Project description:The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of E. coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable. In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli. Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic E. coli can be modulated by deletion of PAIs.
Project description:For the uropathogenic Escherichia coli strain 536 (O6:K15:H31), the DNA sequences of three pathogenicity islands (PAIs) (PAI I(536) to PAI III(536)) and their flanking regions (about 270 kb) were determined to further characterize the virulence potential of this strain. PAI I(536) to PAI III(536) exhibit features typical of PAIs, such as (i) association with tRNA-encoding genes; (ii) G+C content differing from that of the host genome; (iii) flanking repeat structures; (iv) a mosaic-like structure comprising a multitude of functional, truncated, and nonfunctional putative open reading frames (ORFs) with known or unknown functions; and (v) the presence of many fragments of mobile genetic elements. PAI I(536) to PAI III(536) range between 68 and 102 kb in size. Although these islands contain several ORFs and known virulence determinants described for PAIs of other extraintestinal pathogenic E. coli (ExPEC) isolates, they also consist of as-yet-unidentified ORFs encoding putative virulence factors. The genetic structure of PAI IV(536), which represents the core element of the so-called high-pathogenicity island encoding a siderophore system initially identified in pathogenic yersiniae, was further characterized by sample sequencing. For the first time, multiple PAI sequences (PAI I(536) to PAI IV(536)) in uropathogenic E. coli were studied and their presence in several wild-type E. coli isolates was extensively investigated. The results obtained suggest that these PAIs or at least large fragments thereof are detectable in other pathogenic E. coli isolates. These results support our view that the acquisition of large DNA regions, such as PAIs, by horizontal gene transfer is an important factor for the evolution of bacterial pathogens.
Project description:The features of Mycoplasma in human organ such lung and urinary tract are enigmatic. Here, the role of M. hominis in regard to biofilm formation of uropathogenic Escherichia coli (UPEC) strain CFT073 was investigated. Although M. hominis were inferred to not impact on UPEC bacterial fitness including growth and productions of signaling molecules as autoinducer-2 (AI-2) and indole, we found that the presence of M. hominis dramatically decreased biofilm formation of UPEC CFT073 as well as slightly repressed attachment and cytotoxicity of that. Importantly, this activity was observed on UPEC strain specifically, not enterohemorrhagic E. coli (EHEC) strain that exists on intestine. Whole-transcriptome profiling and quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed PhoPQ system and anti-termination protein (encoded by ybcQ) participates on the reduction of biofilm formation by M. hominis (corroborated by qRT-PCR). Furthermore, collaborating with previous report that toxin-antitoxin (TA) system involved in biofilm formation, M. hominis increased on the transcriptions of toxin genes including hha (toxin gene in Hha-TomB TA system) and pasT (toxin part in PasT-PasI TA system). Hence, we propose that one possible role of M. hominis is to influence bacterial biofilm formation in urinary tract. Only fourteen genes were induced (2.5-fold) by the presence of M. hominis in Uropathogenic Escherichia coli (UPEC) biofilm cells. Among upregulated genes, ybcQ (encodes anti-termination protein Q homolog) and phoP/phoQ (encode DNA-binding response regulators in two-component regulatory system), were induced by the presence of M. hominis.
Project description:The sfa(I) determinant encoding the S-fimbrial adhesin of uropathogenic Escherichia coli strains was found to be located on a pathogenicity island of uropathogenic E. coli strain 536. This pathogenicity island, designated PAI III(536), is located at 5.6 min of the E. coli chromosome and covers a region of at least 37 kb between the tRNA locus thrW and yagU. As far as it has been determined, PAI III(536) also contains genes which code for components of a putative enterochelin siderophore system of E. coli and Salmonella spp. as well as for colicin V immunity. Several intact or nonfunctional mobility genes of bacteriophages and insertion sequence elements such as transposases and integrases are present on PAI III(536). The presence of known PAI III(536) sequences has been investigated in several wild-type E. coli isolates. The results demonstrate that the determinants of the members of the S-family of fimbrial adhesins may be located on a common pathogenicity island which, in E. coli strain 536, replaces a 40-kb DNA region which represents an E. coli K-12-specific genomic island.
Project description:Transcriptional analysis of UTI89 - uropathogenic E.coli (UPEC) strain grown in urine/Luria bertani medium culture in vitro as well as during three distinct phases of UPEC bladder infection: intracellular growth, filament formation and filament reversal. UTI89 was used to infect a bladder epithelial cell line cultured within a dynamic flow chamber system and harvested at particular stages of its pathogenecity cascade. Total RNA was processed and cy3 labeled for microarray analysis using Agilent custom Escherichia coli UTI89 arrays designed using E-Array.
Project description:Bacterial choline degradation in the human gut has been associated with cancer and heart disease. In addition, recent studies found that a bacterial microcompartment is involved in choline utilization by Proteus and Desulfovibrio species. However, many aspects of this process have not been fully defined. Here, we investigate choline degradation by the uropathogen Escherichia coli 536. Growth studies indicated E. coli 536 degrades choline primarily by fermentation. Electron microscopy indicated that a bacterial microcompartment was used for this process. Bioinformatic analyses suggested that the choline utilization (cut) gene cluster of E. coli 536 includes two operons, one containing three genes and a main operon of 13 genes. Regulatory studies indicate that the cutX gene encodes a positive transcriptional regulator required for induction of the main cut operon in response to choline supplementation. Each of the 16 genes in the cut cluster was individually deleted, and phenotypes were examined. The cutX, cutY, cutF, cutO, cutC, cutD, cutU, and cutV genes were required for choline degradation, but the remaining genes of the cut cluster were not essential under the conditions used. The reasons for these varied phenotypes are discussed.IMPORTANCE Here, we investigate choline degradation in E. coli 536. These studies provide a basis for understanding a new type of bacterial microcompartment and may provide deeper insight into the link between choline degradation in the human gut and cancer and heart disease. These are also the first studies of choline degradation in E. coli 536, an organism for which sophisticated genetic analysis methods are available. In addition, the cut gene cluster of E. coli 536 is located in pathogenicity island II (PAI-II536) and hence might contribute to pathogenesis.
Project description:While in transit within and between hosts, uropathogenic E. coli (UPEC) encounter multiple stresses, including substantial levels of nitric oxide and reactive nitrogen intermediates. Strains of UPEC become conditioned to high concentrations of acidified sodium nitrite (ASN), a model system used to generate nitrosative stress. We used microarrays to define the expression profile of UPEC that have been conditioned for growth in ASN.
Project description:We previously determined that loss of respiratory quinol oxidase cytochrome bd disrupts biofilm formation in uropathogenic Escherichia coli (UPEC). In this study, we extracted and interrogated the outer membrane and extracellular matrix of colony biofilms formed by UPEC isolate UTI89 and an isogenic mutant lacking cytochrome bd (∆cydAB).