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Appl Environ Microbiol, June 1998, p. 2013-2019, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The gal Genes for the Leloir Pathway of Lactobacillus casei 64H

Katja Bettenbrockdagger and Carl-Alfred Alpert*

Universität Osnabrück, Fachbereich Biologie/Chemie, AG Genetik, 49076 Osnabrück, Germany

Received 21 November 1997/Accepted 6 March 1998

The gal genes from the chromosome of Lactobacillus casei 64H were cloned by complementation of the galK2 mutation of Escherichia coli HB101. The pUC19 derivative pKBL1 in one complementation-positive clone contained a 5.8-kb DNA HindIII fragment. Detailed studies with other E. coli K-12 strains indicated that plasmid pKBL1 contains the genes coding for a galactokinase (GalK), a galactose 1-phosphate-uridyltransferase (GalT), and a UDP-galactose 4-epimerase (GalE). In vitro assays demonstrated that the three enzymatic activities are expressed from pKBL1. Sequence analysis revealed that pKBL1 contained two additional genes, one coding for a repressor protein of the LacI-GalR-family and the other coding for an aldose 1-epimerase (mutarotase). The gene order of the L. casei gal operon is galKETRM. Because parts of the gene for the mutarotase as well as the promoter region upstream of galK were not cloned on pKBL1, the regions flanking the HindIII fragment of pKBL1 were amplified by inverse PCR. Northern blot analysis showed that the gal genes constitute an operon that is transcribed from two promoters. The galKp promoter is inducible by galactose in the medium, while galEp constitutes a semiconstitutive promoter located in galK.

* Corresponding author. Present address: Institut National de la Recherche Agronomique, Unité de Recherches sur la Viande, Jouy-en-Josas, France. Phone: 33 01 34 65 21 06. Fax: 33 01 34 65 21 05. E-mail: Alpert{at}biotec.jouy.inra.fr.

dagger Present address: Universitäts-Klinikum Ulm, Abteilung Medizinische Mikrobiologie und Hygiene, 89081 Ulm, Germany.

Appl Environ Microbiol, June 1998, p. 2013-2019, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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