Detection of recombination among Salmonella enterica strains using the incongruence length difference test

Particular serovars of Salmonella enterica have emerged as significant foodborne pathogens in humans. At the chromosomal level, discrete regions in the Salmonella genome have been identified that are known to play important roles in the maintenance, survival, and virulence of S. enterica within the host. Interestingly, several of these loci appear to have been acquired by horizontal transfer of DNA among and between bacterial species. The profound importance of recombination in pathogen emergence is just now being realized, perhaps explaining the sudden interest in developing novel and facile ways for detecting putative horizontal transfer events in bacteria. The incongruence length difference (ILD) test offers one such means. ILD uses phylogeny to trace sequences that may have been acquired promiscuously by exchange of DNA during chromosome evolution. We show here that the ILD test readily detects recombinations that have taken place in several housekeeping genes in Salmonella as well as genes composing the type 1 pilin complex (14 min) and the inv-spa invasion gene complex (63 min). Moreover, the ILD test indicated that the mutS gene (64 min), whose product helps protect the bacterial genome from invasion by foreign DNA, appears to have undergone intragenic recombination within S. enterica subspecies I. ILD findings were supported using additional tests known to be independent of the ILD approach (e.g., split decomposition analysis and compatibility of sites). Taken together, these data affirm the application of the ILD test as one approach for identifying recombined sequences in the Salmonella chromosome. Furthermore, horizontally acquired sequences within mutS support a model whereby evolutionarily important recombinants of S. enterica are rescued from strains carrying defective mutS alleles via horizontal transfer.     

Spontaneous mutators of salmonella typhimurium LT2 generated by insertion of transposable elements.

Spontaneous mutators of Salmonella typhimurium LT2 were generated by inserting the transposable element Tn5 or Tn10 into the bacterial chromosome. Two mutators mapped at the position of the mutH and mutL loci of S. typhimurium, and two other mutators mapped at positions corresponding to the mutS and uvrD loci of Escherichia coli. A fifth mutator, mutB, did not map at a position corresponding to any of the known mutators of S. typhimurium or E. coli. The mutH,L,S and uvrD alleles increased the frequency of both spontaneous base substitution and frameshift mutations, whereas the mutB allele increased the frequency only of spontaneous base substitution mutations. The increased frequency of base substitution mutations was recA+ independent in the mutH, mutL, and uvrD strains and partially recA+ independent in the mutS strain. The uvrD mutation decreased the resistance of the cells to killing by ultraviolet irradiation. The mutH,L,S and uvrD strains showed an increased sensitivity to mutagenesis by the alkylating agents methyl methane sulfonate and ethyl methane sulfonate, but not to mutagenesis by 4-nitroquinoline-1-oxide.     

Escherichia coli molecular phylogeny using the incongruence length difference test

Molecular phylogeny of the species Escherichia coli using the E. coli reference (ECOR) collection strains has been hampered by (1) the absence of rooting in the commonly used phenogram obtained from multilocus enzyme electrophoresis (MLEE) data and (2) the existence of recombination events between strains that scramble phylogenetic trees reconstructed from the nucleotide sequences of genes. We attempted to determine the phylogeny for E. coli based on the ECOR strain data by extracting from GenBank the nucleotide sequences of 11 chromosomal structural and 2 plasmid genes for which the Salmonella enterica homologous gene sequences were available. For each of the 13 DNA data sets studied, incongruence with a nonnucleotide whole-genome data set including MLEE, random amplified polymorphic DNA, and rrn restriction fragment length polymorphism data was measured using the incongruence length difference (ILD) test of Farris et al. As previously reported, the incongruence observed between the gnd and plasmid gene data and the whole-genome data was multiple, indicating numerous horizontal transfer and/or recombination events. In five cases, the incongruence detected by the ILD test was punctual, and the donor group was identified. Congruence was not rejected for the remaining data sets. The strains responsible for incongruences with the whole-genome data set were removed, leading to a "prior-agreement" approach, i.e., the determination of a phylogeny for E. coli based on several genes, excluding (1) the genes with multiple incongruences with the whole genome data, (2) the strains responsible for punctual incongruences, and (3) the genes incongruent with each other. The obtained phylogeny shows that the most basal group of E. coli strains is the B2 group rather than the A group, as generally thought. The D group then emerges as the sister group of the rest. Finally, the A and B1 groups are sister groups. Interestingly, the most primitive taxon within E. coli in terms of branching pattern, i.e., the B2 group, includes highly virulent extraintestinal strains with derived characters (extraintestinal virulence determinants) occurring on its own branch.      

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli . We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA . These shuA -positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA , so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae . The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA . The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA -positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae , indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae , and it is located in the same relative position on the chromosome.     

Functions of the Mismatch Repair Gene mutS from Acinetobacter sp. Strain ADP1

The genus Acinetobacter encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges. Acinetobacter sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned mutS from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of mutS resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of mutS also abolished the marker-specific variations in transforming efficiency seen in mutS(+) recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual Acinetobacter strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the Acinetobacter amino acid sequences.     

Evolutionary implications of the frequent horizontal transfer of mismatch repair genes

Mutation and subsequent recombination events create genetic diversity, which is subjected to natural selection. Bacterial mismatch repair (MMR) deficient mutants, exhibiting high mutation and homologous recombination rates, are frequently found in natural populations. Therefore, we have explored the possibility that MMR deficiency emerging in nature has left some "imprint" in the sequence of bacterial genomes. Comparative molecular phylogeny of MMR genes from natural Escherichia coli isolates shows that, compared to housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages. This apparent horizontal gene transfer correlates with hyperrecombination phenotype of MMR-deficient mutators. The sequence mosaicism of MMR genes may be a hallmark of a mechanism of adaptive evolution that involves modulation of mutation and recombination rates by recurrent losses and reacquisitions of MMR gene functions.     

Mutagenesis and repair in Bacillus anthracis: the effect of mutators

We have generated mutator strains of Bacillus anthracis Sterne by using directed gene knockouts to investigate the effect of deleting genes involved in mismatch repair, oxidative repair, and maintaining triphosphate pools. The single-knockout strains are deleted for mutS, mutY, mutM, or ndk. We also made double-knockout strains that are mutS ndk or mutY mutM. We have measured the levels of mutations in the rpoB gene that lead to the Rif(r) phenotype and have examined the mutational specificity. In addition, we examined the mutational specificity of two mutagens, 5-azacytidine and N-methyl-N'-nitro-N-nitroso-guanidine. The mutY and mutM single knockouts are weak mutators by themselves, but the combination of mutY mutM results in very high mutation rates, all due to G:C --> T:A transversions. The situation parallels that seen in Escherichia coli. Also, mutS knockouts are strong mutators and even stronger in the presence of a deletion of ndk. The number of sites in rpoB that can result in the Rif(r) phenotype by single-base substitution is more limited than in certain other bacteria, such as E. coli and Deinococcus radiodurans, although the average mutation rate per mutational site is roughly comparable. Hotspots at sites with virtually identical surrounding sequences are organism specific.     

Evolutionary genetics of the proline permease gene (putP) and the control region of the proline utilization operon in populations of Salmonella and Escherichia coli.

Virtually complete sequences (1,467 bp) of the proline permease gene (putP) and complete sequences (416 to 422 bp) of the control region of the proline utilization operon were determined for 16 strains of Salmonella, representing all eight subspecies, and 13 strains of Escherichia coli recovered from natural populations. Strains of Salmonella and E. coli differed, on average, at 16.3% of putP nucleotide sites and 17.5% of control region sites; the average difference between strains was much larger for Salmonella strains (4.6% of putP sites and 3.4% of control region sites) than for E. coli (2.4 and 0.9%, respectively). There was no difference in the distribution of polymorphic amino acid positions between the membrane-spanning and loop regions of the permease molecule, and rates of synonymous nucleotide substitution were virtually the same for the two domains. Statistical analysis yielded evidence of three probable cases of intragenic recombination, including the acquisition of a large segment of putP by strains of Salmonella subspecies VII from an unidentified source, the exchange of a 21-bp segment between two strains of E. coli, and the acquisition by one strain of E. coli of a cluster of 14 unique polymorphic control region sites from an unknown donor. An evolutionary tree for the putP and control region sequences was generally concordant with a tree for the gapA gene and a tree based on multilocus enzyme electrophoresis, thus providing evidence that for neither gene nor for enzyme genes in general has recombination occurred at rates sufficiently high or over regions sufficiently large to completely obscure phylogenetic relationships dependent on mutational divergence. It is suggested that the recombination rate varies among genes in relation to functional type, being highest for genes encoding cell surface and other proteins for which there is an adaptive advantage in structural diversity.     

Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci.     

Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site.

Horizontal transfer of genes responsible for pollutant biodegradation may play a key role in the evolution of bacterial populations and the adaptation of microbial communities to environmental contaminants. However, field evidence for horizontal gene transfer between microorganisms has traditionally been very difficult to obtain. In this study, the sequences of the 16S rRNA and naphthalene dioxygenase iron-sulfur protein (nahAc) genes of nine naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site, as well as a naphthalene-degrading bacterium from a contaminated site in Washington state and two archetypal naphthalene-degrading strains, were compared. Seven strains from the study site had a single nahAc allele, whereas the 16S rRNA gene sequences of the strains differed by as much as 7.9%. No nahAc alleles from the site were identical to those of the archetypal strains, although the predominant allele was closely related to that of Pseudomonas putida NCIB 9816-4, isolated in the British Isles. However, one site-derived nahAc allele was identical to that of the Washington state strain. Lack of phylogenetic congruence of the nahAc and 16S rRNA genes indicates that relatively recent in situ horizontal transfer of the nahAc gene has occurred, possibly as a direct or indirect consequence of pollutant contamination. Alkaline lysis plasmid preparations and pulsed-field gel electrophoresis have revealed the presence of plasmids ranging in size from 70 to 88 kb in all site isolates. Southern hybridizations with a 407-bp nahAc probe have suggested that the nahAc gene is plasmid borne in all the site isolates but one, a strain isolated from subsurface sediment 400 m upstream from the source of the other site isolates. In this strain and in the naphthalene-degrading strain from Washington state, nahAc appears to be chromosomally located. In addition, one site isolate may carry nahAc on both chromosome and plasmid. Within the group of bacteria with identical nahAc sequences the Southern hybridizations showed that the gene was distributed between plasmids of different sizes and a chromosome. This suggests that plasmid modification after transfer may have been effected by transposons. Horizontal transfer of catabolic genes may play a significant role in the acclimation of microbial communities to pollutants.     

Inactivation of mismatch repair increases the diversity of Vibrio parahaemolyticus

Inactivation of mismatch repair (MMR) has been shown to increase the accumulation of spontaneous mutations and frequency of recombination for diverse pathogenic bacteria. Currently, little is known regarding the role of mutator phenotypes for the diversification of natural populations of opportunistic human pathogens in marine environments. In this study, a higher frequency of mutators was detected among V. parahaemolyticus strains obtained from environmental sources compared with clinical sources. Inactivation of the MMR gene mutS caused increased antibiotic resistance and phase variation resulting in translucent colony morphologies. Increased nucleotide diversity in mutS and rpoB alleles from mutator compared with wild-type strains indicated a significant contribution of the mutator phenotype to the evolution of select genes. The results of this study indicate that the inactivation of MMR in V. parahaemolyticus leads to increased genetic and phenotypic diversity. This study is the first to report a higher frequency of natural mutators among Vibrio environmental strains and to provide evidence that inactivation of MMR increases the diversity of V. parahaemolyticus .     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Decreasing the effects of horizontal gene transfer on bacterial phylogeny: the Escherichia coli case study

Phylogenetic reconstructions of bacterial species from DNA sequences are hampered by the existence of horizontal gene transfer. One possible way to overcome the confounding influence of such movement of genes is to identify and remove sequences which are responsible for significant character incongruence when compared to a reference dataset free of horizontal transfer (e.g., multilocus enzyme electrophoresis, restriction fragment length polymorphism, or random amplified polymorphic DNA) using the incongruence length difference (ILD) test of Farris et al. [Cladistics 10 (1995) 315]. As obtaining this "whole genome dataset" prior to the reconstruction of a phylogeny is clearly troublesome, we have tested alternative approaches allowing the release from such reference dataset, designed for a species with modest level of horizontal gene transfer, i.e., Escherichia coli. Eleven different genes available or sequenced in this work were studied in a set of 30 E. coli reference (ECOR) strains. Either using ILD to test incongruence between each gene against the all remaining (in this case 10) genes in order to remove sequences responsible for significant incongruence, or using just a simultaneous analysis without removals, gave robust phylogenies with slight topological differences. The use of the ILD test remains a suitable method for estimating the level of horizontal gene transfer in bacterial species. Supertrees also had suitable properties to extract the phylogeny of strains, because the way they summarize taxonomic congruence clearly limits the impact of individual gene transfers on the global topology. Furthermore, this work allowed a significant improvement of the accuracy of the phylogeny within E. coli.     

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome

A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.     

A phylogenomic analysis of Escherichia coli / Shigella group: implications of genomic features associated with pathogenicity and ecological adaptation

ABSTRACT: BACKGROUND: The Escherichia coli species contains a variety of commensal and pathogenic strains, and its intraspecific diversity is extraordinarily high. With the availability of an increasing number of E. coli strain genomes, a more comprehensive concept of their evolutionary history and ecological adaptation can be developed using phylogenomic analyses. In this study, we constructed two types of whole-genome phylogenies based on 34 E. coli strains using collinear genomic segments. The first phylogeny was based on the concatenated collinear regions shared by all of the studied genomes, and the second phylogeny was based on the variable collinear regions that are absent from at least one genome. Intuitively, the first phylogeny is likely to reveal the lineal evolutionary history among these strains (i.e., an evolutionary phylogeny), whereas the latter phylogeny is likely to reflect the whole-genome similarities of extant strains (i.e., a similarity phylogeny). RESULTS: Within the evolutionary phylogeny, the strains were clustered in accordance with known phylogenetic groups and phenotypes. When comparing evolutionary and similarity phylogenies, a concept emerges that Shigella may have originated from at least three distinct ancestors and evolved into a single clade. By scrutinizing the properties that are shared amongst Shigella strains but missing in other E. coli genomes, we found that the common regions of the Shigella genomes were mainly influenced by mobile genetic elements, implying that they may have experienced convergent evolution via horizontal gene transfer. Based on an inspection of certain key branches of interest, we identified several collinear regions that may be associated with the pathogenicity of specific strains. Moreover, by examining the annotated genes within these regions, further detailed evidence associated with pathogenicity was revealed. CONCLUSIONS: Collinear regions are reliable genomic features used for phylogenomic analysis among closely related genomes while linking the genomic diversity with phenotypic differences in a meaningful way. The pathogenicity of a strain may be associated with both the arrival of virulence factors and the modification of genomes via mutations. Such phylogenomic studies that compare collinear regions of whole genomes will help to better understand the evolution and adaptation of closely related microbes and E. coli in particular.     

A Genomic Island in Salmonella enterica ssp. salamae Provides New Insights on the Genealogy of the Locus of Enterocyte Effacement

The genomic island encoding the locus of enterocyte effacement (LEE) is an important virulence factor of the human pathogenic Escherichia coli. LEE typically encodes a type III secretion system (T3SS) and secreted effectors capable of forming attaching and effacing lesions. Although prominent in the pathogenic E. coli such as serotype O157:H7, LEE has also been detected in Citrobacter rodentium, E. albertii, and although not confirmed, it is likely to also be in Shigella boydii. Previous phylogenetic analysis of LEE indicated the genomic island was evolving through stepwise acquisition of various components. This study describes a new LEE region from two strains of Salmonella enterica subspecies salamae serovar Sofia along with a phylogenetic analysis of LEE that provides new insights into the likely evolution of this genomic island. The Salmonella LEE contains 36 of the 41 genes typically observed in LEE within a genomic island of 49, 371 bp that encodes a total of 54 genes. A phylogenetic analysis was performed on the entire T3SS and four T3SS genes (escF, escJ, escN, and escV) to elucidate the genealogy of LEE. Phylogenetic analysis inferred that the previously known LEE islands are members of a single lineage distinct from the new Salmonella LEE lineage. The previously known lineage of LEE diverged between islands found in Citrobacter and those in Escherichia and Shigella. Although recombination and horizontal gene transfer are important factors in the genealogy of most genomic islands, the phylogeny of the T3SS of LEE can be interpreted with a bifurcating tree. It seems likely that the LEE island entered the Enterobacteriaceae through horizontal gene transfer as a single unit, rather than as separate subsections, which was then subjected to the forces of both mutational change and recombination.     

Microcin H47 system: an Escherichia coli small genomic island with novel features

Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria.Microcin H47 is a ribosomally-synthesized antibacterial peptide that belongs to the group of chromosome-encoded microcins. The 13 kb-genetic system responsible for its production resides in the chromosome of the Escherichia coli H47 strain and is flanked by extensive and imperfect direct repeats. In this work, both excision and integration of the microcin H47 system were experimentally detected. The analyses were mainly performed in E. coli K12 cells carrying the microcin system cloned in a multicopy plasmid. As expected of a site-specific recombination event, the genetic exchange also occurred in a context deficient for homologous recombination. The DNA sequence of the attachment sites resulting from excision were hybrid between the sequences of the direct repeats. Unexpectedly, different hybrid attachment sites appeared which resulted from recombination in four segments of identity between the direct repeats. Genes encoding the trans-acting proteins responsible for the site-specific recombination were shown to be absent in the microcin H47 system. Therefore, they should be provided by the remaining genetic context, not only in the H47 strain but also in E. coli K12 cells, where both excision and integration occurred. Moreover, a survey of the attachment sites in data banks revealed that they are widely spread among E. coli strains. It is concluded that the microcin system is a small island -H47 genomic island- that would employ a parasitic strategy for its mobility.     

Role of Intraspecies Recombination in the Spread of Pathogenicity Islands within the Escherichia coli Species

Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Besides phages and plasmids, pathogenicity islands (PAIs) are subjected to horizontal transfer. The transfer mechanisms of PAIs within a certain bacterial species or between different species are still not well understood. This study is focused on the High-Pathogenicity Island (HPI), which is a PAI widely spread among extraintestinal pathogenic Escherichia coli and serves as a model for horizontal transfer of PAIs in general. We applied a phylogenetic approach using multilocus sequence typing on HPI-positive and -negative natural E. coli isolates representative of the species diversity to infer the mechanism of horizontal HPI transfer within the E. coli species. In each strain, the partial nucleotide sequences of 6 HPI–encoded genes and 6 housekeeping genes of the genomic backbone, as well as DNA fragments immediately upstream and downstream of the HPI were compared. This revealed that the HPI is not solely vertically transmitted, but that recombination of large DNA fragments beyond the HPI plays a major role in the spread of the HPI within E. coli species. In support of the results of the phylogenetic analyses, we experimentally demonstrated that HPI can be transferred between different E. coli strains by F-plasmid mediated mobilization. Sequencing of the chromosomal DNA regions immediately upstream and downstream of the HPI in the recipient strain indicated that the HPI was transferred and integrated together with HPI–flanking DNA regions of the donor strain. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer of a pathogenicity island within the species E. coli.     

Autotransporter-encoding sequences are phylogenetically distributed among Escherichia coli clinical isolates and reference strains

Autotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various autotransporters have been identified among Escherichia coli associated with intestinal or extraintestinal infections; however, the specific distribution of autotransporter sequences among a diversity of E. coli strains has not been investigated. We have validated the use of a multiplex PCR assay to screen for the presence of autotransporter sequences. Herein, we determined the presence of 13 autotransporter sequences and five allelic variants of antigen 43 (Ag43) among 491 E. coli isolates from human urinary tract infections, diarrheagenic E. coli, and avian pathogenic E. coli (APEC) and E. coli reference strains belonging to the ECOR collection. Clinical isolates were also classified into established phylogenetic groups. The results indicated that Ag43 alleles were significantly associated with clinical isolates (93%) compared to commensal isolates (56%) and that agn43K12 was the most common and widely distributed allele. agn43 allelic variants were also phylogenetically distributed. Sequences encoding espC, espP, and sepA and agn43 alleles EDL933 and RS218 were significantly associated with diarrheagenic E. coli strains compared to other groups. tsh was highly associated with APEC strains, whereas sat was absent from APEC. vat, sat, and pic were associated with urinary tract isolates and were identified predominantly in isolates belonging to either group B2 or D of the phylogenetic groups based on the ECOR strain collection. Overall, the results indicate that specific autotransporter sequences are associated with the source and/or phylogenetic background of strains and suggest that, in some cases, autotransporter gene profiles may be useful for comparative analysis of E. coli strains from clinical, food, and environmental sources.