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Y Sadaie

Publications and source records attributed to Y Sadaie.

At least 19 recordsLinked to original sources
PubMed2003

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 approximately 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.

Bacillus subtilis↗
PubMed2000

Synthesis and characterization of the spore proteins of Bacillus subtilis YdhD, YkuD, and YkvP, which carry a motif conserved among cell wall binding proteins.

We have previously reported that YaaH and YrbA are spore proteins of Bacillus subtilis that are required for spore resistance and/or germination and that they have a motif conserved among so-called cell wall binding proteins [Kodama et al. (1999) J. Bacteriol. 181, 4584-4591, Takamatsu et al. (1999) J. Bacteriol. 181, 4986-4994]. In this study, we analyzed the expression of ydhD, ykuD, and ykvP genes, which encode putative proteins containing the same motif. Transcription of ydhD was dependent on SigE, and the mRNA was detectable from 2 h after the cessation of logarithmic growth (T(2) of sporulation). ykuD was transcribed by SigK RNA polymerase from T(4) of sporulation. Both SigK and GerE were essential for ykvP expression, and this gene was transcribed from T(5) of sporulation. Inactivation of these genes by insertion of an erythromycin resistance gene did not affect vegetative growth, spore resistance to heat, chloroform, and lysozyme, or spore germination in the presence of L-alanine or in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride. The His tag fusions of YdhD, YkuD, and YkvP downstream of their natural promoter regions were introduced into a multicopy plasmid. These fusion proteins were produced during sporulation in B. subtilis transformants and were detected in mature spores, indicating that YdhD, YkuD, and YkvP are all proteins intrinsic to spores. Excessive YkuD and YkvP in the sporulating cells did not affect spore resistance or germination. The cells producing excessive YdhD also did not show impaired spore resistance, but their germination properties were changed: the spores revealed reduced response to L-alanine and some of them germinated even without germinants. Escherichia coli b-lactamase, whose signal sequence had been genetically replaced by the cell wall binding motif of YaaH, was produced in sporulating cells, and Western blot analysis indicated that the fused protein was assembled into spores. We speculate that the conserved motif functions as a kind of signal sequence involved in assembly of these proteins on forespores.

Amino Acid Motifs↗
PubMed1998

Rapid isolation of RNA polymerase from sporulating cells of Bacillus subtilis.

A highly ordered program of temporal and spatial gene activation during sporulation in Bacillus subtilis is governed by the principal RNA polymerase, and RNA polymerases containing at least five developmental sigma factors appearing successively during sporulation. This report describes a rapid procedure for extracting RNA polymerase from sporulating B. subtilis cells, which involves the construction of hexahistidine tagged beta' subunit of RNA polymerase and the isolation of RNA polymerase holoenzyme with Ni2+-NTA resin. In in vitro transcription of various promoters with the RNA polymerase thus purified, we observed the temporal change of each RNA polymerase activity during sporulation. This procedure enables isolation of RNA polymerase within 4h, starting with cell pellets. Our results indicated that a principal sigma factor, sigmaA, could be detected in a holoenzyme form during all the stages of growth and sporulation, while the other sigma factors sigmaH, sigmaE, sigmaF, sigmaG, and sigmaK involved in sporulation could be detected sequentially during sporulation. Moreover, Spo0A, the central transcription factor of commitment to sporulation, was also co-purified with RNA polymerase at early stages of sporulation.

Bacillus subtilis↗
PubMed1998

A novel sporulation-control gene (spo0M) of Bacillus subtilis with a sigmaH-regulated promoter.

A novel sporulation-control gene (spo0M) of Bacillus subtilis was cloned, sequenced and analyzed. The spo0M gene is located at the end of large tRNA gene clusters including rrnD and codes for a 257-amino-acid protein with a calculated size of 29.6kDa. The protein Spo0M has a strong negative charge (calculated pI=4.3) and shows no significant sequence homology to any known proteins. Gene disruption experiments revealed that spo0M is not essential for cell viability, but its disruption results in considerable impairments (decreasing by 20- to 100-fold) in sporulation. The morphological stage blocked in sporulation was stage 0 as observed by electron microscopy, and expression analysis using spo0Aps-bgaB fusion revealed an impaired gene expression of spo0A in the spo0M mutant. In contrast, spo0M disruption had no effect on antibiotic productivity. Propagation of the spo0M gene in wild-type cells using a high-copy-number plasmid also impaired sporulation, indicating that overproduction of Spo0M exerts certain negative effects on sporulation. spo0M gene expression is controlled by sigmaH, as demonstrated: (1) by monitoring expression of a bgaB transcriptional fusion integrated into the amyE locus on the chromosome of the wild-type or spo0H mutant cells, and (2) by in-vitro transcription of spo0M gene with EsigmaH.

Amino Acid Sequence↗
PubMed1998

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 sigmaB-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 sigmaH 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 sigmaH in the cell at 45 degrees C after T2, at which time the sigmaH 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 sigmaH 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 degrees C.

Bacillus subtilis↗
PubMed1998

Promoter selectivity of the Bacillus subtilis RNA polymerase sigmaA and sigmaH holoenzymes.

The sigmaH of Bacillus subtilis directs transcription of a large number of early sporulation genes, whereas the principal sigma factor, sigmaA, is essential for the transcription of the genes for vegetative growth and early sporulation. We have purified sigmaA and sigmaH proteins, and characterized their properties. The genes encoding sigmaA or sigmaH were separately cloned into an expression vector under the control of T7 promoter. Both proteins were overproduced in Escherichia coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride. Antigenicities and N-terminal amino acid sequences of the overproduced proteins were used to identify both proteins. Unlike sigmaA protein, sigmaH protein showed a DNA-binding ability. To compare the promoter selectivity of the sigmaA protein with that of the sigmaH protein, transcription in vitro of 16 promoters was performed using RNA polymerase holoenzymes reconstituted from a purified core enzyme with either sigmaH or sigmaA. These holoenzymes correctly recognized each of the cognate promoters; sigmaH-RNA polymerase recognized sigmaH promoters but not sigmaA promoters, and vice versa. A competition experiment for core RNA polymerase using sigmaA and sigmaH revealed that sigmaA had a stronger affinity. We propose that the predicted replacement of a sigma subunit in a holoenzyme from sigmaA to sigmaH in vivo at late logarithmic growth phase may require an additional factor, or the modification of a core enzyme or sigma factor.

Bacillus subtilis↗
PubMed1998

Feedback loops involving Spo0A and AbrB in in vitro transcription of the genes involved in the initiation of sporulation in Bacillus subtilis.

Through mainly in vivo studies, the initiation of sporulation in Bacillus subtilis has been shown to depend on the phosphorylation of the Spo0A transcription factor mediated by the multicomponent phosphorelay via KinAB (C), Spo0F, Spo0B, and Spo0A in this order. RNA polymerase containing sigmaA (EsigmaA) or sigmaH (EsigmaH) transcribes the genes of the phosphorelay components. Phosphorylated Spo0A is also involved in their expression and is required for the induction of sigmaH by repressing its repressor gene abrB. We have examined the effects of phosphorylated Spo0A (Spo0A-P) and AbrB on in vitro transcription of the genes involved in the Spo0A phosphorylation and initiation of sporulation. Spo0A-P repressed EsigmaA-dependent transcription of the kinC and EsigmaH-dependent transcription of spo0A and kinA. EsigmaH-dependent transcription of spo0F was stimulated by Spo0A-P at low concentrations but was repressed by higher amounts of Spo0A-P. On the other hand, AbrB repressed EsigmaA-dependent transcription of spo0H (sigmaH gene), kinC, and abrB, although its effect was not strong. With the present results providing in vitro evidence for the roles of Spo0A-P and AbrB as transcriptional regulators, and other results described in the literature, the positive and negative feedback loops controlling the temporal expression of early sporulation genes are discussed.

Bacillus subtilis↗
PubMed1998

Restricted transcription from sigma H or phosphorylated spo0A dependent promoters in the temperature-sensitive secA341 mutant of Bacillus subtilis.

The temperature-sensitive secA341 mutation of Bacillus subtilis affects sporulation and sporulation-associated events as well as protein secretion and cell septation. With lacZ or bgaB fusion genes, we examined the expression of the early sporulation genes in the mutant strain. Transcriptional expression of delta H dependent kinA, spo0A (Ps), phrC, spoVG, and citG (p2) genes was blocked by the secA341 mutation at 37 degrees C. On the other hand, neither repression of the abrB gene nor induction of the spoH (delta H) gene was affected. Active RNA polymerase containing delta H was, however, found to be produced in the mutant cells. Expression of the phosphorylated Spo0A dependent spoIIG operon was also blocked. Thus the secA341 mutation blocks some step(s) or factor(s) required for delta H-dependent transcription in vivo.

Adenosine Triphosphatases↗
PubMed1997

Sequence analysis of the groESL-cotA region of the Bacillus subtilis genome, containing the restriction/modification system genes.

We have determined a 35-kb sequence of the groESL-gutR-cotA (45 degrees-52 degrees) region of the Bacillus subtilis genome. In addition to the groESL, gutRB and cotA genes reported previously, we have newly identified 24 ORFs including gutA and fruC genes, encoding glucitol permease and fructokinase, respectively. The inherent restriction/modification system genes, hsdMR and hsdMM, were mapped between groESL and gutRB, and we have identified two open reading frames (ORFs) encoding 5-methylcytosine forming DNA methyl transferase and an operon probably encoding a restriction enzyme complex. The unusual genome structure of few ORFs and lower GC content around the restriction/modification genes strongly suggests that the region originated from a bacteriophage integrated during evolution.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of a sporulation initiation mutation in the Bacillus subtilis secA gene.

A Bacillus subtilis secA mutant, secA12, which is blocked at an early stage of sporulation, is able to grow as well as the wild-type strain at all temperatures tested. Experiments with lacZ fusion genes showed that the induction of kinA expression, as well as the sporulation-specific transcription of the spo0A gene, was not observed in the secA12 mutant. However, transcription of the spo0H gene (coding for sigmaH, which is required for the transcription of kinA and spo0A) and accumulation of the sigmaH protein were not affected in secA12. These results suggested that mutations in secA affect a factor required for efficient transcription of kinA as well as for the activation of the phosphorelay pathway.

Adenosine Triphosphatases↗
PubMed1995

Acquisition of azide-resistance by elevated SecA ATPase activity confers azide-resistance upon cell growth and protein translocation in Bacillus subtilis.

We isolated four azide-resistant secA mutants of Bacillus subtilis and found that all of them were the result of a single amino acid replacement of threonine 128 of SecA by alanine or isoleucine. In the presence of 1.5 mM sodium azide, cell growth and protein translocation of the wild-type strain were completely inhibited, but those of the azide-resistant mutant strains were not. Wild-type and two mutant SecA proteins were purified. Both the basal level and the elevated ATPase activity of the mutant SecA proteins were threefold higher than those of the wild-type SecA. The elevated ATPase activity of the SecA mutants was reduced upon the addition of 1.5 mM sodium azide by only 5-10% as compared with 40% for that of the wild-type. These results indicate that the elevated ATPase activity of the SecA mutants is resistant to sodium azide and that is also required for the protein translocation process of B. subtilis.

Adenosine Triphosphatases↗
PubMed1994

A truncated Bacillus subtilis SecA protein consisting of the N-terminal 234 amino acid residues forms a complex with Escherichia coli SecA51(ts) protein and complements the protein translocation defect of the secA51 mutant.

Although wild-type Bacillus subtilis SecA barely complements the growth and protein translocation defect of Escherichia coli secA51(ts) at the non-permissive temperature, an N-terminal peptide of B. subtilis SecA complements the defects. To elucidate the mechanism of this complementation, a series of plasmids encoding truncated SecA proteins was constructed and their products were analyzed in E. coli cells. The truncated B. subtilis SecA protein consisting of the N-terminal 234 amino acid residues (BN234) complemented the growth and protein translocation defects of E. coli secA51 but not those of another secA amber mutant, E. coli secA13(ts). BN234 existed in both a soluble form, possibly as a homodimer, and a higher-molecular-weight complex in E. coli strain MM52 (secA51). The purified complex, consisting of at least BN234, SecA51, and ATP-dependent protease La, was held together by a cross-linking reagent, EDAC. The other truncated proteins consisting of the N-terminal 584 or 396 amino acid residues and the C-terminal 607 residues of B. subtilis SecA did not complement the two E. coli mutants or form a complex with SecA51. These results suggest that BN234 and SecA51 proteins form a functional complex in vivo and complement the defects of E. coli MM52.

Adenosine Triphosphatases↗
PubMed1994

The rapid degradation of mutant SecA protein in the Bacillus subtilis secA341 (ts) mutant causes a protein translocation defect in the cell.

To study the function of SecA protein and the protein translocation system of Bacillus subtilis, wild-type and mutant SecA proteins were characterized in vivo and in vitro. SecA protein was abundant in a wild-type strain (168) and existed in a stable homodimer. In contrast to this, SecA341 (ts) protein having an amino acid replacement from proline to leucine at residue 431 was undetectable by immunoblotting in the cell lysate of a secA341 mutant (TB301) at the nonpermissive temperature, 42 degrees C. Pulse-chase studies using 35S-methionine showed that newly synthesized SecA protein was rapidly degraded in the mutant at 42 degrees C. Purified SecA341 protein was more sensitive to trypsin and subtilisin than purified wild-type SecA protein in the presence of ATP. These results indicate that the secA341 mutation causes the rapid degradation of mutant SecA protein and a concomitant protein translocation defect in the cell.

Adenosine Triphosphatases↗
PubMed1992

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.

Adenosine Triphosphatases↗