The Structure of the Transposable Genetic Element ISBsu2 from the Cryptic Plasmid p1516 of a Soil Bacillus subtilis Strain and the Presence of Homologues of This Element in the Chromosomes of Various Bacillus subtilis Strains

A cryptic plasmid from a soil strain of Bacillus subtiliswas found to contain a sequence having features of an IS element. Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B. subtilis strains.     

Site-Specific Deoxyribonucleases in Bacillus subtilis and Other Bacillus Strains

We systematically studied site-specific deoxyribonucleases in Bacillus strains and detected deoxyribonuclease activities in 20 of 62 strains tested.     

Soil Strain of Bacillus subtilis Harboring a Large Plasmid That Mediates High-Frequency Conjugal Mobilization

The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcal plasmid, pUB110, was studied. The latter plasmid was transferred to the recipient cells of Bacillus subtilis168 at a high frequency (about 10^–2 per recipient cell) both on the filter surface and in liquid medium. Mobilization was initiated 40 min after the beginning of the contact between donor and recipient cells.     

Conjugal Plasmid Transfer in Bacillus subtilis under Conditions of Soil Microcosms

Conjugal transfer of the small plasmid pUB110 betweenBacillus subtilis strains was studied under conditions of microcosms with sterile and nonsterile soil. Plasmid transfer proved to be possible after soil inoculation with vegetative partner cells or with their spores. Plasmid transfer occurred at temperatures of 30 and 22–23°C.     

Construction of a Chimeric Plasmid in Bacillus subtilis

Staphylococcal plasmids pTP4 (2.7 megadaltons encoding resistance to chloramphenicol) and pTP5 (2.6 megadaltons encoding resistance to tetracycline), which replicate and express resistance in B. subtilis, were found to cut by HindIII endonuclease respectively at a single site and three sites. A chimeric plasmid pTA1245 (4.1 megadaltons) was constructed from pTP4 and pTP5 by HindIII digestion and ligation with E. coli DNA ligase. pTA1245 expresses resistances to chloramphenicol and tetracycline in B. subtilis, and pTA1245 is amplified in the presence of tetracycline. A physical map of pTA1245 was constructed.     

The ISBsu2 mobile element is present in a plasmid of a soil strain and in the chromosomes of several other strains of Bacillus subtilis

Chromosomes of several Bacillus subtilis strains were shown to contain homologs of the ISBsu2 mobile genetic element, which was earlier revealed in a cryptic plasmid of a soil strain of B. subtilis.     

The structure of the transposable genetic element ISBsu2 in the cryptic plasmid p1516 from a soil Bacillus subtilis strain and the presence of homologues of this element in the chromosomes of various Bacillus subtilis strains

A cryptic plasmid from a soil strain of Bacillus subtilis was found to contain a sequence having features of IS element. Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B. subtilis strains.     

Mobility of the Native Bacillus subtilis Conjugative Plasmid pLS20 Is Regulated by Intercellular Signaling

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default “OFF” state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.     

Characterization of pTZ12, a Chloramphenicol-resistance Plasmid in Bacillus subtilis

The chloramphenicol-resistance (CP^r) plasmid pTZ12 (2.55 kb) in Bacillus subtilis was genetically analyzed in detail, and the CP^r determinant and the functional unit of replication were mapped. The plasmids pTZ12 and pBR322 were digested with suitable restriction endonucleases and ligated with T4 ligase. The ligated DNAs were introduced into E. coli by transformation and CP-resistant transformants were selected. In conclusion, the CP^r determinant was mapped between a TaqI site and a BclI site (about 900 base pairs) on pTZ12. A set of pTZ12–pBR322 recombinant plasmids isolated from E. coli was introduced into B. subtilis by transformation to test for ability to replicate in B. subtilis. From the results, the region of the functional unit of pTZ12 replication was mapped. It was also proved that the gene product of this CP^r determinant was chloramphenicol acetyltransferase (CAT) and the native CAT in the cells carrying pTZ12 was a dimeric protein with two identical subunits having a molecular weight of approximately 24,000 (24 K).     

The Maceration of Vegetable Tissue by a Strain of Bacillus subtilis

Pectate lyase (PAL EC 4.2.2.2), pectinesterase (PE EC 3.1.1.11), L-arabinanase, D-xylanase, D-galactanase and neutral protease activities were identified in culture filtrates prepared from a strain B3 of Bacillus subtilis isolated from carrot. The PAL was purified by ion-exchange chromatography and iso-electric focusing and its properties examined. PAL had a pI of 9·85 and a molecular weight of 33000. Optimum activity occurred at pH 8–9 and 60–65°C. Calcium and to a lesser extent strontium were stimulatory while ethylenediamine tetraacetic acid led to inactivation. Thin layer chromatography separations of the end products of reactions and viscosity measurements suggested that the enzyme acted in a random manner. When examined over a range of pH values both culture filtrate and the purified PAL produced two distinct peaks of maceration (pH 6–6·5 and 8–9) against carrot or potato tissues. Evidence was obtained that although the presence of lyase was the sole external factor responsible for the maceration of carrot at pH 6·0, it acted in conjunction with a heat-labile, high molecular weight factor extractable from carrot tissue. Carrot extracts were unable to macerate carrot but liberated reducing groups from polygalacturonic acid and it is suggested that the factor may be, in part at least, carrot polygalacturonase. Maceration at pH 8·5 was largely accounted for by PAL and PE activities.     

Structure and Replication of Chromosomes in Competent Cells of Bacillus subtilis

Competent cultures of Bacillus subtilis 168 were fractionated on gradients of Renografin-76 to obtain a population enriched for competent cells. The cells in this fraction contained two nuclear bodies. The competent cell fraction synthesized deoxyribonucleic acid and ribonucleic acid at reduced rates compared to the noncompetent cell fraction and appeared to divide synchronously upon incubation. The state of the chromosome in competent cells was determined by density transfer experiments and marker frequency analyses. The results are consistent with a competent cell possessing two, or a multiple of two, chromosomes, one complete and the other partially duplicated. During subsequent growth the partially completed chromosome replicates preferentially.     

Transformation and Transfection in Lysogenic Strains of Bacillus subtilis 168

Strains of Bacillus subtilis lysogenic for either temperate bacteriophage phi105 or SPO2 were reduced to less than 1.0% of the level of transformation of the nonlysogenic strains. Strains lysogenic for both phi105 and SPO2 are virtually nontransformable, indicating that the effect of lysogeny is additive. Lysogenic cultures transfected at essentially wild-type levels with deoxyribonucleic acid (DNA) isolated from bacteriophages phi29 and SPO1. The residual transformation and transfection achieved by the lysogenic cultures changed dramatically during growth in SPII medium, whereas nonlysogenic strains remained competent for 5 hr in SPII medium. Despite a marked reduction in transformation, lysogenic cultures initially irreversibly bound as much DNA as nonlysogenic cultures. After 60 min in SPII medium, there was a rapid decrease in the capacity of lysogenic cells to bind DNA irreversibly. These results, as discussed, indicate that the inhibition of transformation is probably due to an alteration of the cell surface or a differential inactivation of bacterial genes after lysogenic conversion.     

The Genetically Remote Pathogenic Strain NVH391-98 of the Bacillus cereus Group Is Representative of a Cluster of Thermophilic Strains

Bacteria of the Bacillus cereus group are known to cause food poisoning. A rare phylogenetically remote strain, NVH391-98, was recently characterized to encode a particularly efficient cytotoxin K presumably responsible for food poisoning. This pathogenic strain and its close relatives can be phenotypically distinguished from other strains of the B. cereus group by the inability to grow at temperatures below 17°C and by the ability to grow at temperatures from 48 to 53°C. A temperate phage, phBC391A2, residing in the genome of NVH391-98 allows us to distinguish the three known members of this thermophilic strain cluster.     

枯草芽孢杆菌产胞苷的初步研究

目的:通过对野生型枯草芽孢杆菌NO122的诱变筛选,选育出胞苷产生菌株。方法:以野生型枯草芽孢杆菌为出发菌株进行紫外线、硫酸二乙酯诱变,经3-脱杂氮尿嘧啶、5-氟胞苷结构类似物平板定向筛选产胞苷突变株;研究碳源、氮源、温度、初始pH值等发酵条件对该突变菌株产胞苷的影响。结果:经诱变传代后得到突变株TZM1012,该突变株在发酵温度37℃、初始pH7.2、摇床转速220r/min的条件下,摇瓶发酵72h,发酵液中的胞苷可达1.873g/L,并具有较好的遗传稳定性。结论:获得产胞苷生产菌株,并具有较好的遗传稳定性。     

外源质粒在枯草芽孢杆菌BF7658中的稳定性及其基因表达

通过B.subtilis噬菌体PBSI转导,已将携带热稳定α-淀粉酶基因的质粒pAmy411引进了B.subtilis BF7658.转导频率为10_(-9)转导子/PFU。尽管pAmy 411的诲贝数在B.subtilis BF7658中较在B.subtilis AS 1.1176中高1倍,但其传代稳定性却较后者低。质粒携带的热稳定α-淀粉酶基因的表达水平在B.subtilis BF 7658中较在B.subtilisAS1.1176中高6倍。     

Development of Competence in Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

Tryptophan- and thymine-requiring cells of Bacillus subtilis, emerging from an amino acid starvation treatment which causes arrest of the chromosomes at the terminus, were not transformable. During subsequent incubation in a thymineless medium supplemented with amino acids, the cultures developed competence while retaining chromosome arrest. The competent subpopulation apparently shares the synchronous chromosome arrest of the bulk population. This was shown by different methods. The principal method was marker frequency analysis of the deoxyribonucleic acid extracted from a population enriched for competent cells by a column-chromatographic method. It is concluded that development of the competent state can occur in nondividing cells, and that the presence of a replication fork actively engaged in synthesis of deoxyribonucleic acid is not required for the development of this state.