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Effects of Kasugamycin on the Translatome of Escherichia coli.

ABSTRACT: It is long known that Kasugamycin inhibits translation of canonical transcripts containing a 5'-UTR with a Shine Dalgarno (SD) motif, but not that of leaderless transcripts. To gain a global overview of the influence of Kasugamycin on translation efficiencies, the changes of the translatome of Escherichia coli induced by a 10 minutes Kasugamycin treatment were quantified. The effect of Kasugamycin differed widely, 102 transcripts were at least twofold more sensitive to Kasugamycin than average, and 137 transcripts were at least twofold more resistant, and there was a more than 100-fold difference between the most resistant and the most sensitive transcript. The 5'-ends of 19 transcripts were determined from treated and untreated cultures, but Kasugamycin resistance did neither correlate with the presence or absence of a SD motif, nor with differences in 5'-UTR lengths or GC content. RNA Structure Logos were generated for the 102 Kasugamycin-sensitive and for the 137 resistant transcripts. For both groups a short Shine Dalgarno (SD) motif was retrieved, but no specific motifs associated with resistance or sensitivity could be found. Notably, this was also true for the region -3 to -1 upstream of the start codon and the presence of an extended SD motif, which had been proposed to result in Kasugamycin resistance. Comparison of the translatome results with the database RegulonDB showed that the transcript with the highest resistance was leaderless, but no further leaderless transcripts were among the resistant transcripts. Unexpectedly, it was found that translational coupling might be a novel feature that is associated with Kasugamycin resistance. Taken together, Kasugamycin has a profound effect on translational efficiencies of E. coli transcripts, but the mechanism of action is different than previously described.

SUBMITTER: Lange C

PROVIDER: S-EPMC5230787 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Effects of Kasugamycin on the Translatome of Escherichia coli.

Lange Christian C Lehr Matthias M Zerulla Karolin K Ludwig Petra P Schweitzer Jens J Polen Tino T Wendisch Volker F VF Soppa Jörg J

PloS one 20170112 1

It is long known that Kasugamycin inhibits translation of canonical transcripts containing a 5'-UTR with a Shine Dalgarno (SD) motif, but not that of leaderless transcripts. To gain a global overview of the influence of Kasugamycin on translation efficiencies, the changes of the translatome of Escherichia coli induced by a 10 minutes Kasugamycin treatment were quantified. The effect of Kasugamycin differed widely, 102 transcripts were at least twofold more sensitive to Kasugamycin than average, ...[more]

PMID: 28081129

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Project description:BackgroundEscherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.ResultsThe glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression.We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes, among others, are consistent with these observations.ConclusionWhile the simplest model of CRP*-mediated gene expression assumes insensitivity to glucose (or cAMP), our results show that gene expression in the context of CRP* is very different from that of wild-type in the absence of glucose, and is influenced by the presence of glucose. Most of the transcription changes in response to CRP* expression are difficult to interpret in terms of possible systematic effects on metabolism. Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of crp* strains that extend beyond relief of catabolite repression.

Dataset Information

Effects of Kasugamycin on the Translatome of Escherichia coli.

Publications

Effects of Kasugamycin on the Translatome of Escherichia coli.

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