Horizontal transfer of the high-pathogenicity island of Yersinia pseudotuberculosis

The horizontal transfer of genetic elements plays a major role in bacterial evolution. The high-pathogenicity island (HPI), which codes for an iron uptake system, is present and highly conserved in various Enterobacteriaceae, suggesting its recent acquisition by lateral gene transfer. The aim of this work was to determine whether the HPI has kept its ability to be transmitted horizontally. We demonstrate here that the HPI is indeed transferable from a donor to a recipient Yersinia pseudotuberculosis strain. This transfer was observable only when the donor and recipient bacteria were cocultured at low temperatures in a liquid medium. When optimized conditions were used (bacteria actively growing in an iron-deprived medium at 4 degrees C), the frequency of HPI transfer reached approximately 10(-8). The island was transferable to various serotype I strains of Y. pseudotuberculosis and to Yersinia pestis, but not to Y. pseudotuberculosis strains of serotypes II and IV or to Yersinia enterocolitica. Upon transfer, the HPI was inserted almost systematically into the asn3 tRNA locus. Acquisition of the HPI resulted in the loss of the resident island, suggesting an incompatibility between two copies of the HPI within the same strain. Transfer of the island did not require a functional HPI-borne insertion-excision machinery and was RecA dependent in the recipient but not the donor strain, suggesting that integration of the island into the recipient chromosome occurs via a mechanism of homologous recombination. This lateral transfer also involved the HPI-adjacent sequences, leading to the mobilization of a chromosomal region at least 46 kb in size.     

The Yersinia high-pathogenicity island.

A pathogenicity island present only in highly pathogenic strains of Yersinia (Y. enterocolitica 1B, Y. pseudotuberculosis I and Y. pestis) has been identified on the chromosome of Yersinia spp. and has been designated High-Pathogenicity Island (HPI). The Yersinia HPI carries a cluster of genes involved in the biosynthesis, transport and regulation of the siderophore yersiniabactin. The major function of this island is thus to acquire iron molecules essential for in vivo bacterial growth and dissemination. The presence of an integrase gene and att sites homologous to those of phage P4, together with a G + C content much higher than the chromosomal background, suggests that the HPI is of foreign origin and has been acquired by chromosomal integration of a phage. The HPI can excise from the chromosome of Y. pseudotuberculosis and is found inserted into any of the three copies of the asn tRNA loci present in this species. A unique characteristic of the HPI is its wide distribution in various enterobacteria. Although first identified in Yersinia spp., it has subsequently been detected in other genera such as E. coli, Klebsiella and Citrobacter.     

Widespread occurrence of the restriction endonuclease YenI, an isoschizomer of PstI, in Yersinia enterocolitica serotype O8.

The cold-active restriction endonuclease YenI, an isoschizomer of PstI, was found in 12 of 14 Yersinia enterocolitica serotype O8 strains of different origins, but not in other serotypes of Y. enterocolitica, Yersinia pseudotuberculosis, or Yersinia pestis. In spite of the limited number of strains tested, the result suggests that the detection of YenI endonuclease or the gene might result in more rapid determination of the prominently pathogenic serotype of Y. enterocolitica.     

Widespread occurrence of the restriction endonuclease YenI, an isoschizomer of PstI, in Yersinia enterocolitica serotype O8

The cold-active restriction endonuclease YenI, an isoschizomer of PstI, was found in 12 of 14 Yersinia enterocolitica serotype O8 strains of different origins, but not in other serotypes of Y. enterocolitica, Yersinia pseudotuberculosis, or Yersinia pestis. In spite of the limited number of strains tested, the result suggests that the detection of YenI endonuclease or the gene might result in more rapid determination of the prominently pathogenic serotype of Y. enterocolitica.     

The lytic activity of Yersinia pestis phage P 3d serovar

The lytic activity of plague phage II, serovar 3, with respect to 1,800 bacterial strains has been studied: 760 Yersinia pestis strains, 262 Y. pseudotuberculosis strains, 252 Y. enterocolitica strains, 166 Escherichia coli strains, 90 Shigella strains and 270 strains of other species. The phage has been found to lyse 81.8% of Y. pestis strains, 1 Y. pseudotuberculosis strain and 1 Y. enterocolitica strain. The representatives of other 19 bacterial species have proved to be resistant to the phage. Though having a wide range of action within Y. pestis, the phage does not lyse most of the strains of the causative agent of plague, isolated in certain natural foci. This fact offers promise for using the phage for the differentiation of Y. pestis.     

Gene Transfer in Pasteurella pestis Harboring the F′Cm Plasmid of Escherichia coli

A strain of Pasteurella pestis, harboring the F'Cm plasmid from Escherichia coli, was able to donate its chromosome to auxotrophic recipient strains of P. pestis. The frequency of gene transfer in P. pestis was approximately 10(-6) per donor cell, 100 times less efficient than gene transfer in Pasteurella pseudotuberculosis, but efficient enough to determine entry times for the markers histidine, threonine, and tryptophan and to show linkage to the markers arginine and pigmentation. An attempt to extend the conjugation system to different serotypes of P. pseudotuberculosis and to Yersinia enterocolitica did not succeed.     

Chromosomal-encoded siderophores are required for mouse virulence of enteropathogenic Yersinia species

Abstract Yersinia enterocolitica and Y. pseudotuberculosis are enteropathogenic for humans. Essential virulence functions of these pathogens are determined by a 40-mDa plasmid. Plasmid-bearing Y. pseudotuberculosis strains and Y. enterocolitica strains of serotypes 0 : 8, 0 : 13, 0 : 20 and 0 : 40 are lethal for mice. In contrast, human pathogenic Y. enterocolitica strains of serotype 0 : 3, 0 : 9 and 0 : 5.27 are not mouse-lethal. Using a sensitive siderophore-indicator CAS-agar, we were able to detect siderophore production in all mouse-lethal Y. enterocolitica and Y. pseudotuberculosis strains mentioned above. By Tn5-transposon insertions into the chromosome of a serotype 0 : 8 strain we obtained two siderophore-deficient mutants. Introduction of the virulence plasmid did not render these mutants mouse-lethal, indicating that siderophore production is an essential virulence factor. The human nonpathogenic, aerobactin-producing strains of Y. intermedia, Y. kristensenii and Y. frederiksenii remained avirulent for mice after receiving the virulence plasmid of Y. enterocolitica . Obviously the siderophore aerobactin does not contribute to virulence in the genus Yersinia .     

Evidence for two evolutionary lineages of highly pathogenic Yersinia species.

Sensitivity to Yersinia pestis bacteriocin pesticin correlates with the existence of two groups of human pathogenic yersiniae, mouse lethal and mouse nonlethal. The presence of the outer membrane pesticin receptor (FyuA) in mouse-lethal yersiniae is a prerequisite for pesticin sensitivity. Genes that code for FyuA (fyuA) were identified and sequenced from pesticin-sensitive bacteria, including Y. enterocolitica biotype 1B (serotypes O8; O13, O20, and O21), Y. pseudotuberculosis serotype O1, Y. pestis, two known pesticin-sensitive Escherichia coli isolates (E. coli Phi and E. coli CA42), and two newly discovered pesticin-sensitive isolates, E. coli K49 and K235. A 2,318-bp fyuA sequence was shown to be highly conserved in all pesticin-sensitive bacteria, including E. coli strains (DNA sequence homology was 98.5 to 99.9%). The same degree of DNA homology (97.8 to 100%) was established for the sequenced 276-bp fragment of the irp2 gene that encodes high-molecular-weight protein 2, which is also thought to be involved in the expression of virulence by Yersinia species. Highly conserved irp2 was also found in all pesticin-sensitive E. coli strains. On the basis of the fyuA and irp2 sequence homologies, two evolutionary groups of highly pathogenic Yersinia species can be established. One group includes Y. enterocolitica biotype 1B strains, while the second includes Y. pestis, Y. pseudotuberculosis serotype O1, and irp2-positive Y. pseudotuberculosis serotype O3 strains. E. coli Phi, CA42, K49, and K235 belong to the second group. The possible proximity of these two iron-regulated genes (fyuA and irp2), as well as their high levels of sequence conservation and similar G+C contents (56.2 and 59.8 mol%), leads to the assumption that these two genes may represent part of an unstable pathogenicity island that has been acquired by pesticin-sensitive bacteria as a result of a horizontal transfer.     

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.     

Monoclonal antibodies directed against plasmid-encoded released proteins of enteropathogenic Yersinia

Abstract Yersinia enterocolitica of serotypes O:3, O:8, O:9 and O:5,27 and Yersinia pseudotuberculosis of serotypes I and III release plasmid-encoded proteins into calcium-deficient medium. Mouse monoclonal antibodies were elicited against plasmid-encoded released proteins of Y. enterocolitica of serotype O:9. As shown by immunoblot analysis the monoclonal antibody Mab9–200 recognized the 46-kDa protein of Y. enterocolitica of serotypes O:3, O:9 and O:5,27, the 58-kDa protein of Y. enterocolitica of serotype O:8 and the 67-kDa protein of Y. pseudotuberculosis of serotypes I and III. Mab9–15 reacted with the 36-kDa protein of Y. enterocolitica of serotypes O:9, O:3 and O:8, and the 34-kd protein of Y. enterocolitica of serotype O:5,27 and Y. pseudotuberculosis of serotypes I and III. The 25-kDa proteins of Y. enterocolitica of serotypes O:3, O:9, O:8 and O:5,27, but not those of Y. pseudotuberculosis were recognized by the monoclonal antibody Mab-128. This species-specific recognition of epitopes could not be achieved by mouse polyclonal antibodies.     

Characterization of the ysa Pathogenicity Locus in the Chromosome of Yersinia enterocolitica and Phylogeny Analysis of Type III Secretion Systems

Several Gram negative bacteria use a complex system called "type III secretion system" (TTSS) to engage their host. The archetype of TTSS is the plasmid-encoded "Yop virulon" shared by the three species of pathogenic Yersinia (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica). A second TTSS, called Ysa (for Yersinia secretion apparatus) was recently described in Y. enterocolitica 8081, a strain from serotype O:8. In this study, we describe the ysa locus from A127/90, another strain of serotype O:8, and we extend the sequence to several new genes encoding Ysp proteins which are the substrates of this secretion system, and a putative chaperone SycB. According to the deduced protein sequences, the ysa system from A127/90 is identical to that of 8081. It is different from the chromosome-encoded TTSS of Y. pestis but is instead closely related to the Mxi-Spa TTSS of Shigella and to the SPI-1 encoded TTSS of Salmonella enterica. We further demonstrated that the ysa locus is only present in biotype IB strains of Y. enterocolitica. Including this new Ysa system, a phylogenetic analysis of the 26 known TTSSs was carried out, based on the sequence analysis of three conserved proteins. All the TTSSs fall into five different clusters. The phylogenetic tree of these TTSSs is completely different from the evolutionary tree based on 16S RNA, indicating that TTSSs have been distributed by horizontal transfer.     

Serotype differences and lack of biofilm formation characterize Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.     

A highly efficient electroporation system for transformation of Yersinia

The various pathogenic Yersinia species are not readily and efficiently transformed by classical methods. For this reason, the electroporation technique was applied for genetic transformation of these species. Using optimal conditions, we were able to transform the six Yersinia strains studied with the two most widely used groups of plasmids: pSU2718 (a pACYC184 derivative) and pK19 (a pUC19 derivative). Only Yersinia enterocolitica (Y. e.) serotype 0:8 gave poor results (less than 5 x 10(2) transformants/microgram) DNA). Electrical transformation of the other species resulted in high efficiencies, up to 10(5) transformants/microgram DNA for Y. e. serotypes 0:3 and 0:9, 10(6) for Y. pseudotuberculosis and 10(7) for Y. pestis. The results varied for each strain with the type of plasmid used. Neither the introduced foreign plasmid nor the resident 72-kb virulence plasmid underwent detectable deletions. Transformation was most efficient with supercoiled DNA, decreasing by one and four orders of magnitude for relaxed circular and linearized plasmids, respectively. The ability to easily and efficiently transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA technology for direct cloning and analysis of significant genes into Yersinia.     

Characterization of IS1541-like elements in Yersinia enterocolitica and Yersinia pseudotuberculosis

We characterized Yersinia enterocolitica and Yersinia pseudotuberculosis insertion sequences related to insertion sequence 1541, recently identified in Yersinia pestis. For each of the two species, two insertion sequence copies were cloned and sequenced. Genetic elements from Y. pseudotuberculosis were almost identical to insertion sequence 1541, whereas these from Y. enterocolitica were less related. Phylogenetic analysis of the putative transposases encoded by insertion sequences from the three pathogenic members of the genus Yersinia showed that they clustered with those encoded by Escherichia coli and Salmonella enterica elements belonging to the insertion sequence 200/insertion sequence 605 group. Insertion sequences originating from Y. pestis and Y. pseudotuberculosis constitute a monophyletic lineage distinct from that of Y. enterocolitica.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. IV. Specificity of antigens

The specificity of the lipopolisacharydes and released proteins (Yop) of Yersinia was tested using the sera of rabbits immunised with pathogenic and non-pathogenic strain of Y. enterocolitica and Y. pseudotuberculosis as well as selected sera of patients. The results of this study showed a cross-reactions between the different serotypes of Y. enterocolitica with the strongest reactions between the pathogenic serotypes O:3 and O:9 and pathogenic serotype O:5,27 and non-pathogenic serotype O:5. Sera positive for B. burgdorferi and from patients with Graves' disease showed a slight cross-reactivity with Yop proteins of Yersinia. However, the higher cross-reactivity was observed between the LPS of Yersinia and Salmonella spp. Due to the evidence of cross-reactivity the results of serological investigations should be interpreted with caution.     

Fatty acid composition of lipopolysaccharides of the strains of different species of Yersinia

The fatty acid composition of lipopolysaccharides of the strains of Y. enterocolitica, Y. intermedia, Y. frederiksenii and Y. ruckeri studied during cultivation on meat-peptone agar is characterized by the predominance of 3-hydroxytetradecanoic and dodecanoic acids. Closely related to the mentioned bacteria is the strain of Y. kristensenii which is distinguished only by its higher level of hexadecanoic acid. The strains of Y. pseudotuberculosis and the vaccine strain of Y. pestis have a uniform fatty acid composition of lipopolysaccharides with predominance of 3-hydroxytetradecanoic acid. Their relatively low level of dodecanoic acid conditions the characteristic fatty acid spectrum of lipopolysaccharides which differs from that of the above mentioned group of Yersinia. The peculiarities of the fatty acid composition of lipopolysaccharides of both groups of Yersinia are preserved during growth on meat-peptone broth, but the increase in the level of hexadecanoic acid balances the differences between Y. kristensenii, the other Y. enterocolitica-like bacteria and Y. ruckeri. The obtained results confirm close relationship of Y. pseudotuberculosis and Y. pestis, and also of Y. enterocolitica and Y. enterocolitica-like bacteria, showing propinquity of Y. ruckeri to the latter.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Functional recruitment of the human complement inhibitor C4BP to Yersinia pseudotuberculosis outer membrane protein Ail

Ail is a 17-kDa chromosomally encoded outer membrane protein that mediates serum resistance (complement resistance) in the pathogenic Yersiniae (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis). In this article, we demonstrate that Y. pseudotuberculosis Ail from strains PB1, 2812/79, and YPIII/pIB1 (serotypes O:1a, O:1b, and O:3, respectively) can bind the inhibitor of the classical and lectin pathways of complement, C4b-binding protein (C4BP). Binding was observed irrespective of serotype tested and independently of YadA, which is the primary C4BP receptor of Y. enterocolitica. Disruption of the ail gene in Y. pseudotuberculosis resulted in loss of C4BP binding. Cofactor assays revealed that bound C4BP is functional, because bound C4BP in the presence of factor I cleaved C4b. In the absence of YadA, Ail conferred serum resistance to strains PB1 and YPIII, whereas serum resistance was observed in strain 2812/79 in the absence of both YadA and Ail, suggesting additional serum resistance factors. Ail from strain YPIII/pIB1 alone can mediate serum resistance and C4BP binding, because its expression in a serum-sensitive laboratory strain of Escherichia coli conferred both of these phenotypes. Using a panel of C4BP mutants, each deficient in a single complement control protein domain, we observed that complement control protein domains 6-8 are important for binding to Ail. Binding of C4BP was unaffected by increasing heparin or salt concentrations, suggesting primarily nonionic interactions. These results indicate that Y. pseudotuberculosis Ail recruits C4BP in a functional manner, facilitating resistance to attack from complement.     

Yersinia genome diversity disclosed by Yersinia pestis genome-wide DNA microarray

The genus Yersinia includes 11 species, 3 of which (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) are pathogenic for humans. The remaining 8 species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) are merely opportunistic pathogens found mostly in the environment. In this work, the genomic differences among Yersinia were determined using a Y. pestis-specific DNA microarray. The results revealed 292 chromosomal genes that were shared by all Yersinia species tested, constituting the conserved gene pool of the genus Yersinia. Hierarchical clustering analysis of the microarray data revealed the genetic relationships among all 11 species in this genus. The microarray analysis in conjunction with PCR screening greatly reduced the number of chromosomal genes (32) specific for Y. pestis to 16 genes and uncovered a high level of genomic plasticity within Y. pseudotuberculosis, indicating that its different serotypes have undergone an extensively parallel loss or acquisition of genetic content, which is likely to be important for its adaptation to changes in environmental niches.