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Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli.

ABSTRACT: The Lactococcus lactis adhE gene, which encodes a multifunctional alcohol dehydrogenase, has been cloned and characterized. A DNA fragment encoding the putative alcohol dehydrogenase domain of the AdhE protein was cloned by screening an L. lactis genomic library in a fermentative mutant of Escherichia coli and selecting for the ability to grow anaerobically. Further analysis of the clone obtained allowed the cloning of the entire adhE gene sequence. Analysis of adhE expression in L. lactis during anaerobiosis showed induction at the transcriptional level, especially in medium containing glucose. Constructed mutant strains produced reduced amounts of ethanol under anaerobic conditions. With the L. lactis gene as a probe, adhE homologs were found in other industrially relevant lactic acid bacteria.

SUBMITTER: Arnau J

PROVIDER: S-EPMC107803 | biostudies-literature | 1998 Jun

REPOSITORIES: biostudies-literature

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Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli.

Arnau J J Jørgensen F F Madsen S M SM Vrang A A Israelsen H H

Journal of bacteriology 19980601 12

The Lactococcus lactis adhE gene, which encodes a multifunctional alcohol dehydrogenase, has been cloned and characterized. A DNA fragment encoding the putative alcohol dehydrogenase domain of the AdhE protein was cloned by screening an L. lactis genomic library in a fermentative mutant of Escherichia coli and selecting for the ability to grow anaerobically. Further analysis of the clone obtained allowed the cloning of the entire adhE gene sequence. Analysis of adhE expression in L. lactis durin ...[more]

PMID: 9620952

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Project description:An unusual, spontaneous, phage sk1-resistant mutant (RMSK1/1) of Lactococcus lactis C2 apparently blocks phage DNA entry into the host. Although no visible plaques formed on RMSK1/1, this host propagated phage at a reduced efficiency. This was evident from center-of-infection experiments, which showed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sensitive parental strain, C2. Moreover, viable cell counts 0 and 4 h after infection were not significantly different from those of an uninfected culture. Further characterization showed that phage adsorption was normal, but burst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages. Phage sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterization of the DNA that restored phage sensitivity revealed an open reading frame with similarity to sequences encoding lysozymes (beta-1,4-N-acetylmuramidase) and lysins from various bacteria, a fungus, and phages of Lactobacillus and Streptococcus and also revealed DNA homologous to noncoding sequences of temperate phage of L. lactis, DNA similar to a region of phage sk1, a gene with similarity to tRNA genes, a prophage attachment site, and open reading frames with similarities to sun and to sequences encoding phosphoprotein phosphatases and protein kinases. Mutational analyses of the cloned DNA showed that the region of homology with lactococcal temperate phage was responsible for restoring the phage-sensitive phenotype. The region of homology with DNA of lactococcal temperate phage was similar to DNA from a previously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal temperate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strain. The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with components of the resistance mechanism.

Dataset Information

Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli.

Publications

Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli.

Similar Datasets

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