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, At kB and AtkY, show sequence similarity to the Enterococcus hirae copper P-ty pe ATPase, responsible for copper efflux, and its negative regulator. Chara cterization of AtkB as: a copper P-type ATPase could not be demonstrated si nce an atkB mutant did not show any phenotype, Thus, another strategy was f ollowed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L. sakei, A pla smid was constructed, the use of which allowed gene replacement at the lacL M locus in L. sakei by two successive crossovers. A strain deleted of the l acLM 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 rep orter gene to investigate the regulation of atkYB. Results show that the at kYB operon is induced by small concentrations of CuSO4 (30 to 40 mu M) but not when CuSO4 is omitted or added at higher concentrations.