Institut de Génétique et de Biologie Microbiennes, Université de Lausanne, Rue César-Roux 19, CH-1005 Lausanne, Switzerland1
Author for correspondence: Dimitri Karamata. Tel: +41 21 320 60 75. Fax: +41 21 320 60 78. e-mail: dimitri.karamata{at}igbm.unil.ch
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: teichoic acid, teichuronic acid
Abbreviations: GlcNAc, N-acetylglucosamine; poly(GlcGalNAcP), poly(glucosyl N-acetylgalactosamine 1-phosphate); poly(groP), poly(glycerol phosphate); TA, teichoic acid
a The EMBL accession number for the nucleotide sequence reported in this paper is AJ004803.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The genes identified so far that are known to be specifically involved in TA metabolism are clustered between 308° and 311° on the genetic map of B. subtilis 168 (Lazarevic et al., 1995 ; Kunst et al., 1997
). They form the divergons tagABtagDEF (Mauël et al., 1991
; Honeyman & Stewart, 1989
) and mnaA(formerly orfX or yvyH)gtaB (Soldo et al., 1993
; Soldo et al., 2002
), as well as the operons ggaAB (Freymond, 1995
) and tagGH (Lazarevic & Karamata, 1995
). Thermosensitive strains bearing mutations in the tagB, tagF or tagD genes, involved in the synthesis of poly(groP), are impaired in growth and exhibit a coccoid morphology at the non-permissive temperature (Rogers et al., 1970
; Karamata et al., 1972
; Mauël et al., 1991
; Pooley et al., 1991
, 1992
). These phenotypes are also generated by reduced expression of tagGH, the operon involved in TA translocation (Lazarevic & Karamata, 1995
).
Genes responsible for all but two of the steps involved in the synthesis of TA were identified biochemically and/or predicted from the sequence of B. subtilis 168 (Honeyman & Stewart, 1989 ; Mauël et al., 1991
; Soldo et al., 1993
; Lazarevic & Karamata, 1995
; Lazarevic et al., 1995
). The exceptions are the very first reaction in the linkage unit synthesis and the ultimate one, i.e. the attachment of TA to peptidoglycan. In the present study, we describe tagO, the one-gene operon that is most likely involved in the initiation of TA linkage unit formation and which, surprisingly, affects the synthesis of TAs as well as the synthesis of teichuronic acid.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
DNA preparation.
Plasmid DNA was prepared by the boiling method of Del Sal et al. (1988) .
Sequencing.
Both DNA strands of the EcoRI insert of plasmid p5504 were sequenced by primer walking and the dideoxy-chain termination method, using the Sequenase Version 2.0 Kit (USB) and [-35S]dATP (Amersham). Assembly and analysis of the sequence data were done by using the University of Wisconsin Computer Group software (Devereux et al., 1984
).
PCR conditions.
PCRs were set up in 100 µl of reaction buffer (Pharmacia Biotech) containing 2·5 U Taq DNA polymerase (Pharmacia Biotech), 1 ng of B. subtilis genomic DNA, 100 pmol of each primer and 20 nmol of each of the four dNTPs (Pharmacia Biotech). Reactions were run with denaturation at 95 °C for 2 min, followed by 30 cycles of amplification (95 °C for 30 s, 45 °C for 1 min, 72 °C for 1 min), with a final extension at 72 °C for 10 min.
RNA isolation and primer-extension mapping.
RNA was isolated from B. subtilis 168 cells by using the RNeasy Total RNA Kit (Qiagen). Labelling of the 5' end of the oligonucleotide with [-33P]ATP and cDNA synthesis were performed with the Primer Extension System (Promega). One picomole of the 5'-end-labelled oligonucleotide VL307 (5'-GTTATGATTAAAACTGTCAGCAGCGA-3') was incubated with 20 µg of total RNA at 95 °C for 1 min, at 55 °C for 2 min and then on ice for 15 min. cDNA products were analysed on a 6% (w/v) denaturing polyacrylamide gel, alongside the sequencing ladder.
Enzyme assay.
ß-Galactosidase activity assays were performed according to Nicholson & Setlow (1990) . Cells were grown in LB medium and then pelleted for 2 min in a microcentrifuge. The pellets (from 0·3 to 1 ml samples) were resuspended in 100 µl of Z buffer (Miller, 1972
). Toluene (10 µl) was added to the suspension; the suspension was vortexed for 15 s and then transferred to 900 µl of Z buffer containing 0·89 µg ONPG ml-1, which had been pre-equilibrated for 5 min at 28 °C. For cultures grown in PL medium (Grant, 1979
), 100 µl aliquots were toluene-treated and transferred to 900 µl of ONPG-supplemented Z buffer. Reactions were stopped with 400 µl of 1 M Na2CO3; the samples were then centrifuged. The A420 of the supernatant was read against the reaction that had been stopped at time zero. The activity of the supernatants is expressed in units calculated according to the formula A420x1000/[reaction time (min)xOD595 of culture].
Cell-wall preparation.
Cell walls were prepared as previously described (Soldo et al., 1999 ).
Estimation of cell-wall phosphate.
An aliquot of the culture that had been grown at 37 °C in SA medium supplemented with 20 µg tryptophan ml-1, 5 µg kanamycin ml-1 and 200 µg IPTG ml-1 was centrifuged, washed with the IPTG-free medium and resuspended in either fresh IPTG-free medium or in IPTG-containing medium to an OD595 of 0·020. Cell walls were isolated from the cultures when OD595 0·75 was reached. Lyophilized cell walls were mineralized (Ames, 1966 ), and the phosphate concentration was determined according to Chen et al. (1956)
.
Estimation of cell-wall uronate.
Exponentially growing cells in PL medium supplemented with IPTG were washed and transferred to fresh medium with or without IPTG to an OD595 of 0·1. Walls were isolated from phosphate-starved cells collected 5 h after growth had slowed down. Uronic acid was determined as described by Blumenkrantz & Asboe-Hansen (1973) .
Labelling of cell-wall polymers with [2-3H]glycerol and [1-14C]GlcNAc.
Cells were grown at 37 °C in SA medium supplemented with 20 µg tryptophan ml-1, 5 µg kanamycin ml-1, 100 µM GlcNAc, 5 mM glycerol, 1·5 mM MgCl2 and 200 µg IPTG ml-1. At an OD595 of 0·3, a sample from the exponentially grown culture was washed with the IPTG-free medium and diluted in fresh medium with or without IPTG to an OD595 of 0·060. [1-14C]GlcNAc or [2-3H]glycerol were added at a final concentration of 0·2 mCi ml-1 (7·4 MBq ml-1) and 1 mCi ml-1 (37 MBq ml-1), respectively, and incubation was continued until OD595 0·300 was reached. To determine the incorporation of radioactive glycerol into the cell-wall fraction, 2 ml samples were washed and resuspended in TMS/lysozyme buffer [1 M sucrose, 50 mM Tris/HCl (pH 8), 8 mM MgCl2, 200 µg lysozyme ml-1]. After the conversion of over 95% of the cells into protoplasts (which generally required up to 30 min incubation at 37 °C), the protoplasts were sedimented and the radioactivity of the supernatant, corresponding to that released by wall digestion (Pooley & Karamata, 2000 ), was counted. The incorporation of [1-14C]GlcNAc was measured in 1 ml samples, which were washed, boiled for 5 min in 0·1 M phosphate buffer (pH 7) and then centrifuged. To selectively extract the N-acetylgalactosamine-containing polymer (Estrela et al., 1991
), pellets were resuspended in 1 ml of 0·1 M sodium citrate buffer (pH 4), incubated for 30 min at 100 °C and then centrifuged. The radioactivity present in the supernatants, corresponding to poly(GlcGalNAcP), was counted. The pellets, containing essentially radioactive hexosamine incorporated into the peptidoglycan, were resuspended in 1 ml of water and their radioactivity was counted. Radioactivity was always determined by scintillation counting in 10 ml of Optifluor (Packard).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Insertion of the chloramphenicol-resistance cassette (cat) gene from linearized p5515 into the tagO StuI site or a Campbell-type integration of p5530 (Fig. 1) did not yield viable recombinants. Thus, recombination events generating truncated TagO proteins containing 233 and 182 N-terminal residues, respectively, were apparently lethal. This observation is compatible with the proposed function of TagO in the synthesis of the TA linkage unit.
To control the level of tagO expression and to study the phenotype of conditional tagO deficient strains, the operon was placed under the control of a Pspac promoter (Yansura & Henner, 1984 ). A DNA fragment containing the proximal part of tagO and its RBS was cloned into vector pSGMU441; the resulting plasmid, p5525, was integrated into the chromosome. Proper plasmid integration into the selected kanamycin-resistant recombinant (L16055) was confirmed by PCR (not shown). Strain L16055 harbours a truncated copy of tagO which is under the control of its native promoter, as well as a complete tagO gene whose expression depends on the vector-borne IPTG-inducible Pspac promoter. In the absence of IPTG, the LacI protein represses transcription from the Pspac promoter. Following cell transfer from IPTG-containing to IPTG-free medium, typical rods are progressively converted into aggregates of coccoid cells (Fig. 4
), a morphology characteristic of mutants deficient in TA synthesis. The implication of this result that TA metabolism is impaired by reduced tagO expression was confirmed by determining the amounts of phosphate, glycerol and N-acetylgalactosamine [the cell-wall compounds which correspond to poly(groP) and poly(GlcGalNAcP)] present in the cell walls of the mutant and wild-type strains (Table 2
). In the presence of IPTG, the cell-wall phosphate of the mutant strain L16055 (PspactagO) was close to 0·95 mmol (g cell wall)-1, the value obtained with strain 168. However, in the IPTG-free medium, the amount of cell-wall phosphate in strain L16055 cells was drastically reduced when compared to that of cells grown in the IPTG-supplemented culture and collected at the same optical density (Table 2
). During a relatively limited labelling period, i.e. 2·25 generations after IPTG depletion, the incorporation of [1-14C]GlcNAc and of [2-3H]glycerol into the cell-wall fraction was significantly reduced. The reduced level of cell-wall glycerol is in agreement with an impaired incorporation of the major TA, whereas low levels of acid-extractable hexosamine reflect a deficiency in the synthesis of the secondary polymer. These observations are compatible with the involvement of TagO in the synthesis of the linkage unit that appears to be common to poly(groP) and poly(GlcGalNAcP) (Pooley & Karamata, 1994
; Freymond, 1995
; Lazarevic & Karamata, 1995
). Incidentally, the reduced level of incorporation of TA radioactive precursors due to tagO repression parallels the reduction in incorporation obtained upon the addition of tunicamycin to the growth medium (Pooley & Karamata, 2000
). Tunicamycin is an antibiotic whose precise target is the reaction involved in the formation of undecaprenyl-PP-GlcNAc (Hancock et al., 1976
), the reaction most likely mediated by TagO. Impaired tagO expression during labelling with [1-14C]GlcNAc was accompanied by a slight reduction in the radioactivity count in the cell-wall fraction that was non-extractable at pH 4, i.e. the fraction which essentially corresponds to peptidoglycan. However, this reduction is probably due to the absence of the TA linkage unit. Indeed, under our experimental conditions (mild acid treatment of the cell wall) the linkage unit, when synthesized, remains attached to the cell wall and, due to labelled GlcNAc and N-acetylmannosamine, contributes to the overall radioactivity count.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the precise enzymic activity of TagO has not been confirmed by an assay, nucleotide sequence homologies strongly suggest that tagO encodes a UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme that mediates the first step in the synthesis of the TA linkage unit. This is in full agreement with the observation that interference in the expression of tagO can impair the synthesis of poly(groP) as well as poly(GlcGalNAcP), since both polymers have a common linkage unit (Freymond, 1995 ; Pooley & Karamata, 1994
). The absence of tagO expression in phosphate-depleted conditions, in which teichuronic acid normally replaces TA, is accompanied by inhibition of uronate incorporation, which is less severe but nevertheless comparable to the reduction of phosphate incorporation into TA in the absence of tagO expression (Table 1
). This unexpected result, revealing an involvement of TagO in teichuronic acid synthesis, implies (Soldo et al., 1999
) that, in B. subtilis 168, teichuronic acid either has a more complex chemical composition than previously reported (Wright & Heckels, 1975
) or that it is hooked to peptidoglycan through a linkage unit, a situation observed in Micrococcus luteus (Araki & Ito, 1989
). Unlike the TA genes tagA, tagB, tagD, tagE and tagF, whose expression is turned off by phosphate starvation (Mauël et al., 1994
), tagO is efficiently transcribed for several hours following phosphate exhaustion (Fig. 2
), formally in agreement with the possibility that tagO is involved in teichuronic acid synthesis. Incidentally, in phosphate-depleted conditions, the replacement of TAs by teichuronic acids is at least partly mediated by PhoP/R, a two-component regulatory system (Seki et al., 1988
). The binding of PhoP to the Pho boxes in the tagAtagD and tuaA regulatory regions results in repression of the relevant TA genes (Liu et al., 1998
) and induction of the teichuronic acid operon genes (Liu & Hulett, 1998
). Inspection of the tagO regulatory region did not reveal repeats of TTAACA-like motifs corresponding to the B. subtilis Pho box (Liu & Hulett, 1998
). Therefore, the balance between TA and teichuronic acid does not seem to include PhoP binding to the tagO regulatory region. This is in agreement with the expression pattern of tagO, which is not significantly altered by phosphate exhaustion, as well as with the involvement of TagO in the synthesis of both phosphate-containing and phosphate-free anionic cell-wall polymers.
Sequence analysis and primer-extension experiments revealed that the expression of the tagO operon takes place from a regulatory region organized in a way similar to the regulatory region that controls the expression of the structural gene of the A factor. Indeed, several features of the tagO regulatory region resemble those of the three-gene sigA (rpoD) operon (Wang & Doi, 1987
): (i) the -10 region of P1 overlaps the -35 region of P2 by 2 nt, (ii) P2 is a much stronger promoter than P1, and (iii) the relevant transcriptional starts lie within short direct repeats, which are, in the case of tagO, represented by the AAGGA pentamer. Overlapping promoters and direct repeats could also contribute to a complex regulation of tagO expression, probably in response to the cell growth rate. To our knowledge, TagO is the first example of a gene apparently co-regulated with sigA, a gene whose product is intimately associated with cell growth.
Should chemical analyses confirm the presence of GlcNAc in a putative teichuronic acid linkage unit, the pivotal structural role, as well as the regulatory role, of TagO in the balanced growth of B. subtilis would appear very likely. For instance, TagO, a membrane-anchored protein, may have the potential to associate itself with either TagG and TagH, the ABC transporter for TA (Lazarevic & Karamata, 1995 ), or with TuaB, the transmembrane protein possibly responsible for the translocation(s) related to teichuronic acid synthesis (Soldo et al., 1999
). Assembling the other, relevant, tag or tua gene products around their respective pivotal blocks would generate multi-enzyme complexes responsible for anionic polymer synthesis. Analogies at the level of their regulatory regions would suggest that, like
A, TagO too is not dissociable from balanced cell growth which, as clearly shown, cannot take place without the concomitant synthesis of anionic polymers (Mauël et al., 1989
). By controlling the very first step of anionic polymer synthesis, tagO would be the rate-limiting factor in this synthetic process in B. subtilis 168. The significant homology between the sequences of TagO and MraY, a protein capable of recognizing the peptidoglycan subunit, leaves open the possibility that TagO may also take part in the hooking of complete TA chains to peptidoglycan, a reaction for which no candidate enzyme has so far been identified.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anagnostopoulos, C. & Spizizen, J. (1961). Requirements for transformation in Bacillus subtilis. J Bacteriol 81, 741-746.
Araki, Y. & Ito, E. (1989). Linkage units in cell walls of gram-positive bacteria. Crit Rev Microbiol 17, 121-135.[Medline]
Archibald, A. R., Hancock, I. C. & Harwood, C. R. (1993). Cell wall structure, synthesis and turnover. In Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology and Molecular Genetics , pp. 381-410. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC. American Society for Microbiology.
Blumenkrantz, N. & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Anal Biochem 54, 484-489.[Medline]
Boylen, C. W. & Ensign, J. C. (1968). Ratio of teichoic acid and peptidoglycan in cell walls of Bacillus subtilis following spore germination and during vegetative growth. J Bacteriol 96, 421-427.[Medline]
Chambers, S. P., Prior, S. E., Barstow, D. A. & Minton, N. P. (1988). The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68, 139-149.[Medline]
Chen, P. S., Toribara, T. Y. & Warner, H. (1956). Microdetermination of phosphorus. Anal Chem 18, 1756-1758.
Chung, C. T. & Miller, R. H. (1988). A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Res 16, 3580.[Medline]
Daniel, R. A. & Errington, J. (1993). DNA sequence of the murEmurD region of Bacillus subtilis 168. J Gen Microbiol 139, 361-370.[Medline]
Del Sal, G., Manfioletti, G. & Schneider, C. (1988). A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Res 16, 9878.[Medline]
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395.[Abstract]
Estrela, A.-I., Pooley, H. M., de Lencastre, H. & Karamata, D. (1991). Genetic and biochemical characterization of Bacillus subtilis 168 mutants specifically blocked in the synthesis of the teichoic acid, poly(3-O-ß-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate); gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity. J Gen Microbiol 137, 943-950.[Medline]
Freymond, P.-P. (1995). Génétique et Biochimie des Acides Teichoïques Secondaires de Bacillus subtilis 168 et W23. PhD Thesis, University of Lausanne, Switzerland.
Grant, W. D. (1979). Cell wall teichoic acid as a reserve phosphate source in Bacillus subtilis. J Bacteriol 137, 35-43.[Medline]
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[Medline]
Hancock, I. C., Wiseman, G. & Baddiley, J. (1976). Biosynthesis of the unit that links teichoic acid to the bacterial wall: inhibition by tunicamycin. FEBS Lett 69, 75-80.[Medline]
Helmann, J. D. (1995). Compilation and analysis of Bacillus subtilis A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res 23, 2351-2360.[Abstract]
Honeyman, A. L. & Stewart, G. C. (1989). The nucleotide sequence of the rodC operon of Bacillus subtilis. Mol Microbiol 3, 1257-1268.[Medline]
Karamata, D. & Gross, J. D. (1970). Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in DNA synthesis. Mol Gen Genet 108, 277-287.[Medline]
Karamata, D., McConnell, M. & Rogers, H. J. (1972). Mapping of rod mutants of Bacillus subtilis. J Bacteriol 111, 73-79.[Medline]
Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249256.[Medline]
Lazarevic, V. & Karamata, D. (1995). The tagGH operon of Bacillus subtilis 168 encodes a two-component ABC transporter involved in the metabolism of two wall teichoic acids. Mol Microbiol 16, 345-355.[Medline]
Lazarevic, V., Mauël, C., Soldo, B., Freymond, P.-P., Margot, P. & Karamata, D. (1995). Sequence analysis of the 308° to 311° segment of the Bacillus subtilis 168 chromosome, a region devoted to cell wall metabolism, containing non-coding grey holes which reveal chromosomal rearrangements. Microbiology 141, 329-335.[Abstract]
Liu, W. & Hulett, F. M. (1998). Comparison of PhoP binding to the tuaA promoter with PhoP binding to other Pho-regulon promoters establishes a Bacillus subtilis Pho core binding site. Microbiology 144, 1443-1450.[Abstract]
Liu, W., Eder, S. & Hulett, F. M. (1998). Analysis of Bacillus subtilis tagAB and tagDEF expression during phosphate starvation identifies a repressor role for PhoP-P. J Bacteriol 180, 753-758.
Maki, H., Yamaguchi, T. & Murakami, K. (1994). Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus. J Bacteriol 176, 4993-5000.[Abstract]
Mauël, C., Young, M., Margot, P. & Karamata, D. (1989). The essential nature of teichoic acids in Bacillus subtilis as revealed by insertional mutagenesis. Mol Gen Genet 215, 388-394.[Medline]
Mauël, C., Young, M. & Karamata, D. (1991). Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units. J Gen Microbiol 137, 929-941.[Medline]
Mauël, C., Young, M., Monsutti-Grecescu, A., Marriott, S. A. & Karamata, D. (1994). Analysis of Bacillus subtilis tag gene expression using transcriptional fusions. Microbiology 140, 2279-2288.[Abstract]
Meier-Dieter, U., Barr, K., Starman, R., Hatch, L. & Rick, P. D. (1992). Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rferff gene cluster. J Biol Chem 267, 746-753.
Miller, J. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Nicholson, W. L. & Setlow, P. (1990). Sporulation, germination and outgrowth. In Molecular Biological Methods for Bacillus , pp. 391-450. Edited by C. R. Harwood & S. M. Cutting. Chichester: Wiley.
Pooley, H. M. & Karamata, D. (1994). Teichoic acid synthesis in Bacillus subtilis: genetic organization and biological roles. In Bacterial Cell Wall , pp. 187-198. Edited by J.-M. Ghuysen & R. Hakenbeck. Amsterdam: Elsevier Science.
Pooley, H. M. & Karamata, D. (2000). Incorporation of [2C-3H]glycerol into cell surface components of Bacillus subtilis 168 and thermosensitive mutants affected in wall teichoic acid synthesis: effect of tunicamycin. Microbiology 146, 797-805.
Pooley, H. M., Abellan, F.-X. & Karamata, D. (1991). A conditional-lethal mutant of Bacillus subtilis 168 with a thermosensitive glycerol-3-phosphate cytidylyltransferase, an enzyme specific for the synthesis of the major wall teichoic acid. J Gen Microbiol 137, 921-928.[Medline]
Pooley, H. M., Abellan, F.-X. & Karamata, D. (1992). CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase, which is involved in the synthesis of the major wall teichoic acid in Bacillus subtilis 168, is encoded by tagF (rodC). J Bacteriol 174, 646-649.[Abstract]
Rogers, H. J., McConnell, M. & Burdett, I. D. J. (1970). The isolation and characterization of mutants of Bacillus subtilis and Bacillus licheniformis with disturbed morphology and cell division. J Gen Microbiol 61, 155-171.[Medline]
Seki, T., Yoshikawa, H., Takahashi, H. & Saito, H. (1988). Nucleotide sequence of the Bacillus subtilis phoR gene. J Bacteriol 170, 5935-5938.[Medline]
Shibaev, V. N., Duckworth, M., Archibald, A. R. & Baddiley, J. (1973). The structure of a polymer containing galactosamine from walls of Bacillus subtilis 168. Biochem J 135, 383-384.[Medline]
Soldo, B., Lazarevic, V., Margot, P. & Karamata, D. (1993). Sequencing and analysis of the divergon comprising gtaB, the structural gene of UDP-glucose pyrophosphorylase of Bacillus subtilis 168. J Gen Microbiol 139, 3185-3195.[Medline]
Soldo, B., Lazarevic, V., Mauël, C. & Karamata, D. (1996). Sequence of the 305°307° region of the Bacillus subtilis chromosome. Microbiology 142, 3079-3088.[Abstract]
Soldo, B., Lazarevic, V., Pagni, M. & Karamata, D. (1999). Teichuronic acid operon of Bacillus subtilis 168. Mol Microbiol 31, 795-805.[Medline]
Soldo, B., Lazarevic, V., Pooley, H. M. & Karamata, D. (2002). Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase. J Bacteriol 184 (in press).
Sonnhammer, E. L., von Heijne, G. & Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6, 175-182.[Medline]
Wang, L. F. & Doi, R. H. (1987). Promoter switching during development and the termination site of the 43 operon of Bacillus subtilis. Mol Gen Genet 207, 114-119.[Medline]
Wright, J. & Heckels, J. E. (1975). The teichuronic acid of cell walls of Bacillus subtilis W23 grown in a chemostat under phosphate limitation. Biochem J 147, 187-189.[Medline]
Yansura, D. G. & Henner, D. J. (1984). Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci USA 81, 439-443.[Abstract]
Yokoyama, K., Mizuguchi, H., Araki, Y., Kaya, S. & Ito, E. (1989). Biosynthesis of linkage units for teichoic acids in gram-positive bacteria: distribution of related enzymes and their specificities for UDP-sugars and lipid-linked intermediates. J Bacteriol 171, 940-946.[Medline]
Received 12 December 2001;
revised 6 February 2002;
accepted 20 March 2002.