Osmosensitivity associated with insertions in argP (iciA) or glnE in glutamate synthase-deficient mutants of Escherichia coli.
An ampicillin enrichment strategy following transposon insertion mutagenesis was employed to obtain NaCl-sensitive mutants of a gltBD (glutamate synthase [GOGAT]-deficient) strain of Escherichia coli. It was reasoned that the gltBD mutation would sensitize the parental strain even to small perturbations affecting osmotolerance. Insertions conferring an osmosensitive phenotype were identified in the proU, argP (formerly iciA), and glnE genes encoding a glycine betaine/proline transporter, a LysR-type transcriptional regulator, and the adenylyltransferase for glutamine synthetase, respectively. The gltBD+ derivatives of the strains were not osmosensitive. The argP mutation, but not the glnE mutation, was associated with reduced glutamate dehydrogenase activity and a concomitant NH4+ assimilation defect in the gltBD strain. Supplementation of the medium with lysine or a lysine-containing dipeptide phenocopied the argP null mutation for both osmosensitivity and NH4+ assimilation deficiency in a gltBD background, and a dominant gain-of-function mutation in argP was associated with suppression of these lysine inhibitory effects. Osmosensitivity in the gltBD strains, elicited either by lysine supplementation or by introduction of the argP or glnE mutations (but not proU mutations), was also correlated with a reduction in cytoplasmic glutamate pools in cultures grown at elevated osmolarity. We propose that an inability to accumulate intracellular glutamate at high osmolarity underlies the osmosensitive phenotype of both the argP gltBD and glnE gltBD mutants, the former because of a reduction in the capacity for NH4+ assimilation into glutamate and the latter because of increased channeling of glutamate into glutamine.
Nandineni
Madhusudan R
MR
Centre for Cellular and Molecular Biology, Hyderabad, India.
Laishram
Rakesh S
RS
Gowrishankar
J
J
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000502:physiology*; D005970:Glutamate Synthase / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism; D008239:Lysine / Q000494:pharmacology; D009713:Nucleotidyltransferases / Q000502:physiology*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology*
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000644:Quaternary Ammonium Compounds; C068863:argP protein, E coli; D018698:Glutamic Acid; D012965:Sodium Chloride; D005970:Glutamate Synthase; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; D008239:Lysine