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Département de Microbiologie Fondamentale, Bâtiment Biophore, Université de Lausanne, Quartier UNIL-Sorge, CH-1015 Lausanne, Switzerland
Correspondence
Dimitri Karamata
dimitri.karamata{at}unil.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A-controlled promoter. The incorporation of the poly(GlcGalNAc 1-P) precursors by various mutants deficient in the synthesis of poly(glycerol phosphate), which is the most abundant WTA of strain 168, revealed that both WTAs were most likely to be attached to peptidoglycan (PG) through the same linkage unit (LU). The incorporation of poly(GlcGalNAc 1-P) precursors by protoplasts confirmed the existence of this LU, and provided further evidence that incorporation takes place at the outer surface of the protoplast membrane. The data presented here strengthen the view that biosynthesis of the LU, and the hooking of the LU-endowed polymer to PG, offer distinct widespread targets for antibiotics specific to Gram-positive bacteria.
Present address: 33, rue Prévost-Martin, CH-1205 Genève, Switzerland.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pooley et al., 1987; Shibaev et al., 1973; Soldo et al., 2003). During exponential growth in phosphate-replete medium, the phosphorus contained in this polymer is equivalent to 10 % of that present in poly(glycerol phosphate) [poly(GroP)], the most abundant and essential WTA of strain 168 (H. M. Pooley, personal communication).
Under laboratory conditions, the synthesis of poly(GlcGalNAc 1-P) is not essential for cell growth (Estrela et al., 1991), and its only known function is to serve as adsorption site for bacteriophages 
3T and 
11 (Estrela et al., 1991). The isolation and analysis of 3T-resistant mutants has led to the identification of two linkage groups specifically involved in poly(GlcGalNAc 1-P) synthesis: gne (gneA), the structural gene of the UDP-N-acetylglucosamine (UDP-GlcNAc) 4-epimerase (Soldo et al., 2003), and gga, a locus to which most mutants conferring 3T-resistance have been mapped, and which is assumed to be involved in the polymerization of poly(GlcGalNAc 1-P) (Estrela et al., 1991). The nucleotide sequence of the gga locus has been determined within the B. subtilis genome sequencing project (Lazarevic et al., 1995).
The synthesis of poly(GlcGalNAc 1-P) is inhibited by tunicamycin (Tm) (Pooley & Karamata, 2000), an antibiotic interfering with the coupling of N-acetylglucosamine 1-phosphate (GlcNAc 1-P) to undecaprenyl phosphate (UP), i.e. the first step of the synthesis of the poly(GroP) linkage unit (LU) carrier (Araki & Ito, 1989). The LU, consisting of phospho-N-acetylglucosaminyl-N-acetylmannosaminyl (glycerol phosphate)1–2, [P-GlcNAc-ManNAc-(GroP)1–2], joins the poly(GroP) chain to peptidoglycan (PG). Furthermore, the deficiency in TagO, the UDP-GlcNAc : UP GlcNAc 1-P transferase, prevents the synthesis of poly(GlcGalNAc 1-P) (Soldo et al., 2002). These observations suggest that poly(GlcGalNAc 1-P) is attached to PG by a LU containing elements of the poly(GroP) LU.
In this study, we analyse the ggaAB operon responsible for poly(GlcGalNAc 1-P) polymerization and discuss the relevance of the incorporation of poly(GlcGalNAc 1-P) precursors by whole cells and protoplasts to the mode of polymerization and attachment of this polymer to PG.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Chromosomal fragments of B. subtilis subcloned in plasmid vectors were maintained in Escherichia coli DH5
or JM83.
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60 mCi mmol–1; 2.22 GBq mmol–1), [1-14C]GlcNAc (2.22 GBq mmol–1), [-35S]dATP (>1000 Ci mmol–1; >37 TBq mmol–1) and [
-32P]ATP (>5000 Ci mmol–1; >185 TBq mmol–1) were from Amersham.
Media and growth conditions.
E. coli strains were routinely grown in Luria–Bertani (LB) medium (Difco) or on LB agar (Difco) containing, when appropriate, ampicillin (50 µg ml–1). B. subtilis cells were grown in liquid LB, SA+trp [(0.2 % (NH4)2SO4, 1.4 % K2HPO4, 0.6 % KH2PO4, 0.1 % trisodium citrate.2H2O, 0.02 % MgSO4.7H2O, 5 µM MnSO4.H2O, 0.5 % glucose, 1 % casein acid hydrolysate (Difco), 20 µg tryptophan ml–1)] or SAT2T (SA+trp, 40 µg thymine ml–1) medium, and on LB agar. When required, SA+trp and SAT2T media were supplemented with adenine (100 µg ml–1). Chloramphenicol and kanamycin were added at final concentrations of 3 and 5 µg ml–1, respectively. Amylase deficiency, due to amyE locus disruption, was demonstrated as described previously by Soldo et al. (1993). Growth was followed by measuring nephelometric density (ND) or OD600; for B. subtilis; an ND of 100 corresponds to about 108 cells ml–1.
Phage susceptibility test.
3T and 11 phage stocks were obtained as described previously by Soldo et al. (2003). Phage susceptibility was tested by spotting 5 µl of the phage stock (109–1010 phage ml–1) onto fresh streaks of B. subtilis strains on LB agar plates. Plates were incubated for 8 h at 30 °C.
Transformation.
E. coli competent cells were prepared and transformed by the procedure of Chung & Miller (1988). Transformation of B. subtilis was performed as described previously by Karamata & Gross (1970). For the selection of kanamycin-resistant recombinants, the transformation mixture was incubated for an additional 90 min with a sublethal concentration (0.1 µg ml–1) of the antibiotic prior to plating.
DNA preparation and sequencing.
Plasmid DNA was prepared by the boiling miniprep method (Del Sal et al., 1988). DNA sequencing was performed with a Sequenase Version 2.0 Kit (USB) and [-35S]dATP, according to the supplier's recommendations.
RNA isolation, and primer extension.
B. subtilis strain L5047 was grown in SA+trp medium at 37 °C, and harvested at an ND of 60. Total cellular RNA was isolated as previously described (Soldo et al., 1999). Oligonucleotide 5'-labelling with [32-P]ATP, and the primer extension reaction, were carried out as described previously by Lazarevic et al. (1992). Extension products were separated on a 6 % polyacrylamide sequencing gel, alongside a sequencing ladder obtained with the same primer on plasmid p6360.
-Galactosidase assay.
The assay was performed as described by Mauël et al. (1994).
Polymer synthesis by protoplasts.
The protocol was essentially that described by Bertram et al. (1981). B. subtilis cells were grown in SAT2T. At an ND of 60, the cells were harvested by centrifugation, concentrated 40-fold, and incubated in protoplasting medium (50 mM Tris/HCl, pH 7.5, 0.625 M sucrose, 10 mM MgCl2, and 0.5 mg lysozyme ml–1) for 30 min at 30 °C. Polymer synthesis was assayed at 30 °C on a 0.2 ml sample of protoplasts, to which substrates 500 µM UDP-Glc, 500 µM UDP-GlcNAc, 400 µM CDP-Gro, 2.5 µM UDP-GalNAc and 1.6 µM UDP-[1-14C]GalNAc were added in different combinations. The final volume of the assay mixture was 0.25 ml. The reaction was stopped by immersion in boiling water for 2 min. Following separation of polymerized material from unincorporated precursors by descendent paper chromatography (Bertram et al., 1981), radioactivity was determined by scintillation counting in 10 ml Optifluor (Packard).
Labelling and extraction of poly(GlcGalNAc 1-P).
Cells were grown in SAT2T medium containing 100 µM GlcNAc at 30, 37, 42 and 45 °C. At an ND of 6, [1-14C]GlcNAc was added at a final concentration of 0.1 µCi ml–1 (3.7 kBq ml–1), and the incubation was continued until the ND reached 100. Selective acid extraction of poly(GlcGalNAc 1-P) was as described previously by Soldo et al. (2002).
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RESULTS |
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3T and 11, have been mapped between tagF and gtaB loci (Estrela et al., 1991; Lazarevic et al., 1995; Soldo et al., 2003). Analysis of the nucleotide sequence of this region has revealed two ORFs, ggaA and ggaB. The 26 nt sequence located 34 nt downstream of the stop codon of ggaB may form a hairpin structure corresponding to a terminator. Absence of any obvious terminator-like structure in the ggaA–ggaB intergenic segment implies that the two ORFs form an operon. The operon is separated from the upstream tagH and the downstream gtaB gene by non-coding regions, which consist of about 2.2 and 0.7 kbp, respectively (Lazarevic et al., 1995). The 2.2 kbp non-coding region exhibits a high degree of similarity to strain W23 tar genes (Lazarevic et al., 2002a). The G+C contents of ggaA and ggaB are 28.2 and 28.8 %, respectively, i.e. very low compared with 43.5 %, which is the mean G+C content of the B. subtilis 168 chromosome (Kunst et al., 1997). The low G+C content of the ggaAB operon, as well as its location between two non-coding regions, leave little doubt that poly(GlcGalNAc 1-P)-encoding genes are acquired through horizontal transfer.
The 320 residue N-terminal regions of GgaA and GgaB share 34 % identity, and contain the pfam00535 domain (Marchler-Bauer et al., 2003), which is characteristic of enzymes transferring a sugar from its NDP-form. The C-terminal moiety of GgaB (residues 520–894) corresponds to the glucosyl/glycerophosphate transferase domain COG1887 (Marchler-Bauer et al., 2003). This domain is present in the WTA biosynthetic enzymes TagB and TagF, which are the putative CDP-Gro : undecaprenyl pyrophosphoryl-N-acetylglucosaminyl-N-acetylmannosamine (UPP-GlcNAc-ManNAc) GroP transferase (Pooley et al., 1992) and the CDP-Gro : poly(GroP) GroP transferase, respectively (Mauël et al., 1991).
The involvement of the ggaAB operon in poly(GlcGalNAc 1-P) synthesis was confirmed by insertional mutagenesis with plasmids p6361 (strain L4664) and p6362 (strain L4665) containing fragments of ggaB (Fig. 1), as well as by a partial deletion of ggaA (strain L4660). All of these mutants exhibited resistance to bacteriophages 3T and 11, and apparently normal morphology and growth rate. The poly(GlcGalNAc 1-P)-deficient phenotype, i.e. considerably reduced amount of acid-extractable cell wall hexosamines, was confirmed on strain L4660 with a partly deleted ggaA (Fig. 2).
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), RNA was prepared from cultures of strain L5047 growing exponentially in SA+trp medium at 30 and 45 °C. Total RNAs were incubated with a series of 32P-labelled primer oligonucleotides at a stepwise increasing distance from the ggaA start codon. A start signal was obtained with oligonucleotide 5'-TCCTTTGTAGACAATCGTACGATT-3', spanning residues –905 to –928 upstream of the ggaA start codon. The transcriptional starts (Fig. 3) were located on a DNA segment almost identical to the regulatory region of the strain W23 tarD gene (Lazarevic et al., 2002a) (Fig. 1), and corresponded to a sequence differing from that of the A-dependent promoter consensus (Helmann & Moran, 2002) in 3 nt only (Figs 1 and 3). The innermost start point was separated from the ggaA start codon by 1114 nt, which is a considerable distance. Visual inspection of the gel revealed that the intensity of the start signal at 45 °C was significantly weaker than that at 30 °C (Fig. 3). This thermosensitivity was examined with a lacZ reporter gene inserted into the amyE locus. The relevant construct was obtained by transforming B. subtilis competent cells with pAZ8102, which is a plasmid with the ggaAB promoter region inserted upstream of the lacZ gene, and selecting chloramphenicol-resistant and amylase-deficient mutants. -Galactosidase activity was measured on cultures growing at 30, 37, 42 and 45 °C. The reporter gene was expressed at a constant rate until the OD600 reached about 0.6. It appeared that the relatively weak activity of this ggaAB promoter was comparable during growth at 30 and 37 °C, but decreased with increasing temperature (Fig. 4). In conclusion, the expression of the ggaAB operon was thermosensitive, and took place from a promoter located over 1 kbp away from the ggaA start codon.
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), as well as its dependence on TagO, the UDP-GlcNAc : UP GlcNAc 1-P transferase (Soldo et al., 2002), strongly suggest the existence of a LU that shares certain elements with the poly(GroP) LU. To characterize this LU, we resorted to a genetic approach by examining whether other tag genes are involved in the synthesis of poly(GlcGalNAc 1-P). For that purpose, since thermosensitivity of poly(GlcGalNAc 1-P) synthesis (see above) prevents a straightforward utilization of tag thermosensitive mutants, we placed the ggaAB operon of strains L6601 tagD11, L6476 tagB1 and L6477 tagF1 under an IPTG-inducible Pspac promoter. The constructs obtained, as well as the reference thermoresistant strain L4674, were incubated in SAT2T medium supplemented with [1-14C]GlcNAc. For each strain, two cultures were grown: one with 100 µg IPTG ml–1, and the other without IPTG. At an ND of 15, cultures were shifted from 30 to 45 °C, and the cells were harvested at an ND of 100. It appeared (Fig. 5) that deficiency of tagF, encoding the CDP-Gro : poly(GroP) GroP transferase (Pooley et al., 1992), did not affect the poly(GlcGalNAc 1-P) synthesis, while that of either tagD, the structural gene encoding glycerol-3-phosphate cytidylyltransferase (Pooley et al., 1991), or tagB, encoding the enzyme putatively involved in the completion of the poly(GroP) LU, i.e. attachment of GroP to UPP-GlcNAc-ManNAc (Mauël et al., 1991), allowed only residual synthesis of poly(GlcGalNAc 1-P). The incorporation of 14C in tagB- or tagD-deficient strains was comparable with that obtained with ggaA- (see above) or gne-deficient strains (Soldo et al., 2003). These observations strongly suggest that poly(GroP) and poly(GlcGalNAc 1-P) are linked to PG through the same LU. Thermosensitivity of strain L4677 Pspac–ggaA tagF1 (not presented) revealed, in addition, that synthesis of poly(GlcGalNAc 1-P), expressed from the Pspac promoter, cannot compensate for the missing poly(GroP).
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. Here, the incorporation of [14C]GalNAc, supplied as UDP-[1-14C]GalNAc, was measured. Protoplasts of strains derived from 168 were obtained by lysozyme treatment. Assay mixtures containing 40-fold concentrated protoplast suspensions were incubated with UDP-[1-14C]GalNAc, and an excess of cold UDP-GlcNAc, to prevent UDP-GalNAc 4-epimerization, and incorporation of the label through UDP-GlcNAc (Bertram et al., 1981). Protoplasts of the parent strain incorporated significant amounts of [14C]GalNAc at a nearly steady rate, an incorporation that was attributed to the extension of existing chains of poly(GlcGalNAc 1-P) (Fig. 6). The absolute requirement of UDP-Glc for [14C]GalNAc incorporation (not presented) left no doubt that the label was incorporated into poly(GlcGalNAc 1-P). This conclusion was confirmed by the absence of [14C]GalNAc incorporation into the ggaA-deficient strain, as well as by a higher rate of incorporation into protoplasts of strain L4661 (Fig. 6), in which expression of ggaB was increased through a promoter of the erythromycin-resistance gene from the integrated plasmid. Attempts to increase the rate of incorporation by adding CDP-Gro and UDP-GlcNAc to the assay mixture, with the aim to de novo generate LUs (Bertram et al., 1981), failed (Table 2). This result is in apparent contradiction with the reduced incorporation of [1-14C]GalNAc from exogenously supplied [1-14C]GlcNAc into cells of tagB- and tagD-deficient mutants at the non-permissive temperature (Fig. 5), and with the observed stimulating effect of CDP-Gro on WTA synthesis by protoplasts of strain W23 (Bertram et al., 1981). However, it should be recalled that the most abundant WTA of W23 is poly(ribitol phosphate) [poly(RboP)], which contains GroP in the LU only, whereas in strain 168, CDP-Gro is overwhelmingly drained into poly(GroP) synthesis. Therefore, since the stimulating effect of CDP-Gro was observed with poly(RboP) synthesis (Bertram et al., 1981), we resorted to hybrids between strains 168 and W23, in which the poly(GroP)-synthesizing genes had been replaced by poly(RboP)-synthesizing genes, but which retained the capacity to synthesize poly(GlcGalNAc 1-P). In protoplasts of these so-called mixed-type hybrids (Karamata et al., 1987), the incorporation of [1-14C]GalNAc from UDP-[1-14C]GalNAc was hardly detected. This was not surprising in view of the low quantities of GalNAc in walls of whole cells, relative to amounts present in strain 168 (H. M. Pooley, personal communication). However, with these hybrid strains, addition of CDP-Gro and UDP-GlcNAc to the assay mixture did increase the rate of incorporation by a factor of 10 (Table 2), a relative increase comparable to that obtained for poly(RboP) (Bertram et al., 1981). This increase was most likely due to newly synthesized LU, since it was completely inhibited by Tm, an antibiotic known to interfere with the synthesis of the LU by blocking the coupling of GlcNAc 1-P to the lipid carrier.
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, Fig. 6). It also appeared that the excess incorporation relative to that obtained with strain L5047 was insensitive to Tm, i.e. it was achieved on an excess of apparently idle LUs. Again, as with the parent L5047 strain, addition of CDP-Gro and UDP-GlcNAc, two out of three LU precursors, did not increase the rate of incorporation of [1-14C]GalNAc supplied as UDP-[1-14C]GalNAc by protoplasts of the gtaB-deficient strain (Table 2). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We have obtained strong evidence that the main chain of this WTA is attached to PG through the same LU as poly(GroP), which is the most abundant WTA of strain 168. First, the synthesis of poly(GlcGalNAc 1-P) requires TagO (Soldo et al., 2002), and is sensitive to Tm (Pooley & Karamata, 2000), revealing that GlcNAc is the first element of the LU. Second, requirement of TagD, the glycerol-3-phosphate cytidylyltransferase, and TagB, which most likely couples GroP to UPP-GlcNAc-ManNAc, shows that both ManNAc and GroP, the second and the third elements of the poly(GroP) LU, are part of the poly(GlcGalNAc 1-P) LU. Third, incorporation of [1-14C]GalNAc, supplied as UDP-[1-14C]GalNAc, by protoplasts of 168/W23 hybrid strains was strongly stimulated by addition of both CDP-Gro and UDP-GlcNAc to the assay mixture. The two latter precursors provide a complete set of LU constituents, since UDP-ManNAc is obtained from UDP-GlcNAc through the action of the UDP-GlcNAc 2-epimerase, a cytoplasmic enzyme that is released to the medium following lysis of a small proportion of protoplasts. This is substantiated by Harrington & Baddiley (1985) who reported that a very small contamination of B. subtilis membrane preparations by UDP-GlcNAc 2-epimerase allows in vitro synthesis of the LU. The polymerization of the poly(GlcGalNAc 1-P) chain is most likely to be achieved by alternate incorporation GalNAc-P and glucose, mediated by GgaB and GgaA, which are two proteins whose N-terminal moieties contain similar motifs characteristic of sugar transferases (Marchler-Bauer et al., 2003). Despite an exhaustive search, no other gene specifically involved in poly(GlcGalNAc 1-P) has been found (Estrela et al., 1991). Therefore, it appears that the large GgaB protein endowed with the motif characteristic of glycerol- and ribitol-metabolizing enzymes (Marchler-Bauer et al., 2003) is bifunctional; its roles being the initiation of the main polymer chain, as well as the addition of one of its elements in alternation with GgaA. According to the structural formula of the GlcGalNAc 1-P disaccharide (Shibaev et al., 1973), the precursor phosphate remains attached to the C-1 of GalNAc, strongly suggesting that the main chain begins by the coupling of GalNAc 1-P to the C-1 of the LU glycerol. Finally, the poly(GlcGalNAc 1-P) chain polymerized on the LU is translocated to the outer membrane surface by the TagGH ABC transporter (Lazarevic & Karamata, 1995), and hooked to the nascent PG chain.
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strongly suggest that the incorporation by protoplasts of WTA components, supplied as NDP precursors, takes place at the outer surface of the cytoplasmic membrane. Our experiments provide additional evidence, by revealing that incorporation of label, supplied as UDP-[1-14C]GalNAc, into poly(GlcGalNAc1-P) has an absolute requirement for UDP-Glc. Had the label been transported into the cytoplasm, some incorporation would have taken place during residual synthesis of poly(GlcGalNAc 1-P), without any exogenously supplied UDP-Glc, since UDP-Glc is present in the cytoplasm. It should be recalled that, in cases examined to date, addition of Tm to the reaction mixture reduces the incorporation by 35 % (Bertram et al., 1981) and about 50 % (this work), revealing that a residual synthesis of new LUs and chains does take place in protoplasts. Therefore, it would seem that the export of the polymer by the TagGH transporter is coupled with a temporary exposal of the polymerizing enzymes at the outer surface (Bertram et al., 1981; Lazarevic & Karamata, 1995). The latter enzymes could incorporate exogenously supplied precursors, and further elongate the exported chains, which, due to lysozyme-mediated removal of PG, would remain idle. This would be the case for poly(GlcGalNAc 1-P), as well as for poly(RboP) (Bertram et al., 1981).
The relatively high [14C]GalNAc incorporation rate into protoplasts of L5054, a gtaB-deficient strain (Fig. 6), was likely to be due to accumulation of idle LU destined for the synthesis of poly(GlcGalNAc 1-P). First, in absolute terms, the inhibitory effect of Tm on the gtaB-deficient strain was comparable with that obtained with protoplasts of the reference strain L5047. This implies that the increased rate of synthesis of the gtaB-deficient mutant is due to accumulated idle elements on which synthesis can readily begin when appropriate precursors are provided. Second, since gtaB-deficient cells can supply UDP-GalNAc through UDP-GlcNAc 4-epimerization (Soldo et al., 2003), and since the synthesis of the poly(GlcGalNAc 1-P) chain probably begins with GalNAc (see above), it seems that the LUs accumulated during growth of L5054 gtaB are endowed with GalNAc, and therefore cannot serve for poly(GroP) synthesis. It is most likely that the same mechanism is responsible for a higher rate of poly(GlcGalNAc 1-P) synthesis by protoplasts of strains with increased expression of ggaB (Fig. 6).
One of the main roles of WTAs is the maintenance of the global negative charge of the cell surface. As previously discussed (Estrela et al., 1991), whenever the rate of synthesis of poly(GroP) is reduced, that of poly(GlcGalNAc 1-P) is increased (Rosenberger, 1976; F.-X. Abellan & H. M. Pooley, personal communication). However, at 45 °C, synthesis of the poly(GlcGalNAc 1-P) from a Pspac promoter can not compensate for the absence of poly(GroP) (see above), either because of an insufficient rate of expression of the ggaAB operon, or because the main polymer may have a more specific role.
It has been shown that WTAs are essential for growth of B. subtilis strains 168 and W23 (Mauël et al., 1991; unpublished), as well as for Staphylococcus epidermidis ATCC 14990 (Fitzgerald & Foster, 2000). Therefore, the presence of a LU common to many Gram-positive bacteria (Araki & Ito, 1989) suggests that it represents an interesting and a relatively widespread target for antibiotics (Pooley & Karamata, 1988). This conclusion is strengthened by the recently reported evidence that in B. subtilis, under laboratory conditions, teichuronic acid cannot substitute for a deficiency in poly(GroP) (Bhavsar et al., 2004).
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ACKNOWLEDGEMENTS |
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Received 26 December 2005;
revised 30 January 2006;
accepted 3 February 2006.
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); L4660 
ggaA (
).
(amyE : : cat PggaA–lacZ) were grown in LB medium at 30 (
), 37 (
). Samples were withdrawn at regular intervals of time, and processed and assayed for 
