Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80

Israelsen, Hans; Madsen, Søren; Vrang, Astrid; Hansen, Egon Bech; Johansen, Eric

doi:10.1128/aem.61.7.2540-2547.1995

Cited by 179 publications

(68 citation statements)

References 39 publications

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“…The pH-and growth phase-regulated promoter P170 from L. lactis MG1363 was originally isolated on a 9.7 kb DNA fragment (Israelsen et al, 1995). This former study also showed that low pH and stationary phase co-dependency were required for high promoter activity.…”

Section: Resultsmentioning

confidence: 88%

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Molecular characterization of the pH‐inducible and growth phase‐dependent promoter P170 of Lactococcus lactis

Madsen¹,

Arnau²,

Vrang³

et al. 1999

Molecular Microbiology

107

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SummaryIn a previous study, we described the use of transposon Tn917-LTV1 for identi®cation of environmentally regulated promoters in Lactococcus lactis. Here, we report the molecular analysis of one of these promoters, P170, that is upregulated at low pH during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus À35 promoter region, but it contained an`extended' À10 promoter region. When a 28 bp segment, containing the consensus À35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase. This demonstrates that the P170 segment contains a cis-acting sequence involved in the control of promoter regulation. Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis. Analysis of total RNA from L. lactis grown at constant pH con®rmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase. Deletion analysis and chemical mutagenesis of P170 de®ned a speci®c region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter. Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150-to 200-fold increase in the level of gene expression, without affecting the regulation. The functionality was con®rmed by introducing this modulating element downstream of other lactococcal promoters.

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Section: Resultsmentioning

confidence: 88%

“…To investigate whether a gene was transcribed from the P170 promoter, inverse PCR was used to amplify the DNA region located downstream of the Tn917 insertion point of integrant PA170 (Israelsen et al, 1995) Fig. 3.…”

Section: Resultsmentioning

confidence: 99%

Molecular characterization of the pH‐inducible and growth phase‐dependent promoter P170 of Lactococcus lactis

Madsen¹,

Arnau²,

Vrang³

et al. 1999

Molecular Microbiology

107

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“…The 358-bp PCR fragment corresponding to 257 bp upstream and 98 bp downstream the ATG codon of ytgB, which is the ¢rst gene of the operon containing ytgH was cloned into the SmaI site of the pAK80 plasmid [6]. This transcriptional fusion was transformed in L. lactis IL1403 resulting in strain NVlac.…”

Section: Construction Of the Transcriptional Fusionmentioning

confidence: 99%

“…No obvious 335 box was present in this promoter region. A 358-bp fragment including this promoter was cloned in front of the reporter genes lacL^lacM (encoding the L-galactosidase of Leuconostoc mesenteroides) in the low copy number plasmid pAK80 [6]. Fig.…”

Section: Mapping the Transcriptional Start Sites Of Ytgh Operonmentioning

confidence: 99%

Characterization of genes homologous to the general stress-inducible gene gls24 in Enterococcus faecalis and Lactococcus lactis

Giard

2002

FEMS Microbiology Letters

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Three genes (Ef0604, ymgG, and ytgH) were identified as homologous to the gls24 gene (encoding the general stress protein Gls24) in Enterococcus faecalis V583 and Lactococcus lactis IL1403 genomes sequences, and therefore are part of the`gls24 family'. The other proteins encoded by the different surrounding genes in each of their respective operons are also highly similar. Our transcriptional analysis showed that Ef0604 and ymgG are not induced under the stress conditions tested. On the other hand, ytgH is induced at the onset of glucose starvation and by heat, ethanolic, osmotic, and CdCl 2 stresses. The transcriptional start site of this operon was identified and the promoter region appears to contain an`extended 310 Box'. Moreover, the over-expression under several stress conditions of the YtgH protein observed on 2D gel electrophoresis confirms that it corresponds to a general stress protein in L. lactis. ß

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“…After electroporation into L. lactis MG1363, growth of transformants was accompanied in both cases by the development of a deep blue colour on GM17 plates containing Xgal, indicating b-gal activity and, therefore, the presence of active promoters. To quantify the levels of b-gal activity, Miller assays were performed on liquid cultures of both strains according to the method of Israelsen et al (1995). The levels of b-gal were in the same range for both cultures, with high levels of expression from both promoters ( Fig.…”

Section: Overexpression Of Ltnr Results In An Increase In Sensitivitymentioning

confidence: 99%

Regulation of immunity to the two‐component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR

McAuliffe

O'Keeffe

Hill

et al. 2001

Molecular Microbiology

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SummaryLacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA 1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA 1 A 2 M 1 TM 2 D and ltnRIFE respectively. Although the promoter controlling biosynthesis (P bac ) appears to be constitutive, characterization of a downstream b-galactosidase (bgal) fusion beyond an intragenic stem±loop structure in ltnM 1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ltnRIFE operon (P imm ) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the P imm promoter region, and fusion of this promoter to the b-gal gene of pAK80 revealed that expression from P imm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.

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Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80

Cited by 179 publications

References 39 publications

Molecular characterization of the pH‐inducible and growth phase‐dependent promoter P170 of Lactococcus lactis

Molecular characterization of the pH‐inducible and growth phase‐dependent promoter P170 of Lactococcus lactis

Characterization of genes homologous to the general stress-inducible gene gls24 in Enterococcus faecalis and Lactococcus lactis

Regulation of immunity to the two‐component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR

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