Expression and Purification of Glutamine Synthetase Cloned from Bacteroides fragilis

State Vaccine Institute, Alexandra Road, Pinelands, 7405, South Africa Department of Microbiology, University of Cape Town, Rondebosch 7700, South Africa ABSTRACT SUMMARY: A glutamine synthetase (GS) gene, glnA , from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH 4 ) 2 SO 4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B. fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli , but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified: it had an apparent subunit M r of approximately 75000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.