Genome sequencing of Bacillus subtilis SC-8, antagonistic to the Bacillus cereus group, isolated from traditional Korean fermented-soybean food

Bacillus subtilis SC-8 is a Gram-positive bacterium displaying narrow antagonistic activity for the Bacillus cereus group. B. subtilis SC-8 was isolated from Korean traditional fermented-soybean food. Here we report the draft genome sequence of B. subtilis SC-8, including biosynthetic genes for antibiotics that may have beneficial effects for control of food-borne pathogens.     

枯草芽胞杆菌基因组删减对异源酶表达影响的研究进展

枯草芽胞杆菌作为一种遗传背景清晰、基因编辑成熟的革兰氏阳性菌,是多种重要工业酶的生产宿主.随着转录组、蛋白质组、代谢组等多组学测序和分析技术的发展,通过合理设计简化枯草芽胞杆菌基因组,减少细胞内冗余的调控和代谢网络,使得细胞更精简且便于控制,展现出了枯草芽胞杆菌作为异源酶表达宿主细胞的应用潜力.本文简要综述了枯草芽胞杆...     

Ecology and genomics of Bacillus subtilis

Bacillus subtilis is a remarkably diverse bacterial species that is capable of growth within many environments. Recent microarray-based comparative genomic analyses have revealed that members of this species also exhibit considerable genomic diversity. The identification of strain-specific genes might explain how B. subtilis has become so broadly adapted. The goal of identifying ecologically adaptive genes could soon be realized with the imminent release of several new B. subtilis genome sequences. As we embark upon this exciting new era of B. subtilis comparative genomics we review what is currently known about the ecology and evolution of this species.     

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein gamma-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.     

Crystal protein synthesis is dependent on early sporulation gene expression in Bacillus sphaericus

Insertional mutations in the spo0A and spoIIAC genes of Bacillus sphaericus 2362 were prepared by conjugation with Escherichia coli using a suicide plasmid containing cloned portions of the target genes. The mutants resembled their Bacillus subtilis counterparts phenotypically and were devoid of crystal proteins as determined by electron microscopy, SDS-PAGE and Western blots. The mutants had greatly reduced toxicity to anopheline mosquito larvae compared to the parental strain. We conclude that crystal protein synthesis in this bacterium is dependent on expression of early sporulation genes.     

生防工程菌株BS-17A稳定性与抑菌活性初步研究

目的:BS-17是内生细菌,对番茄灰霉病菌、叶霉病菌和枯萎病菌具有显著抑菌活性,为了跟踪研究野生型菌株BS-17在番茄根围和叶围的定殖情况,构建了1株带有黄绿荧光蛋白基因标记的生防菌株BS-17A.方法:采用NYD连续培养的方法和平皿抑菌试验的方法对工程菌株的遗传稳定性和抑菌活性进行了初步研究.结果:该工程菌在无选择压力培养基中连续培养50h,质粒遗传稳定性为94%,对番茄灰霉病菌Botrytis cinerea、叶霉病菌Cladosporiumfulvum和枯萎病菌Fusarium oxysporum的抑制作用与野生菌无显著差异,平皿抑菌率分别为85.5%、86.5%和89.8%.结论:该工程菌具有较强的遗传稳定性和抑菌活性.     

Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism

Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates, enabling the bacterium to survive in a continuously changing environment. These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market. Unfortunately, the secretion of heterologous proteins, originating from Gram-negative bacteria or from eukaryotes, is often severely hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed. Nevertheless, research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host. This review focuses on studies that aimed at optimizing B. subtilis as cell factory for commercially interesting heterologous proteins.     

Genome sequence of Bacillus subtilis subsp. spizizenii gtP20b, isolated from the Indian ocean

Bacillus subtilis is an aerobic spore-forming Gram-positive bacterium that is a model organism and of great industrial significance as the source of diverse novel functional molecules. Here we present, to our knowledge, the first genome sequence of Bacillus subtilis strain gtP20b isolated from the marine environment. A subset of candidate genes and gene clusters were identified, which are potentially involved in production of diverse functional molecules, like novel ribosomal and nonribosomal antimicrobial peptides. The genome sequence described in this paper is due to its high strain specificity of great importance for basic as well as applied researches on marine organisms.     

Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile Aquifex aeolicus

The terminal two heme biosynthetic pathway enzymes, protoporphyrinogen oxidase and ferrochelatase, of the hyperthermophilic bacterium Aquifex aeolicus have been expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Ferrochelatase and protoporphyrinogen oxidase of this organism are both monomeric, as was found for the corresponding enzymes of Bacillus subtilis. However, unlike the B. subtilis proteins, both A. aeolicus enzymes are membrane-associated. Both proteins have temperature optima over 60 degrees C. This is the first demonstration of functional heme biosynthetic enzymes in an extreme thermophilic bacterium.     

Expression and characterization of the genes encoding azoreductases from Bacillus subtilis and Geobacillus stearothermophilus

Azoreductases have been characterized as enzymes that can decolorize azo dyes by reducing azo groups. In this study, genes encoding proteins having homology with the azoreductase gene of Bacillus sp. OY1-2 were obtained from Bacillus subtilis ATCC6633, B. subtilis ISW1214, and Geobacillus stearotherophilus IFO13737 by polymerase chain reaction. All three genes encoded proteins with 174 amino acids. The deduced amino acid sequences of azoreductase homologs from B. subtilis ISW1214, B. subtilis ATCC6633, and G. stearotherophilus IFO13737 showed similarity of 53.3, 53.9, and 53.3% respectively to that of Bacillus sp. OY1-2.All three genes were expressed in Escherichia coli, and were characterized as having the decolorizing activity of azo dyes in a beta-NADPH dependent manner. The transformation of several azo dyes into colorless compounds by recombinant enzymes was demonstrated to have distinct substrate specificity from that of azoreductase from Bacillus sp. OY1-2.     

Characterization of pyrimidine-repressible and arginine-repressible carbamyl phosphate synthetases from Bacillus subtilis.

The number and properties of carbamyl phosphate synthetases in Bacillus subtilis have been uncertain because of conflicting genetic results and instability of the enzyme in extracts. The discovery of a previously unrecognized requirement of B. subtilis carbamyl phosphate synthetases for a high concentration of potassium ions for activity and stability permitted unequivocal demonstration that this bacterium elaborates two carbamyl phosphate synthetases. Carbamyl phosphate synthetase A was shown to be repressed by arginine, to have a molecular weight of about 200,000, and to be coded for by a gene that maps near argC4. This isozyme was insensitive to metabolites of the arginine and pyrimidine biosynthetic pathways. Carbamyl phosphate synthetase P was found to be repressed by uracil, to have a molecular weight of 90,000 to 100,000, and to be coded for by a gene that maps near the other pyr genes. This isozyme was activated by phosphoridine nucleotides. Other kinetic properties of the two isozymes were compared. Bacillus thus resembles eucaryotic microbes in producing two carbamyl phosphate synthetases, rather than the enteric bacteria, which produce a single carbamyl phosphate synthetase.     

Bacillus subtilis and its relatives: molecular biological and industrial workhorses.

The non-pathogenic bacterium Bacillus subtilis, since its first reported genetic transformation in 1959, has become a model system for the study of many aspects of the biochemistry, genetics and physiology of Gram-positive bacteria, and particularly of sporulation and associated metabolism. Extensive knowledge of the molecular biology of B. subtilis has led to the recent development of this bacterium as a host for the industrial production of heterologous proteins. Although difficulties have been encountered, these are being systematically addressed and overcome.     

A disulfide bond-containing alkaline phosphatase triggers a BdbC-dependent secretion stress response in Bacillus subtilis

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for alpha-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.     

Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes

We have established the co-linear regions of Bacillus licheniformis, an industrially important bacterium, and Bacillus subtilis, a model bacterium. In the co-linear regions, revealed by PCR, gene content and order are presumed to be conserved. These regions constitute approximately 60% of the compared chromosomes. Sequencing of the competence genes of B. licheniformis allowed us to validate the approach, and to demonstrate how it can be used for the comparative analysis of complex genetic systems. A new insertion sequence, designated IS3Bli1, was discovered in the competence region of the analyzed B. licheniformis strain.     

The Bacillus subtilis ochre suppressor sup-3 is located in an operon of seven tRNA genes.

Most Bacillus subtilis tRNA genes have been isolated from lambda libraries by use of probes that hybridize to tRNA or rRNA sequences. None of those genes map to the region of the sup-3 mutation. By cloning of the sup-3 allele, a cluster of seven tRNA genes (the trnS operon) that had not been isolated by other methods was identified. In principle, this approach could be used to isolate at least one more predicted tRNA-containing operon in this bacterium. The trnS operon was shown to contain tRNA genes for Asn (GUU), Ser (GCU), Glu (UUC), Gln (UUG), Lys (UUU), Leu (UAG), and Leu (GAG). The sup-3 mutation was found to be a T-to-A transversion that changes the anticodon of the lysine tRNA from 5'-UUU-3' to 5'-UUA-3'. This result agrees with previous work that determined that the sup-3 mutation causes lysine to be inserted at ochre nonsense mutations.     

The Bacillus subtilis cytochrome-c oxidase. Variations on a conserved protein theme.

The structural genes of cytochrome-c oxidase in Bacillus subtilis have been isolated and sequenced. Five genes, ctaB-F, are closely spaced. ctaC, ctaD, ctaE and ctaF are the genes for subunits II, I, III and IVB, respectively, ctaB, which may encode an assembly factor, is separated and upstream from the others. In comparison to its mitochondrial counterparts, subunit I has an extended C-terminus with two additional transmembrane segments, whereas subunit III has lost two such segments from its N-terminus. The C-terminal extension in subunit II is a covalent cytochrome-c domain, previously characterized only in the thermophilic oxidases. Subunit IVB, a small hydrophobic protein, is a novel subunit. These predictions suggest that the B. subtilis cytochrome-c oxidase is structurally more related to the four-subunit Escherichia coli cytochrome-bo complex than, for instance, to the Paracoccus denitrificans enzyme. Cytochrome aa3, which was previously isolated from B. subtilis [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103] is not encoded by the ctaC-F genes; thus, there seems to be two different cytochrome-aa3-type oxidases in this Gram-positive bacterium.