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.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among Ϸ4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillinbinding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two D,L-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the D,L-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these D,L-endopeptidases showed LytF and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of D,L-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed.

DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18-and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A 580 , and 72% dipicolinic acid release compared with that of the wild-type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 ؋ 10 Bacillus subtilis produces several autolysins (9), including two major autolysins (CwlB [LytC] and CwlG [LytD]) (23,28,31,40). CwlB is a 50-kDa N-acetylmuramoyl-L-alanine amidase (amidase) which cleaves the amide bond between the lactyl group of muramic acid and the ␣-amino group of Lalanine (23,28), and CwlG is a 90-kDa endo-␤-N-acetylglucosaminidase which cleaves the glycosyl bond between glucosamine and muramic acid (31,40).During sporulation and germination, the action of autolysins is assumed to be required for asymmetric septum peptidoglycan hydrolysis, which is a morphogenic transition between sporulation stages II and III, cortex maturation, mother cell lysis, and cortex hydrolysis during germination (5,6,13,42,45). The spore cortex, with a chemical structure slightly distinct from that of vegetative cell wall peptidoglycan, is apparently responsible for the maintenance of spore dormancy (6, 9). At the onset of germination, the cortex is selectively hydrolyzed, leaving a thin layer of vegetative cell peptidoglycan which forms the basis of the new vegetative cell wall (11). Germination-specific cortex-lytic enzymes which are apparently responsible for hydrolysis of the spore cortex during the germination response have been purified from spores of Bacillus megaterium KM (11, 12) and Bacillus cereus (30). It has proved difficult to solubilize autolysins from spores of B. subtilis (5, 9), although several sporulation-specific lytic activities have been identified by means of synthetic substrates (16) or by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (9). We recently cloned a sporulation-specific cell wall hydrolase gene (cwlC) from B. subtilis (20). CwlC degraded spore cortex peptidoglycan, but its function is still obscure.We report here that a new gene exhibiting sporulation phase-specific gene expression, cwlD, encodes a putative cell wall hydrolase and that spores from a mutant having an insertionally inactivated cwlD gene are deficient in germination. MATERIALS AND METHODSBacterial strains, phages, and plasmids. The strains of B. subtilis used in this s...

LytF, LytE, and LytC are vegetative cell wall hydrolases in Bacillus subtilis. Immunofluorescence microscopy showed that an epitope-tagged LytF fusion protein (LytF-3xFLAG) in the wild-type background strain was localized at cell separation sites and one of the cell poles of rod-shaped cells during vegetative growth. However, in a mutant lacking both the cell surface protease WprA and the extracellular protease Epr, the fusion protein was observed at both cell poles in addition to cell separation sites. This suggests that LytF is potentially localized at cell separation sites and both cell poles during vegetative growth and that WprA and Epr are involved in LytF degradation. The localization pattern of LytE-3xFLAG was very similar to that of LytF-3xFLAG during vegetative growth. However, especially in the early vegetative growth phase, there was a remarkable difference between the shape of cells expressing LytE-3xFLAG and the shape of cells expressing LytF-3xFLAG. In the case of LytF-3xFLAG, it seemed that the signals in normal rod-shaped cells were stronger than those in long-chain cells. In contrast, the reverse was found in the case of LytE-3xFLAG. This difference may reflect the dependence on different sigma factors for gene expression. The results support and extend the previous finding that LytF and LytE are cell-separating enzymes. On the other hand, we observed that cells producing LytC-3xFLAG are uniformly coated with the fusion protein after the middle of the exponential growth phase, which supports the suggestion that LytC is a major autolysin that is not associated with cell separation.

A major BaciUus subtlis 168S autolysin (N-acetylmuramoyl-L-alanine amidase [EC 3.5.1.28]) was purified and then cleaved with cyanogen bromide. The N-terminal amino acid sequence of one of the resultant peptides was determined in order to make synthetic oligonucleotides. A 2.5-kb EcoRI fragment was cloned into Escherichia coli JM109 and detected by colony hybridization by using the oligonucleotides as probes. Sequencing of the insert showed the presence of an open reading frame (designated cwlB), starting at a UUG codon, which encodes a polypeptide of 496 amino acids with a molecular mass of 52,623 Da. CWLB had a presumed signal peptide which is processed after Ala at position 24. Insertional inactivation of the cwlB gene of the B. subtilis chromosome led to an approximately 90% decrease in the total cell wall hydrolytic activity of stationary-phase cells and extraordinary resistance to cell lysis, even after 6 days of incubation at 37C. No apparent changes in cell morphology, motility, competence, sporulation, or germination were observed.

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, M r ‫؍‬ 50,513) is a unique 6-phosphoryl-O-␣-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-␣-glucosidase) that requires both NAD(H) and divalent metal (Mn 2؉ , Fe 2؉ , Co 2؉ , or Ni 2؉) for activity. 6-Phospho-␣-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the M r of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-␣-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-␣-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp 41 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-␣-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn 2؉ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-␣-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.The serendipitous discovery in 1964 (1, 2) of the bacterial phosphoenol pyruvate-dependent sugar phosphotransferase system (PEP-PTS) 1 by Roseman and colleagues represents a landmark in our understanding of carbohydrate transport by microorganisms (3, 4). Since the initial description in Escherichia coli, this phosphoryl group-transfer system (5, 6) has been established as the primary mechanism for the accumulation of sugars by bacteria from both Gram-negative (7, 8) and Gram-positive genera (9 -12). Operationally, the multi-component PEP-PTS (13) comprises both membrane-localized and cytoplasmic proteins that in concert catalyze the simultaneous phosphorylation and vectorial translocation of sugar across the cytoplasmic membrane. Catalytically, each PEP-PTS requires two general components (Enzyme I and HPr) that, allied with sugar-specific proteins (IIA, -B, and -C; for discussion, see Ref. 14), promote the sequential transfer of the high energy, phosphoryl moiety from PEP to the incoming sugar. Prior to catabolism via energy-yielding pathways, the intracellular disaccharide phosphates must first be hydrolyzed to their constituent hexose 6-phosphate and aglycone moieties. Several phosphoglycosylhydrolases (whose ge...

A new peptidoglycan hydrolase, Bacillus subtilis YojL (cell wall-lytic enzyme associated with cell separation, renamed CwlS), exhibits high amino acid sequence similarity to LytE (CwlF) and LytF (CwlE), which are associated with cell separation. The N-terminal region of CwlS has four tandem repeat regions (LysM repeats) predicted to be a peptidoglycan-binding module. The C-terminal region exhibits high similarity to the cell wall hydrolase domains of LytE and LytF at their C-terminal ends. The C-terminal region of CwlS produced in Escherichia coli could hydrolyze the linkage of D-␥-glutamyl-meso-diaminopimelic acid in the cell wall of B. subtilis, suggesting that CwlS is a D,L-endopeptidase. ␤-Galactosidase fusion experiments and Northern hybridization analysis suggested that the cwlS gene is transcribed during the late vegetative and early stationary phases. A cwlS mutant exhibited a cell shape similar to that of the wild type; however, a lytE lytF cwlS triple mutant exhibited aggregated microfiber formation. Moreover, immunofluorescence microscopy showed that FLAG-tagged CwlS was localized at cell separation sites and cell poles during the late vegetative phase. The localization sites are similar to those of LytF and LytE, indicating that CwlS is involved in cell separation with LytF and LytE. These specific localizations may be dependent on the LysM repeats in their N-terminal domains. The roles of CwlS, LytF, and LytE in cell separation are discussed.