Arginine biosynthesis inCampylobacter jejuniTGH9011: determination of theargCOBDcluster

Abstract

Arginine biosynthetic genes from Campylobacter jejuni TGH9011 were cloned by functional complementation of the respective Escherichia coli arginine biosynthetic mutants. Complementation of argA, argB, argC, argD, argE, argF, and argH auxotrophs was accomplished using a pBR322-based C. jejuni TGH9011 plasmid library. By cross-complementation analyses, the first four steps of arginine biosynthesis were shown to be closely linked on the genome. Two additional clones complementing the first (ArgA) and fifth (ArgE) steps in arginine biosynthesis were obtained. Neither recombinant showed linkage to the arg cluster, to each other, nor to other arginine biosynthetic functions by cross-complementation. Genes argF and argH were not linked to other arginine biosynthetic genes by cross-complementation analysis. Restriction enzyme patterns of recombinant plasmids fell into five groups. Group I contained the arg(ABCD) complementing locus. Group II and Group III were the two genetic loci corresponding to the argA and argE complementing genes. Group II contains the hipO gene encoding N-benzoylglycine-amino-acid amidohydrolase, also known as hippurate hydrolase. Group III contains the hipO homolog of C. jejuni. Group IV represents the argF gene. GroupV is the argH gene. Functional complementation of mutations in the first four steps of the arginine biosynthetic pathway was obtained on recombinant plasmid pARGC2. The predicted order of gene complementation was argCargA(argBargD). The sequence of the insert in plasmid pARGC2 revealed direct homologs for argC, argB, and argD. However, sequence analysis of the gene complementing ArgA function in two separate E. coli argA mutants determined that the C. jejuni gene was not a canonical argA gene. The gene complementing the argA defect, which we call argO, showed limited homology to the streptothricin acetyltransferase gene (sat) of Escherichia coli. The flanking open reading frames in pARGC2 showed no homologies to arginine biosynthetic genes. The structure of the argCOBD gene arrangement is discussed with reference to the presence and location of other arginine biosynthetic genes on the genome of C. jejuni and other bacterial organisms.Key words: arginine synthesis, Campylobacter jejuni, arginine biosynthetic genes, gene sequence, gene arrangement.