The long polar fimbriae (lpf) operon and its flanking regions in bovine Escherichia coli O157:H43 and STEC O136:H12 strains

Long polar fimbriae (Lpf) are intestinal adhesins and important virulence factors of pathogenic Escherichia coli strains. We cloned and sequenced the lpf2-1 operon (lpf2ABCD) and its flanking regions of an intimin- and Shiga toxin-negative E. coli O157:H43 strain from bovine origin, and also sequenced the lpf2-1 operon of 6 additional atypical O157 bovine Escherichia coli strains of various serotypes Nucleotide sequence comparison of these lpf operons showed sequence conservation as they contain only four polymorphic nucleotide positions. Investigation of these O157 strains as well as 13 Escherichia coli Reference Collection (ECOR) strains carrying the lpf2-1 allele revealed high degree of sequence conservation in the lpf2 flanking regions. The lpf2-1 allele is also present in a bovine Shiga toxin-producing E. coli STEC O136:H12 strain and in vitro adherence assays revealed that the absence of lpf2-1 in this strain did not affect its host cell-binding properties. Our data indicate that lpf2 loci is highly conserved in E. coli isolates, but its role in adherence might be masked by other uncharacterized adhesins

Structure and Function Relationship of the Autotransport and Proteolytic Activity of EspP from Shiga Toxin-Producing Escherichia coli

BACKGROUND: The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPalpha, EspPbeta, EspPgamma, and EspPdelta), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown. METHODOLOGY/PRINCIPAL FINDINGS: The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the beta-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His(127), Asp(156) and Ser(263) in Domain 1 form the catalytic triad of EspP. CONCLUSIONS/SIGNIFICANCE: Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPbeta and EspPdelta

Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic Escherichia coli in vitro support the role of conjugation for horizontal transfer of genomic islands

<p>Abstract</p> <p>Background</p> <p>A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs) - including pathogenicity islands (PAIs) - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far.</p> <p>Results</p> <p>To study mobilisation of such large genomic regions in prototypic uropathogenic <it>E. coli </it>(UPEC) strain 536, PAI II₅₃₆was supplemented with the <it>mob</it>_RP4region, an origin of replication (<it>oriV</it>_<it>R6K</it>), an origin of transfer (<it>oriT</it>_<it>RP4</it>) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II₅₃₆construct occured from strain 536 into an <it>E. coli </it>K-12 recipient. In transconjugants, PAI II₅₃₆existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the <it>leuX </it>tRNA gene. This locus is the chromosomal integration site of PAI II₅₃₆in UPEC strain 536. From the <it>E. coli </it>K-12 recipient, the chromosomal PAI II₅₃₆construct as well as the CIs could be successfully remobilised and inserted into <it>leuX </it>in a PAI II₅₃₆deletion mutant of <it>E. coli </it>536.</p> <p>Conclusions</p> <p>Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.</p

Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae

Horizontal gene transfer is a key step in the evolution of Enterobacteriaceae. By acquiring virulence determinants of foreign origin, commensals can evolve into pathogens. In Enterobacteriaceae, horizontal transfer of these virulence determinants is largely dependent on transfer by plasmids, phages, genomic islands (GIs) and genomic modules (GMs). The High Pathogenicity Island (HPI) is a GI encoding virulence genes that can be transferred between different Enterobacteriaceae. We investigated the HPI because it was present in an Enterobacter hormaechei outbreak strain (EHOS). Genome sequence analysis showed that the EHOS contained an integration site for mobile elements and harbored two GIs and three putative GMs, including a new variant of the HPI (HPI-ICEEh1). We demonstrate, for the first time, that combinatorial transfers of GIs and GMs between Enterobacter cloacae complex isolates must have occurred. Furthermore, the excision and circularization of several combinations of the GIs and GMs was demonstrated. Because of its flexibility, the multiple integration site of mobile DNA can be considered an integration hotspot (IHS) that increases the genomic plasticity of the bacterium. Multiple combinatorial transfers of diverse combinations of the HPI and other genomic elements among Enterobacteriaceae may accelerate the generation of new pathogenic strains

Crenarchaeal Biofilm Formation under Extreme Conditions

Background: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments. Methodology: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells. Conclusion: The study gives first insights into formation and development of crenarchaeal biofilms in extrem

Phylogenetic Group –Associated Differences in Regulation of the Common Colonization Factor Mat Fimbria in Escherichia coli

Peer reviewe

Fast identification of <i>Escherichia coli</i> in urinary tract infections using a virulence gene based PCR approach in a novel thermal cycler

Uropathogenic Escherichia coli (UPEC) is the most common causal agent of urinary tract infections (UTIs) in humans. Currently, clinical detection methods take hours (dipsticks) to days (culturing methods), limiting rapid intervention. As an alternative, the use of molecular methods could improve speed and accuracy, but their applicability is complicated by high genomic variability within UPEC strains. Here, we describe a novel PCR-based method for the identification of E. coli in urine. Based on in silico screening of UPEC genomes, we selected three UPEC-specific genes predicted to be involved in pathogenesis (c3509, c3686 (yrbH) and chuA), and one E. coli-specific marker gene (uidA). We validated the method on 128 clinical (UTI) strains. Despite differential occurrences of these genes in uropathogenic E. coli, the method, when using multi-gene combinations, specifically detected the target organism across all samples. The lower detection limit, assessed with model UPEC strains, was approximately 104 CFU/ml. Additionally, the use of this method in a novel ultrafast PCR thermal cycler (Nextgen PCR) allowed a detection time from urine sampling to identification of only 52 min. This is the first study that uses such defined sets of marker genes for the detection of E. coli in UTIs. In addition, we are the first to demonstrate the potential of the Nextgen thermal cycler. Our E. coli identification method has the potential to be a rapid, reliable and inexpensive alternative for traditional methods

Complete Genome Sequences of Escherichia coli Strains 1303 and ECC-1470 Isolated from Bovine Mastitis

Escherichia coli is the leading causative agent of acute bovine mastitis. Here, we report the complete genome sequence of E. coli O70:H32 strain 1303, isolated from an acute case of bovine mastitis, and E. coli Ont:Hnt strain ECC-1470, isolated from a persistent infection