Junichi Sekiguchi

Essential Bacillus subtilis genes

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 ≈4,100 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.

Lipase production of Aspergillus oryzae

Aspergillus oryzae produced a small amount of lipase (0.05–0.8 U/wet-g of solid medium) in solid cultures, in contrast to the larger amount (0.46 U/ml) in a shake-flask culture in a modified GYP medium containing 2% glucose, 1% yeast extract and 2% Polypepton. Optimum conditions of lipase production in the submerged culture of A. oryzae were determined in terms of pH, composition of medium, and temperature. In a shake-flask culture at 28°C, the maximum amount of lipase increased to 0.78 U/ml upon the addition of 3% soybean oil to the modified GYP medium. In a jar fermentor culture, 30 U/ml lipase activity was obtained after 72 h at 28°C under appropriate conditions. Lipase production was greatly influenced by the culture temperature, and the optimum temperature for lipase production was about 24°C with a narrow temperature range, which was 10 degrees lower than that for the growth. In the submerged cultures, two kinds of lipase at least exhibiting different substrate specificities were also suggested.

The CitST two‐component system regulates the expression of the Mg‐citrate transporter in Bacillus subtilis

citS and citT genes encoding a new two‐component system were identified in the 71° region between the pel and citM loci on the Bacillus subtilis chromosome. citS‐ and citT‐deficient strains were unable to grow on minimal plates including citrate as a sole carbon source. In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg‐citrate complex, exhibited the same phenotype on this medium. Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM–yflN operon. Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant. Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose. We found a sequence exhibiting homology to a catabolite‐responsive element (cre) in the citM promoter region. Glucose repression was lost in ccpA and citM–cre mutants. From the result of a citM–promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from −62 to −74 and from −149 to −189, relative to the citM start point. Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from −36 to −84 and from −168 to −194. From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA.

Localization of the Vegetative Cell Wall Hydrolases LytC, LytE, and LytF on the Bacillus subtilis Cell Surface and Stability of These Enzymes to Cell Wall-Bound or Extracellular Proteases

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.

Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene

We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241-248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.

Bacillus minimum genome factory: effective utilization of microbial genome information

In 1997, the complete genomic DNA sequence of Bacillus subtilis (4.2 Mbp) was determined and 4100 genes were identified [Kunst, Ogasawara, Moszer, Albertini, Alloni, Azevedo, Bertero, Bessieres, Bolotin, Borchert, S. et al. (1997) Nature 90, 249–256]. In addition, B. subtilis, which shows an excellent ability to secrete proteins (enzymes) and antibiotics in large quantities outside the cell, plays an important role in industrial and medical fields. It is necessary to clarify the genes involved in the production of compounds by understanding the network of these 4100 genes and the proceeding analysis of genes of unknown functions. In promoting such a study, it is expected that the regulatory system of B. subtilis can be simplified by the creation of a Bacillus strain with a reduced genome by discriminating genes unnecessary for the production of proteins from essential genes, and deleting as many of these unnecessary genes as possible, which may help to understand this complex network of genes. We have previously distinguished essential and non‐essential genes by evaluating the growth and enzyme‐producing properties of strains of B. subtilis in which about 3000 genes (except 271 essential genes) have been disrupted or deleted singly, and have successfully utilized the findings from these studies in creating the MG1M strain with an approx. 1 Mbp deletion by serially deleting 17 unnecessary regions from the genome. This strain showed slightly reduced growth in enzyme‐production medium, but no marked morphological changes. Moreover, we confirmed that the MG1M strain had cellulase and protease productivity comparable with that of the B. subtilis 168 strain, thus demonstrating that genome reduction does not contribute to a negative influence on enzyme productivity.

Stabilization of cell wall proteins in Bacillus subtilis: a proteomic approach.

Even though cell wall proteins of Bacillus subtilis are characterized by specific cell wall retention signals, some of these are also components of the extracellular proteome. In contrast to the majority of extracellular proteins, wall binding proteins disappeared from the extracellular proteome during the stationary phase and are subjected to proteolysis. Thus, the extracellular proteome of the multiple protease‐deficient strain WB700 was analyzed which showed an increased stability of secreted WapA processing products during the stationary phase. In addition, stabilization of the WapA processing products was observed also in a sigD mutant strain which is impaired in motility and cell wall turnover. Next, we analyzed if proteins that can be extracted from B. subtilis cell walls are stabilized in the WB700 strain as well as in the sigD mutant. Thus, the cell wall proteome of B. subtilis wild type was defined showing most abundantly cell wall binding proteins (CWBPs) resulting from the WapA and WprA precursor processing. The inactivation of extracellular proteases as well as SigmaD caused an increase of CWBP105 and a decrease of CWBP62 in the cell wall proteome. We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover.

Glucosaminidase of Bacillus subtilis: cloning, regulation, primary structure and biochemical characterization

The 90 kDa glucosaminidase protein was purified to apparent homogeneity from vegetative cells of Bacillus subtilis AC327, and then the corresponding gene was cloned into Escherichia coli in two inactive forms by standard procedures. Nucleotide sequencing of the glucosaminidase region revealed a monocistronic operon, (designated lytD = cwIG) encoding a 95.6 kDa protein, comprising 880 amino acid residues, which has a typical signal peptide. Moreover, another monocistronic operon (designated pmi = orfX), encoding a 35.4 kDa protein, was found upstream of the glucosaminidase gene. Expression of a lytD-lacZ fusion gene, driven by lytD regulatory sequences, was observed during the exponential growth phase. The introduction of a sigD null mutation greatly reduced (by about 95%) the expression of the fusion. Amino acid sequence analysis of the glucosaminidase showed two types of direct repeats, each type being present twice, in the N-terminal-to-central region of the glucosaminidase: these repeats probably represent the cell-wall-binding domain. Zymographic analysis revealed that the 90 kDa glucosaminidase is partly processed to several smaller proteins (35-39 kDa), retaining lytic activity. Processing of these proteins occurred between the N-terminal cell-wall-binding and C-terminal catalytic domains of the glucosaminidase, the site being located between the 569th and 606th codons of the glucosaminidase. Serial deletions from the N-terminus of the glucosaminidase revealed that the loss of more than one repeating unit drastically reduces its lytic activity toward cell walls. The lytD gene product, in either an intact or a truncated form, was found to be lethal for E. coli, and the N-terminally truncated glucosaminidase proteins, produced in E. coli, were very unstable. The partially purified glucosaminidase from B. subtilis was found to be very unstable at low ionic strength at 37 degrees C, but this instability was overcome by the addition of either SDS-purified cell wall or protease inhibitor (PMSF) to the enzyme or after purification of the glucosaminidase to apparent homogeneity.

A New d,l-Endopeptidase Gene Product, YojL (Renamed CwlS), Plays a Role in Cell Separation with LytE and LytF in Bacillus subtilis

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-gamma-glutamyl-meso-diaminopimelic acid in the cell wall of B. subtilis, suggesting that CwlS is a d,l-endopeptidase. beta-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.

A Polysaccharide Deacetylase Gene (pdaA) Is Required for Germination and for Production of Muramic δ-Lactam Residues in the Spore Cortex of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. Beta-galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by E(sigma)(G) RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of L-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic delta-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic delta-lactam is discussed.