Yoshito Sadaie

In vitro and host-mediated "Rec-assay" procedures for screening chemical mutagens ; and phloxine, a mutagenic red dye detected

Summary Recombinationless mutant cells of Bacillus subtilis are more sensitive to the cell-killing action of typical chemical mutagens, such as ethyl methanesulfonate, hydroxylamine, mitomycin C , N -methyl- N ′-nitrosoguanidine or4-nitroquinoline- N -oxide than are the wild-type bacteria. Based on this relation, a simple method was devised for screening new chemical mutagens by examining their increased lethal action on Rec − over Rec + bacteria instead of assessing their mutagenic capacities directly. We call this method “rec-assay”. When it was applied to a number of dyes, phloxine was found to be positive. Though this rec-assay procedure did not show the mutagenicity of phloxine directly, successive experiments using E. coli cells showed that this dye really was mutagenic. Thus a correlation between the rec-assay result and the genetic data was confirmed. The rec-assay was also carried out in the peritoneal cavity of mice that had received intramuscular injections of phloxine.

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.

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.

A novel sporulation-control gene (spo0M) of Bacillus subtilis with a σH-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.

Nucleotide sequence and analysis of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome

A 36 kb sequence of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome at around 55 degrees has been determined. The sequenced region contains 36 ORFs including the phoB and groESL genes, and the whole rrnE operon. The phoB gene is transcribed in the direction opposite to that of chromosome replication, while most ORFs, including groESL and the rrnE operon, are transcribed in the same direction. Two newly identified tRNA genes upstream of the rrnE operon were those for Arg-tRNA and Gly-tRNA. The sequenced region contains an operon consisting of genes for degradation and uptake of mannan. The rrnE operon and its downstream ORFs are well conserved among Mycoplasma genitalium, Haemophilus influenzae, Synechocystis sp. and Methanococcus jannaschii. delta H consensus sequences are present in the promoter regions of three ORFs, including groESL.

Effect of septum-initiation mutations on sporulation and competent cell formation in Bacillus subtilis

SummarySporulation and competent cell formation have been studied in four Bacillus subtilis strains, carrying septum-initiation mutations of different loci, div-31, div-341, div-12 and div-355 which exhibit filamentous growth at 45° C. The div-31 mutant was found to be defective in competence development at 30°–40°C whereas the div-12 mutant was affected only slightly. The div-341 and div-355 mutants showed lower competence, particularly at the higher temperatures. The four div mutant strains all showed poor sporulation at higher temperatures compared to the wild-type strain. We propose that some of the initial steps of septation are involved both in sporulation (possibly in forespore septum formation) and in competent cell formation and that these two processes share certain common features distinct from those in vegetative cell division.

Rad-2-dependent repair of radiation-induced chromosomal aberrations in Caenorhabditis elegans

A method involving light microscopy was developed and utilized for the observation of gamma- or ultraviolet-induced aberrations of the chromosomes of Caenorhabditis elegans var. Bristol (N2). Gravid worms were irradiated and the chromosomes were examined in the early embryos derived from eggs fertilized after the irradiation. The frequency of gamma-induced aberrations in the early embryonic cells of C. elegans increased proportionally with the dosage of gamma-rays. It decreased greatly following incubation of the irradiated gravid worms for 2 days. This decrease was blocked by the rad-2 mutation but not by the rad-1 mutation of the same epistasis group. Both mutations make worms sensitive to radiation and chemicals. In addition, the hatchability of eggs laid by the rad-2 mutant after irradiation was restored very quickly as was that of the wild-type strain. Ultraviolet irradiation, on the other hand, induced few aberrations in both the wild-type and rad-1 strains, but it caused an elevated frequency of aberrations in the rad-2 strain. Ultraviolet irradiation strongly blocked the separation of chromosomes of the rad-2 strain. Furthermore, hatchability was very low in eggs laid by ultraviolet-irradiated rad-2 worms. These results suggest the existence of a rad-2-dependent mechanism for gonadal repair of chromosomal aberrations, including chromosomal non-separation, and indicate that gamma-induced chromosome aberrations are not fatal to the hatching of Caenorhabditis elegans which has holocentric chromosomes.

Analysis of HutP-dependent transcription antitermination in the Bacillus subtilis hut operon: identification of HutP binding sites on hut antiterminator RNA and the involvement of the N-terminus of HutP in binding of HutP to the antiterminator RNA

We investigated HutP‐dependent transcription antitermination of the Bacillus subtilis hut operon. In vitro transcription assays with the B. subtilisσA‐containing RNA polymerase indicated that HutP inhibits transcription termination at the internal terminator by binding to the antiterminator on hut mRNA in the presence of histidine. Ethylnitrosourea modification interference assays and mutational analyses of the interference sites showed that interaction of HutP with a region containing three UAG trinucleotide sequences, which is located on top of the antiterminator structure, is critical for hut antitermination in vivo. Results from kinetic analysis of binding of HutP to RNA containing various portions of the antiterminator sequences indicated that secondary structure is required for binding of HutP to the region containing three UAG triplets in the antiterminator. The in vivo HutP antiterminator activity was reduced by the mutations in the N‐terminal region of HutP. The HutP variants with H4A, R7A, I9A and Q26A mutations exhibited reduced binding affinities to the antiterminator RNA in vitro. A 25‐mer peptide consisting of amino acid residues 2–26 of HutP bound to the antiterminator RNA. These results indicated that the N‐terminus of HutP is involved in binding of HutP to the antiterminator RNA.

Differential effects of a DNA-synthesis mutation on UV-induced mutation yields in vegetative cells and spores of Bacillus subtilis.

The temperature-sensitive DNA synthesis mutation, dna-8132, which is located near the replication origin of Bacillus subtilis, stops DNA synthesis at a restricted temperature (48°C) and affects neither initiation nor continuation of chromosome replication (Hara and Yoshikawa, 1973). Recently we have found that strains carrying the dna-8132 gene exhibit, at a permissive temperature of 30C, elevated spontaneous mutability and enhanced sensitivity to inactivation by ultraviolet light (UV), gamma rays and some radiomimetic chemicals (Sadaie and Narui, 1976). This prompted us to test whether the dna-8132 gene affects the UV-induced mutagenesis in Bacillus subtilis. This Note describes UV-mutability characteristics of strains carrying t h i s mutation and/or a polymerase I-deficient mutation.

secA341 Mutation Inhibition of Expression of the Bacillus subtilis Protease Gene, aprE

The production of extracellular protease(s) of Bacillus subtilis is induced in the post-exponential growth phase, and is severely reduced in a strain carrying a temperature sensitive mutation (secA341(ts)) in the secA gene, the product of which is required for protein secretion. The expression of the extracellular serine protease gene, aprE, as monitored by the β-galactosidase activity of an aprE-lacZ translational fusion, was inhibited in the secA341 mutant cell and restored by the introduction of the degU32(Hy) mutation, which makes the phosphorylated DegU response regulator of a two-component system refractory to dephosphorylation and causes the cell to become a strong producer of AprE. The secA341 mutation appears to block some activation step required for aprE expression.