Role of pathogenicity island-associated integrases in the genome plasticity of uropathogenic Escherichia coli strain 536

The genome of uropathogenic Escherichia coli isolate 536 contains five well-characterized pathogenicity islands (PAIs) encoding key virulence factors of this strain. Except PAI IV(536), the four other PAIs of strain 536 are flanked by direct repeats (DRs), carry intact integrase genes and are able to excise site-specifically from the chromosome. Genome screening of strain 536 identified a sixth putative asnW-associated PAI. Despite the presence of DRs and an intact integrase gene, excision of this island was not detected. To investigate the role of PAI-encoded integrases for the recombination process the int genes of each unstable island of strain 536 were inactivated. For PAI I(536) and PAI II(536), their respective P4-like integrase was required for their excision. PAI III(536) carries two integrase genes, intA, encoding an SfX-like integrase, and intB, coding for an integrase with weak similarity to P4-like integrases. Only intB was required for site-specific excision of this island. For PAI V(536), excision could not be abolished after deleting its P4-like integrase gene but additional deletion of the PAI II(536)-specific integrase gene was required. Therefore, although all mediated by P4-like integrases, the activity of the PAI excision machinery is most often restricted to its cognate island. This work also demonstrates for the first time the existence of a cross-talk between integrases of different PAIs and shows that this cross-talk is unidirectional.     

Instability of pathogenicity islands in uropathogenic Escherichia coli 536

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.     

Delineation of the recombination sites necessary for integration of pathogenicity islands II and III into the Escherichia coli 536 chromosome

In uropathogenic Escherichia coli strain 536, six pathogenicity islands (PAIs) encode key virulence factors. All PAIs except PAI IV₅₃₆ are flanked by direct repeats and four of them encode integrases responsible for their chromosomal excision. To study recombination sites used for the integration by PAI II₅₃₆ and III₅₃₆ integrases, we measured site-specific recombination between the chromosomal integration site attB , and the PAI-specific attachment site attP . We show that PAI III₅₃₆ IntB, but not IntA, mediates PAI III₅₃₆ integration. Studies of integrative recombination sites of both PAIs show that, when using a large cognate attP site (839 bp for PAI II₅₃₆ and 268 bp for PAI III₅₃₆), PAI II₅₃₆ and III₅₃₆ attB sites could be reduced to 16 bp and 20 bp, respectively, without affecting recombination. Further reduction to 14 bp for PAI II₅₃₆ and 13 bp for PAI III₅₃₆ diminished recombination efficiency. Surprisingly, attP sites could also be reduced to 14 bp (PAI II₅₃₆) and 20 bp (PAI III₅₃₆). The integration host factor (IHF) and the DNA-bending HU protein do not influence PAI II₅₃₆ recombination, but IHF enhances PAI-III₅₃₆ excision and negatively affects its integration. These data suggest that PAI intasomes differ from those of lambda and P4 integrase paradigms.     

Bacterial genomic islands: organization, function, and role in evolution

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are extended DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA genes, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.     

Pathogenicity island markers, virulence determinants malX and usp, and the capacity of Escherichia coli to persist in infants' commensal microbiotas

Virulence-associated genes in bacteria are often located on chromosomal regions, termed pathogenicity islands (PAIs). Several PAIs are found in Escherichia coli strains that cause extraintestinal infections, but their role in commensal bowel colonization is unknown. Resident strains are enriched in adhesins (P fimbriae and type 1 fimbriae), capsular antigens (K1 and K5), hemolysin, and aerobactin and mostly belong to phylogenetic group B2. Here, we investigated whether six pathogenicity islands and the virulence determinants malX and usp are associated with fitness of E. coli in the infant bowel microbiota. E. coli strains isolated from stools of 130 Swedish infants during the first year of life were examined for their carriage of PAI markers, malX, and usp by PCR. Carriage was related to strain persistence: long-term colonizers (≥12 months) carried significantly more of PAI II from strain CFT703 (II(CFT703)), IV(536,) and II(J96) and malX and usp than intermediate colonizers (1 to 11 months) and transient strains (<3 weeks). The accumulation of PAI markers in each individual strain correlated positively with its time of persistence in the colon. Phylogenetic group B2 accounted for 69% of long-term colonizers, 46% of intermediate colonizers and 14% of transient strains. These results support the hypothesis that some bacterial traits contributing to extraintestinal infections have in fact evolved primarily because they increase the fitness of E. coli in its natural niche, the colon; accordingly, they may be regarded as fitness islands in the gut.     

Bacterial Genomic Islands: Organization,Function, and Evolutionary Role

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are long DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA gene loci, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.     

Molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) pathogenicity islands in F165-positive E. coli strain from a diseased animal

Septicemic Escherichia coli 4787 (O115: K-: H51: F165) of porcine origin possess gene clusters related to extraintestinal E. coli fimbrial adhesins. This strain produces two fimbriae: F165(1) and F165(2). F165(1) (Prs-like) belongs to the P fimbrial family, encoded by foo operon and F165(2) is a F1C-like encoded by fot operon. Data from this study suggest that these two operons are part of two PAIs. PAI I(4787) includes a region of 20 kb, which not only harbors the foo operon but also contains a potential P4 integrase gene and is located within the pheU tRNA gene, at 94 min of the E. coli chromosome. PAI II(4787) includes a region of over 35 kb, which harbors the fot operon, iroBCDEN gene clusters, as well as part of microcin M genes and nonfunctional mobility genes. PAI II(4787) is found between the proA and yagU at 6 min of the E. coli chromosome.     

A novel integrative and conjugative element (ICE) of Escherichia coli: the putative progenitor of the Yersinia high-pathogenicity island

Diversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high-pathogenicity island (HPI) carrying genes involved in siderophore-mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA-processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4-like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.     

Molecular evolution of pathogenicity-island genes in Pseudomonas viridiflava

The bacterial pathogen Pseudomonas viridiflava possesses two pathogenicity islands (PAIs) that share many gene homologs, but are structurally and phenotypically differentiated (T-PAI and S-PAI). These PAIs are paralogous, but only one is present in each isolate. While this dual presence/absence polymorphism has been shown to be maintained by balancing selection, little is known about the molecular evolution of individual genes on the PAIs. Here we investigate genetic variation of 12 PAI gene loci (7 on T-PAI and 5 on S-PAI) in 96 worldwide isolates of P. viridiflava. These genes include avirulence genes (hopPsyA and avrE), their putative chaperones (shcA and avrF), and genes encoding the type III outer proteins (hrpA, hrpZ, and hrpW). Average nucleotide diversities in these genes (pi = 0.004-0.020) were close to those in the genetic background. Large numbers of recombination events were found within PAIs and a sign of positive selection was detected in avrE. These results suggest that the PAI genes are evolving relatively freely from each other on the PAIs, rather than as a single unit under balancing selection. Evolutionarily stable PAIs may be preferable in this species because preexisting genetic variation enables P. viridiflava to respond rapidly to natural selection.     

Characterization and evidence of mobilization of the LEE pathogenicity island of rabbit-specific strains of enteropathogenic Escherichia coli

We have characterized the LEE pathogenicity islands (PAIs) of two rabbit-specific strains of enteropathogenic E. coli (REPEC), 83/39 (serotype O15:H-) and 84/110-1 (O103:H2), and have compared them to homologous loci from the human enteropathogenic and enterohaemorrhagic E. coli strains, E2348/69 and EDL933, and another REPEC strain, RDEC-1. All five PAIs contain a 34 kb core region that is highly conserved in gene order and nucleotide sequence. However, the LEE of 83/39 is significantly larger (59 540 basepairs) than those of the human strains, which are less than 44 kb, and has inserted into pheU tRNA. The regions flanking the 34 kb core of 83/39 contain homologues of two putative virulence determinants, efa1/lifA and senA. The LEE of 84/110-1 is approximately 85 kb and is located at pheV tRNA. Its core is almost identical to those of 83/39 and RDEC-1, apart from a larger espF gene, but its flanking regions contain trcA, a putative virulence determinant of EPEC. All three REPEC LEE PAIs contain a gene for an integrase, Int-phe. The LEE PAI of 84/110-1 is also flanked by short direct repeats (representing the 3'-end of pheV tRNA), suggesting that it may be unstable. To investigate this possibility, we constructed a LEE::sacB derivative of 84/110-1 and showed that the PAI was capable of spontaneous deletion. We also showed that Int-phe can mediate site-specific integration of foreign DNA at the pheU tRNA locus of E. coli DH1. Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI.     

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.     

Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes

The genetic differences between the human pathogen, Shigella flexneri, and the non-pathogenic Escherichia coli were investigated in an attempt to identify pathogenicity islands (PAIs) in the S. flexneri genome. Genomic subtraction identified a large unique region of DNA which was present in S. flexneri serotype 2a but absent from E. coli K-12. This 42-kb DNA segment was localised to the S. flexneri chromosome and was found to contain a number of elements often associated with PAIs including: insertion sequence elements, bacteriophage genes, and a previously identified Shigella virulence gene (criR). These findings indicate that this region may form a new PAI in the S. flexneri genome.     

Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island

We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 micro M in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.     

Distribution of pathogenicity island markers and virulence factors in new phylogenetic groups of uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates

The present study was aimed at investigating the relationship between the new Clermont’s phylogenetic groups, virulence factors, and pathogenicity island markers (PAIs) among uropathogenic Escherichia coli (UPEC) in Iran. This cross-sectional study was carried out on 140 UPEC isolates collected from patients with urinary tract infections in Bushehr, Iran. All isolates were subjected to phylogenetic typing using a new quadruplex-PCR method. The presence of PAI markers and virulence factors in UPEC strains was evaluated by multiplex PCR. The most predominant virulence gene was fimH (85%), followed by iucC (61.4%), papC (38.6%), hlyA (22.1%), cnf-1 (18.6%), afa (10.7%), papG and neuC (each 9.3%), ibeA (3.6%), and sfa/foc (0.7%). The most common phylogenetic group was related to B2 (39.3%), and the least common to A (0.7%). The most prevalent PAI marker was PAI IV536 (77.14%), while markers for PAI III536 (13.57%), PAI IIJ96 (12.86%), and PAI II536 (12.14%) were the least frequent among the UPEC strains. Meanwhile, the PAI IJ96 marker was not detected. There was a significant association between the phylogenetic group B2 and all the studied virulence genes and PAI markers. To our knowledge, this is the first study to compare the relationship between new phylogenetic groups, virulence genes and PAI markers in UPEC strains in Iran. The phylogenetic group B2 was predominantly represented among the studied virulence genes and PAI markers, indicating the preference of particular strains to carry virulence genes.     

Plasticity of Corynebacterium diphtheriae pathogenicity islands revealed by PCR

Despite the existence of a vaccine against diphtheria, this disease remains endemic and is reemerging in several regions due to many factors, including variations in genes coding for virulence factors. One common feature of virulence factors is their high concentration in pathogenicity islands (PAIs), very unstable regions acquired via horizontal gene transfer, which has lead to the emergence of various bacterial pathogens. The 13 putative PAIs in Corynebacterium diphtheriae NCTC 13129 and the reemergence of this disease point to the great variability in the PAIs of this species, which may reflect on bacterial life style and physiological versatility. We investigated the relationships between the large number of PAIs in C. diphtheriae and the possible implications of their plasticity in virulence. The GenoFrag software was used to design primers to analyze the genome plasticity of two pathogenicity islands of the reference strain (PiCds 3 and 8) in 11 different strains. We found that PiCd 3 was absent in only two strains, showing genes playing putative important roles in virulence and that only one strain harbored PiCd 8, due to its location in a putative "hotspot" for horizontal gene transfer events.     

Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems

Pathogenicity islands (PAIs) are large DNA segments in the genomes of bacterial pathogens that encode virulence factors. Five PAIs have been identified in the Gram-negative bacterium Salmonella enterica. Two of these PAIs, Salmonella pathogenicity island (SPI)-1 and SPI-2, encode type III secretion systems (TTSS), which are essential virulence determinants. These 'molecular syringes' inject effectors directly into the host cell, whereupon they manipulate host cell functions. These effectors are either encoded with their respective TTSS or scattered elsewhere on the Salmonella chromosome. Importantly, SPI-1 and SPI-2 are expressed under distinct environmental conditions: SPI-1 is induced upon initial contact with the host cell, whereas SPI-2 is induced intracellularly. Here, we demonstrate that a single PAI, in this case SPI-5, can encode effectors that are induced by distinct regulatory cues and targeted to different TTSS. SPI-5 encodes the SPI-1 TTSS translocated effector, SigD/SopB. In contrast, we report that the adjacently encoded effector PipB is part of the SPI-2 regulon. PipB is translocated by the SPI-2 TTSS to the Salmonella-containing vacuole and Salmonella-induced filaments. We also show that regions of SPI-5 are not conserved in all Salmonella spp. Although sigD/sopB is present in all Salmonella spp., pipB is not found in Salmonella bongori, which also lacks a functional SPI-2 TTSS. Thus, we demonstrate a functional and regulatory cross-talk between three chromosomal PAIs, SPI-1, SPI-2 and SPI-5, which has significant implications for the evolution and role of PAIs in bacterial pathogenesis.     

Describing ancient horizontal gene transfers at the nucleotide and gene levels by comparative pathogenicity island genometrics

MOTIVATION: Lateral gene transfer is a major mechanism contributing to bacterial genome dynamics and pathovar emergence via pathogenicity island (PAI) spreading. However, since few of these genomic exchanges are experimentally reproducible, it is difficult to establish evolutionary scenarios for the successive PAI transmissions between bacterial genera. Methods initially developed at the gene and/or nucleotide level for genomics, i.e. comparisons of concatenated sequences, ortholog frequency, gene order or dinucleotide usage, were combined and applied here to homologous PAIs: we call this approach comparative PAI genometrics. RESULTS: YAPI, a Yersinia PAI, and related islands were compared with measure evolutionary relationships between related modules. Through use of our genometric approach designed for tracking codon usage adaptation and gene phylogeny, an ancient inter-genus PAI transfer was oriented for the first time by characterizing the genomic environment in which the ancestral island emerged and its subsequent transfers to other bacterial genera.