Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously we showed that EHEC produces long polar fimbriae (LPF) and that maximum expression is observed during the exponential phase of growth at 37°C and pH 6.5. In this study, we analyzed the roles of several regulators in the expression of LPF using the β-galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an antisilencer of LPF expression. Interestingly, deletion of the hns and ler genes in EHEC caused constitutive expression of the fusion reporter protein. Semiquantitative reverse transcription (RT)-PCR was also used to analyze LPF expression in the EHEC ler or hns mutant strain. The hns mutant exhibited an increase in lpf mRNA expression, while expression in the ler mutant was decreased, compared to that in the wild-type strain. Using primer extension analysis, we identified two potential transcriptional start sites within the regulatory region of lpf and located consensus hexamers of −10 (CAAGAT) and −35 (TTCAAA), which are commonly found in σ ⁷⁰ -dependent promoters. Further, we determined whether H-NS and Ler interact directly with the lpf promoter region by using purified His-tagged proteins and electrophoretic mobility shift assays. Our data are the first to show direct binding interactions between the H-NS and Ler proteins within the regulatory sequence of the lpf operon. Based on the electrophoretic mobility shift assay, RT-PCR, primer extension, and β-galactosidase assay results, we concluded that the E. coli O157:H7 lpf operon possesses a promoter dependent on σ ⁷⁰ , that H-NS binds to the regulatory sequence of lpfA and “silences” the transcription of lpf , and that Ler binds to the regulatory sequence and inhibits the action of the H-NS protein.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Gram-positive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface.

Background/Purpose: Uropathogenic E. coli (UPEC) is the main cause of urinary tract infection (UTI) and it is known that pregnant women have a higher risk for UTI. UPEC has a variety of virulence and antibiotic resistance factors that facilitate its pathogenic success and it is crucial to know which are the susceptibility patterns, Extended-Spectrum-β-Lactamase (ESBL) production, virulence genes, pathogenicity islands (PAI), phylogenetic groups and serotypes among strains isolated from pregnant and non-pregnant women. Methods: One hundred fifty UPEC strains were isolated from pregnant and non-pregnant women from two different Mexican states (Sonora and Puebla). Strains were analyzed using the Kirby-Bauer method for the determination of antibiotic susceptibility and ESBL. Virulence genes, PAIs and phylogenetic groups were determined using a multiplex PCR. Strains were serotyped by an agglutination assay. Blood agar and CAS agar were used for phenotypic assays. Results: 92.7% of UPEC strains showed multidrug-resistant (MDR), 6.7% extremely-resistant (XDR) and 0.6% pandrug-resistant (PDR). The highest resistance was determined to be for β-lactam antibiotics (> 72% in both states) and 44.5% of the UPEC strains were ESBL⁺. The predominant virulence genes found were fimH (100%), iucD (85%) and iha (60%). The strains isolated from pregnant women from Puebla presented a large percentage of genes associated with upper urinary tract infections. PAIs were found in 51% and 68% of the strains from Sonora and Puebla, respectively. All the strains were siderophores producers and 41.5% produced hemolysis. The serotypes found were diverse and belonged to phylogroups A, B2 and C. Conclusion: The UPEC strains from this study are MDR with tendency to XDR or PDR, they can cause upper UTIs and are serotypically and phylogenetically diverse, which supports the need to develop new strategies for UTI treatment in pregnant and non-pregnant Mexican women.

Long polar fimbriae 1 (Lpf1) of Escherichia coli O157:H7 is a tightly regulated adhesin, with H-NS silencing the transcriptional expression of the lpf1 operon while Ler (locus of enterocyte effacement-encoded regulator) acts as an antisilencer. We mapped the minimal regulatory region of lpf1 required for H-NS- and Ler-mediated regulation and found that it is 79% AT rich. Three putative sites for H-NS binding were identified. Two of them, named silencer regulatory sequence 1 (SRS1) and SRS2, are located on a region that covers both of the lpf1 promoters (P1 and P2). The third putative H-NS binding site is located within the lpfA1 gene in a region extending from +258 bp to +545 bp downstream of ATG; however, this site does not seem to play a role in lpfA1 regulation under the conditions tested in this work. Ler was also found to interact with Ler binding sites (LBSs). Ler binding site 1 (LBS1) and LBS2 are located upstream of the two promoters. LBS1 overlaps SRS1, while LBS3 overlaps the P1 promoter and SRS2. Based on the experimental data, we propose that H-NS silences lpf1 expression by binding to both of the SRSs on the promoter region, forming an SRS-H-NS complex that prevents RNA polymerase-mediated transcription. A model of the regulation of the lpfA1 operon of E. coli O157:H7 by H-NS and Ler is discussed.

The genus Klebsiella belongs to Enterobacteriaceae family, and currently is considered to be non-motile and non-flagellated. In the present work, 25 Klebsiella strains isolated from nosocomial infections were assessed for motility under different growth conditions. One Klebsiella isolate, KpBuap021 demonstrated a swim-like motility phenotype. The K. pneumoniae genotype was confirmed by 16S rRNA and rpoB gene sequence analysis. MLST analysis also revealed that the KpBuap021 strain places it in the ST345 sequence type, and belongs to the phylogenetic Kpl Group. Transmission Electron Microscopy and the Ryu staining technique revealed that KpBuap021 expresses polar flagella. Finally, the presence of fliC, fliA, and flgH genes in this K. pneumoniae strain was confirmed. This report presents the first evidence for flagella-mediated motility in a K. pneumoniae clinical isolate, and represents an important finding related to its evolution and pathogenic potential.