Cloning and characterization of brnQ, a gene encoding a low-affinity, branched-chain amino acid carrier in Lactobacillus delbrückii subsp. lactis DSM7290.
A gene (brnQ), encoding a carrier for branched-chain amino acids in Lactobacillus delbrückii subsp. lactis DSM7290 was cloned in the low-copy-number vector pLG339 by complementation of a transport-deficient Escherichia coli strain. The plasmid carrying the cloned gene restored growth of an E. coli strain mutated in 4 different branched-chain amino acid transport genes at low concentrations of isoleucine, and increased its sensitivity to valine. Transport assays showed that leucine, isoleucine and valine are transported by this carrier and that transport is driven by the proton motive force. Nucleotide sequence analysis revealed 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 extension analysis using mRNA from Lactobacillus delbrückii subsp. lactis DSM7290. The hydropathy profile suggests the existence of at least 12 hydrophobic domains that probably form membrane-associated alpha-helices. Comparisons of the nucleotide sequence of brnQ from Lactobacillus delbrückii subsp. lactis DSM7290, the amino acid sequence of its product and the topology of the hydrophobic domains with those of the respective carrier genes and proteins of Salmonella typhimurium and Pseudomonas aeruginosa revealed extensive homology.
Stucky
K
K
Universität Kaiserslautern, Fachbereich Biologie, Abteilung Mikrobiologie, Kaiserslauterm, Germany.
Hagting
A
A
Klein
J R
JR
Matern
H
H
Henrich
B
B
Konings
W N
WN
Plapp
R
R
D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007778:Lactobacillus / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013045:Species Specificity
D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D002352:Carrier Proteins; C029752:branched-chain amino acid-binding proteins