Yasutaro Fujita

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.

Additional Targets of the Bacillus subtilis Global Regulator CodY Identified by Chromatin Immunoprecipitation and Genome-Wide Transcript Analysis

Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.

Regulation of fatty acid metabolism in bacteria

In Escherichia coli, the main player in transcription regulation of fatty acid metabolism is the FadR protein, which is involved in negative regulation of fatty acid degradation and in positive and negative regulation of the cellular processes related to it, as well as in positive regulation of the biosynthesis of unsaturated fatty acids in a concerted manner with negative regulation of FabR. On the other hand, Bacillus subtilis possesses two global transcriptional regulators, FadR (YsiA) and FapR. B. subtilis FadR represses fatty acid degradation, whereas FapR represses almost all the processes in the biosynthesis of saturated fatty acids and phospholipids. Furthermore, Streptococcus pneumoniae FabT represses the genes of fatty acid biosynthesis that are clustered in its genome. Long‐chain acyl‐CoAs appear to be metabolic signals for fatty acid degradation by bacteria in general, and antagonize the FadR protein from either E. coli or B. subtilis. However, malonyl‐CoA is a metabolic signal for fatty acid and phospholipid biosynthesis by Gram‐positive low‐GC bacteria, and it antagonizes FapR. These would be the primary aspects for understanding the elaborate and complex regulation of fatty acid metabolism in bacteria to maintain membrane lipid homeostasis.

Specific recognition of the Bacillus subtilis gnt cis‐acting catabolite‐responsive element by a protein complex formed between CcpA and seryl‐phosphorylated HPr

Catabolite repression of various Bacillus subtilis catabolic operons which carry a cis‐acting catabolite‐responsive element (ORE), such as the gnt operon, is mediated by CcpA, a protein belonging to the GalR‐Lacl family of bacterial transcriptional repressors/activators, and the seryl‐phosphorylated form of HPr, a phosphocarrier protein of the phosphoenol‐pyruvate:sugar phosphotransferase system. Footprinting experiments revealed that the purified CcpA protein interacted with P‐ser‐HPr to cause specific protection of the gnt CRE against DNase I digestion. The specific recognition of the gnt CRE was confirmed by the results of footprinting experiments using mutant gnt CREs carrying one of the following base substitutions within the CRE consensus sequence: G to T at position +149 or C to Tat position +154 (+1 is the gnt transcription initiation nucleotide). The two mutant CREs causing a partial relief from catabolite repression were not protected by the CcpA/P‐ser‐HPr complex in footprinting experiments. Based on these and previous findings, we propose a molecular mechanism underlying catabolite repression in B. subtilis mediated by CcpA and P‐ser‐HPr.

Carbon Catabolite Control of the Metabolic Network in Bacillus subtilis

The histidine-containing protein (HPr) is the energy coupling protein of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS), which catalyzes the transport of carbohydrates in bacteria. In Bacillus subtilis and close relatives, global regulation of carbon catabolite control occurs on the binding of the complex of CcpA (catabolite control protein A) and P-Ser-HPr (seryl-phosphorylated form of HPr) to the catabolite responsive elements (cre) of the target operons, the constituent genes of which are roughly estimated to number 300. The complex of CcpA and P-Ser-HPr triggers the expression of several genes involved in the formation of acetate and acetoin, major extracellular products of B. subtilis grown on glucose. It also triggers the expression of an anabolic operon (ilv-leu) involved in the biosynthesis of branched-chain amino acids, which subsequently leads to cell propagation. On the other hand, this complex represses many genes and operons, which include an entrance gene for the TCA cycle (citZ), several transporter genes for TCA cycle-intermediates, some respiration genes, and many catabolic and anabolic genes involved in carbon, nitrogen, and phosphate metabolism, as well as for certain extracellular enzymes and secondary metabolites. Furthermore, these bacteria have CcpA-independent catabolite regulation systems, each of which involves a transcriptional repressor of CggR or CcpN. CggR and CcpN are derepressed under glycolytic and gluconeogenic growth conditions, and enhance glycolysis and gluconeogenesis respectively. Another CcpA-independent catabolite repression system involves P-His-HPr (histidyl-phosphorylated form of HPr). P-His-HPr phosphorylates and activates glycerol kinase, whose product is necessary for antitermination of the glycerol utilization operon through GlpP, the antiterminators (LicT and SacT, Y) of several operons for the utilization of less-preferred PTS-sugars, and some transcriptional activators such as LevR for the levan utilization operon. This phosphorylation is reduced due to the decreased level of P-His-HPr during active transport of a preferred PTS-carbohydrate such as glucose, resulting in catabolite repression of the target operons. Thus CcpA-dependent and independent networks for carbon metabolism play a major role in the coordinate regulation of catabolism and anabolism to ensure optimum cell propagation in the presence and the absence of a preferred PTS-carbohydrate.

Whole-Genome Analysis of Genes Regulated by the Bacillus subtilis Competence Transcription Factor ComK

The Bacillus subtilis competence transcription factor ComK is required for establishment of competence for genetic transformation. In an attempt to study the ComK factor further, we explored the genes regulated by ComK using the DNA microarray technique. In addition to the genes known to be dependent on ComK for expression, we found many genes or operons whose ComK dependence was not known previously. Among these genes, we confirmed the ComK dependence of 16 genes by using lacZ fusions, and three genes were partially dependent on ComK. Transformation efficiency was significantly reduced in an smf disruption mutant, although disruption of the other ComK-dependent genes did not result in significant decreases in transformation efficiency. Nucleotide sequences similar to that of the ComK box were found for most of the newly discovered genes regulated by ComK.

Comprehensive DNA Microarray Analysis of Bacillus subtilis Two-Component Regulatory Systems

It has recently been shown through DNA microarray analysis of Bacillus subtilis two-component regulatory systems (DegS-DegU, ComP-ComA, and PhoR-PhoP) that overproduction of a response regulator of the two-component systems in the background of a deficiency of its cognate sensor kinase affects the regulation of genes, including its target ones. The genome-wide effect on gene expression caused by the overproduction was revealed by DNA microarray analysis. In the present work, we newly analyzed 24 two-component systems by means of this strategy, leaving out 8 systems to which it was unlikely to be applicable. This analysis revealed various target gene candidates for these two-component systems. It is especially notable that interesting interactions appeared to take place between several two-component systems. Moreover, the probable functions of some unknown two-component systems were deduced from the list of their target gene candidates. This work is heuristic but provides valuable information for further study toward a comprehensive understanding of the B. subtilis two-component regulatory systems. The DNA microarray data obtained in this work are available at the KEGG Expression Database website (http://www.genome.ad.jp/kegg/expression).

Elaborate transcription regulation of the Bacillus subtilis ilv‐leu operon involved in the biosynthesis of branched‐chain amino acids through global regulators of CcpA, CodY and TnrA

The Bacillus subtilis ilv‐leu operon involved in the biosynthesis of branched‐chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis‐elements located upstream of the ilv‐leu promoter. This operon is known to be under CcpA‐dependent positive regulation. We have currently identified a catabolite‐responsive element (cre) for this positive regulation (bases −96 to −82; +1 is the ilv‐leu transcription initiation base) by means of DNase I‐footprinting in vitro, and deletion and base‐substitution analyses of cre. Under nitrogen‐rich growth conditions in glucose‐minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation of ilv‐leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA‐dependent positive regulation did not overcome the CodY‐dependent negative regulation. Furthermore, under nitrogen‐limited conditions in glucose‐minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA‐dependent positive regulation occurred without the TnrA‐dependent negative regulation. However, the TnrA‐dependent negative regulation did not occur without the CcpA‐dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA‐dependent positive regulation. The physiological role of this elaborate transcription regulation of the B. subtilis ilv‐leu operon in overall metabolic regulation in this organism is discussed.

Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a TnrA box

Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen‐limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome‐wide screening for TnrA‐regulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome‐wide search for TnrA boxes allowed us to find 36 TnrA‐regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well‐studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.

Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite‐responsive elements

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis‐acting catabolite‐responsive element (cre) by a complex of CcpA, which is a member of the GalR‐LacI family of bacterial regulatory proteins, and the seryl‐phos‐phorylated form of HPr (P‐ser‐HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (credown), this operon contains another ere located in the promoter region (creup). A translational gntR‐lacZ fusion expressed under the control of various combinations of wild‐type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of p‐galac‐tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P‐ser‐HPr, and catabolite repression exerted by credown was partially independent of P‐ser‐HPr. DNase I footprinting experiments indicated that a complex of CcpA and P‐ser‐HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose‐6‐phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.