Efficient transformation of <i>Lactococcus lactis</i> IL1403 and generation of knock‐out mutants by homologous recombination

Gerber, Simon; Solioz, Marc

doi:10.1002/jobm.200610297

Cited by 26 publications

(20 citation statements)

References 36 publications

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“…L. lactis IL1403 was transformed with pGH9:ISS1 (Maguin et al, 1996), which contains a temperaturesensitive origin of replication and a transposon insertion sequence with an erythromycin resistance cassette, as described previously (Gerber & Solioz, 2007). Transformed cells were grown fermentatively at 30 uC to stationary phase in the presence of 10 mg erythromycin ml 21 .…”

Section: Methodsmentioning

confidence: 99%

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403

et al. 2013

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Lactococcus lactis possesses a pronounced extracellular Cu 2+ -reduction activity which leads to the accumulation of Cu + in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductasedeficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The DmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the DmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the DmenE mutant, due to the production of Cu + ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu + was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu + toxicity in L. lactis.

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Section: Methodsmentioning

confidence: 99%

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403

et al. 2013

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“…L. lactis electrocompetent cells were prepared and transformed essentially as described by Gerber & Solioz (2007).…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712

et al. 2012

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The widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, D-lactate dehydrogenase and a-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus.

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“…Plasmid integrity was checked by restriction analysis. For gene knockout, L. lactis IL1403 was transformed with pTELO1 by electroporation as described previously (4). Transformants were selected at 30°C on plates with brain heart infusion medium (Difco Laboratories, Detroit, MI) containing 5 g/ml of erythromycin.…”

Section: Methodsmentioning

confidence: 99%

Copper Induction of Lactate Oxidase of Lactococcus lactis : a Novel Metal Stress Response

2007

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Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 M copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5 region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

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Efficient transformation of Lactococcus lactis IL1403 and generation of knock‐out mutants by homologous recombination

Cited by 26 publications

References 36 publications

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403

Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712

Copper Induction of Lactate Oxidase of Lactococcus lactis : a Novel Metal Stress Response

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