Cloning, characterization, and nucleotide sequence analysis of the argH gene from Campylobacter jejuni TGH9011 encoding argininosuccinate lyase

Abstract

The complete structural gene for argininosuccinate lyase (argH) from Campylobacter jejuni TGH9011 has been cloned into Escherichia coli by complementation of an E. coli argH auxotrophic mutant. The gene has been subcloned for sequencing on a 4.1-kb DNA segment and localized by the complementing activity of deletion mutants. The complete DNA sequence of the C. jejuni argH gene was determined. The transcription start point for argH mRNA was determined by primer extension analysis and found to be within the coding sequence of the upstream gene, identified as the phosphoenolpyruvate carboxykinase gene (ppc). The argininosuccinate lyase and the phosphoenolpyruvate carboxykinase reading frames overlap by one base, the second example of this phenomenon in C. jejuni chromosomal genes. The enzyme has a deduced subunit molecular weight of 51,831. Recombinant plasmids containing the argH gene generate a 56-kDa protein and a 43-kDa protein in E. coli maxicells. An alternate translation initiation producing a polypeptide with a deduced molecular mass of 42 kDa may account for the smaller protein observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C. jejuni argH gene shows nucleotide homology to both yeast and human argininosuccinate lyase genes, and conserved amino acid domains are evident between the corresponding proteins.

Computational Design of Auxotrophy-Dependent Microbial Biosensors for Combinatorial Metabolic Engineering Experiments

“…Specifically, for 10 amino-acids, the predicted knockouts match those of the known auxotrophic strain. For 5 amino-acids the predicted knockouts differ from the ones described in the CGSC database, though further literature search revealed experimental evidence showing that all predicted knockouts also lead to the desired auxotrophy [40] , [41] , [42] . Simulating the knockouts that give rise to these 5 auxotrophic strains in CGSC showed that they indeed lead to substrate auxotrophy, though not to the desired ultra-auxotrophy – i.e.…”

Expression and characterization of Campylobacter jejuni benzoylglycine amidohydrolase (Hippuricase) gene in Escherichia coli

“…Two of the theoretical open reading frames overlap by 16 nucleotides (ORFU2 and ORFL1) on complementary strands. Overlapping genes have already been described for C. jejuni (7,10). The intergenic regions between the coding sequences are small, which reflects the size of the genome of C. jejuni (17).…”

Mechanisms for Intragenic Complementation at the Human Argininosuccinate Lyase Locus

“…The ability of plasmid-borne ASL mutants to complement an E. coli strain with its endogenous ASL gene deleted served as a simple in vivo assay for ASL activity. E. coli strain W3678 (∆galT, LAM-, IN(rrnD-rrNE)1, ∆argH, F-) was obtained from V. L. Chan (25) and lysogenized using Novagen's λDE3 Lysogenization Kit. The resulting ∆ASL cells transformed with various ASL-expressing plasmids were assayed on plates containing M9 minimal media (24) with and without arginine supplementation.…”