Development of Genetic Tools for Lactobacillus sakei : Disruption of the β-Galactosidase Gene and Use of lacZ as a Reporter Gene To Study Regulation of the Putative Copper ATPase, AtkB

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Abstract
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 β-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 μM) but not when CuSO 4 is omitted or added at higher concentrations.