Beyond Nitrogen Limitation – Novel Mechanisms Regulating Glutamine Synthetase Expression in Escherichia coli and a Possible Alternative Pathway of Glutamine Synthesis

dc.contributor.advisorReitzer, Lawrence J.
dc.contributor.advisorConlon, Ronan
dc.contributor.committeeMemberPalmer, Kelli
dc.contributor.committeeMemberSpiro, Stephen
dc.contributor.committeeMemberKim, Tae Hoon
dc.creatorUrs, Karthik
dc.date.accessioned2023-10-11T14:21:38Z
dc.date.available2023-10-11T14:21:38Z
dc.date.created2023-05
dc.date.issuedMay 2023
dc.date.submittedMay 2023
dc.date.updated2023-10-11T14:21:43Z
dc.description.abstractThe expression of glnA (ammonia-assimilating glutamine synthetase) is high for uropathogenic E. coli grown in urine. Because glnA is part of an operon that codes for regulators of the nitrogen-regulated (Ntr) response, high glnA expression has been interpreted to suggest nitrogen limitation, which is unexpected because of the high urinary ammonia concentration which should suppress glnA expression. We present evidence that glnA expression does not result from nitrogen limitation. First, in the presence of ammonia, urea induced expression of glnA from the cAMP receptor protein (Crp)- dependent glnAp1 promoter, which circumvents control from the nitrogen-regulated glnAp2 promoter. This urea effect on glnA expression has not been previously described. Second, the most abundant amino acids in urine inhibited GS activity, based on reversal of the inhibition by glutamate and glutamine, and increased glnA expression. The relevance of these inhibitory amino acids in natural environments has not been previously demonstrated. Third, urea and the inhibitory amino acids did not induce other Ntr genes, i.e., high glnA expression can be independent of other Ntr genes. Finally, the urea- dependent induction did not result in GlnA synthesis because of a previously undescribed translational control. We conclude that glnA expression in urea-containing environments does not imply growth rate-limiting nitrogen restriction and is consistent with rapid growth of uropathogenic E. coli. ΔglnA mutants are glutamine auxotrophs, however, UTI89ΔglnA mutants, were unexpectedly able to grow in a synthetic urine medium. This phenotype was conditional and required the presence of both glutamate and ammonia, the substrates for glutamine synthetase. Additionally, overexpression of proA, which is part of the proline biosynthesis pathway, whose product catalyzes the formation of glutamate-5-semialdehyde, improved growth. In contrast, an increase in proC expression, which directs pyrroline-5-carboxylate, the cyclized form of glutamate-5-semialdehyde to proline, impaired growth. We describe a possible alternate route of glutamine production in these mutants involving the enzymes of proline synthesis and the substrates, glutamate and ammonia, via the intermediate glutamate-5-semialdehyde. The pathway may be facilitated by a putative secondary activity of the ProA enzyme – the reduction of a proposed imine intermediate to ultimately form glutamine. Members of the B2 clade of E. coli exhibit high glnA transcript levels in nitrogen-rich glucose-tryptone medium. This increased expression did not translate to increased protein production and enzyme activity as evidenced by low translation levels and glutamine synthetase activity in these strains. Transcriptomic analysis revealed an inverse correlation between phoB, a phosphate-dependent transcriptional regulator, and glnA expression. Consistent with this, overexpression of phoB, reduced glnA transcription levels. The effect was not a complete repression of glnA transcription. Additionally, translational expression appeared to be stabilized upon phoB overexpression. Our findings suggest a novel mechanism of glnA regulation at both the transcriptional and translational levels that involves PhoB operating either directly or indirectly, and possibly in combination with other unidentified factors.
dc.format.mimetypeapplication/pdf
dc.identifier.uri
dc.identifier.urihttps://hdl.handle.net/10735.1/9910
dc.language.isoEnglish
dc.subjectBiology, Microbiology
dc.subjectBiology, Molecular
dc.subjectBiology, Cell
dc.titleBeyond Nitrogen Limitation – Novel Mechanisms Regulating Glutamine Synthetase Expression in Escherichia coli and a Possible Alternative Pathway of Glutamine Synthesis
dc.typeThesis
dc.type.materialtext
thesis.degree.collegeSchool of Natural Sciences and Mathematics
thesis.degree.departmentBiology
thesis.degree.grantorThe University of Texas at Dallas
thesis.degree.nameDoctor of Philosophy

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