Plasmid vectors for Gram-positive bacteria switching from high to low copy number

Renault, Pierre; Corthier, Gérard; Goupil, Nathalie; Delorme, Christine; Ehrlich, S. Dusko

doi:10.1016/s0378-1119(96)00554-9

Cited by 68 publications

(61 citation statements)

References 18 publications

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“…As readout, we used RNA levels expressed from each gene, and thereby we investigated: i) the effects of one module cloned on a plasmid (10 molcules per cell). 42 on the expression of the other present at its chromosomal locus (i.e. trans-effect), and ii) the changes in the expression of one module cloned on a plasmid in comparison to its expression at the chromosomal locus (i.e., cis-effect).…”

Section: Resultsmentioning

confidence: 99%

“…Sequences of inserts were verified and cloned into the shuttle vector pJIM2246 at the EcoRI restriction site. 42 Direct repeated sequences, drm1 and drm2, were specifically amplified from plasmid constructs harboring mazEF and txpAratA modules alone, and then cloned into the vector pJIM2246. Punctual mutants of the ¡10 promoter boxes for ratA and txpA were obtained directly from the plasmid bearing TAI-II (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

et al. 2015

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We discovered a chromosomal locus containing 2 toxin-antitoxin modules (TAs) with an antisense transcriptional organization in the E. faecalis clinical isolate V583. These TAs are homologous to the type I txpA-ratA system and the type II mazEF, respectively. We have shown that the putative MazF is toxic for E. coli and triggers RNA degradation, and its cognate antitoxin MazE counteracts toxicity. The second module, adjacent to mazEF, expresses a toxin predicted to belong to the TxpA type I family found in Firmicutes, and the antisense RNA antidote, RatA. Genomic analysis indicates that the cis-association of mazEF and txpA-ratA modules has been favored during evolution, suggesting a selective advantage for this TA organization in the E. faecalis species. We showed regulatory interplays between the 2 modules, involving transcription control and RNA stability. Remarkably, our data reveal that MazE and MazEF have a dual transcriptional activity: they act as autorepressors and activate ratA transcription, most likely in a direct manner. RatA controls txpA RNA levels through stability. Our data suggest a pivotal role of MazEF in the coordinated expression of mazEF and txpA-ratA modules in V583. To our knowledge, this is the first report describing a crosstalk between type I and II TAs.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

et al. 2015

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“…cremoris MG1363 (7) as a host. The pILnew plasmid can be converted into a low-copy-number one by removal of a 22-bp KpnI fragment inserted inside the plasmid copy number regulatory gene (28). The low-copy-number plasmid derivative with the cspB-lacZ fusion insert (named pTIL67) was introduced into the MG1363 strain by electroporation (12).…”

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confidence: 99%

Characterization of cspB, a cold-shock-inducible gene from Lactococcus lactis, and evidence for a family of genes homologous to the Escherichia coli cspA major cold shock gene

et al. 1997

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Upon temperature downshift, the major cold shock protein CspA is highly induced in Escherichia coli. This protein being conserved in other bacteria, we used a PCR-based approach with a pair of degenerate primers derived from highly conserved regions of the CspA-related proteins to evidence the presence of at least three related genes in Lactococcus lactis. One of them, cspB, was cloned and sequenced. It encodes a 66-residue protein which possesses 60% sequence identity with E. coli CspA. Following a cold shock from 30 to 15°C, the level of the cspB mRNA transcript increased, as shown by Northern blot hybridization. In addition, induction of cspB-directed ␤-galactosidase activity was observed. These results indicate that the L. lactis cspB gene is cold shock inducible.

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“…Moreover, a DNA sequence similar to the highly conserved cre box involved in glucose repression was found in front of one of them (Doman-Pytka et al, manuscript in preparation). The hypothesis of glucose repression was further tested using transcriptional fusion between the pul and luxAB-reporter genes [5,13], located on the parental plasmid in the L. lactis IBB500 strain (Tab. II).…”

Section: Glucose Repression Of the Pullulanase Genementioning

confidence: 99%

Article

Pytka

Renault

Bardowski

2004

Lait

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-The generally accepted opinion is that the natural niche for lactococci are plants. Several genes reminiscent of the environmental adaptation of these bacteria to the plant habitat were found as a result of our work on the pullulanase coding region in the Lactococcus lactis IBB500 strain. All genes were located within an 11-kb DNA fragment of a 35-kb plasmid. Analysis of the nucleotide sequence of the 11-kb DNA fragment showed three regions: (i) a middle region -encoding the potential pullulanase operon (ii); a pullulanase upstream region -encoding two cold shock proteins; and (iii) a pullulanase downstream region -encoding two cadmium resistance proteins. Analysis of the phylogenetic relationships of these regions through inspection of their G+C content and codon preference, as well as homology of their protein products and constructions of phylogenetic trees strongly supported the hypothesis of their acquisition through a horizontal gene transfer. Focusing further research on the middle region we have found that the pullulanase gene, belonging to the putative pul operon, was preceded by a long, non-translated region in which five putative promoter sequences were identified. Moreover, a DNA sequence similar to the highly conserved cre box involved in glucose repression was found in front of one of them. The hypothesis of glucose repression was further tested using transcriptional fusion between the pul and luxAB-reporter genes, located on the parental plasmid in the L. lactis IBB500 strain. Lactococcus lactis / adaptation / gene-cassette / horizontal gene transfer / pullulanaseRésumé -Une cassette de gènes pour l'adaptation de Lactococcus lactis à l'environnement végétal. Une opinion largement acceptée est que les plantes constituent une niche naturelle pour les lactocoques. Nos recherches ont montré que plusieurs gènes impliqués dans une possible adaptation environnementale de ces bactéries aux plantes sont localisés dans la région codant une pullulanase chez la souche Lactococcus lactis IBB500. Tous ces gènes se trouvent sur un fragment d'ADN d'une taille de 11 kb, situé sur un plasmide de 35 kb. L'analyse de la séquence nucléotidique de ce fragment de 11 kb a révélé 3 régions : (i) une région centrale -avec la présence d'un opéron potentiel de pullulanase, (ii) une région en amont -codant 2 protéines du choc au froid et (iii) une région en aval -codant 2 protéines de la résistance au cadmium. L'analyse phylogénique de ces régions suggère que ces gènes ont été acquis par un mécanisme de transfert horizontal. L'étude de la région centrale a montré que le gène de la pullulanase est précédé par une longue région non codante, dans laquelle nous avons identifié 5 promoteurs potentiels. De plus, une séquence nucléotidique similaire à la boîte cre, très conservée et impliquée dans la répression par le glucose, * Corresponding author: jacek@ibb.waw.pl 34 M. Doman-Pytka et al.

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Plasmid vectors for Gram-positive bacteria switching from high to low copy number

Cited by 68 publications

References 18 publications

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

Characterization of cspB, a cold-shock-inducible gene from Lactococcus lactis, and evidence for a family of genes homologous to the Escherichia coli cspA major cold shock gene

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