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.

We have analyzed the regulons of the Bacillus subtilis two-component regulators DegU, ComA and PhoP by using whole genome DNA microarrays. For these experiments we took the strategy that the response regulator genes were cloned downstream of an isopropyl-beta-D-thiogalactopyranoside-inducible promoter on a multicopy plasmid and expressed in disruptants of the cognate sensor kinase genes, degS, comP and phoR, respectively. The feasibility of this experimental design to detect target genes was demonstrated by the following two results. First, expression of lacZ fusions of aprE, srfA and ydhF, the target genes of DegU, ComA and PhoP, respectively, was stimulated in their cognate sensor kinase-deficient mutants upon overproduction of the regulators. Secondly, by microarray analysis most of the known target genes for the regulators were detected and, where unknown genes were found, the regulator dependency of several of them was demonstrated. As the mutants used were deficient in the kinase genes, these results show that target candidates can be detected without signal transduction. Using this experimental design, we identified many genes whose dependency on the regulators for expression had not been known. These results suggest the applicability of the strategy to the comprehensive transcription analysis of the B.subtilis two-component systems.

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.

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).

Endothelin-l is a novel endothelium-derived vasoconstrictive peptide. Using a highly specific and sensitive radioimmunoassay for endothelin-l, plasma levels of immunoreactive endothelin-I were measured in 32 research subjects with normal renal function (21 normal subjects and II patients with essential hypertension), 24 patients with nondialyzed chronic renal failure, and 51 patients undergoing maintenance hemodialysis. Although there was no significant difference in plasma immunoreactive endothelin-l levels among the three groups, patients with essential hypertension had significantly higher plasma endothelin-l levels than normal subjects (2.29±1.09 vs. 1.41+0.50 pg/ml,p<0.025). When nondialyzed and hemodialyzed patients were divided into hypertensive and normotensive groups, the nondialyzed hypertensive group (n=17) had higher plasma endothelin-l levels than the comparable normotensive group (n=7) (3.08±3.43 vs. 0.73±0.34 pg/ml, p<0.05), and the hemodialyzed hypertensive group (n=18) had higher plasma endothelin-l levels than the comparable normotensive group (n=33) (2.66±1.92 vs. 1.35±0.73 pg/ml,p<0.005). Plasma atrial natriuretic factor, arginine vasopressin, renin activity, and aldosterone concentration did not show significant differences between hypertensive and normotensive individuals or a correlation with plasma endothelin-l levels. These data suggest that circulating endothelin-l may be partly involved in the development or maintenance of hypertension in humans. We recently demonstrated the presence of ET-1-like immunoreactivity (ET-LI) in normal human plasma with a highly sensitive and specific radioimmunoassay (RIA), 8 suggesting its potential role as a circulating vasoconstrictor. However, no information From the Second Department of Internal Medicine, Tokyo Medical and Dental University, Kitasato Biochemical Laboratories, SMI-Bristol Ltd. Sagamihara, Kanagawa, Tokyo Metropolitan Tama Geriatric Medical Center, Tokyo, and Shimoochiai Clinic, Shinjuku-ku, Tokyo, Japan.Address for correspondence: Yukio Hirata, MD, Endocrine Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan.Received October 6, 1989; accepted in revised form January 23, 1990.is yet available as to its pathophysiological role in hypertension. Therefore, the present study was designed to determine plasma ET-LI in normal subjects and in patients with essential hypertension as well as those with chronic renal failure with or without hypertension. Methods SubjectsThe study population consisted of 21 normal individuals (11 men and 10 women), 11 patients with essential hypertension (seven men and four women), 24 patients with nondialyzed chronic renal failure (18 men and six women), and 51 patients undergoing maintenance hemodialysis (34 men and 17 women). Informed consent was obtained from each subject. Essential hypertension was defined as elevated blood pressure while in a sitting position, exceeding 160/95 mm Hg, for three consecutive measurements over a pe...

SummaryWe screened the putative rap-phr ( r esponse regulator a spartyl-phosphate p hosphatase-ph osphatase r egulator) 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; NH 2 -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.

Plasmid pLS32 is a relatively large (approximately 70 kbp), cryptic, low copy-number plasmid present in Bacillus natto. We isolated and analyzed the replication region of the plasmid in B. subtilis, and the following results were obtained: the replication region contained an open reading frame encoding a 287-amino acid protein (RepN), whose amino acid sequence was partially homologous with those of the Rep proteins encoded on plasmids pAD1 and pLJ1 isolated from Enterococcus faecalis and Lactobacillus helveticus, respectively; the replication origin (oriN) was located in the repN-coding region; the copy number of a pLS32 derivative, pBET131, was 2 to 3 per chromosome; replication of pBET131 required polI. These features show that pLS32 is a novel plasmid capable of replication in B. subtilis.z 1998 Federation of European Biochemical Societies.

We constructed Bacillus subtilis strains in which chromosome replication initiates from the minimal replicon of a plasmid isolated from Bacillus natto, independently of oriC. Integration of the replicon in either orientation at the proA locus (115؇ on the genetic map) suppressed the temperature-sensitive phenotype caused by a mutation in dnaA, a gene required for initiation of replication from oriC. In addition, in a strain with the plasmid replicon integrated into the chromosome, we were able to delete sequences required for oriC function. These strains were viable but had a slower growth rate than the oriC ؉ strains. Marker frequency analysis revealed that both pyrD and metD, genes close to proA, showed the highest values among the markers (genes) measured, and those of other markers decreased symmetrically with distance from the site of the integration (proA). These results indicated that the integrated plasmid replicon operated as a new and sole origin of chromosome replication in these strains and that the mode of replication was bidirectional. Interestingly, these mutants produced anucleate cells at a high frequency (about 40% in exponential culture), and the distribution of chromosomes in the cells was irregular. A change in the site and mechanism (from oriC to a plasmid system) of initiation appears to have resulted in a drastic alteration in coordination between chromosome replication and chromosome partition or cell division.