Uropathogenic Escherichia coli (UPEC) are capable of occupying physiologically distinct intracellular and extracellular niches within the urinary tract. This feat requires the timely regulation of gene expression and small RNAs (sRNAs) are known to mediate such rapid adjustments in response to changing environmental cues. This study aimed to uncover sRNA-mediated gene regulation in the UPEC strain UTI89, during infection of bladder epithelial cells. Hfq is an RNA chaperone known to facilitate and stabilize sRNA and target mRNA interactions with bacterial cells. The co-immunoprecipitation and high throughput RNA sequencing of Hfq bound sRNAs performed in this study, revealed distinct sRNA profiles in UPEC in the extracellular and intracellular environments. Our findings emphasize the importance of studying regulatory sRNAs in a biologically relevant niche. This strategy also led to the discovery of a novel virulence-associated trans-acting sRNA—PapR. Deletion of papR was found to enhance adhesion of UTI89 to both bladder and kidney cell lines in a manner independent of type-1 fimbriae. We demonstrate PapR mediated posttranscriptional repression of the P-fimbriae phase regulator gene papI and postulate a role for such regulation in fimbrial cross-talk at the population level in UPEC. Our results further implicate the Leucine responsive protein (LRP) as a transcriptional activator regulating PapR expression. Our study reports, for the first time, a role for sRNAs in regulation of P-fimbriae phase variation and emphasizes the importance of studying pathogenesis-specific sRNAs within a relevant biological niche.

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BackgroundDuring infection of the urinary tract, uropathogenic Escherichia coli (UPEC) are exposed to different environments, such as human urine and the intracellular environments of bladder epithelial cells. Each environment elicits a distinct bacterial environment-specific transcriptional response. We combined differential fluorescence induction (DFI) with next-generation sequencing, collectively termed DFI-seq, to identify differentially expressed genes in UPEC strain UTI89 during growth in human urine and bladder cells.ResultsDFI-seq eliminates the need for iterative cell sorting of the bacterial library and yields a genome-wide view of gene expression. By analysing the gene expression of UPEC in human urine we found that genes involved in amino acid biosynthesis were upregulated. Deletion mutants lacking genes involved in arginine biosynthesis were outcompeted by the wild type during growth in human urine and inhibited in their ability to invade or proliferate in the J82 bladder epithelial cell line. Furthermore, DFI-seq was used to identify genes involved in invasion of J82 bladder epithelial cells. 56 genes were identified to be differentially expressed of which almost 60% encoded hypothetical proteins. One such gene UTI89_C5139, displayed increased adhesion and invasion of J82 cells when deleted from UPEC strain UTI89.ConclusionsWe demonstrate the usefulness of DFI-seq for identification of genes required for optimal growth of UPEC in human urine, as well as potential virulence genes upregulated during infection of bladder cell culture. DFI-seq holds potential for the study of bacterial gene expression in live-animal infection systems. By linking fitness genes, such as those genes involved in amino acid biosynthesis, to virulence, this study contributes to our understanding of UPEC pathophysiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-1008-4) contains supplementary material, which is available to authorized users.

The lac promoter is one of the most commonly used promoters for expression control of recombinant genes in E. coli. In the absence of galactosides, the lac promoter is repressed by its repressor protein LacI. Since the lac promoter is regulated by a repressor, overexpression of LacI is necessary for regulation when the promoter is introduced on a high-copy plasmid. For that purpose, a modified variant of LacI, a LVA-tagged LacI, was submitted to the Registry of Standard Biological Parts and has been used for more than 500 constructs since then. We have found, however, that natural LacI is superior to the LVA-tagged LacI as controller of expression.