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 ␤-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 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 ␤-galactosidase assay results, we concluded that the E. coli O157:H7 lpf operon possesses a promoter dependent on 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.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a serious food-borne pathogen causing diarrhea that is often bloody and accompanied by severe abdominal cramps and can result in a life-threatening condition known as the hemolyticuremic syndrome (reviewed in reference 33). The organism can be found living in the intestines of healthy cattle, and eating contaminated meat, especially ground beef, has resulted in multiple outbreaks worldwide (23). Recent E. coli O157:H7 outbreaks have drawn attention to food-borne illnesses, and though official sources were saying that the overall number of cases is on the decline in the United States, consumption of produce, particularly leafy vegetables, is becoming increasingly associated with human infections. This alternate source of infection has set up new challenges for the scientific community in trying to identify novel determinants and regulatory mechanisms implicated in the colonization, survival, and/or pathogenic processes (9,10,40).During the infectious process, EHEC adheres to the intestinal epithelium, where it produces Shiga toxins responsible for the hemorrhagic symptoms. Adhesion of E. coli O157:H7 t...

Summary Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion and horizontal gene transfer. However, many Gram-positive species, including C. difficile, also produce Type IV pili. Here, we identify the major subunit of the Type IV pili of C. difficile, PilA1, and describe multiple three-dimensional structures of PilA1, demonstrating the diversity found in three strains of C. difficile. We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative Type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the Type IVb pilins from Gram-negative bacteria that suggest that the Type IV pili of C. difficile are involved in microcolony formation.

Background: Type IV pili are non-covalently assembled appendages, characterized now in both Gram-negative and Grampositive bacteria. Results: Clostridium difficile produces Type IV pili containing PilJ, a pilin with a novel dual-pilin fold. Conclusion: Models suggest that the C-terminal pilin domain is exposed in pili, providing a unique interaction surface. Significance: The novel fold of PilJ suggests a new mode for Type IV pilus function.

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 βlactam 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 nonpregnant Mexican women.