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Title:Molecular analysis of small RNA and small protein regulation of Escherichia coli stress responses
Author(s):Lloyd, Chelsea Royal
Director of Research:Vanderpool, Carin K.
Doctoral Committee Chair(s):Vanderpool, Carin K.
Doctoral Committee Member(s):Cronan, John E.; Metcalf, William W.; Orlean, Peter A.B.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
small protein
glucose-phosphate stress
Abstract:Small RNA (sRNA) regulators control gene expression throughout all domains of life. In bacteria, they typically affect virulence, metabolism, and stress response genes posttranscriptionally through imperfect antisense pairing with their mRNAs. While most sRNAs are non-coding, a small number act as mRNAs themselves by encoding functional proteins. This study examines the regulatory and physiological effects of both a non-coding sRNA, DicF, and the protein product of a dual-function sRNA, SgrS in Eschericha coli. The sRNA SgrS encodes the small 43-amino acid protein SgrT. Both molecules are expressed during glucose-phosphate stress - a bacteriostatic condition in which phosphosugars accumulate in the cell either because of mutations in glycolysis or because of the transport of non-metabolizable glucose analogs such as αMG or 2DG. While both SgrT and SgrS base pairing can independently mitigate glucose-phosphate stress, they do so through distinct mechanisms. SgrS base pairing destabilizes the mRNA of the respective major and minor glucose transporters PtsG and ManXYZ, thereby inhibiting synthesis of additional glucose permeases and restricting further influx of non-metabolizable sugars. In this study we demonstrate that SgrT acts to specifically inhibit the transport activity of preexisting PtsG transporters, but does not affect ManXYZ. We reveal that by targeting PtsG transport activity SgrT not only prevents influx of non-metabolizable αMG, but also overrides inducer exclusion allowing alternative carbon sources to be transported and metabolized during stress. We also uncover the regions of PtsG that are required for SgrT regulation. Although the precise nature of glucose-phosphate stress is not well understood, this work establishes that sugar phosphates are not inherently toxic, but most likely inhibit growth by depleting glycolytic intermediates, as these are expended to transport sugars and not are replenished due to a blockage in glycolysis. Sugar phosphate accumulation is, however, problematic and cells cope by effluxing excess sugars out of the cell. Phosphosugar efflux must be preceded by dephosphorylation and previous studies found that the phosphatase YigL is posttranscriptionally stabilized by SgrS under stress to promote this process. However, we still have yet to identify the efflux pump through which these sugars are flushed out of the cell. Here we provide evidence that the multidrug efflux channel TolC may be involved as tolC mutants exhibit impaired αMG efflux and more impaired growth when combined with an sgrS mutation. While the benefits of SgrS and SgrT to E. coli physiology are clear, the role of the sRNA DicF remains elusive. DicF is produced from the cryptic prophage Qin and while we have yet to identify the conditions under which it is naturally produced, ectopic expression of DicF leads to growth inhibition and filamentation. Previous work has identified a few DicF targets including ftsZ, which causes filamentation, no single target can be attributed to the growth defect we observe. Here we use a combination of approaches to uncover the novel targets ahpC and mdfA. While mutating ahpC has no effect on growth, mdfA (which encodes a proton/sodium/potassium antiporter) mutants partially rescue cell growth. Additionally, under alkaline conditions mdfA mutants expressing DicF are able to outcompete wild type cells. While there are more targets to uncover that contribute to growth inhibition and we have more to learn about DicF regulation and why its maintenance is beneficial, this study provides new insights into E. coli physiology during DicF expression and highlights the challenges associated with characterizing sRNA targetomes.
Issue Date:2018-07-10
Rights Information:Copyright 2018 Chelsea Lloyd
Date Available in IDEALS:2018-09-27
Date Deposited:2018-08

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