Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis

Mongkolsuk, Skorn; Chiang, Yungping; Reynolds, Robert B.; Lovett, Paul S.

doi:10.1128/jb.155.3.1399-1406.1983

Cited by 60 publications

(19 citation statements)

References 29 publications

(25 reference statements)

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“…Hence, we decided to assay the 740-bp PvuII-HindIII fragment for repressor activity. The B. subtilis vector pPL7O3 [10] containing unique HindIII and PvuII sites, was digested with both enzymes and ligated to HindIII+PvuII-digested pUC4/F. A large number of kanamycin-resistant B. subtilis transformants were subsequently tested for sensitivity to 4105.…”

Section: Resultsmentioning

confidence: 99%

“…These strains, as well as E. coli C600 rk mk, were routinely grown at 37°C in LB medium supplemented with the appropriate antibiotics. Selective concentrations were : ampicillin (Ap), 200 pg/ml; erythromycin (Em), 10 pg/ml; kanamycin (Km), 10 pg/ml. Plasmids constructed during this work are described in the text.…”

Section: Materials and Methods Bacterial Strains Phage And Plasmidsmentioning

confidence: 99%

“…Plasmids constructed during this work are described in the text. They are all derived either from the E. coli replicon pUC4 (ampicillin-resistant; [8]), or the B. subtilis vectors pE194cop-6 (erythromycin-resistant; [9]), and pPL703 (kanamycin-resistant; [10]). Plasmid pCGV14, containing the 2.3-kb PstI fragment I of 4105 [11] inserted in the PstI site of pE194cop-6, has been described earlier [3].…”

Section: Materials and Methods Bacterial Strains Phage And Plasmidsmentioning

confidence: 99%

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Nucleotide sequence and mutational analysis of an immunity repressor gene fromBacillus subtilistemperate phage ø105

et al. 1985

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We have identified and sequenced a bacteriophage phi 105 gene encoding an immunity repressor, the first to be characterized from a temperate phage infecting a Gram-positive host. Using superinfection immunity as an assay for repressor function, the phi 105 repressor gene was located within a 740-bp PvuII-HindIII subfragment near the left end of the phi 105 EcoRI-F fragment. We show that the repressor is specified by the 5'-proximal coding sequence of a translationally overlapping gene pair, transcribed from right to left on the conventional phi 105 map. Comparison of its amino acid sequence (146 residues) with that of a large number of Gram-negative bacterial and phage repressors revealed a putative DNA-binding region between positions 20 and 39. The coding region is preceded by a strong Shine-Dalgarno sequence 5' AAAGGAG 3'. Deletion analysis of the 5'-flanking DNA allowed to identify transcriptional control elements. Their structure, 5' TTGTAT 3' at -35 and 5' TATAAT 3' at -10, strongly suggests that the phi 105 repressor gene is transcribed by the major vegetative form of B. subtilis RNA polymerase, as would be expected for an early phage gene.

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Section: Resultsmentioning

confidence: 99%

Section: Materials and Methods Bacterial Strains Phage And Plasmidsmentioning

confidence: 99%

Section: Materials and Methods Bacterial Strains Phage And Plasmidsmentioning

confidence: 99%

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Nucleotide sequence and mutational analysis of an immunity repressor gene fromBacillus subtilistemperate phage ø105

et al. 1985

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“…E. coli cells were grown in LB at 37³C. Lysate preparation and Cat enzyme assay were performed spectrophotometrically as described by Mongkolsuk et al [6].…”

Section: Media Growth Conditions and Enzyme Assaymentioning

confidence: 99%

Construction and characterization of regulated ?-arabinose-inducible broad host range expression vectors in Xanthomonas

Sukchawalit¹

1999

FEMS Microbiology Letters

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Several versions of broad host range (BHR), L-arabinose-inducible expression vectors were constructed. These expression vectors were based on a high copy number BHR pBBR1MCS-4 replicon that could replicate in both enteric and non-enteric Gram-negative bacteria. Two versions of expression cassettes containing multiple cloning sites either with or without a ribosome binding site were placed under transcriptional control of the Escherichia coli BAD promoter and araC gene. Three versions of vectors containing ampicillin or kanamycin or tetracycline resistance genes as selectable markers were constructed. In all six new L-arabinose-inducible BHR expression vectors containing many unique cloning sites, selectable markers were made to facilitate cloning and expression of genes in various Gram-negative bacteria. A Tn9 chloramphenicol acetyl transferase (cat) gene was cloned into an expression vector, resulting in pBBad18Acat that was used to establish optimal expression conditions (addition of 0.02% L-arabinose to mid-exponential phase cells for at least 1 h) in a Xanthomonas campestris pv. phaseoli. Comparison of the Cat enzyme activities between uninduced and a 180-min L-arabinose-induced culture showed a greater than 150-fold increased Cat specific activity. In addition, L-arabinose induction of exponential phase cells harboring pBBad18Acat gave a higher amount of Cat than similarly treated stationary phase cells. The usefulness of the expression vector was also demonstrated in both enteric and non-enteric Gram-negative bacteria. ß Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 0 9 7 ( 9 9 ) 0 0 5 4 1 -8

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“…Rl lacks promoter activity but contains sequences necessary for Cm inducibility of cat-86 (4). Derivatives of pPL531 deleted for the P3 fragment (yielding plasmid pPL603) (14,25) or deleted for both P3 and P1 (yielding plasmid pPL703) (4,14) are useful as promoter cloning vectors in B. subtilis (14).…”

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confidence: 99%

Regulatory regions that control expression of two chloramphenicol-inducible cat genes cloned in Bacillus subtilis

Duvall¹,

Williams²,

Mongkolsuk³

et al. 1984

J Bacteriol

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Plasmid pPL603 is a promoter cloning vector for Bacillus subtilis and consists of a 1.1-kilobase fragment of Bacillus pumilus DNA inserted between the EcoRI and BamHI sites of pUB110. The gene cat-86, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, is located on the 1.1-kilobase cloned DNA. When pPL603 is present in B. subtilis, cat-86 is unexpressed during vegetative growth but expressed during sporulation. The regulation of cat-86 in pPL603 is due to sequences within two restriction fragments, designated P1 and Rl, that precede the main coding portion of the gene. The P1 fragment promotes transcription of cat-86 only during sporulation, whereas the adjacent Rl fragment lacks promoter function but contains sequences essential to chloramphenicol inducibility. A second B. pumilus gene, cat-66, was cloned in B. subtilis and is expressed throughout the vegetative growth and sporulation cycle. The cat-66 coding region is preceded by two adjacent restriction fragments designated as P2 and R2. P1 and P2 are identical in size and share 95% conservation of base sequence. Rl and R2 are also identical in size and share 91% conservation of base sequence. Fragment substitution experiments demonstrate that R2 can functionally replace Rl. The substitution of P2 for P1 promotes cat-86 expression throughout vegetative growth and sporulation. Analysis of a derivative of pPL603 in which P2 has replaced P1 demonstrates that P2 promotes transcription of cat-86 during vegetative growth and that P2 contains the start site for transcription of cat-86. Thus, P1 and P2 differ strikingly in vegetative promoter function, yet they differ by single-base substitutions at only 11 positions of 203.

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Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis

Cited by 60 publications

References 29 publications

Nucleotide sequence and mutational analysis of an immunity repressor gene fromBacillus subtilistemperate phage ø105

Nucleotide sequence and mutational analysis of an immunity repressor gene fromBacillus subtilistemperate phage ø105

Construction and characterization of regulated ?-arabinose-inducible broad host range expression vectors in Xanthomonas

Regulatory regions that control expression of two chloramphenicol-inducible cat genes cloned in Bacillus subtilis

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