 M NaCl. RNA was isolated from these cultures, blotted onto a nitrocellulose membrane and probed with a radioactive probe pTW12. This probe consisted of all of the rodD coding region, and 950 bp of the 5' end of the rodC gene. The dot blots (Fig. 3A) indicate the steady-state levels of rodC operon mRNA are higher in phosphate excess conditions than they are in phosphate-limiting conditions. In addition, the expression of the rodC operon is higher when the cells are grown in phosphate-limiting medium in the presence of 0.6 M NaCl than when the cells are grown in phosphatelimiting conditions (Fig. 3B). Effect of rodCl on spore germination and outgrowth. B. subtilis W23 and 168 have been shown to have extensive DNA homology (22). One region of variance between these two strains is in genes encoding the enzymes involved in teichoic acid synthesis. Strain W23 produces a ribitol-based teichoic acid, while 168 produces a glycerol-based teichoic acid (22). With B. subtilis W23, glucosylated teichoic acid was shown to be essential for spore outgrowth (25). If the genes encoded in the rodC operon are involved in the glucosylation of the glycerol teichoic acid of 168 as indicated by the mapping data, then the rodCl mutation could also have an effect on the germination efficiency of the ROD113 cells. To determine if the rodC gene is essential for outgrowth of the cells from spores, a spore stock of the rodC mutant strain ROD113 was generated. This spore suspension was diluted and plated in duplicate on TSA plates. One plate of each dilution was incubated at 30°C, while the other was incubated at 42°C. The remainder of the spore suspension was incubated with aeration in TSB at 30°C. Samples of this TSB suspension were removed at 3 h and at 6 h (a time when all spores should be germinated), and plated at 30 and 42°C.

The results shown in Table 3 indicate the rodC mutant cells cannot achieve their outgrowth potential at the higher temperature of 420C. ROD113 cells did not reach full outgrowth potential until 6 h, when all of the spores had most likely germinated prior to plating. The control B. subtilis 168 trpC2 cells were not affected in their ability to germinate at the higher temperature. To determine whether the block in outgrowth observed in the ROD113 mutant cells occurred at the outgrowth phase of the spores or at the actual germination stage, a minicycle of growth was established (7). The spores were placed in medium which would only allow germination to occur, but not outgrowth from that point. A sample of the spore stocks was diluted and plated on TSA plates and incubated at 30°C. This plating represented time T = 0. The remainder of the spore stocks was divided and incubated with shaking at 30°C or at 42°C in the minicycle medium. Samples (1 ml) were removed from these suspensions at various time points and heated at 85°C for 15 min. This incubation killed any spores that had germinated but would not harm the ungerminated spores present. The heat-treated samples were then diluted, plated on TSA, and incubated at 30°C. A decrease in the number of cells which were able to grow on the plate then indicated to what extent germination had occurred. Neither the ROD113 cells nor the 168 cells showed any inhibition of germination (Table 4). Therefore, the rodC mutation affects cells in their outgrowth ability once germination is complete. Introduction of the cloned rodC and rodD genes into B. subtilis. As the absence of RodC is lethal to the cells, it was of interest to determine the effect that multiple copies of this gene would have on B. subtilis. A clone bearing the rodC region of the operon was generated by using the multicopy plasmid pDH5060, which confers Cmr (23). This clone, pTW873, contained all of rodC, 1 kb of DNA 3' to rodC, and 1,389 bp of the 3' end of rodD. pTW873 was introduced into competent B. subtilis 168 trpC2 cells. The presence of multiple copies of rodC had no apparent effect on the B. subtilis cells, as transformants displayed normal growth rates on chloramphenicol-containing medium and displayed a normal morphology when viewed microscopically. Plasmid DNA was isolated and shown not to have undergone any TABLE 4. Efficiency of germination (%) at time T = 2a

Temp (°C)

168 trpC2

ROD113

ROD113 + pTW873

42 30

99.8

99.0 82.0

99.9 98.6

99.8

aNumbers given represent percentage of germination observed after a 2-h incubation relative to values obtained without prior incubation (T = 0). T = 2 is the time of incubation in germination medium following heat shock of spores.

VOL. 173, 1991

deletion event. Because rodC was cloned without its promoter in this construct, it was possible that rodC was not expressed from the plasmid. To test for expression of rodC, pTW873 was introduced into competent ROD113 cells, and transformants were incubated at 42°C, a temperature at which ROD113 cells are spherical because of the rodCl mutation. Microscopic observation of these transformants indicated that the mutant phenotype had been corrected, and, therefore, rodC+ was being expressed from pTW873. Expression was most likely due to read-through transcription from a promoter on the plasmid vector pDH5060. Because the presence of this plasmid (pTW873) caused no morphological or growth-rate change in wild-type cells, multiple copies of the rodC gene have no apparent deleterious effect on B. subtilis. The effect of multiple copies of rodD on B. subtilis was also examined. The rodD containing DNA was cloned into the vector pDH5060 as a BamHI-KpnI fragment that contained all of the rodD coding region and 570 bp of the 5' region of rodC. This construct, pTW1250, was utilized to transform competent B. subtilis. In this case, transformants were only isolated after 48 h of incubation at 37°C. These transformants could not be subcultured on antibiotic-containing media. This inability to maintain growth in the presence of pTW1250 suggests that the presence of rodD in multiple copies is in some way detrimental to the cells. Duplication of the rodD sequences was tolerated by the cells, since the BamHI-KpnI fragment could be successfully integrated into the chromosome at a second site, adjacent to rrnB (6). The putative promoter region sequences 5' to rodD (the 598-bp BamHI to Sall fragment) caused no deleterious effects when introduced into B. subtilis. The two genes rodD and rodC were cloned as a single contiguous fragment in the multicopy plasmid pDH5060. This clone, pTW2056 (Table 1 and Fig. 1), was used to transform competent B. subtilis, and transformants were obtained within 24 h. In this case, the transformants could be maintained. Thus, the presence of multiple copies of the rodD gene was not lethal to B. subtilis in this context. These results suggest that there is an interaction between RodD and RodC which is necessary for the cells to maintain viability. DISCUSSION The rodC operon had been previously cloned and sequenced (17, 18). These genes map to a region of the chromosome where many of the genes involved in teichoic acid synthesis reside (22, 24). Because it had been previously shown that the enzymes involved in teichoic acid synthesis are not present under conditions of phosphate limitation, SEM studies were performed on the mutant ROD113 cells. These SEM studies confirmed that the mutant morphology of ROD113 is not displayed by ROD113 cells when they are grown in phosphate-limiting conditions at 42°C. However, this effect could be reversed in phosphate-limiting medium by the addition of NaCl, suggesting the absence of mutant morphology could be dependent simply upon the ionic strength of the media. However, RNA dot blot experiments were performed which determined the level of expression of the rodC operon under the various growth conditions. These studies demonstrated that the rodC operon is expressed at lower levels when phosphate is limiting than when phosphate is in excess. In addition, the level of rodC expression increases in low-phosphate medium supplemented with NaCl.

EXPRESSION OF rodC OF B. SUBTILIS

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Our results correlate well with the suggestion of Baddiley (5). He determined that cells will switch from the synthesis of teichuronic acid to the synthesis of teichoic acid in response to the availability of magnesium ions to the cells. Therefore, in the presence of the excess of another cation (in this case, Na+), the cells sense a limitation of magnesium ions. When this occurs, the cells preferentially synthesize teichoic acid, the more negatively charged of the two polymers, regardless of the phosphate concentration of the media. This added negativity of the teichoic acid moiety supplies the cells with a strong binding site for cations and increases the cells' ability to bind much needed magnesium ions. The RNA dot blots indicate that the cells do indeed increase expression of the rodC operon under low-phosphate, high-salt conditions. Thus, the alteration in the morphology of ROD113 in low-phosphate medium observed in the SEM studies results from the cells no longer utilizing the mutant RodC protein. The reduction in expression of the mutant RodC protein, coincident with a switch to teichuronic acid synthesis, allows the cells to maintain a normal morphology. However, when conditions exist which allow the expression of mutant RodC proteins (phosphate excess or low-phosphate plus NaCl minimal media), teichoic acid synthesis and the mutant phenotype again prevail. Therefore, the SEM results correlate well with the RNA studies described. In addition, it was noted that when the mutant ROD113 cells were grown in phosphate-limiting medium supplemented with 0.6 M NaCl in the presence of excess magnesium ions, the mutant morphology was not displayed at 42°C (not shown). These results support the theory that the availability of magnesium ions plays a role in the expression of teichoic acid synthesis genes. The reduced expression of the rodC operon correlates with the absent mutant morphology of ROD113 in low-phosphate minimal medium at 42°C. Because the rodC operon was mapped to a region of the chromosome encoding enzymes involved in the glucosylation of teichoic acid, and because these enzymes had been shown in B. subtilis to be present during germination from spores, studies were done to determine the effect the rodC mutation had on the germination of ROD113 spores. The inability of the mutant ROD113 cells to reach their full outgrowth potential indicates that rodC is essential for germination of spores to cells. The presence of a wild-type rodC gene on a multicopy plasmid in the mutant ROD113 allows it to germinate efficiently once again. rodC thus appears to encode an enzyme involved in the glucosylation of the glycerol teichoic acid of B. subtilis 168. In further support of this idea, mutants which have been mapped to the rodD locus lack the ability to bind bacteriophage, a phenotype associated with cells with a nonglucoslylated teichoic acid (31, 32). Because a mutation in rodC has deleterious effects on the normal growth of B. subtilis, this fragment was introduced on the multiple copy plasmid vector pDH5060 (16, 23). This construct had no detrimental effect on B. subtilis. The fact that this construct could correct the rodCl mutation when introduced into ROD113 cells indicated that rodC was being expressed from this construct. Curing of the plasmid from a sample of these transformants restored the rodCl phenotype (not shown). This is indicative of rodC+ expression in trans rather than recombination between the chromosomal and plasmid alleles. The presence of rodD on a multicopy plasmid appeared to be lethal to B. subtilis cells. This lethality was not apparent if both rodD and rodC were introduced into B. subtilis on the same plasmid.

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This work

was

ACKNOWLEDGMENT supported by Public Health Service grant

16.

GM37990 from the National Institutes of Health.

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17. 18. 19. 20. 21. 22.

23. 24.

25. 26.

27.

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32.

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Role and expression of the Bacillus subtilis rodC operon.

The role of the rodC operon in Bacillus subtilis was investigated. The operon encodes two genes (rodD and rodC) necessary for the synthesis of the cel...
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