Poly(glucosyl-N-acetylgalactosamine 1-phosphate), a wall teichoic acid of Bacillus subtilis 168: its biosynthetic pathway and mode of attachment to peptidoglycan.

The ggaAB operon of Bacillus subtilis 168 encodes enzymes responsible for the synthesis of poly(glucosyl N-acetylgalactosamine 1-phosphate) [poly(GlcGalNAc 1-P)], a wall teichoic acid (WTA). Analysis of the nucleotide sequence revealed that both GgaA and GgaB contained the motif characteristic of sugar transferases, while GgaB was most likely to be bifunctional, being endowed with an additional motif present in glucosyl/glycerophosphate transferases. Transcription of the operon was thermosensitive, and took place from an unusually distant sigma(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.

Genometrics as an essential tool for the assembly of whole genome sequences: the example of the chromosome of Bifidobacterium longum NCC2705.

BACKGROUND: Analysis of the first reported complete genome sequence of Bifidobacterium longum NCC2705, an actinobacterium colonizing the gastrointestinal tract, uncovered its proteomic relatedness to Streptomyces coelicolor and Mycobacterium tuberculosis. However, a rapid scrutiny by genometric methods revealed a genome organization totally different from all so far sequenced high-GC Gram-positive chromosomes. RESULTS: Generally, the cumulative GC- and ORF orientation skew curves of prokaryotic genomes consist of two linear segments of opposite slope: the minimum and the maximum of the curves correspond to the origin and the terminus of chromosome replication, respectively. However, analyses of the B. longum NCC2705 chromosome yielded six, instead of two, linear segments, while its dnaA locus, usually associated with the origin of replication, was not located at the minimum of the curves. Furthermore, the coorientation of gene transcription with replication was very low. Comparison with closely related actinobacteria strongly suggested that the chromosome of B. longum was misassembled, and the identification of two pairs of relatively long homologous DNA sequences offers the possibility for an alternative genome assembly proposed here below. By genometric criteria, this configuration displays all of the characters common to bacteria, in particular to related high-GC Gram-positives. In addition, it is compatible with the partially sequenced genome of DJO10A B. longum strain. Recently, a corrected sequence of B. longum NCC2705, with a configuration similar to the one proposed here below, has been deposited in GenBank, confirming our predictions. CONCLUSION: Genometric analyses, in conjunction with standard bioinformatic tools and knowledge of bacterial chromosome architecture, represent fast and straightforward methods for the evaluation of chromosome assembly.

Bacillus subtilis alpha-phosphoglucomutase is required for normal cell morphology and biofilm formation.

Mutations designated gtaC and gtaE that affect alpha-phosphoglucomutase activity required for interconversion of glucose 6-phosphate and alpha-glucose 1-phosphate were mapped to the Bacillus subtilis pgcA (yhxB) gene. Backcrossing of the two mutations into the 168 reference strain was accompanied by impaired alpha-phosphoglucomutase activity in the soluble cell extract fraction, altered colony and cell morphology, and resistance to phages phi29 and rho11. Altered cell morphology, reversible by additional magnesium ions, may be correlated with a deficiency in the membrane glycolipid. The deficiency in biofilm formation in gtaC and gtaE mutants may be attributed to an inability to synthesize UDP-glucose, an important intermediate in a number of cell envelope biosynthetic processes.

The Bacillus subtilis Gne (GneA, GalE) protein can catalyse UDP-glucose as well as UDP-N-acetylglucosamine 4-epimerisation.

Mutations in the Bacillus subtilis gene that affect the activity of the uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 4-epimerase (EC 5.1.3.7) were shown to map to galE, the structural gene of the UDP-glucose (Glc) 4-epimerase (EC 5.1.3.2). This genetic evidence that the same enzyme can catalyse the epimerisation of hexoses as well as of their N-acetylated forms is confirmed by in vitro assays with purified enzyme. It appears that in B. subtilis, Gne (GneA, GalE) is involved in two distinct and essential functions, i.e., cell detoxification under certain growth conditions and the biosynthesis of anionic cell wall polymers. We discuss the evidence that such enzymes capable of utilizing both UDP-hexoses and UDP-N-acetylhexosamines are present in other organisms.

Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase.

The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit.

tagO is involved in the synthesis of all anionic cell-wall polymers in Bacillus subtilis 168.

Sequence homologies suggest that the Bacillus subtilis 168 tagO gene encodes UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme responsible for catalysing the first step in the synthesis of the teichoic acid linkage unit, i.e. the formation of undecaprenyl-PP-N-acetylglucosamine. Inhibition of tagO expression mediated by an IPTG-inducible P(spac) promoter led to the development of a coccoid cell morphology, a feature characteristic of mutants blocked in teichoic acid synthesis. Indeed, analyses of the cell-wall phosphate content, as well as the incorporation of radioactively labelled precursors, revealed that the synthesis of poly(glycerol phosphate) and poly(glucosyl N-acetylgalactosamine 1-phosphate), the two strain 168 teichoic acids known to share the same linkage unit, was affected. Surprisingly, under phosphate limitation, deficiency of TagO precludes the synthesis of teichuronic acid, which is normally induced under these conditions. The regulatory region of tagO, containing two partly overlapping sigma(A)-controlled promoters, is similar to that of sigA, the gene encoding the major sigma factor responsible for growth. Here, the authors discuss the possibility that TagO may represent a pivotal element in the multi-enzyme complexes responsible for the synthesis of anionic cell-wall polymers, and that it may play one of the key roles in balanced cell growth.

Comparison of ribitol and glycerol teichoic acid genes in Bacillus subtilis W23 and 168: identical function, similar divergent organization, but different regulation.

The tar genes directing the synthesis of poly(ribitol phosphate), the main teichoic acid in Bacillus subtilis strain W23, were sequenced. They are organized in two divergently transcribed operons, tarABIJKL and tarDF, as are the tag genes specifying poly(glycerol phosphate) synthesis in B. subtilis 168. The features of the tar genes as well as the putative participation of their products in the proposed biosynthesis pathway of poly(ribitol phosphate) are presented. The tarA and tarD genes, which are most likely involved in the synthesis of the linkage unit (the entity coupling teichoic acid to peptidoglycan), are separated by 508 nt. Sequences of the outer segments of this regulatory region are similar to the two divergent promoter regions identified upstream of the tagA and tagD genes in strain 168. However, in W23, these regions, which also included functional promoters, are separated by an additional DNA segment of about 100 nt, on which two new mRNA starts, one in each direction, were identified. The regulatory regions of teichoic acid divergons of Bacillus globigii, Bacillus licheniformis and eight strains of B. subtilis were cloned and sequenced. In four B. subtilis strains and in B. globigii, their length and sequence are similar to the regulatory region of W23. In the others, including B. licheniformis, they are of the 168-type. Analysis of nucleotide sequences of a non-coding grey hole, present in the tag region of strain 168, revealed higher similarities to tar than to tag entities. This suggests that at least part of the tag genes specifying the synthesis of glucosylated poly(glycerol phosphate) in strain 168 was introduced by horizontal gene transfer into a strain originally synthesizing a ribitol-phosphate-containing teichoic acid.

Comparative Genometrics (CG): a database dedicated to biometric comparisons of whole genomes.

The ever increasing rate at which whole genome sequences are becoming accessible to the scientific community has created an urgent need for tools enabling comparison of chromosomes of different species. We have applied biometric methods to available chromosome sequences and posted the results on our Comparative Genometrics (CG) web site. By genometrics, a term coined by Elston and Wilson [GENET: Epidemiol. (1990), 7, 17-19], we understand a biometric analysis of chromosomes. During the initial phase, our web site displays, for all completely sequenced prokaryotic genomes, three genometric analyses: the DNA walk [Lobry (1999) Microbiology Today, 26, 164-165] and two complementary representations, i.e. the cumulative GC- and TA-skew analyses, capable of identifying, at the level of whole genomes, features inherent to chromosome organization and functioning. It appears that the latter features are taxon-specific. Although primarily focused on prokaryotic chromosomes, the CG web site contains genometric information on paradigm plasmids, phages, viruses and eukaryotic organelles. Relevant data and methods can be readily used by the scientific community for further analyses as well as for tutorial purposes. Our data posted at the CG web site are freely available on the World Wide Web at http://www.unil.ch/comparativegenometrics.

Incorporation of [2-3H]glycerol into cell surface components of Bacillus subtilis 168 and thermosensitive mutants affected in wall teichoic acid synthesis: effect of tunicamycin.

A method is described for measuring the synthesis of poly(glycerol phosphate) [poly(groP)], the major wall teichoic acid (WTA), lipoteichoic acid (LTA) and phospholipid (P-lipid), through fractionation of [2-3H]glycerol ([2-3H]gro)-labelled Bacillus subtilis cells. When cultures of certain temperature-sensitive mutants defective in one of several tag genes, encoding enzymes involved in WTA synthesis, were transferred to the restrictive temperature, the synthesis of WTA underwent a specific, immediate, block, while that of LTA or P-lipid proceeded unimpeded. These results, in addition to confirming the role of tag genes, demonstrated, reciprocally, the specificity of the fractionation procedure used to distinguish label in WTA from that in LTA or P-lipid. Results of analysis of other, less severely affected, tag-deficient mutants, as well as of another genetically unrelated mutant developing comparable morphological phenotypes in non-permissive conditions, are discussed in relation to a possible mechanism generating the latter phenotype. Fractionation of B. subtilis 168 cells labelled either with [2-3H]gro or with [1-14C]N-acetylglucosamine, to which tunicamycin was added at 0.5 microg ml(-1) (the MIC) revealed a specific and marked inhibition of poly(groP) as well as of poly(3-O-beta-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate), the minor WTA. However, for 60 min at least, the syntheses of PG, LTA and P-lipid were barely affected.

Assay for UDPglucose 6-dehydrogenase in phosphate-starved cells: gene tuaD of Bacillus subtilis 168 encodes the UDPglucose 6-dehydrogenase involved in teichuronic acid synthesis.

A novel assay permitting the detection of UDPglucose 6-dehydrogenase activity in cell-free extracts obtained from phosphate-starved cultures of Bacillus subtilis is described. The critical step, the separation of phosphate-starvation-induced exo-enzymes, phosphatases and phosphodiesterases from the cytoplasmic fraction containing the UDPglucose dehydrogenase, was achieved by protoplasting and removal of the periplasmic fraction by protoplast washing. Using this method, the following were unambiguously demonstrated: (i) the presence in the cytoplasm of an enzymic activity oxidizing UDPglucose to UDPglucuronic acid, and (ii) that detection of the activity in whole-cell-free extracts is prevented by the presence of 'periplasmic' enzymes catalysing the degradation of the sugar nucleotides. With this method, several B. subtilis 168 mutants unable to synthesize teichuronic acid were examined. Strains inactivated in gene tuaD, whose product shares homology with UDPglucose 6-dehydrogenase and GDPmannose 6-dehydrogenase from other organisms, were shown to lack UDPglucose 6-dehydrogenase activity. Anion exchange chromatography revealed that mutants deficient in tuaD lacked a cytoplasmic UDPglucuronate pool.

Nucleotide sequence of the Bacillus subtilis temperate bacteriophage SPbetac2.

The Bacillus subtilis 168 chromosomal region extending from 184 degrees to 195 degrees, corresponding to prophage SPbeta, has been completely sequenced using DNA of the thermoinducible SPbetac2 mutant. This 134416 bp segment comprises 187 putative ORFs which, according to their orientation, were grouped into three clusters. Compared to its host, SPbetac2 is characterized by a lower G+C content, shorter mean ORF length, as well as a different usage of start codons. Nearly 75% of predicted ORFs do not share significant homologies to sequences in available databases. The only highly similar proteins to SPbetac2-encoded ones are host paralogues. SPbetac2 promoter regions contain SOS box consensus sequences and a repeated motif, designated SPbeta repeated element (SPBRE), that is absent from the host genome. Gene sspC, encoding the small acid-soluble protein C, that has been previously sequenced and mapped to the vicinity of the SPbeta region, was found to be part of the prophage.

Bacillus subtilis 168 gene lytF encodes a gamma-D-glutamate-meso-diaminopimelate muropeptidase expressed by the alternative vegetative sigma factor, sigmaD.

A gamma-D-glutamate-meso-diaminopimelate muropeptidase was detected in the vegetative growth phase of Bacillus subtilis 168. It is encoded by the monocistronic lytF operon expressed by the alternative vegetative sigma factor, sigmaD. Sequence analysis of LytF revealed two domains, an organization common to exoproteins of B. subtilis as well as to those from other organisms. The N-terminal domain contains a fivefold-repeated motif attributed to cell wall binding, whilst the C-terminal domain is probably endowed with the catalytic activity. Overexpression of LytF allowed its purification and biochemical characterization. Inactivation of lytF led to the loss of the cell-wall-bound protein 49' (CWBP49') and of the corresponding lytic activity as revealed by renaturation gel assay. Native cell walls prepared from the multiple lytC lytD lytE lytF-deficient mutant did not exhibit any autolysis, whereas walls prepared from a strain endowed with LytF but not with the other three enzymes underwent a slight lysis. Analysis of degradation products of cell wall devoid of teichoic-acid-bound O-esterified D-alanine unambiguously confirmed that LytF cuts the gamma-D-glutamate-mesodiaminopimelate bond.

The product of the yvoC (gerF) gene of Bacillus subtilis is required for spore germination.

All known gerF mutations affecting Bacillus subtilis spore germination have been mapped, by a combination of recombination and complementation analysis, to yvoC (Igt), a gene belonging to the yvoB (ptsK) yvoC (Igt) yvoDEF operon. Examination of the properties of null mutants confirmed that the only gene in the operon that affects germination is yvoC, which encodes a homologue of known prelipoprotein diacylglyceryl transferases. As several germination proteins (GerAC, GerBC, GerKC, GerD) are predicted lipoproteins, it is not unreasonable to assume that a defect in prelipoprotein processing will affect spore germination. Two other null mutants in this chromosomal region showed a clear phenotype: the nagA gene is required for growth on N-acetylglucosamine, whereas a null mutation in yvoB (ptsK) affects colony formation from single cells.

The N-acetylmuramoyl-L-alanine amidase encoded by the Bacillus subtilis 168 prophage SP beta.

Heat shock of Bacillus subtilis CU1147, a strain lysogenic for SP beta c2, a prophage with a thermosensitive repressor, results in phage induction and subsequent cell lysis. Cloning in Escherichia coli and sequencing of a DNA fragment of prophage SP beta led to the identification of blyA, the gene encoding a 367 amino acid polypeptide with a molecular mass of 39.6 kDa. Purified BlyA obtained from the E. coli clone exhibited an N-acetylmuramoyl-L-alanine amidase activity. Insertional mutagenesis confirmed that the latter enzyme was associated with SP beta-phage-mediated cell lysis. Analysis of the neighbouring sequence suggested that the two ORFs immediately downstream of blyA and belonging to the same operon encode polypeptides which may be involved in the release of the endolysin. The presence on the chromosomes of B. subtilis or related Bacillus spp. of other, similar genes, and their possible relationship, is discussed.