Fujio Kawamura

Potential use of bacillus subtilis for secretion and production of foreign proteins

Abstract The successful use of Bacillus subtilis for the secretion of foreign gene products will require a host lacking extracellular proteases, the development of genetic regulatory elements and nutritional conditions that allow expression of foreign genes during growth, and additional understanding of the hosts secretory mechanism to allow it to be made more efficient.

Isolation and mapping of a new suppressor mutation of an early sporulation gene spo0F mutation in Bacillus subtilis

SummaryWe constructed an spo0F deletion (spo0FΔS) mutant of Bacillus subtilis by inserting a chromosomal segment carried by plasmid pUBSFΔS. This plasmid carries a 0.7-kilobase pair deletion that removes the spo0F promoter and a part of the structural gene. We used the spo0F deletion mutant to isolate a new intergenic suppressor of the spo0F phenotype, designated sof1. The sof1 suppressor completely restores the sporulation ability of all spo0F defective mutants tested, including spo0F77, spo0F221 and spo0FΔS. The sof1 suppressor maps to the left of lys1 on the B. subtilis chromosome, in a region rich in sporulation markers and distant from the spo0F locus.

The effect ofspo0 mutations on the expression ofspo0A- andspo0F-lacZ fusions

SummaryWe have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo+ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.

A specialized transducing phage constructed from Bacillus subtilis phage φ105

Chromosomal DNA of Bacillus subtilis 168 (trpC2) prepared from defective phage P BSX was digested by restriction endonuclease Eco RI and ligated in vitro with DNA fragments of page phi 105C digested by the same endonuclease. The ligated DNA was used to transform a competent culture of B. subtilis (trpC2 lys3 metB10) which was lysogenic for phi 105, and transformants of the auxotroph markers were selected. The bacterial DNA ligated to the phage DNA fragments could be integrated into the prophage genome by transformation. The transformants in toto were treated with mitomycin C and the lysate was used to transduce B. subtilis (trpC2 lys3 metB10). Among metB+ transductants, one clone appeared to be a double lysogen carrying both plaque forming and metB+ transducing phage genomes. The latter defective phage was designated phi 105dmetB. Physical mapping of these phages was carried out by agarose gel electrophoresis of the restriction endonuclease digests and also by electron microscopic analysis of heteroduplex DNA. These results indicate that two adjacent fragments Eco RI-G and E of phi 105 DNA had been substituted with a foreign fragment Eco RI-M in phi 105dmetB DNA. Transformation experiments showed that the metB+ gene resided on the fragment Eco RI-M. This fragment was found to have a BamHI-sensitive site. The transforming activity for the metB marker, however, was not affected by the treatmment with BamHI.

Bacteriophage ϕ1 as a gene-cloning vector in Bacillus subtilis

SummaryWe attempted to use Bacillus subtilis phage ϕ1 as a gene-cloning vector since the ϕ1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A ϕ1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, ϕ1E1 and ϕ1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant ϕ1E2Δ1 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage ρ11 DNA, that ϕ1E2Δ1 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten ϕ1 clones isolated from independent transfectants and found that six of them carried ρ11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the ρ11 DNA portion, whereas the parental ϕ1E2Δ1 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.

Patch clamp analysis of the respiratory chain in Bacillus subtilis

Bacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, we developed the patch clamp method to analyze the respiratory chain in B. subtilis. First, we prepared giant protoplasts (GPs) from B. subtilis cells. Electron micrographs and fluorescent micrographs revealed that GPs of B. subtilis had a vacuole-like structure and that the intravacuolar area was completely separated from the cytoplasmic area. Acidification of the interior of the isolated and purified vacuole-like structure, due to H(+) translocation after the addition of NADH, revealed that they consisted of everted cytoplasmic membranes. We called these giant provacuoles (GVs) and again applied the patch clamp technique. When NADH was added as an electron donor for the respiratory system, a significant NADH-induced current was observed. Inhibition of KCN and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) demonstrated that this current is certainly due to aerobic respiration in B. subtilis. This is the first step for more detailed analyses of respiratory chain in B. subtilis, especially H(+) translocation mechanism.

Frequent deletion of Bacillus subtilis chromosomal fragment in artificially constructed phage ρ11phisA

A novel gene-cloning method named ‘prophage transformation’ has been developed for constructing specialized transducing phages, pl 1 [l] and

Selection for restriction-induced in vivo deletion in phage vector Φ1E1 of Bacillus subtilis

105 [2]. Using this method, several genes of Bacillus sub tilis, such as hid, lys [ 11, amyE, nrol [3], spoOB [4,5] and SPOOF [6,7] have been successfully cloned. We have studied the unusual instability of histidine-transducing phage, pl lphisA+, with regard to His’-transducing ability and found that -5% of the total phages obtained by Induction of either Rec+or recE4 lysogen carrying p 11 phisA * did not have His+-transducing ability [8]. The loss of I&‘-transducing ability was not ascribed to the recombination between the prophage and the host chromosomal hisA 1 gene, since I-I&+-transduction was not restored when a His’ Ret’ lysogen carrying the non-transducer phage was constructed and treated with inducer. These results strongly suggest that p 11 phiti’ prophages, upon induction, lost the hisA’moiety by deletion. The instability of gene-cloning vectors has been a serious problem in DNA recombination, but the mechanism has remained unclear. Here we show that the loss of the His*-transducing ability of p 1 lphisA+ is caused by deletion of the nucleotide sequence for the hisA’ gene on the p 1lphisA’ genome. 2.2. DNA preparations Plasmid DNA was isolated using low melting-point agarose in vertical gels. Phages were induced by treatment with mitomycin C, purified, and their DNA isolated as in [ 111.

Differential regulation of spo0A transcription in Bacillus subtilis: glucose represses promoter switching at the initiation of sporulation.

Recombinant phage phi 1E1metB, which contains the 4.5-kb EcoRI fragment of Bacillus subtilis DNA, has no HaeIII cleavage sites within the vector phi 1E1 genome but only in the metB insert. When phi 1E1metB was grown in B. subtilis ISR11, which produces BsuR, the isoschizomer of HaeIII, it was restricted and survived with an efficiency of approx. 10(-5). All the survivors were deletion mutants of phi 1E1metB, and only various segments of the insert DNA delineated by HaeIII sites were deleted. The Met+ transforming activities of the DNAs from phi 1E1metB and its deletion derivatives were examined, and the restriction maps of the deletion mutants were correlated with five metB- mutation sites.