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Applied and Environmental Microbiology, October 2000, p. 4272-4278, Vol. 66, No. 10
0099-2240/00/$04.00+0

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

Régis Stentz, Christophe Loizel, Christine Malleret, and Monique Zagorec^*

Laboratoire de Recherches sur la Viande, INRA-CRJ, F-78350 Jouy en Josas, France

Received 8 May 2000/Accepted 10 July 2000

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 -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 CuSO₄ (30 to 40 µM) but not when CuSO₄ is omitted or added at higher concentrations.

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^* Corresponding author. Mailing address: Laboratoire de Recherches sur la Viande, INRA-CRJ, Domaine de Vilvert, F-78350 Jouy en Josas, France. Phone: 33 (0)1 34-65-22-89. Fax: 33 (0)1 34-65-21-05. E-mail: zagorec{at}biotec.jouy.inra.fr.

Present address: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom.

Present address: Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.

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Applied and Environmental Microbiology, October 2000, p. 4272-4278, Vol. 66, No. 10
0099-2240/00/$04.00+0

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