CLONING OF AN EF-P HOMOLOG FROM BACTEROIDES-FRAGILIS THAT INCREASES BACTEROIDES-FRAGILIS GLUTAMINE-SYNTHETASE ACTIVITY IN ESCHERICHIA-COLI

Investigations of possible regulators of Bacteroides fragilis glutamin e synthetase (GS) activity were done in Escherichia coli using a compa tible dual-plasmid system. The B. fragilis glnA gene, together with up stream and downstream flanking regions, was cloned onto the low copy n umber plasmid pACYC184 and expressed in the E. coli glnA ntrB ntrC del etion strain, YMC11. GS activity was monitored following cotransformat ion with a B. fragilis genomic library carried on the compatible plasm id pEcoR251. A gene was cloned that caused a twofold increase in B. fr agilis GS activity but did not affect the activity of the E. coli GS e nzyme or the B. fragilis sucrase (ScrL). Deletion of the B. fragilis g lnA downstream region decreased basal levels of GS activity, but did n ot affect the ability of the cloned gene to increase the B. fragilis G S activity. Reporter gene analysis, using the B. fragilis glnA promote r region fused to the promoterless Clostridium acetobutylicum endogluc anase gene, showed no increase in reporter gene activity. This demonst rated that the increase in GS activity was not regulated at the transc riptional level, and that the cloned gene product was not affecting th e copy number of the plasmid in trans. Sequence data indicated that th e cloned gene had good amino acid identity to a range of elongation fa ctor P (EF-P) proteins, the highest beings to that of a Synechocystis sp (48%), and the least to Mycobacterium genitalium (27%). Amino acid identity to the E. coli EF-P was intermediate (37%). A possible role f or EF-P in enhancing translation of the B. fragilis glnA mRNA is propo sed.