Kei Asai

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.

A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition

We have analysed the function of a gene of Bacillus subtilis, the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23°C but not at 37°C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23°C and in the conditional null mutant grown in low concentrations of IPTG at 37°C. In addition, immunofluorescence microscopy showed that subcellular localization of the Spo0J partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild‐type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.

Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis

GTP-binding proteins are found in all domains of life and are involved in various essential cellular processes. With the recent explosion of available genome sequence data, a widely distributed bacterial subfamily of GTP-binding proteins was discovered, represented by the Escherichia coli Era and the Bacillus subtilis Obg proteins. Although only a limited number of the GTP-binding proteins belonging to the subfamily have been experimentally characterized, and their function remains unknown, the available data suggests that many of them are essential to bacterial growth. When the complete genomic sequence of B. subtilis was surveyed for genes encoding GTP-binding proteins of the Era/Obg family, nine such genes were identified. As a first step in elucidating the functional networks of those nine GTP-binding proteins, data presented here indicates that six of them are essential for B. subtilis viability. Additionally, it is shown that the six essential proteins are able to specifically bind GTP and GDP in vitro. Experimental depletion of the essential GTP-binding proteins was examined in the context of cell morphology and chromosome replication, and it was found that two proteins, Bex and YqeH, appeared to participate in the regulation of initiation of chromosome replication. Collectively, these results suggest that members of the GTP-binding Era/Obg family are important proteins with precise, yet still not fully understood, roles in bacterial growth and viability.

The essential two-component regulatory system encoded by yycF and yycG modulates expression of the ftsAZ operon in Bacillus subtilis

Essential two-component systems are now being identified in bacteria. The Bacillus subtilis yycF gene encoding a response regulator, and its orthologue in Staphylococcus aureus, were reported recently to be essential for cell growth, although genes under their control have yet to be identified. The essential nature of the yycF regulator gene and its cognate kinase gene, yycG, in B. subtilis was also noted during the course of construction of a knockout mutant bank of newly identified genes in the genome sequence project. It was found that yycG could be deleted in the presence of an active form of the YycF protein, thereby suggesting direct interaction between YycG and YycF. Production of mini-cells and reduction in cell length occurred when the YycF regulator was overproduced in B. subtilis. These observations led to the finding that YycF overproduction up-regulated the expression from the P1 promoter of the cell division operon, ftsAZ. In addition, the YycF protein binds to the P1 promoter region in vitro. These results clearly indicate that the essential two-component regulatory system encoded by yycF and yycG genes has the potential to modulate expression of the ftsAZ operon in B. subtilis.

Binding of response regulator DegU to the aprE promoter is inhibited by RapG, which is counteracted by extracellular PhrG in Bacillus subtilis

We screened the putative rap‐phr (response regulator aspartyl‐phosphate phosphatase‐phosphatase regulator) systems identified in the Bacillus subtilis genome for a rap gene that affects aprE (alkaline protease gene) expression by using a multicopy plasmid. We found that rapG was involved in the regulation of aprE, which belongs to the regulon of DegU, the response regulator of the DegS‐DegU two‐component system. Disruption of rapG and phrG resulted in enhancement and reduction of aprE‐lacZ expression, respectively, suggesting that PhrG inhibits RapG activity. Addition of 1–30 nM of a synthetic pentapeptide (PhrG; NH2‐EKMIG‐COOH) to the phrG disruptant completely rescued aprE‐lacZ expression, indicating that the PhrG peptide is indeed involved in aprE‐lacZ expression. Surprisingly, either introduction of multicopy phrG or addition of the PhrG peptide at high concentrations (100–300 nM) to the phrG cells decreased aprE‐lacZ expression. These results are reminiscent of the previous observation that at higher concentrations the PhrC peptide inhibits srfA‐lacZ expression directed by ComA, the regulator of the ComP‐ComA two‐component system. Because the Rap proteins belong to a family of aspartyl protein phosphatases, we tried to investigate the possible influence of RapG on dephosphorylation of DegU‐P (phosphorylated DegU) in vitro. RapG, however, did not affect dephosphorylation of DegU‐P under the adopted experimental conditions. Therefore, we hypothesized that RapG might inhibit the binding activity of DegU to the target promoters. We analysed the interaction of DegU and RapG using the aprE promoter and another target, a comK promoter. Gel shift analysis revealed that RapG served as the inhibitor of DegU binding to the promoter regions of aprE and comK and that this inhibition was counteracted by the PhrG peptide.

Regulation of the transport system for C4-dicarboxylic acids in Bacillus subtilis

Transport systems for C4-dicarboxylates, such as malate, fumarate and succinate, are poorly understood in Gram-positive bacteria. The whole genome sequence of Bacillus subtilis revealed two genes, ydbE and ydbH, whose deduced products are highly homologous to binding proteins and transporters for C4-dicarboxylates in Gram-negative bacteria. Between ydbE and ydbH, genes ydbF and ydbG encoding a sensor-regulator pair, were located. Inactivation of each one of the ydbEFGH genes caused a deficiency in utilization of fumarate or succinate but not of malate. Expression of ydbH, encoding a putative transporter, was stimulated in a minimal salt medium containing 0-05% yeast extract but repressed by the addition of malate to the medium. Inactivation of the putative sensor-regulator pair or solute-binding protein, ydbFG or ydbE, caused complete loss of ydbH expression. The utilization of fumarate and stimulation of ydbH expression resumed in a ydbE null mutant in which ydbFGH were overproduced. Based on these observations, together with analysis of the sequence similarities of the deduced product, we conclude that YdbH is a C4-dicarboxylate-transport protein and its expression is regulated by a C4-dicarboxylate sensor kinase-regulator pair, YdbF and YdbG. Furthermore, it is suggested that YdbE does not directly participate in transport of C4-dicarboxylates, but plays a sensory role in the ydbF-ydbG two-component system, giving rise to specificity or increased efficiency to the system. Deletion analysis of the promoter region of ydbH revealed that a direct repeat sequence was required for the activation of ydbH expression. A catabolite-responsive element (CRE) was also found in the -10 region of the promoter, suggesting negative regulation by a CRE-binding protein.

ClpC regulates the fate of a sporulation initiation sigma factor, sigmaH protein, in Bacillus subtilis at elevated temperatures.

Using a strain carrying a clpC–bgaB transcriptional fusion at the amyE locus, we found that the expression of a clpC operon was induced at the end of exponential growth in a σB‐independent manner and ceased around T3.5 in the wild type but not in a spo0H mutant. This suggests that some gene product(s) whose expression is dependent on σH function is required for the turn‐off of clpC transcription during an early stage of sporulation. A clpC deletion mutant showed a temperature‐sensitive sporulation phenotype and exhibited an abnormally large accumulation of σH in the cell at 45°C after T2, at which time the σH level in the wild type had begun to decrease. These results, together with the fact that spo0H transcription in the clpC deletion mutant was similar to that of the wild type, suggested that ClpC may be responsible for the degradation of σH after the accomplishment of its role in sporulation. Moreover, as expected from these results, overproduction of Spo0A was also observed after the initiation of sporulation in the clpC deletion mutant at 45°C.

The Bacillus subtilis yabQ gene is essential for formation of the spore cortex.

An extensive screening for transcripts with probes specific for the genes in a 108 kb region from rrnO to spo0H of the Bacillus subtilis chromosome led to identification of an operon, yabP--yabQ--divIC--yabR, the expression of which was initiated at the second hour of sporulation and in a sigma(E)-dependent manner. Among three y genes in the operon, deletion of the yabQ gene, which is predicted to encode a protein product of 468 residues with five membrane-spanning domains, resulted in a large decrease in numbers of chloroform-, lysozyme- and heat-resistant spores, compared to findings with the wild-type strain. Electron microscopy revealed that development of the spore cortex was blocked in the yabQ mutant. In addition, although the spore coat was visible, the inner coat layer of the mutant seemed partially detached from the outer coat. In sporangia of the strains harbouring an in-frame fusion of the green fluorescent protein gene to yabQ, fluorescence was detected around the forespore. This localization did not depend on SpoIVA or on CotE functions, both of which determine proper localization of coat proteins and cortex formation. The yabQ deletion did not affect expression of genes involved in cortex synthesis. These results suggest that the YabQ protein localizes in the membrane of the forespore and plays an important role in cortex formation.

Thermo-labile stability of σH (Spo0H) in temperature-sensitive spo0H mutants of Bacillus subtilis can be suppressed by mutations in RNA polymerase β subunit

Abstract We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5, having E61K and G30E amino-acid substitutions within the σH protein, respectively, and located in the highly conserved region, “2”, among prokaryotic sigma factors that participates in binding to core enzyme of RNA polymerase. These mutants showed a sporulation-deficient phenotype at 43°C. Moreover, we successfully isolated suppressor mutants that were spontaneously generated from the spo0H mutants. Our genetic analysis of these suppressor mutations revealed that the suppressor mutations are within the rpoB gene coding for the β subunit of RNA polymerase. The mutations caused single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas the σH-dependent expression of a spo0A–bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the suppressor mutants at 43°C. Western blot analysis showed that the level of σH protein in the wild type increased between T0 and T2 and decreased after T3, while the level of σH protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant σH proteins were rapidly degraded by some unknown proteolytic enzyme(s). The analysis of the half-life of σH protein showed that the short life of σH in spo0H mutants is prolonged in the suppressor mutants. These findings suggest that, at least to some extent, the process of E-σH formation may be involved in stabilization of σH at the onset of sporulation.