Transferable antibiotic resistance elements in Haemophilus influenzae share a common evolutionary origin with a diverse family of syntenic genomic islands

Transferable antibiotic resistance in Haemophilus influenzae was first detected in the early 1970s. After this, resistance spread rapidly worldwide and was shown to be transferred by a large 40- to 60-kb conjugative element. Bioinformatics analysis of the complete sequence of a typical H. influenzae conjugative resistance element, ICEHin1056, revealed the shared evolutionary origin of this element. ICEHin1056 has homology to 20 contiguous sequences in the National Center for Biotechnology Information database. Systematic comparison of these homologous sequences resulted in identification of a conserved syntenic genomic island consisting of up to 33 core genes in 16 beta- and gamma-Proteobacteria. These diverse genomic islands shared a common evolutionary origin, insert into tRNA genes, and have diverged widely, with G+C contents ranging from 40 to 70% and amino acid homologies as low as 20 to 25% for shared core genes. These core genes are likely to account for the conjugative transfer of the genomic islands and may even encode autonomous replication. Accessory gene clusters were nestled among the core genes and encode the following diverse major attributes: antibiotic, metal, and antiseptic resistance; degradation of chemicals; type IV secretion systems; two-component signaling systems; Vi antigen capsule synthesis; toxin production; and a wide range of metabolic functions. These related genomic islands include the following well-characterized structures: SPI-7, found in Salmonella enterica serovar Typhi; PAP1 or pKLC102, found in Pseudomonas aeruginosa; and the clc element, found in Pseudomonas sp. strain B13. This is the first report of a diverse family of related syntenic genomic islands with a deep evolutionary origin, and our findings challenge the view that genomic islands consist only of independently evolving modules.     

Genome-wide identification of novel genomic islands that contribute to Salmonella virulence in mouse systemic infection

Salmonella pathogenicity islands are inserted into the genome by horizontal gene transfer and are required for expression of full virulence. Here, we performed tRNA scanning of the genome of Salmonella enterica serovar Typhimurium and compared it with that of nonpathogenic Escherichia coli in order to identify genomic islands that contribute to Salmonella virulence. Using deletion analysis, we identified four genomic islands that are required for virulence in the mouse infection model. One of the newly identified pathogenicity islands was the pheV- tRNA-located genomic island, which is comprised of 26 126 bp, and encodes 22 putative genes, including STM3117–STM3138. We also showed that the pheV tRNA-located genomic island is widely distributed among different nontyphoid Salmonella serovars. Furthermore, genes including STM3118–STM3121 were identified as novel virulence-associated genes within the pheV- tRNA-located genomic island. These results indicate that a Salmonella -specific pheV- tRNA genomic island is involved in Salmonella pathogenesis among the nontyphoid Salmonella serovars.     

Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13

Genomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICE clc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICE clc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICE clc can target a number of different tRNA ^Gly genes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICE clc based on the attR sequence, and we could show that ICE clc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICE clc is excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, including Bordetella petri , Pseudomonas aeruginosa and Burkholderia .     

Self-transmissibility of the integrative and conjugative element ICEPm1 between clinical isolates requires a functional integrase, relaxase, and type IV secretion system

Integrative and conjugative elements (ICEs), which are chromosomal mobile elements, can conjugatively transfer between bacteria. Recently, we identified a genomic island of Proteus mirabilis, a common agent of catheter-associated urinary tract infection (UTI), that possesses all the properties consistent with an ICE. This element, designated ICEPm1, is highly conserved in other causative agents of UTI, suggesting its mobility. We demonstrate that ICEPm1 can actively excise from the chromosome in a clonal population of bacteria and that this excision is integrase dependent. Although in P. mirabilis HI4320, ICEPm1 is annotated as integrated into the phenylalanine tRNA gene pheV, we show that ICEPm1 can integrate into either pheV or pheU. We determined that ICEPm1 transfers at a frequency of 1.35 × 10(-5) transconjugants/donor to ICEPm1-deficient P. mirabilis using plate mating assays with clinical isolates. Insertional inactivation of a putative integrase gene on ICEPm1 decreased transfer frequencies of ICEPm1 to below the limit of detection. Mutation of the relaxase of ICEPm1 also eliminates transfer and demonstrates that this element is indeed self-transmissible and not transferred in trans, as are some mobilizable genomic islands. Together, these findings clearly demonstrate that ICEPm1 can actively excise from the chromosome in an integrase-dependent manner, dynamically integrate into both phenylalanine tRNA genes, and transfer into clinical strains using its own conjugation machinery.     

Composition,acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7

Vi capsular polysaccharide production is encoded by the viaB locus, which has a limited distribution in Salmonella enterica serovars. In S. enterica serovar Typhi, viaB is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNA(pheU) sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of viaB-associated DNA in Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin isolates revealed the presence of similar SPI-7 islands. In S. enterica serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNA(pheU) site were absent. In S. enterica serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of S. enterica serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other Salmonella serovars examined, 24 contained no insertions at the equivalent tRNA(pheU) site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA(pheU)-phoN junction, which was not analyzed further. Sequence analysis of the SPI-7 region from S. enterica serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including Pseudomonas aeruginosa and Xanthomonas axonopodis pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.     

Sequence and functional analyses of Haemophilus spp. genomic islands

Background

A major part of horizontal gene transfer that contributes to the diversification and adaptation of bacteria is facilitated by genomic islands. The evolution of these islands is poorly understood. Some progress was made with the identification of a set of phylogenetically related genomic islands among the Proteobacteria, recognized from the investigation of the evolutionary origins of a Haemophilus influenzae antibiotic resistance island, namely ICEHin1056. More clarity comes from this comparative analysis of seven complete sequences of the ICEHin1056 genomic island subfamily.

Results

These genomic islands have core and accessory genes in approximately equal proportion, with none demonstrating recent acquisition from other islands. The number of variable sites within core genes is similar to that found in the host bacteria. Furthermore, the GC content of the core genes is similar to that of the host bacteria (38% to 40%). Most of the core gene content is formed by the syntenic type IV secretion system dependent conjugative module and replicative module. GC content and lack of variable sites indicate that the antibiotic resistance genes were acquired relatively recently. An analysis of conjugation efficiency and antibiotic susceptibility demonstrates that phenotypic expression of genomic island-borne genes differs between different hosts.

Conclusion

Genomic islands of the ICEHin1056 subfamily have a longstanding relationship with H. influenzae and H. parainfluenzae and are co-evolving as semi-autonomous genomes within the 'supragenomes' of their host species. They have promoted bacterial diversity and adaptation through becoming efficient vectors of antibiotic resistance by the recent acquisition of antibiotic resistance transposons.     

Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa

ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNA(Lys) gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of beta- and gamma-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNA(Lys)-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNA(Lys) gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.     

Novel type IV secretion system involved in propagation of genomic islands

Type IV secretion systems (T4SSs) mediate horizontal gene transfer, thus contributing to genome plasticity, evolution of infectious pathogens, and dissemination of antibiotic resistance and other virulence traits. A gene cluster of the Haemophilus influenzae genomic island ICEHin1056 has been identified as a T4SS involved in the propagation of genomic islands. This T4SS is novel and evolutionarily distant from the previously described systems. Mutation analysis showed that inactivation of key genes of this system resulted in a loss of phenotypic traits provided by a T4SS. Seven of 10 mutants with a mutation in this T4SS did not express the type IV secretion pilus. Correspondingly, disruption of the genes resulted in up to 100,000-fold reductions in conjugation frequencies compared to those of the parent strain. Moreover, the expression of this T4SS was found to be positively regulated by one of its components, the tfc24 gene. We concluded that this gene cluster represents a novel family of T4SSs involved in propagation of genomic islands.     

PIPS: pathogenicity island prediction software

The adaptability of pathogenic bacteria to hosts is influenced by the genomic plasticity of the bacteria, which can be increased by such mechanisms as horizontal gene transfer. Pathogenicity islands play a major role in this type of gene transfer because they are large, horizontally acquired regions that harbor clusters of virulence genes that mediate the adhesion, colonization, invasion, immune system evasion, and toxigenic properties of the acceptor organism. Currently, pathogenicity islands are mainly identified in silico based on various characteristic features: (1) deviations in codon usage, G+C content or dinucleotide frequency and (2) insertion sequences and/or tRNA genetic flanking regions together with transposase coding genes. Several computational techniques for identifying pathogenicity islands exist. However, most of these techniques are only directed at the detection of horizontally transferred genes and/or the absence of certain genomic regions of the pathogenic bacterium in closely related non-pathogenic species. Here, we present a novel software suite designed for the prediction of pathogenicity islands (pathogenicity island prediction software, or PIPS). In contrast to other existing tools, our approach is capable of utilizing multiple features for pathogenicity island detection in an integrative manner. We show that PIPS provides better accuracy than other available software packages. As an example, we used PIPS to study the veterinary pathogen Corynebacterium pseudotuberculosis, in which we identified seven putative pathogenicity islands.     

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.     

Comparative sequence analysis of the symbiosis island of Mesorhizobium loti strain R7A

The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.     

In silico comparison of pKLC102-like genomic islands of Pseudomonas aeruginosa

The genomic island pKLC102 first detected in Pseudomonas aeruginosa clone C strains can cross species barriers and exhibits the highest mobilization rate of a genomic island known to date. Homologous genomic islands of 81-108 kb in size were identified in the completely sequenced P. aeruginosa strains PA7, PA14, 2192, C3719 and PACS2, but not in strains PAO1 and LES. All pKLC102-like genomic islands are integrated in chromosomal tRNA(Lys) genes and share a syntenic set of more than 70 homologous ORFs, part of which are related to DNA replication or mobility genes. The conserved backbone has predilection sites for the uptake of island-specific gene cassettes. A major difference between the islands is the organization of the origin of replication oriV.     

Occurrence of islands in genomes of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> native isolates

Genomes of 184 Sinorhizobium meliloti native isolates were studied to test the occurence of islands Sme21T, Sme19T, and Sme80S previously described in the model strain Rm1021. This analysis was conducted using PCR methodology involving specific primers. It was demonstrated that, in the examined geographically distinct populations of S. meliloti from the Northern Caucasus (NCG) and the Aral Sea region (PAG), the strains containing genomic islands were observed with similar frequency (0.55 and 0.57, respectively). Island Sme80S, denoted as an island of “environmental adaptivity,” was identified predominantly (frequency of 0.38) in genomes of strains which exhibited a lower level of salt tolerance and was isolated in PAG, a modern center of introgressive hybridization of alfalfa subjected to salinity. Island Sme21T designated as “ancestral” was observed in genomes of strains isolated in NCG, the primary center of host-plant biodiversity, 10-fold more often than in strains from PAG. An island Sme19T, which predominantly carries genes encoding transposases, was observed in genomes of strains in both populations with average frequency of 0.10. The analysis of linkage disequilibrium (LD) based on the assessment of probability for detection of different islands combinations in genomes revealed an independent inheritance of islands in salt-sensitive strains of various geographic origin. In contrast, the absence of this trend was noted in the majority of the examined combinations of salt-tolerant strains. It was concluded that the structure of chromosome in PAG strains which predominantly possessed a salt-sensitive phenotype was subjected to active recombinant processes, which could predetermine the intensity of microevolutionary processes in bacterial populations and facilitate an adaptation of bacteria in adverse environmental effect.     

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.     

致病菌基因组岛的研究进展

细菌基因组岛是细菌基因组上的特定区域,和水平基因转移相关,具有一定的结构特点,常携带致病、耐药及与适应性等功能相关的基因。通过基因组岛在细菌间的移动,可以造成相关基因在细菌间的传播,在细菌生存和致病等过程中具有重要作用。目前已经可通过生物信息和分子生物学实验等方法对基因组岛进行预测和验证。通过对致病菌基因组岛的研究,可以阐释细菌致病性和耐药等重要功能的获得,对疾病进行溯源,在传染病预防控制中具有重要意义。     

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.     

The Pseudomonas aeruginosa Pathogenicity Island PAPI-1 Is Transferred via a Novel Type IV Pilus

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. The notable ability of P. aeruginosa to inhabit a broad range of environments, including humans, is in part due to its large and diverse genomic repertoire. The genomes of most strains contain a significant number of large and small genomic islands, including those carrying virulence determinants (pathogenicity islands). The pathogenicity island PAPI-1 of strain PA14 is a cluster of 115 genes, and some have been shown to be responsible for virulence phenotypes in a number of infection models. We have previously demonstrated that PAPI-1 can be transferred to other P. aeruginosa strains following excision from the chromosome of the donor. Here we show that PAPI-1 is transferred into recipient P. aeruginosa by a conjugative mechanism, via a type IV pilus, encoded in PAPI-1 by a 10-gene cluster which is closely related to the genes in the enterobacterial plasmid R64. We also demonstrate that the precursor of the major pilus subunit, PilS2, is processed by the chromosomally encoded prepillin peptidase PilD but not its paralog FppA. Our results suggest that the pathogenicity island PAPI-1 may have evolved by acquisition of a conjugation system but that because of its dependence on an essential chromosomal determinant, its transfer is restricted to P. aeruginosa or other species capable of providing a functional prepilin peptidase.The genomes of a number of microorganisms, primarily those that have a capability of changing and adapting to a wide range of environments, evolve by acquisition of novel genetic information in blocks of genes via a process referred to as horizontal gene transfer (HGT). Other bacterial species change their genetic repertoire minimally, principally those that have adapted to a particular environment and, in the case of pathogenic bacteria, to a specific host. For HGT-mediated acquisition of genes to occur, a recipient has to be in an environment where donor genetic material is available, such as different strains of the same species cohabitating a shared niche or growing in a large and diverse community of several hundred different microorganisms. Moreover, for bacteria to become successful recipients of foreign genetic material, they have to posses one of three mechanisms of HGT: natural competence for uptake of foreign DNA (transformation), the ability to be infected by transducing bacteriophages (transduction), or serving as recipients during conjugation of plasmids or mobilized chromosomal DNA (conjugation). Acquired genetic material can consist of individual genes, where they recombine into homologous sequences in the recipient genome and thus increase the genetic diversity. However, large blocks of hundreds of contiguous genes in elements called genomic islands can be also transferred between bacteria, allowing the recipient microorganisms to acquire a number of new traits by a single HGT event.Previous studies comparing genomes of the opportunistic pathogen Pseudomonas aeruginosa pointed toward HGT as an important factor in its evolution (23). The genomes of all strains sequenced to date contain a significant fraction of horizontally acquired genes, in genomic islands and prophages, consisting of a few to several hundred. These islands can be recognized by the presence of certain signature features, such as an atypical nucleotide composition relative to the rest of the genome, location within predicted sites of chromosomal integration (att sites), and the presence of genes encoding bacteriophages and conjugation machineries. We have recently demonstrated that PAPI-1, a large P. aeruginosa genomic (pathogenicity) island, can be excised from its tRNA att site and that a copy can be transferred into a recipient, where it integrates into the same tRNA gene (27). Inspection of the genes in PAPI-1 and features of the transfer process, namely, an integrase-dependent excision and formation of a circular intermediate, suggested that PAPI-1 is an integrative and conjugative element and that it is likely transferred by a conjugative mechanism.Here we extended our analysis of PAPI-1 by testing its transfer from a preselected group of P. aeruginosa PA14 mutants with insertions in each of the genes on the island. Among those mutants that were defective in PAPI-1 transfer, one group of genes encode homologs of type IV pilus proteins. While type IV pili have been found to be involved primarily in bacterial adhesion and twitching motility (24), the PAPI-1-encoded pilus is closely related to the conjugative apparatus of plasmid R64 (14). Moreover, we show that an essential posttranslational modification reaction, converting the precursor of the major pilin subunit encoded in PAPI-1 into a mature protein, is carried out by an enzyme encoded in the chromosome of the donor cells. The acquisition and adaptation of groups of genes and subsequent loss of an essential function may represent a novel evolutionary strategy, limiting horizontal transfer to a specific bacterial species.     

Excision and transfer of the Mesorhizobium loti R7A symbiosis island requires an integrase IntS, a novel recombination directionality factor RdfS, and a putative relaxase RlxS

The Mesorhizobium loti strain R7A symbiosis island is an Integrative Conjugative Element (ICE), herein termed ICEMlSymR7A, which integrates into a phetRNA gene. Integration reconstructs the phetRNA gene at one junction with the core chromosome, and a direct repeat of the 3-prime 17 bp of the gene is formed at the other junction. We show that the ICEMlSymR7AintS gene, which encodes an integrase of the phage P4 family, is required for integration and excision of the island. Excision also depended on a novel recombination directionality factor encoded by msi109 (rdfS). Constitutive expression of rdfS resulted in curing of ICEMlSymR7A. The rdfS gene is part of an operon with genes required for conjugative transfer, allowing co-ordinate regulation of ICEMlSymR7A excision and transfer. The excised form of ICEMlSymR7A was detectable during exponential growth but occurred at higher frequency during stationary phase. ICEMlSymR7A encodes homologues of the traR and traI genes of Agrobacterium tumefaciens that regulate Ti plasmid transfer via quorum sensing. The presence of a plasmid with cloned island traR traI2 genes resulted in excision of ICEMlSymR7A in all cells regardless of culture density, indicating that excision may be similarly regulated. Maintenance of ICEMlSymR7A in these cells depended on msi106 (rlxS) that encodes a putative relaxase. Transfer of the island to non-symbiotic mesorhizobia required intS, rlxS and rdfS. The rdfS and rlxS genes are conserved across a diverse range of alpha-, beta- and gamma-proteobacteria and identify a large family of genomic islands with a common transfer mechanism.     

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.     

Diversity of the abundant pKLC102/PAGI-2 family of genomic islands in Pseudomonas aeruginosa

The known genomic islands of Pseudomonas aeruginosa clone C strains are integrated into tRNA(Lys) (pKLC102) or tRNA(Gly) (PAGI-2 and PAGI-3) genes and differ from their core genomes by distinctive tetranucleotide usage patterns. pKLC102 and the related island PAPI-1 from P. aeruginosa PA14 were spontaneously mobilized from their host chromosomes at frequencies of 10% and 0.3%, making pKLC102 the most mobile genomic island known with a copy number of 30 episomal circular pKLC102 molecules per cell. The incidence of islands of the pKLC102/PAGI-2 type was investigated in 71 unrelated P. aeruginosa strains from diverse habitats and geographic origins. pKLC102- and PAGI-2-like islands were identified in 50 and 31 strains, respectively, and 15 and 10 subtypes were differentiated by hybridization on pKLC102 and PAGI-2 macroarrays. The diversity of PAGI-2-type islands was mainly caused by one large block of strain-specific genes, whereas the diversity of pKLC102-type islands was primarily generated by subtype-specific combination of gene cassettes. Chromosomal loss of PAGI-2 could be documented in sequential P. aeruginosa isolates from individuals with cystic fibrosis. PAGI-2 was present in most tested Cupriavidus metallidurans and Cupriavidus campinensis isolates from polluted environments, demonstrating the spread of PAGI-2 across habitats and species barriers. The pKLC102/PAGI-2 family is prevalent in numerous beta- and gammaproteobacteria and is characterized by high asymmetry of the cDNA strands. This evolutionarily ancient family of genomic islands retained its oligonucleotide signature during horizontal spread within and among taxa.