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.     

Horizontal transfer of Yersinia high-pathogenicity island by the conjugative RP4 attB target-presenting shuttle plasmid

The high-pathogenicity island (HPI) encodes a highly efficient yersiniabactin system of iron acquisition responsible for mouse lethality in Yersinia. Although the HPI is widely disseminated among Enterobacteriaceae it lacks functions necessary for its replication and transmission. Therefore, the mechanism of its horizontal transfer and circulation is completely obscure. On the other hand, the HPI is a genetically active island in the bacterial cell. It encodes a functional recombinase and is able to transpose to new targets on the chromosome. Here we report on a possible mechanism of the HPI dissemination based on site-specific recombination of the excised HPI with the attB-presenting (asn tRNA gene) RP4 promiscuous conjugative shuttle plasmid. The resulting cointegrate can be transferred by conjugation to a new host, where it dissociates, and the released HPI integrates into any unoccupied asn tRNA gene target in the genome. This mechanism has been proven both with the 'mini' island carrying only the attP recognition site and genes coding for recombination enzymes and with the complete HPI labelled with an antibiotic resistance marker. After acquisition of the mobilized complete form of the HPI, the ability of the HPI-cured Yersinia enterocolitica WA-TH(-) strain to produce yersiniabactin has been restored. Such 'trapping' of pathogenicity islands and subsequent shuffling to new hosts by a conjugative replicon carrying a suitable attB site could be applied to other functional integrative elements and explain wide dissemination of PAIs.     

Organised Genome Dynamics in the Escherichia coli Species Results in Highly Diverse Adaptive Paths

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the ∼18,000 families of orthologous genes, we found ∼2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome''s long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome.     

Pathogenicity island integrase cross-talk: a potential new tool for virulence modulation

Instability and excision of pathogenicity islands (PAIs) have already been described in Escherichia coli 536. In this edition of Molecular Microbiology, Bianca Hochhut and colleagues from the University of Würzburg in Germany have shown that the instability of four of the E. coli 536 PAIs is mediated by a P4-type integrase encoded within the specific PAI by a site-specific recombination mechanism. The integrase encoded on PAI II(536) is able to mediate excision and integration of both PAI II(536), and also PAI V(536). The att sites of both these PAIs have a region of sequence similarity, which is also found in several other PAIs and in tRNA genes in several bacterial species. The cross-PAI activity of this integrase (Int(PAI II)) suggests that it plays an important role in both genome evolution and horizontal transfer of pathogenicity elements, possibly even across species barriers. Deletion of PAIs that carry genes for adhesins and other traits might lead to a phase variation-like phenomenon. Differential regulation of integrase activity or production might add a further level of fine-tuning during bacterial infection.     

Overexpression of the Lactobacillus plantarum peptidoglycan biosynthesis murA2 gene increases the tolerance of Escherichia coli to alcohols and enhances ethanol production

A major challenge in producing chemicals and biofuels is to increase the tolerance of the host organism to toxic products or byproducts. An Escherichia coli strain with superior ethanol and more generally alcohol tolerance was identified by screening a library constructed by randomly integrating Lactobacillus plantarum genomic DNA fragments into the E. coli chromosome via Cre-lox recombination. Sequencing identified the inserted DNA fragment as the murA2 gene and its upstream intergenic 973-bp sequence, both coded on the negative genomic DNA strand. Overexpression of this murA2 gene and its upstream 973-bp sequence significantly enhanced ethanol tolerance in both E. coli EC100 and wild type E. coli MG1655 strains by 4.1-fold and 2.0-fold compared to control strains, respectively. Tolerance to n-butanol and i-butanol in E. coli MG1655 was increased by 1.85-fold and 1.91-fold, respectively. We show that the intergenic 973-bp sequence contains a native promoter for the murA2 gene along with a long 5′ UTR (286 nt) on the negative strand, while a noncoding, small RNA, named MurA2S, is expressed off the positive strand. MurA2S is expressed in E. coli and may interact with murA2, but it does not affect murA2’s ability to enhance alcohol tolerance in E. coli. Overexpression of murA2 with its upstream region in the ethanologenic E. coli KO11 strain significantly improved ethanol production in cultures that simulate the industrial Melle-Boinot fermentation process.     

Isolation and Characterization of Supercoiled Circular Deoxyribonucleic Acid from Beta-Hemolytic Strains of Escherichia coli

Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 ± 0.1 μm, 24.0 ± 0.3 μm, and 29.5 ± 0.5 μm. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed.     

DNA modification and functional delivery into human cells using Escherichia coli DH10B

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a ~200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd^– rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.     

Phylogenetic Groups and Pathogenicity Island Markers in Fecal Escherichia coli Isolates from Asymptomatic Humans in China

The study of phylogenetic groups and pathogenicity island (PAI) markers in commensal Escherichia coli strains from asymptomatic Chinese people showed that group A strains are the most common and that nearly half of all fecal strains which were randomly selected harbor PAIs.Escherichia coli is a well-diversified commensal species in the intestine of healthy humans but also includes intestinal or extraintestinal pathogens. It has been reported that pathogenic E. coli may be derived from fecal strains by acquisition of virulence determinants (11). The relationship between the E. coli genetic background and the acquisition of virulence factors is now better understood (1, 5). Extraintestinal E. coli strains may harbor several virulence factors, such as adhesins, fimbriae, and hemolysin, which can contribute to bacterial pathogenesis. These traits are usually encoded on pathogenicity islands (PAIs), which have been studied in pathogenic E. coli previously (15). The E. coli population includes 4 major phylogroups (A, B₁, B₂, and D) (2). Pathogenic strains belong mainly to groups B₂ and D, while most fecal isolates belong to groups A and B₁. Strains of groups B₂ and D often carry virulence factors that are lacking in group A and B₁ strains (3, 9, 13).In this study, we examined the distribution of phylogroups and the prevalence of PAIs in commensal E. coli strains isolated from asymptomatic persons in one region of China.     

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.     

A new O-antigen gene cluster has a key role in the virulence of the Escherichia coli meningitis clone O45:K1:H7

A new highly pathogenic clone of Escherichia coli meningitis strains harboring the unusual serogroup O45 has recently emerged in France. To gain insight into the pathogenicity of this new clone, we investigated the possible role of antigen O45 in the virulence of strain S88 (O45:K1:H7), representative of this emerging clone. We first showed that the S88 O-antigen gene cluster sequence differs from that of O45 in the reference strain E. coli 96-3285, suggesting that the two O45 polysaccharides, while probably sharing a community of epitopes, represent two different antigens. The unique functional organization of the two O-antigen gene clusters and the low DNA sequence homology of the orthologous genes suggest that the two loci originated from a common ancestor and have since undergone multiple recombination events. Phylogenetic analysis based on the flanking gene gnd sequences indicates that the S88 antigen O45 (O45_S88) gene cluster may have been acquired, at least in part, from another member of the Enterobacteriaceae. Mutagenesis of the O45_S88 antigen gene cluster was used for functional analysis of the loci and revealed the crucial role of the O polysaccharide in S88 virulence in a neonatal rat meningitis model. We also developed a PCR method to specifically identify the O45_S88 antigen gene cluster. Together, our findings suggest that horizontal acquisition of a new O-antigen gene cluster, at least partly from another species, may have been a key event in the emergence and virulence of the E. coli O45:K1:H7 clone in France.     

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.     

Evolution of Pan-Genomes of Escherichia coli,Shigella spp., and Salmonella enterica

Multiple sequencing of genomes belonging to a bacterial species allows one to analyze and compare statistics and dynamics of the gene complements of species, their pan-genomes. Here, we analyzed multiple genomes of Escherichia coli, Shigella spp., and Salmonella enterica. We demonstrate that the distribution of the number of genomes harboring a gene is well approximated by a sum of two power functions, describing frequent genes (present in many strains) and rare genes (present in few strains). The virtual absence of Shigella-specific genes not present in E. coli genomes confirms previous observations that Shigella is not an independent genus. While the pan-genome size is increasing with each new strain, the number of genes present in a fixed fraction of strains stabilizes quickly. For instance, slightly fewer than 4,000 genes are present in at least half of any group of E. coli genomes. Comparison of S. enterica and E. coli pan-genomes revealed the existence of a common periphery, that is, genes present in some but not all strains of both species. Analysis of phylogenetic trees demonstrates that rare genes from the periphery likely evolve under horizontal transfer, whereas frequent periphery genes may have been inherited from the periphery genome of the common ancestor.     

Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4′-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies.     

Variation of the Ribosomal Operon 16S-23S Gene Spacer Region in Representatives of Salmonella enterica Subspecies

The 16S-23S spacer regions of two ribosomal operons (rrnA and rrnE) have been sequenced in seven representatives of the Salmonella enterica subspecies. Isolated nucleotide substitutions were found at the same sites as in Escherichia coli but the number of polymorphic sites was much larger, as could be expected for a more heterogeneous species. Still, as in E. coli, most of the variation found was due to insertions and/or deletions affecting blocks of nucleotides generally located at equivalent regions of the putative secondary structure for both species. Isolated polymorphic sites generated phylogenetic trees generally consistent with the subspecies structure and the accepted relationships among the subspecies. However, the sequences of rrnE put subspecies I closer to E. coli K-12 than to the other S. enterica subspecies. The distribution of polymorphisms affecting blocks of nucleotides was much more random, and the presence of equivalent sequences in distantly related subspecies, and even in E. coli, could reflect relatively frequent horizontal transfer. The smallest 16S-23S spacers in other genera of the family Enterobacteriaceae were also sequenced. As expected, the level of variation was much larger. Still, the phylogenetic tree inferred is consistent with those of 16S rRNA or housekeeping genes.     

Conjugal Transfer of the Pathogenicity Island ROD21 in Salmonella enterica serovar Enteritidis Depends on Environmental Conditions

Unstable pathogenicity islands are chromosomal elements that can be transferred from one bacterium to another. Salmonella enterica serovar Enteritidis (S. Enteritidis) is a pathogenic bacterium containing such unstable pathogenicity islands. One of them, denominated ROD21, is 26.5 kb in size and capable of excising from the chromosome in certain culture conditions, as well as during bacterial infection of phagocytic cells. In this study we have evaluated whether ROD21 can be effectively transferred from one bacterium to another. We generated a donor and several recipient strains of S. Enteritidis to carry out transfer assays in liquid LB medium. These assays showed that ROD21 is effectively transferred from donor to recipient strains of S. Enteritidis and S. Typhimurium. When Escherichia coli was used as the recipient strain, ROD21 transfer failed to be observed. Subsequently, we showed that a conjugative process was required for the transfer of the island and that changes in temperature and pH increased the transfer frequency between Salmonella strains. Our data indicate that ROD21 is an unstable pathogenicity island that can be transferred by conjugation in a species-specific manner between Salmonellae. Further, ROD21 transfer frequency increases in response to environmental changes, such as pH and temperature.     

The dnd operon for DNA phosphorothioation modification system in Escherichia coli is located in diverse genomic islands

Background

Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd⁺) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd⁺E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands.

Results

The dndBCDE genes (dnd operon) were detected in all Dnd⁺E. coli strains by PCR. The addition of thiourea improved the typeability of Dnd⁺E. coli strains to 100% using PFGE and the Dnd⁺ phenotype can be observed in both clonal and genetically diverse E. coli strains.Genomic analysis of 101 dnd operons from genome sequences of Enterobacteriaceae revealed that the dnd operons of the same bacterial species were generally clustered together in the phylogenetic tree. Further analysis of dnd operons of 52 E. coli genomes together with their respective immediate genetic environments revealed a total of 7 types of genetic organizations, all of which were found to be associated with genomic islands designated dnd-encoding GIs. The dnd-encoding GIs displayed mosaic structure and the genomic context of the 7 islands (with 1 representative genome from each type of genetic organization) were also highly variable, suggesting multiple recombination events. This is also the first report where two dnd operons were found within a strain although the biological implication is unknown. Surprisingly, dnd operons were frequently found in pathogenic E. coli although their link with virulence has not been explored.

Conclusion

Genomic islands likely play an important role in facilitating the horizontal gene transfer of the dnd operons in E. coli with 7 different types of islands discovered so far.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1421-8) contains supplementary material, which is available to authorized users.     

Movement of DNA sequence recognition domains between non-orthologous proteins

Comparisons of proteins show that they evolve through the movement of domains. However, in many cases, the underlying mechanisms remain unclear. Here, we observed the movements of DNA recognition domains between non-orthologous proteins within a prokaryote genome. Restriction–modification (RM) systems, consisting of a sequence-specific DNA methyltransferase and a restriction enzyme, contribute to maintenance/evolution of genomes/epigenomes. RM systems limit horizontal gene transfer but are themselves mobile. We compared Type III RM systems in Helicobacter pylori genomes and found that target recognition domain (TRD) sequences are mobile, moving between different orthologous groups that occupy unique chromosomal locations. Sequence comparisons suggested that a likely underlying mechanism is movement through homologous recombination of similar DNA sequences that encode amino acid sequence motifs that are conserved among Type III DNA methyltransferases. Consistent with this movement, incongruence was observed between the phylogenetic trees of TRD regions and other regions in proteins. Horizontal acquisition of diverse TRD sequences was suggested by detection of homologs in other Helicobacter species and distantly related bacterial species. One of these RM systems in H. pylori was inactivated by insertion of another RM system that likely transferred from an oral bacterium. TRD movement represents a novel route for diversification of DNA-interacting proteins.