Cloning Vectors Derived from Plasmids and Phage of Bacillus

Kreft, Jürgen

doi:10.1007/978-3-642-68315-2_1

Cited by 23 publications

(14 citation statements)

References 59 publications

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“…To explain this behavior, we envisaged three possibilities: (i) the FtsH protein modified at its C terminus is inactive under these conditions; (ii) transcription of the ftsH gene is impaired by the P Tc promoter located near the insertion site and reading towards the 5Ј end of ftsH (see Fig. 1A; it has been reported that this promoter is active in B. subtilis [19]); and (iii) a combination of both possibilities. To distinguish between these alternatives, we decided to delete the P Tc promoter from the chromosome, resulting in strain ED1.…”

Section: Resultsmentioning

confidence: 99%

The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift

1995

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The ftsH gene of Bacillus subtilis has been identified as a salt-sensitive insertion mutation in strain UG1. Here, we show that UG1 has an insertion near the 3 end of ftsH. The salt sensitivity of this mutant was caused by reduction of ftsH mRNA levels by the synthesis of an artificial antisense RNA originating at a promoter located within the insertion and reading backwards into the ftsH gene. The salt-sensitive phenotype could be overcome by deleting the promoter from which the antisense RNA was transcribed. A physiological analysis of the isogenic wild-type strain in minimal medium revealed unimpaired growth at up to 1 M NaCl, and growth above 1.2 M NaCl was observed only after addition of the osmoprotectant proline or glycine betaine. In contrast, growth of strain UG1 was reduced at a salt concentration above 0.2 M, which could be rescued by the two compatible solutes already mentioned and also by trehalose. Primer extension revealed one potential transcription start site downstream of a putative vegetative promoter, which was activated after osmotic or temperature upshift. Northern (RNA blot) experiments led to the detection of a 2.1-kb transcript, suggesting that ftsH is monocistronic. A transcriptional fusion between ftsH and the gus reporter gene exhibited a twofold increase in ␤-glucuronidase activity after osmotic upshift. To further confirm the need for an enhanced level of FtsH protein after osmotic upshift, the promoter was replaced by the sucrose-inducible promoter P sacB . Whereas this mutant strain could grow in the absence of inducer in LB medium, it stopped growth immediately after addition of 1.1 M NaCl. We conclude that an increased amount of FtsH protein is essential for B. subtilis to cope with an increase in osmolarity or temperature.

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Section: Resultsmentioning

confidence: 99%

The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift

1995

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“…1. Plasmid pMG32-1 suffered a deletion encompassing approximately 1.6 kb of the original B. cereus insert and most of the pBR322-derived portion of the shuttle vector (26). Direct cloning of the 2.1-kb BamHI DNA fragment preserved in pMG32-1 into pUC8 and pUC9 (31) (yielding constructions pMG8-128 and pMG9-128, respectively) resulted in hemolytic E. coli transformants possessing the cytolysin insert in both orientations relative to the vector lac promoter (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage

et al. 1989

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A cloned cytolytic determinant from the genome of Bacillus cereus Bacillus cereus, a common soil saprophyte, has been recognized as an opportunistic pathogen of increasing importance (reviewed in reference 41). Although food-borne gastroenteritis is the most common malady attributed to B. cereus (41), the most devastating is B. cereus endophthalmitis (1, 4, 17). B. cereus elaborates a variety of extracellular membrane-active enzymes and cytolytic toxins. These membrane-active proteins include a phospholipase C (34), sphingomyelinase (22), phosphatidylinositol phospholipase C (23), cereolysin (7; a cytolysin of the streptolysin 0, thiol-activated class), and a second, heat stabile cytolysin about which little is known (8, 10, 37). Phospholipase C, sphingomyelinase, and cereolysin have been highly purified and used in studies of membrane structure (6,29,43) and in studies on the evolution of cytolysins produced by diverse genera of gram-positive bacteria (13,38). Phospholipase C is a Zn metalloenzyme of 23,000 daltons (34) which shows a high degree of stability in chaotropic agents and surfactants (27,28). The sphingomyelinase produced by B. cereus is a protein of between 41,000 and 23,300 daltons, depending on the method of analysis used (40), and requires divalent cations for activity (21).As a first step in determining the contribution that extracellular membrane-active proteins make to the ecology and virulence of B. cereus, a gene bank was established. Identification of a cloned cytolytic determinant from this B. cereuls GP-4 library has been reported (25). To gain insight into the relationship of the cloned cytolysin determinant to membrane-active proteins of B. cereus, nucleotide sequence and enzyme activity analyses were undertaken. The results reported here show that the cytolytic determinant cloned from B. cereus is composed of tandemly arranged genes for two distinct protein products, the activities of both being required to effect target cell lysis (hemolysis as tested). Moreover, the individual cytolysin components possess * Corresponding author. phospholipase C and sphingomyelinase activity, respectively. These data suggest that although the sphingomyelinase and phospholipase C of B. cereus have been studied in detail individually, their function in nature appears to be as a cytolytic unit representing the heat-stabile hemolysin previously observed. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in these experiments are listed in Table 1. Escherichia coli strains were routinely cultured in 2XYT medium (31) with aeration. Bacillus subtilis cultures were grown in HGP broth as previously described (25). Tetracycline (Sigma Chemical Co., St. Louis, Mo.) was incorporated into liquid and solid (1.2% agar) media at concentrations of 10 ,ug/ml for selection of resistant B. subtilis and E. coli strains. Ampicillin (Sigma) was used at 100 pRg/ml to select for recombinants cloned into the vectors pUC8,45). In addition, to screen for insertional ina...

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“…E. coli HBlOl (Boyer & Roulland-Dussoix, 1969) and 15. coli JM 101 (Messing, 1983) were used as hosts for cloning and sequencing experiments. The shuttle plasmid pJK302, consisting of the vectors pBR322 (4.36 kb) and pBC16-1 (2.84 kb) fused at the EcoRI restriction sites (Kreft & Hughes, 1982), and the plasmids pBR322 (Bolivar et ul., 1977), pIB130, and pIBI31 (IBI) were used as vectors.…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581

Kühn

Fortnagel²

1993

Journal of General Microbiology

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The gene npvM encoding the calcium-dependent extracellular proteinase from Bacillus megatevium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HBlO1. The DNA sequence of the cloned 3.7 kb fragment revealed only one open reading frame consisting of 1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation start site (ATG). A possible promoter sequence (TAGACG for the -35 region and TATAAT for the -10 region) was found about 69 bp upstream of the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide 'pro' sequence of 221 amino acids preceding the putative mature protein of 317 amino acid residues. Amino acid sequence comparison revealed 84.5 % homology between the mature protein and that of a thermolabile neutral protease from B. cereus. It also shares 73% homology with the thermostable neutral proteases of B. thermoproteolyticus and B. stearothermophilus. The zinc-binding sites and the catalytic residues are completely conserved in all four proteases. NprM has a temperature optimum of 58 "C, a pH optimum of between 6.4 and 7-2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.

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Cloning Vectors Derived from Plasmids and Phage of Bacillus

Cited by 23 publications

References 59 publications

The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift

The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift

A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage

Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581

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