Energetics of bacterial growth: balance of anabolic and catabolic reactions.

Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications.

RyaA is a small non-coding RNA in Escherichia coli that was identified by its ability to bind tightly to the RNA chaperone Hfq. This study reports the role of RyaA in mediating the cellular response to glucose-specific phosphoenolypyruvate phosphotransferase system (PTS)-dependent phosphosugar stress. Aiba and co-workers have shown that a block in the metabolism of glucose 6-phosphate causes transient growth inhibition and post-transcriptional regulation of ptsG, encoding the glucose-specific PTS transporter. We found that RyaA synthesis was induced by a non-metabolizable glucose phosphate analogue and was necessary for relief of the toxicity of glucose phosphate stress. Expression of RyaA was sufficient to cause a rapid loss of ptsG mRNA, probably reflecting degradation of the message mediated by RyaA:ptsG pairing. The ryaA gene was renamed sgrS, for sugar transport-related sRNA. Expression of sgrS is regulated by a novel transcriptional activator, SgrR (formerly YabN), which has a putative DNA-binding domain and a solute-binding domain similar to those found in certain transport proteins. Our results suggest that under conditions of glucose phosphate accumulation, SgrR activates SgrS synthesis, causing degradation of ptsG mRNA. Decreased ptsG mRNA results in decreased production of glucose transport machinery, thus limiting further accumulation of glucose phosphate.

Involvement of a novel transcriptional activator and small RNA in post‐transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system

SgrS is a 227-nt small RNA that is expressed in Escherichia coli during glucose-phosphate stress, a condition associated with intracellular accumulation of glucose-6-phosphate caused by disruption of glycolytic flux. Under stress conditions, SgrS negatively regulates translation and stability of the ptsG mRNA, encoding the major glucose transporter, by means of a base pairing-dependent mechanism requiring the RNA chaperone Hfq. SgrS activity mitigates the effects of glucose-phosphate stress, and the present study has elucidated a function of SgrS that is proposed to contribute to the stress response. The 5' end of SgrS, upstream of the nucleotides involved in base pairing with the ptsG mRNA, contains a 43-aa ORF, sgrT, that is conserved in most species that contain SgrS-like small RNAs. The sgrT gene is translated in E. coli under conditions of glucose-phosphate stress. Analysis of alleles that separate the base pairing function of SgrS from the sgrT coding sequence revealed that either of these functions alone are sufficient for previously characterized SgrS phenotypes. SgrS-dependent down-regulation of ptsG mRNA stability does not require SgrT and SgrT by itself has no effect on ptsG mRNA stability. Cells expressing sgrT alone had a defect in glucose uptake even though they had nearly wild-type levels of PtsG (IICB(Glc)). Together, these data suggest that SgrS represents a previously unrecognized paradigm for small RNA (sRNA) regulators as a bifunctional RNA that encodes physiologically redundant but mechanistically distinct functions contributing to the same stress response.

https://www.pnas.org/content/pnas/104/51/20454.full.pdf

A dual function for a bacterial small RNA: SgrS performs base pairing-dependent regulation and encodes a functional polypeptide

Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis.

Methylglyoxal production in bacteria: suicide or survival?

The Escherichia coli uhpT gene encodes an active transport system for sugar phosphates. When the uhpT gene was carried on a multicopy plasmid, amplified levels of transport activity occurred, and growth of these strains was inhibited upon the addition of various sugar phosphates. Two different mechanisms for this growth inhibition were distinguished. Exposure to glucose-6-phosphate, fructose-6-phosphate or mannose-6-phosphate, which enter directly into the glycolytic pathway, resulted in cessation of growth and substantial loss of viability. Cell killing was correlated with the production of the toxic metabolite, methylglyoxal. In contrast, addition of 2-deoxyglucose-6-phosphate, galactose-6-phosphate, glucosamine-6-phosphate or arabinose-5-phosphate, which do not directly enter the glycolytic pathway, resulted in growth inhibition without engendering methylglyoxal production or cell death. Inhibition of growth could result from excessive accumulation of organophosphates in the cell or depletion of inorganic phosphate pools as a result of the sugar-P/Pi exchange process catalysed by UhpT. The phosphate-dependent uptake of glycerol-3-phosphate by the GlpT antiporter was strongly inhibited under conditions of elevated sugar-phosphate transport. There are thus two separate toxic effects of elevated sugar-phosphate transport, one of which was lethal and related to increased flux through glycolysis. It is likely that the control of uhpT transcription by catabolite repression exists to limit the level of UhpT transport activity and thereby prevent the toxic events that result from elevated uptake of its substrates.

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G. Patrick Murphy

Papers

Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli