Shu Ishikawa

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.

Sequence-specific error profile of Illumina sequencers

We identified the sequence-specific starting positions of consecutive miscalls in the mapping of reads obtained from the Illumina Genome Analyser (GA). Detailed analysis of the miscall pattern indicated that the underlying mechanism involves sequence-specific interference of the base elongation process during sequencing. The two major sequence patterns that trigger this sequence-specific error (SSE) are: (i) inverted repeats and (ii) GGC sequences. We speculate that these sequences favor dephasing by inhibiting single-base elongation, by: (i) folding single-stranded DNA and (ii) altering enzyme preference. This phenomenon is a major cause of sequence coverage variability and of the unfavorable bias observed for population-targeted methods such as RNA-seq and ChIP-seq. Moreover, SSE is a potential cause of false single-nucleotide polymorphism (SNP) calls and also significantly hinders de novo assembly. This article highlights the importance of recognizing SSE and its underlying mechanisms in the hope of enhancing the potential usefulness of the Illumina sequencers.

A widespread family of bacterial cell wall assembly proteins

Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR–Cps2A–Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid‐linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall.

Noc protein binds to specific DNA sequences to coordinate cell division with chromosome segregation

Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis, the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP‐on‐Chip and bioinformatics, we have identified a consensus Noc‐binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc–YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc–YFP and conferred a severe Noc‐dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo. We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division.

A new FtsZ-interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis

The assembly of ring‐like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ‐binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z‐ring formation were chemically cross‐linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two‐hybrid analysis and fluorescence microscopy of YFP–YlmF in B. subtilis cells indicated YlmF localizes to the division site in an FtsZ‐dependent manner. A single disruption of YlmF resulted in a slight elongation of cells; however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell division due to the defect in Z‐ring formation. In contrast, the ftsA‐null mutant phenotype, caused by inefficient Z‐ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.

Genome-wide binding profiles of the Bacillus subtilis transition state regulator AbrB and its homolog Abh reveals their interactive role in transcriptional regulation

AbrB is a global transcriptional regulator of Bacillus subtilis that represses the expression of many genes during exponential growth. Here, we demonstrate that AbrB and its homolog Abh bind to hundreds of sites throughout the entire B. subtilis genome during exponential growth. Comparison of regional binding of AbrB and Abh in wild-type, ΔabrB and Δabh backgrounds revealed that they bind as homomer and/or heteromer forms with different specificities and affinities. We found four AbrB and Abh binding patterns were major. Three of these contain pairs of TGGNA motifs connected by A/T-rich sequences, differing in arrangement and spacing. We also assessed the direct involvement of these complexes in the control of gene expression. Our data indicate that AbrB usually acts as a repressor, and that the ability of Abh to act as a transcriptional regulator was limited. We found that changes to AbrB/Abh levels affect their binding at several promoters and consequently transcriptional regulation. Surprisingly, most AbrB/Abh binding events had no impact on transcription, suggesting an interesting possibility that AbrB/Abh binding is analogous to nucleoid-associated protein binding in Escherichia coli.

Synergistic regulation of competence development in Bacillus subtilis by two Rap-Phr systems.

The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulators ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Mol. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and RapF-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity.

Distribution of Stable DnaA-Binding Sites on the Bacillus Subtilis Genome Detected using a Modified ChIP-chip Method

We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors—the local density of DnaA boxes and their affinities for DnaA—are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication.

Structural and genetic analyses reveal the protein SepF as a new membrane anchor for the Z ring

Significance A key step in bacterial cell division is the polymerization of FtsZ at midcell into a ring-like structure. This so-called Z ring forms a scaffold for the other cell division proteins. FtsA anchors the Z ring to the cell membrane; however, many bacterial species do not have FtsA. Here, we show that the conserved protein SepF, which forms large protein rings, also functions as a membrane anchor for the Z ring. We determined the molecular structure of the FtsZ-binding and ring-forming domain of SepF and show that the membrane-binding domain is located at the very beginning of the protein. These results explain why FtsA and SepF can fulfill similar functions in bacterial cell division. A key step in bacterial cell division is the polymerization of the tubulin homolog FtsZ at midcell. FtsZ polymers are anchored to the cell membrane by FtsA and are required for the assembly of all other cell division proteins. In Gram-positive and cyanobacteria, FtsZ filaments are aligned by the protein SepF, which in vitro polymerizes into large rings that bundle FtsZ filaments. Here we describe the crystal structure of the only globular domain of SepF, located within the C-terminal region. Two-hybrid data revealed that this domain comprises the FtsZ binding site, and EM analyses showed that it is sufficient for ring formation, which is explained by the filaments in the crystals of SepF. Site-directed mutagenesis, gel filtration, and analytical ultracentrifugation indicated that dimers form the basic units of SepF filaments. High-resolution structured illumination microscopy suggested that SepF is membrane associated, and it turned out that purified SepF not only binds to lipid membranes, but also recruits FtsZ. Further genetic and biochemical analyses showed that an amphipathic helix at the N terminus functions as the membrane-binding domain, making SepF a unique membrane anchor for the FtsZ ring. This clarifies why Bacillus subtilis grows without FtsA or the putative membrane anchor EzrA and why bacteria lacking FtsA contain SepF homologs. Both FtsA and SepF use an amphipathic helix for membrane binding. These helices prefer positively curved membranes due to relaxed lipid density; therefore this type of membrane anchor may assist in keeping the Z ring positioned at the strongly curved leading edge of the developing septum.

Differential Binding Profiles of StpA in Wild-Type and hns Mutant Cells: a Comparative Analysis of Cooperative Partners by Chromatin Immunoprecipitation-Microarray Analysis

We performed chromatin immunoprecipitation-microarray analysis to investigate differences in function between StpA and H-NS in Escherichia coli cells. StpA binding regions essentially overlap those of H-NS in wild-type cells, while they are reduced to one-third in the hns mutant. The H-NS binding profile was unaffected by stpA inactivation.