Comprehensive mutagenesis of thefimSpromoter regulatory switch reveals novel regulation of type 1 pili in uropathogenicEscherichia coli

Abstract

Type 1 pili (T1P) are major virulence factors for uropathogenic Escherichia coli (UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for the fim operon encoding T1P. Inversion of fimS is stochastic but may be biased by environmental conditions and other signals that ultimately converge at fimS itself. Previous studies of fimS sequences important for T1P phase variation have focused on laboratory-adapted E. coli strains and have been limited in the number of mutations or by alteration of the fimS genomic context. We surmounted these limitations by using saturating genomic mutagenesis of fimS coupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasing fimS inversion, our method also identifies a previously unknown pair of 5′ UTR inverted repeats that act by altering the relative fimA levels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems.urinary tract infection | fimS | type 1 pili | phase variation | saturating chromosomal mutagenesis U rinary tract infections (UTIs) are common infections that mostly affect women with an annual cost of billions of dollars (1). They are caused mainly by uropathogenic Escherichia coli (UPEC), which rely on several virulence factors to cause disease (2); type 1 pili (T1P) are perhaps the most important of these (3). T1P present a tip adhesin, FimH, that binds specifically to mannose, which is found on several glycosylated surface proteins, such as uroplakins and α1-and β3-integrins (4, 5), in the mammalian bladder. Binding to mannosylated proteins initiates a cascade of events including UPEC invasion of host tissues (2), formation of intracellular structures (6), and activation of the host immune response (7). The interplay among these events determines the outcome of infection (8), ranging from full clearance to chronic active cystitis. T1P are important for most of these UTI stages and demonstrate dynamic regulation of expression that can be seen directly in human infections and urine (9, 10).Thus, understanding T1P regulation is fundamental to understanding UTI. T1P have been well studied (11), mostly in laboratory-adapted E. coli strains. T1P are encoded by the fim operon; their expression is phase variable via inversion of fimS, a chromosomal DNA segment containing the fim promoter (12). This binary epigenetic switch results in bacteria switching between fimbriated and nonfimbriated states. Inversion is mediated by the FimB and FimE recombinases, whose genes are located immediately upstream of fimS (13) and are found in most E. coli strains (14). Inversion is influenced by numerous signals, such as temperatu...

Genome-wide detection of conservative site-specific recombination in bacteria

“…Additional cellular factors or cis -acting elements might also participate in the DNA inversion process as was recently demonstrated for the fim switch in uropathogenic E . coli [ 69 ].…”

Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria

“…Microarray-synthesized promoter libraries and measurement of expression from barcoded transcripts using RNA-Seq instead of flow cytometry can be used to allow multiple loci to be studied simultaneously ( 14 , 18 ). Landing pad technologies for chromosomal integration ( 58 – 60 ) should enable massively parallel reporter assays to be performed in chromosomes instead of on plasmids. Techniques that combine these assays with transcription start site readout ( 61 ) may provide additional resolution, further allowing the molecular regulators of overlapping RNAP binding sites to be deconvolved or the contributions from separate RNAP binding sites, like those observed on the dgoR promoter, to be better distinguished.…”

Comprehensive Identification of Fim-Mediated Inversions in Uropathogenic Escherichia coli with Structural Variation Detection Using Relative Entropy

“…Positive verification of a new inversion locus is relatively straightforward once the locus is known, and two recent studies have used whole-genome sequencing (with Illumina and PacBio data) to achieve accurate quantification of fimS inversion percentages under different conditions (42, 43). However, to truly establish the specificity of the fim recombinases, a strong negative predictive value is required when analyzing whole-genome sequencing data (alternatively, a low noise level).…”