STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE GENE-CLUSTER ENCODING THE ENZYMES OF THE ARGININE DEIMINASE PATHWAY OF LACTOBACILLUS-SAKE

Lactobacillus sake can use arginine via the arginine deiminase (ADI) p athway. We designed degenerate primers based on an alignment of known sequences of ornithine transcarbamoylase (OTC)-encoding genes in order to amplify the L. sake counterpart sequences by PCR. Screening a geno mic library oft. sake in lambda EMBL3 allowed us to isolate a clone co ntaining a 10-kb L. sake genomic DNA insert. Sequence analysis reveale d that the genes involved in arginine catabolism were clustered and en coded ADI (arcA), OTC (arcB), carbamate kinase (arcC), and a putative carrier with high similarity to the arginine/ornithine antiporter of P seudomonas aeruginosa (arcD), Additionally, a putative transaminase en coding gene (arcT) was located in this region. The genes followed the order arcA arcB arcC arcT arcD, which differs from that found in other microorganisms. arcA, arcB, arcC, and arcD mutants were constructed, and the ADI pathway was impaired in all of them. Transcriptional studi es indicated that arcA gene is subject to catabolite repression, and u nder the conditions used, several transcripts could be detected, sugge sting the existence of different initiation sites or processing of a l arger mRNA.