Cloning of an EF-P homologue from Bacteroides fragilis that increases B. fragilis glutamine synthetase activity in Escherichia coli.
Investigations of possible regulators of Bacteroides fragilis glutamine synthetase (GS) activity were done in Escherichia coli using a compatible dual-plasmid system. The B. fragilis glnA gene, together with upstream and downstream flanking regions, was cloned onto the low copy number plasmid pACYC184 and expressed in the E. coli glnA ntrB ntrC deletion strain, YMC11. GS activity was monitored following co-transformation with a B. fragilis genomic library carried on the compatible plasmid pEcoR251. A gene was cloned that caused a twofold increase in B. fragilis GS activity but did not affect the activity of the E. coli GS enzyme or the B. fragilis sucrase (ScrL). Deletion of the B. fragilis glnA downstream region decreased basal levels of GS activity, but did not affect the ability of the cloned gene to increase the B. fragilis GS activity. Reporter gene analysis, using the B. fragilis glnA promoter region fused to the promoterless Clostridium acetobutylicum endoglucanase gene, showed no increase in reporter gene activity. This demonstrated that the increase in GS activity was not regulated at the transcriptional level, and that the cloned gene product was not affecting the copy number of the plasmid in trans. Sequence data indicated that the cloned gene had good amino acid identity to a range of elongation factor P (EF-P) proteins, the highest being 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 for EF-P in enhancing translation of the B. fragilis glnA mRNA is proposed.
Abratt
V R
VR
Department of Microbiology, University of Cape Town, Rondebosch, South Africa. val@molbiol.uct.ac.za
Mbewe
M
M
Woods
D R
DR
D000595:Amino Acid Sequence; D001441:Bacteroides fragilis / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004269:DNA, Bacterial; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; C020746:factor EF-P; D005974:Glutamate-Ammonia Ligase