Harnessing the Potential of the Escherichia coli RpoS Phenotype via an Inducible Small RNA Regulatory Platform

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Carter, Karen
Bentley, William E
Recent recognition of the pervasiveness of non-coding RNAs, in both prokaryotic and eukaryotic systems, has prompted metabolic engineers to reevaluate the role of RNAs in a traditionally protein dominated realm. More specifically, bacterial trans-encoded sRNAs have been implicated in the regulation of genes in several critical pathways from quorum sensing to stress responses. The task of responding to stressful conditions, as well as stationary phase, in a comprehensive manner falls to the <italic>Escherichia</italic> coli global stress regulator, RpoS. Genes transcribed by RpoS are involved in motility, biofilm formation and nutrient limitations. One of the challenges modulating RpoS control is its polymorphic nature. We think this can be addressed using an inducible sRNA regulatory platform. Recent studies have confirmed RpoS to be post-transcriptionally regulated by at least four sRNAs: three activators, DsrA, RprA and ArcZ, and one repressor OxyS. Each of these senses different stress conditions, allowing RpoS synthesis to increase or decrease in response to various stressors. This work investigates the potential of a genetically engineered interchangeable small RNA based gene regulation platform as a switch to affect the expression profiles and metabolic behavior of RpoS. RprA and OxyS were put under the control of an arabinose inducible promoter to test the ability to increase/decrease RpoS protein levels and subsequent changes in RpoS-dependent genes. We then assessed gene expression and phenotypic changes using RT-PCR, Western blotting, microarray and motility and biofilm assays. Positive modulation of RpoS using the pRprA platform resulted in a 2-fold decrease in motility in Top10 cells. This difference in motility improved biofilm formation levels up to 12-fold when compared to direct overexpression of RpoS protein. The positive effect of biofilm formation was further supported by the upregulation of other genes essential for biofilms. Conversely, negative modulation of RpoS using the pOxyS platform resulted in an increase in the transcription of the motility gene,<italic> flhD</italic>. Both systems were capable of positively and negatively regulating bacterial RpoS protective genes. The ability to deliberately and purposefully control RpoS protective genes, in conjunction with motility and biofilm formation, can potentially have broad impact on biotechnology applications.