CLONING AND CHARACTERIZATION OF BRNQ, A GENE ENCODING A LOW-AFFINITY,BRANCHED-CHAIN AMINO-ACID CARRIER IN LACTOBACILLUS-DELBRUCKII SUBSP LACTIS DSM7290

A gene (brnQ), encoding a carrier for branched-chain amino acids in La ctobacillus delbruckii subsp. lactis DSM7290 was cloned in the low-cop y-number vector pLG339 by complementation of a transport-deficient Esc herichia coli strain. The plasmid carrying the cloned gene restored gr owth of an E. coli strain mutated in 4 different branched-chain amino acid transport genes at low concentrations of isoleucine, and increase d its sensitivity to valine. Transport assays showed that leucine, iso leucine and valine are transported by this carrier and that transport is driven by the proton motive force. Nucleotide sequence analysis rev ealed an open reading frame of 1338 bp encoding a hydrophobic protein of 446 amino acids with a calculated molecular mass of 47864 Daltons. The start site of brnQ transcription was determined by primer extensio n analysis using mRNA from Lactobacillus delbruckii subsp. lactis DSM7 290. The hydropathy profile suggests the existence of at least 12 hydr ophobic domains that probably form membrane-associated alpha-helices. Comparisons of the nucleotide sequence of brnQ from Lactobacillus delb ruckii subsp. lactis DSM7290, the amino acid sequence of its product a nd the topology of the hydrophobic domains with those of the respectiv e carrier genes and proteins of Salmonella typhimurium and Pseudomonas aeruginosa revealed extensive homology.