Development of genetic tools for Lactobacillus sakei: disruption of the beta-galactosidase gene and use of lacZ as a reporter gene To study regulation of the putative copper ATPase, AtkB.
Downstream from the ptsHI operon of Lactobacillus sakei, the genes atkY and atkB, organized in an operon, were observed. The two putative proteins, AtkB and AtkY, show sequence similarity to the Enterococcus hirae copper P-type ATPase, responsible for copper efflux, and its negative regulator. Characterization of AtkB as a copper P-type ATPase could not be demonstrated since an atkB mutant did not show any phenotype. Thus, another strategy was followed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L. sakei. A plasmid was constructed, the use of which allowed gene replacement at the lacLM locus in L. sakei by two successive crossovers. A strain deleted of the lacLM operon encoding the beta-galactosidase of L. sakei was constructed by this method, and the Escherichia coli lacZ gene could then be used as a reporter gene to investigate the regulation of atkYB. Results show that the atkYB operon is induced by small concentrations of CuSO(4) (30 to 40 microM) but not when CuSO(4) is omitted or added at higher concentrations.
Stentz
R
R
Laboratoire de Recherches sur la Viande, INRA-CRJ, F-78350 Jouy en Josas, France.
Loizel
C
C
Malleret
C
C
Zagorec
M
M
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D003434:Crossing Over, Genetic; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon; D016133:Polymerase Chain Reaction / Q000379:methods; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000235:genetics*
D001426:Bacterial Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; C416125:AtkB protein, Lactobacillus sakei; C416126:AtkY protein, Lactobacillus sakei