Institut de Génétique et Biologie Microbiennes, Université de Lausanne, Rue César-Roux 19, CH-1005 Lausanne, Switzerland1
Author for correspondence: Catherine Mauël. Tel: +41 21 320 60 75. Fax: +41 21 320 60 78. e-mail: catherine.mauel{at}igbm.unil.ch
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ABSTRACT |
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Keywords: poly(glycerol phosphate) origin, poly(ribitol phosphate) sequence, Bacillus evolution, anionic cell-wall polymers
Abbreviations: -P, -phosphate; poly(GroP), poly(glycerol phosphate); poly(RboP), poly(ribitol phosphate); poly(GlcGalNAcP), poly(glucopyranosyl N-acetylgalactosamine 1-phosphate)
The EMBL accession numbers for the nucleotide sequences reported in this paper are AJ313428, AJ318465, AJ318466, AJ318467, AJ318468, AJ318469 and AJ318470.
a Present address: Biokema SA, Chemin de la Chatanerie 2, CH-1023 Crissier, Switzerland.
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INTRODUCTION |
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The expression of tag genes is subject to an elaborate control mechanism. First, there is evidence of an interdependence between teichoic acid and peptidoglycan synthesis (Ward, 1981 ). Second, under phosphate limitation, teichoic acid is replaced by teichuronic acid, a phosphate-free anionic polymer (Ellwood & Tempest, 1969
; Liu et al., 1998
; Soldo et al., 1999
). Transcriptional fusions to tagA and tagD, the first genes of the tag operons (Mauël et al., 1994
), confirmed that phosphate limitation was accompanied by reduced tag gene expression. In addition, these constructs revealed that, (i) under all experimental conditions used, tagA and tagD were apparently coordinately expressed, the level of tagD always being higher than that of tagA; (ii) following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II; and (iii) during germination, this activity was detectable before the rise in culture turbidity associated with spore outgrowth. Finally, the rate of tagA and tagB transcription increases at the end of the exponential phase of growth. Experiments in which transcription was monitored in media of different richness confirmed that higher growth rates were paralleled by lower transcription rates. This behaviour, characteristic of genes involved in septation (Vicente et al., 1991
), suggested that tag gene transcription may also respond to cell-cycle-specific signals coupling chromosome replication and cell division (Mauël et al., 1995
).
Hybrids containing teichoic acid determinants specific to strain W23 in the 168 genetic background were constructed (Karamata et al., 1987 ). Southern hybridization analyses of nine such hybrids revealed that, in all of them, integral substitution of strain 168 tag genes by strain W23 tar genes had occurred (Young et al., 1989
). Surface growth, flagellar motility, transformability and sporulation were normal (Karamata et al., 1987
), suggesting that the signals regulating teichoic acid synthesis as a function of the environment, including hypothetical signals related to the cell cycle, were identical in the parent W23 and 168 strains. Therefore, sequence comparison of the regulatory region governing teichoic acid synthesis in these strains was expected to allow recognition of common cis-regulatory transcriptional signals.
In this contribution, we report the sequence and the genetic organization of the tar genes specifying the synthesis of poly(RboP) in B. subtilis W23, as well as the main features and presumed roles of their products. Their regulation appears more complex than that of the tag divergon in strain 168. The regulatory regions of the tar and tag divergons are analysed and compared to the equivalent region from ten other Bacillus strains. Finally, we present a new analysis of an untranslated grey hole in the tag region (Lazarevic et al., 1995 ).
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METHODS |
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DNA extraction and purification.
Chromosomal DNA was extracted according to the method of Marmur (1961) and purified on CsCl density gradients in the presence of ethidium bromide (Sambrook et al., 1989
). DNA bands were collected and dye-extracted with 1-butanol prior to precipitation (Sambrook et al., 1989
). Plasmid DNA was prepared by the boiling method (Del Sal et al., 1988
).
DNA was obtained as described by Grossberger (1987)
. Phage was propagated in Escherichia coli strain P2392.
PCR amplification.
PCRs were set up with 10 ng chromosomal DNA, 200 pmol of each primer and 20 nmol of each dNTP (Biofinex) in 100 µl reaction buffer containing 2·5 units AmpliTaq DNA polymerase (Perkin Elmer). Reactions were run with denaturation for 2 min at 95 °C, followed by 30 or 40 cycles of amplification (95 °C for 1 min, 37 °C for 1 min, 72 °C for 1 min) and final extension for 15 min at 72 °C. Teichoic acid regulatory regions were amplified with the following four oligonucleotide pairs: (i) 168-1D (5'-GCAGTAAATCAAAAGTTCCATATGTGATAACTTTTTTCAT-3') and 168-1A (5'-TGTTAAGTTACTGTTAACATAAGGAATATTGTGAATAGTC-3'); (ii) 168-2D (5'-GCCTTCTGTTCTTGGGAGGTAAACAACCTC-3') and 168-2A (5'-CCTAAAGCTACGAATACCATGTCAGGATTTGCT-3'); (iii) W23-1D (5'-AGCTTCATATGTCCGTAGTGAAATAAATCAAATGTTCCGT-3') and W23-1A (5'-CTGTCTACAAAATCTAATTGATTAATGGGCTTTGTTTGC A-3'); (iv) W23-2D (5'-GCCATCTGTTCTTGGAAGGTAAATGACCTCGCA-3') and W23-2A (5'-CCTAACGCGCAAATACCATATCAGGAGTTGA-3'). Letters A and D refer to the genes in which the oligos were chosen, i.e. tagA/tarA or tagD/tarD.
Cloning.
PCR products were recovered from agarose gels (QIAquick gel extraction kit, Qiagen) and cloned in pUC18 (SureClone ligation kit, Pharmacia).
Transformation.
E. coli DH5 competent cells were prepared and transformed by the procedure of Chung & Miller (1988)
.
Primer extension.
RNA preparation and primer extension experiments were performed as previously described (Lazarevic et al., 1992 ) using 20 pmol of each of the W23-1D and W23-1A oligonucleotides (see above).
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RESULTS |
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Sequencing of the regulatory region of the teichoic acid divergon from 10 strains of Bacillus: a DNA segment present in W23-like strains is absent from 168-like strains
The regulatory regions of tagABtagDEF and tarABIJKLtarDF divergons consist of 399 and 508 nt, respectively. To explore the biological significance of this difference and to locate largely conserved sequences probably playing an important role in regulation, the teichoic acid regulatory region of a number of Bacillus strains was cloned and sequenced. Thirteen strains, including Bacillus globigii and Bacillus licheniformis, known, or predicted, to contain a linkage unit of either the 168 or the W23 type (Araki & Ito, 1989 ), were screened by PCR for the presence of a tagAtagD or a tarAtarD regulatory region, with oligonucleotide pairs corresponding to the relevant genes (Table 3
). For each strain, the PCR product, when obtained, was cloned and sequenced; six of them appeared to be of interest (see below and Table 3
). The sequences of strains BS41, BS121 and BS129 were identical to that of strain 168, whereas the sequence of strain BSG66 did not differ from that of strain BSG150. B. subtilis var. natto, B. subtilis var. niger and strain BSG40 did not yield a PCR product, suggesting that the homologies with tagAD or tarAD are much lower, or that their teichoic acid genes are organized differently.
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The tar divergon is controlled by four promoters, two of which are located on the additional 100 nt DNA segment
To confirm that sequences homologous to the divergent promoters in strain 168 were functional in strain W23, mRNA starts were mapped by primer-extension analyses with total RNA extracted from cells grown in LB medium, to OD600 1·4. Surprisingly, four promoters, two in each direction, were identified (Fig. 4). The outer ones, corresponding to those expected from sequence comparison with 168, were designated PtarA-ext and PtarD-ext (ext, external with regard to the regulatory region). Two additional promoters, located in between, were designated PtarA-int and PtarD-int (int, internal with regard to the regulatory region). Their corresponding start sites were situated 107 nt and 123 (127) nt upstream from the outer tarA and tarD mRNA starts, respectively.
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In strain 168 a 2·2 kb grey hole contains remnants of tar genes from a W23-like strain
The possibility of chromosomal rearrangements in strain 168 was already evoked when two non-coding segments of 0·7 and 2·2 kb, designated grey holes, were found on each side of ggaAB, the operon specifying the synthesis of the so-called secondary, or minor teichoic acid, poly(glucopyranosyl N-acetylgalactosamine 1-phosphate) [poly(GlcGalNAcP)] (Freymond, 1995 ; Lazarevic et al., 1995
). Analyses revealed that these, apparently untranslated, regions comprised segments similar to neighbouring teichoic acid determinants. The nucleotide sequence of strain W23 teichoic acid genes sheds new light on the 2·2 kb grey hole which apparently consists of three contiguous domains. One of them, 0·64 kb long, was reported to be 94% identical to the gtaB gene (Lazarevic et al., 1995
). It is followed by a 0·75 kb segment that is 89% identical to the W23 region comprising the equivalent of the tar, not the tag, regulatory region and apparent remnants of the tarD gene (Fig. 5
). The remaining 0·7 kb of the grey hole presents 65 and 62% identity to tarF and tagF genes, respectively. Remnants of tarD-like and tarF/tagF-like genes have obviously suffered large deletions amounting to 195 and 463 nt, respectively.
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DISCUSSION |
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Expression of tar genes identified so far is regulated by four promoters, two in each direction, whereas the regulatory region of the tag divergon contains only two promoters (Mauël et al., 1995 ). The additional promoters in W23 are located on a DNA segment of about 100 nt, situated between homologues of the two 168 promoters. Sequence analysis of the teichoic acid regulatory regions of B. globigii, B. licheniformis and eight strains of B. subtilis revealed a clear-cut difference between W23- and 168-type strains. The latter, which include B. licheniformis, were devoid of the central part of the regulatory region present in W23-like strains and corresponding to the two internal promoters of strain W23. So far, available evidence would suggest that this difference, i.e. the absence of the internal promoters, may be correlated with the nature of the synthesized anionic polymer. On the one hand, walls of S31, a W23-type strain, contain poly(RboP). On the other hand, although the nature of the anionic polymers of B. licheniformis BGSC 5A24 is not known, it was reported that the cell wall of B. licheniformis ATCC 9945, as well as that of strain AHU 1371, contain poly(GroP) (Chin et al., 1968
; Araki & Ito, 1989
). B. globigii appears as an intermediate, since its teichoic acid regulatory region resembles that of W23, from which, however, part of the segment corresponding to PtarD-int is missing, while its susceptibility to B. subtilis defective phages, i.e. PBSX resistant, PBSY and PBSZ sensitive (Mauël, unpublished), is identical to that of strain 168 (Karamata et al., 1987
), suggesting that its wall polymer may be poly(GroP).
In the gene region for the teichoic acids of strain 168, two non-coding grey holes, of 0·7 and 2·2 kb, were found on each side of ggaAB, the operon which specifies the synthesis of poly(GlcGalNAcP), the minor teichoic acid. They comprise remnants of genes involved in the synthesis of the major teichoic acid and possibly reflect chromosome rearrangements generated by horizontal acquisition of the ggaAB operon (Lazarevic et al., 1995 ). Sequence determination of the tar divergon sheds new light on the 2·2 kb grey hole, apparently the longest non-coding region of the B. subtilis 168 chromosome (Kunst et al., 1997
). Indeed, at variance with a previous conclusion, a 1·5 kb segment of this grey hole bears more resemblance to the tar regulatory region as well as to tar, rather than tag, genes. These observations suggest that, like the ggaA operon, at least some of the genes encoding poly(GroP) synthesis in strain 168 were acquired by horizontal transfer (Lazarevic et al., 1995
), possibly displacing all or part of the resident W23-like polyribitol-synthesizing genes. This event could have generated the observed chromosome rearrangements and deletions. It is also possible that the acquisition of a cassette allowing polymerization of glycerol-P residues, originally designed for the synthesis of the linkage unit only, represented a distinct selective advantage (e.g. the economy realized by the substitution of a 5-carbon for a 3-carbon molecule) which might have culminated in the replacement of poly(RboP) by poly(GroP) synthesis, and the elimination of tarIJKL genes. A more extensive comparison between the complete tag and tar regions of 168 and W23, as well as their comparison to equivalent regions of other Bacillus strains, in particular strains containing poly(GroP) in their wall, may yield new elements and possibly allow a partial reconstitution of the events which, in strain 168, led to the disturbed genetic organization of the teichoic acid locus and its interruption by two grey holes (Lazarevic et al., 1995
).
The major teichoic acids in strains 168 and W23 perform analogous functions. Not only do conditional lethal mutants affected in their synthesis exhibit the same phenotype, but also exchange of the 168 poly(GroP) for the poly(RboP) entity (Karamata et al., 1987 ) does not entail any obvious phenotypic alteration. In these hybrids, the synthesis of the major anionic polymer apparently goes hand in hand with that of peptidoglycan, decreasing under phosphate limitation, while apparently remaining closely related to the cell cycle. However, the regulation of the tar and tag divergons is different. As revealed by primer-extension mapping, the synthesis of poly(RboP) requires four promoters, whereas only two are needed for the synthesis of poly(GroP). In our opinion, these observations raise an interesting question, i.e. what is the strategy used by 168 to apparently achieve, with two promoters, the same, finely tuned, regulation as W23, endowed with four functional promoters? Investigating the role of the internal promoters in the latter strain should help in recognizing the regulatory devices elaborated by strain 168 to palliate their absence in the tag regulatory region.
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ACKNOWLEDGEMENTS |
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Received 3 August 2001;
revised 8 October 2001;
accepted 22 October 2001.