New thermosensitive plasmid for gram-positive bacteria

Maguin, Emmanuelle; Duwat, Patrick; Hégé, Timothée; Ehrlich, Dusko; Gruss, Alexandra

doi:10.1128/jb.174.17.5633-5638.1992

Cited by 399 publications

(296 citation statements)

References 49 publications

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“…Recombinant pORI 19-1 plasmids carrying the JH2-2 chromosomal mutated DNA fragment were used to transform Ent. faecalis JH2-2 in which plasmid pG + host 3 (pVE6007) (Maguin et al, 1992) encoding a thermosensitive RepA protein had previously been introduced. After electroporation, the two plasmids were maintained together at the permissive temperature of 30 uC by plating cells on GM17 agar medium containing erythromycin (Em) and chloramphenicol (Cm).…”

Section: Methodsmentioning

confidence: 99%

Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages

et al. 2006

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The gene encoding the manganese-containing superoxide dismutase (MnSOD) of Enterococcus faecalis was characterized. It is transcribed monocistronically from an upstream promoter identified by rapid amplification of cDNA ends (RACE)-PCR. A sodA mutant was constructed and characterized. Growth of the mutant strain was not significantly different from that of its wild-type counterpart in standing and aerated cultures. However, the mutant was more sensitive towards menadione and hydroperoxide stresses. The response to H2O2 stress was analysed in more detail, and the mode of killing of this oxidant was different under anaerobic and aerobic conditions. Cultures grown and challenged under anaerobic conditions were highly sensitive to treatment with 35 mM H2O2. They were largely protected by the iron chelator deferoxamine, which suggested that killing was mainly due to an enhanced Fenton reaction. In contrast, neither strain was protected by the iron chelators deferoxamine and diethylenetriaminepentaacteic acid when grown and challenged under aerobic conditions, which suggested that inactivation of the cells by H2O2 was due to another killing mode. The sodA mutant was more sensitive under these conditions, showing that MnSOD is also important for protecting the cells from damage under aerobic conditions. Finally, the MnSOD of Ent. faecalis may be considered to be a virulence factor, since survival of the corresponding mutant strain was highly affected inside mouse peritoneal macrophages.

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Section: Methodsmentioning

confidence: 99%

Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages

et al. 2006

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“…Since bglR, like E. coli bglG, is controlled by P-glucoside sugars, we suspected that BglR might be involved in regulating ,-glucoside utilization. To test this hypothesis, bglR was disrupted by insertion of a 10-nucleotide HindIII linker at the unique Swal site and replacement of the wild-type gene by the disrupted gene, using a recently described strategy (9,23). Clones containing the disrupted bglR were distinguished from those containing the wild-type bglR by comparing the profiles of their HindIIIcleaved DNAs hybridized with the bglR DNA.…”

Section: Methodsmentioning

confidence: 99%

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis

1994

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The bgl operon of Escherichia coli is involved in the utilization of P-glucoside sugars (25,36). This operon is cryptic in wild-type strains but can be rendered functional by a variety of spontaneous mutations (30,31,35). When functional, this operon is inducible by 3-glucosides. This control is exerted through transcription antitermination mediated by the BglG protein (3,22,25,33,34). Transcription initiates constitutively at the bgl promoter, but in the absence of 3-glucosides, most transcripts terminate at a p-independent terminator located in the leader region, immediately upstream of the bglG gene. In the presence of P-glucosides, the BglG protein binds to a specific sequence of the mRNA, overlapping part of the transcription terminator, thus preventing RNA polymerase from terminating. The binding of BglG is modulated by its phosphorylation by EIIBgi, an enzyme of the phosphoenolpyruvate:p-glucoside phosphotransferase system, which is involved in uptake and phosphorylation of P-glucosides (36). In the absence of 0-glucosides, P-EIIBgl phosphorylates BglG, which thus becomes unable to bind to RNA and act as an antiterminator (3, 4). If 3-glucosides are present, they are phosphorylated by P-EII'9' and P-BglG is dephosphorylated. The protein can thus bind to RNA, which results in transcription antitermination.Based on a strong conservation of the amino acid sequences of the regulatory proteins and of their putative RNA systems. These systems include the control of the Bacillus subtilis levansucrase sacB gene by SacY (7, 13), of the B. subtilis sacPA operon by SacT (6, 14, 39), of B. subtilis 13-glucan utilization by licT (40), of Lactobacillus casei lactose utilization (1), and of the Erwinia chrysanthemi P-glucoside phosphotransferase gene arbF by ArbG (15).In this article, we describe the Lactococcus lactis bglR gene, whose product shares homology with the BglG family of antiterminators. bglR is preceded by a transcription terminator partially overlapped by the conserved target sequence of the BglG antiterminators. Constitutive expression of bglR in E. coli increased the expression of a bglG::lacZ transcriptional fusion. The fact that the expression of BglG is autoregulated in E. coli suggests that BglG and BglR are functionally related. In L.lactis, the expression of bglR is positively controlled by P-glucosides and its disruption results in a deficiency in P-glucoside utilization. Taken together, these results indicate that BglR is a lactococcal counterpart of the E. coli BglG regulator of 3-glucoside utilization. MATERIALS AND METHODSBacterial strains and media. L. lactis subsp. lactis IL1403 (11) and derivatives, E. coli TG1 (18) and MA152 (24), and B. subtilis MT119 (41) were grown as previously described (8). Growth on various carbohydrates in a chemically defined broth (28,29,38), supplemented with the appropriate sugar at 1% final concentration, was monitored. The medium was inoculated with 1% of a culture grown overnight and then washed twice.DNA manipulations. Plasmids and chromosomal DNAs were e...

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“…The fragment was subcloned into the vector pCR2.1-TOPO using the TOPO-TA cloning system. Plasmid pED202 was constructed by ligating pED102 and pGhost + 4 (Maguin et al, 1992) together at the XbaI site, which is unique in both plasmids. After transformation into E. coli XL1-Blue, the fusion plasmid was obtained by selection at 37 uC on LB agar plates containing both 100 mg ampicillin ml 21 and 150 mg erythromycin ml 21 .…”

Section: Methodsmentioning

confidence: 99%

The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker

2007

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The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker A new lactococcal plasmid, pDBORO, was isolated from the Lactococcus lactis subsp. lactis biovar diacetylactis strain DB0410. This plasmid is responsible for the sensitivity of DB0410 to the toxic pyrimidine analogue 5-fluoroorotate. The complete nucleotide sequence has been determined and amounts to 16 404 bp. Of 15 ORFs encountered, three were found to be insertion sequence (IS) elements, identified as two IS946 and one IS982. Two ORFs are incomplete due to the insertion of an IS element in their C-terminal region. Homologues for four ORFs were found in the IL1403 sequence: the copB gene, coding for a copperpotassium-transporting ATPase B, and the ysbA, ysbB and ysbC genes. The structural organization of the pDBORO replication region is highly similar to other theta-replicating plasmids in both the cis-(repA) and trans-acting (repB and orfX) sequences. By plasmid deletion analysis and molecular cloning, a single locus on pDBORO was found to confer sensitivity to 5-fluoroorotate. It was identified as ysbC, but renamed oroP in order to reflect its function. The oroP gene was found to be essential for the utilization of orotate as the sole pyrimidine source in a strain deficient in pyrimidine de novo synthesis. The amino acid sequence encoded by the ORF showed the characteristic features of a membrane protein. Therefore, oroP most probably encodes an orotate transporter. Surprisingly, homologues of oroP could be identified in the genomes of both L. lactis MG1363 and L. lactis IL1403 despite the fact that these strains were unable to significantly utilize orotate. Cloning of oroP in Escherichia coli and Bacillus subtilis showed that the orotate transport phenotype could be transformed to both organisms. The findings presented indicate that oroP can be used as a powerful, food-grade selection/ counterselection marker in many different organisms. INTRODUCTIONLactococcus lactis strains are widely used in the dairy industry as starter cultures in cheese fermentation. All L. lactis species isolated so far from starter cultures contain a number of different plasmids. These plasmids are thought to have been acquired during adaptation, and many plasmid-encoded functions have been related to or are essential for growth in milk. Examples are genes involved in lactose transport and metabolism , genes encoding proteinases necessary for casein degradation McKay et al., 1976) and di-and oligopeptide transport genes (Yu et al., 1995a(Yu et al., , 1996.Pyrimidine metabolism has been extensively studied in L. lactis in our laboratory (Andersen et al., 1996;Jorgensen et al., 2003Jorgensen et al., , 2004Kilstrup & Martinussen, 1998;Martinussen et al., , 2001Martinussen et al., , 2003Wadskov-Hansen et al., 2000, 2001, and a review of nucleotide metabolism in lactic acid bacteria has recently been published (Kilstrup et al., 2005). In L. lactis, pyrimidines can be synthesized de novo or ...

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New thermosensitive plasmid for gram-positive bacteria

Cited by 399 publications

References 49 publications

Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages

Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis

The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker

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