Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

The regulatory unit of Bociffus subtifis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons fytABC and lytR. Proteins LytA, LytB and LytC are endowed with export signal peptides. Mature LytA is a 9-4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein. LytB and LytC, the modifier and the amidase, are highly basic. After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively. These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases. The C-terminal moiety of LytB exhibits homology to the product of spoZZD. LytR is a 35 kDa protein which acts as an attenuator of the expression of both fytABC and fytR operons. Transcription of the fytABC operon proceeds from two promoters: PD, identified as P2*-, (Gilman et d , 1984), and an upstream PA. The former only is subject to LytR attenuation. Translational initiation of fytB and fytC is directed by UUG start codons, suggesting that fytA, B and C undergo coupled translation. Transcription of fytR is initiated at two start sites, one of which corresponds to a highly intense PA promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences. Both promoters are attenuated by LytR. It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e. that it belongs to thefra regulon and that its activity, or part of it, is co-regulated with flagellar motility. The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the fytABC operon is discussed.

Mutations designated gtaC and gtaE that affect ␣-phosphoglucomutase activity required for interconversion of glucose 6-phosphate and ␣-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 ␣-phosphoglucomutase activity in the soluble cell extract fraction, altered colony and cell morphology, and resistance to phages 29 and 11. 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.Peptidoglycan and wall teichoic acids (WTAs) are major constituents of the cell wall in many gram-positive bacteria. It has been proposed that lipoteichoic acids (LTAs), polymers anchored in the membrane, and WTAs contribute to the cell wall electrolyte properties, modulate the activity of peptidoglycan-degrading enzymes, and maintain cation homeostasis (24). The biology of WTAs and LTAs has been reviewed recently (17,24).In Bacillus subtilis 168, poly(glycerolphosphate) [poly(Gro-P)], known as the major WTA, is glucosylated and D-alanylated at the C-2 position of the 1,3-phosphodiester-linked glycerol units (17,24). Absence of either of these substituents does not affect cell viability, whereas the polymer backbone is an essential cell constituent (20). Glucosylation of poly(Gro-P) plays an essential role in the attachment of phage 29 to B. subtilis 168 (34). Mutations associated with a 29-resistant phenotype were mapped to three loci, gtaA, gtaB, and gtaC (35). Subsequently, analyses of gtaB-deficient mutants established that gtaB is the structural gene of the UTP:␣-glucose-1-phosphate uridylyltransferase (28, 31), the enzyme that catalyzes the formation of UDP-glucose (UDP-Glc) from ␣-glucose 1-phosphate (␣-Glc 1-P) and UTP. gtaA (rodD), which has been renamed tagE, encodes the enzyme for the transfer of glucosyl groups from UDP-Glc to the poly(Gro-P) moiety of the major WTA (10, 19). In addition, UDP-Glc is required for the polymerization of poly(glucosyl N-acetylgalactosamine phosphate), the minor WTA (17), as well as for the YpfP-governed synthesis of diglucosyldiacylglycerol, the membrane anchor for poly(Gro-P) which forms the main chain of LTA (12).Mutations leading to an ␣-phosphoglucomutase (␣-PGM) deficiency (i.e., mutations associated with the inability to convert glucose 6-phosphate [Glc 6-P] to ␣-Glc 1-P) were mapped to the gtaC locus at 77°on the B. subtilis genome (1, 28). The gtaC mutants were, however, split into two subgroups; the PBSZ-sensitive mutants retained the designation gtaC, whereas the PBSZ-resistant mutants were renamed gtaE (28). Inspection of the B. subtilis 168 chromosome sequence (16) In the present study we found that gtaC and gtaE mutations map to yhxB (designated pgcA in this paper). Below we present evidence that the...

The Bacillus subtilis 168 chromosomal region extending from 184" to 195", corresponding to prophage SPP, has been completely sequenced using DNA of the thermoinducible SPPc2 mutant. This 134416 bp segment comprises 187 putative ORFs which, according to their orientation, were grouped into three clusters. Compared to its host, SPPc2 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 SPPc2-encoded ones are host paralogues. SPPc2 promoter regions contain 5 0 5 box consensus sequences and a repeated motif, designated SPP repeated element (SPBRE), that is absent from the host genome. Gene sspC, encoding the small acidsoluble protein C, that has been previously sequenced and mapped to the vicinity of the SPP region, was found to be part of the prophage.

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 IPTGinducible 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 σ A -controlled promoters, is similar to that of sigA, the gene encoding the major σ 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.