The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival.

Pathogenicity islands are chromosomal clusters of horizontally acquired virulence genes that are often found at tRNA loci. The selC tRNA locus of Escherichia coli has served as the site of integration of two distinct pathogenicity islands which are responsible for converting benign strains into uro- and enteropathogens. Because virulence genes are targeted to the selC locus of E.coli, we investigated the homologous region of the Salmonella typhimurium chromosome for the presence of horizontally acquired sequences. At this site, we identified a 17 kb DNA segment that is both unique to Salmonella and necessary for virulence. This segment harbors a gene, mgtC, that is required for intramacrophage survival and growth in low Mg2+ media. The mgtC locus is regulated by the PhoP/PhoQ two-component system, a major regulator of virulence functions present in both pathogenic and non-pathogenic bacterial species. Cumulatively, our experiments indicate that the ability to replicate in low Mg2+ environments is necessary for Salmonella virulence, and suggest that a similar mechanism is responsible for the dissemination and acquisition of pathogenicity islands in enteric bacteria.     

Excision of the Shigella resistance locus pathogenicity island in Shigella flexneri is stimulated by a member of a new subgroup of recombination directionality factors

Pathogenicity islands are capable of excision and insertion within bacterial chromosomes. We describe a protein, Rox, that stimulates excision of the Shigella resistance locus pathogenicity island in Shigella flexneri. Sequence analysis suggests that Rox belongs to a new subfamily of recombination directionality factors, which includes proteins from P4, enterohemorrhagic Escherichia coli, and Yersinia pestis.     

The SHI-3 Iron Transport Island of Shigella boydii 0-1392 Carries the Genes for Aerobactin Synthesis and Transport

In Shigella boydii 0-1392, genes encoding the synthesis and transport of the hydroxamate siderophore aerobactin are located within a 21-kb iron transport island between lysU and the pheU tRNA gene. DNA sequence analysis of the S. boydii 0-1392 island, designated SHI-3 for Shigella island 3, revealed a conserved aerobactin operon associated with a P4 prophage-like integrase gene and numerous insertion sequences (IS). SHI-3 is present at the pheU tRNA locus in some S. boydii isolates but not in others. The map locations of the aerobactin genes vary among closely related species. The association of the aerobactin operon with phage genes and mobile elements and its presence at different locations within the genomes of enteric pathogens suggest that these virulence-enhancing genes may have been acquired by bacteriophage integration or IS element-mediated transposition. An S. boydii aerobactin synthesis mutant, 0-1392 iucB, was constructed and was similar to the wild type in tissue culture assays of invasion and intercellular spread.     

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.     

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.     

Nested Deletions of the SRL Pathogenicity Island of Shigella flexneri 2a

In this study, we determined the boundaries of a 99-kb deletable element of Shigella flexneri 2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the Shigella resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS91 elements present at the S. flexneri homologs of the Escherichia coli genes putA and mdoA on NotI fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS91 elements, resulting in a single IS91 element spanning the two original IS91 loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10(-5) per cell in the wild type, and mutation of a P4-like integrase gene (int) at the left end of the SRL PAI revealed that int mediates precise deletion of the PAI.     

Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications.

The genes (selC) coding for the selenocysteine-inserting tRNA species (tRNA(Sec)) from Clostridium thermoaceticum and Desulfomicrobium baculatum were cloned and sequenced. Although they differ in numerous positions from the sequence of the Escherichia coli selC gene, they were able to complement the selC lesion of an E. coli mutant and to promote selenoprotein formation in the heterologous host. The tRNA(Sec) species from both organisms possess all of the unique primary, secondary, and tertiary structural features exhibited by E. coli tRNA(Sec) (C. Baron, E. Westhof, A. Böck, and R. Giegé, J. Mol. Biol. 231:274-292, 1993). The structural and functional properties of the tRNA(Sec) species from prokaryotes analyzed thus far support the notion that tRNA(Sec) may be an evolutionarily conserved structure whose function in the primordial genetic code was to decode UGA with selenocysteine.     

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 Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae

A pathogenicity island termed high-pathogenicity island (HPI) is present in pathogenic Yersinia. This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin. Recently, the HPI was also detected in various strains of Escherichia coli. In this study, the distribution of the HPI in the family Enterobacteriaceae was investigated. Among the 67 isolates pertaining to 18 genera and 52 species tested, nine (13.4%) harbored the island. These isolates were three E. coli, one Citrobacter diversus and five Klebsiella of various species (Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Klebsiella planticola, and Klebsiella oxytoca). As in Yersinia sp., all nine isolates synthesized the HPI-encoded iron-repressible proteins HMWP1 and HMWP2. In the K. oxytoca strain, the right-end portion of the HPI was deleted, whereas the entire core region of the island was present in the eight other enterobacteria strains analyzed. In most of these isolates, the HPI was bordered by an asn tRNA locus, as in Yersinia sp. This report thus demonstrates the spread of the HPI among various members of the family Enterobacteriaceae.     

Structure and genetics of Shigella O antigens

This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.     

Localization of the insertion site and pathotype determination of the locus of enterocyte effacement of shiga toxin-producing Escherichia coli strains

Of 220 Shiga toxin-producing Escherichia coli (STEC) strains collected in central France from healthy cattle, food samples, and asymptomatic children, 12 possessed the eae gene included in the locus of enterocyte effacement (LEE) pathogenicity island. Based on gene typing, we observed 7 different eae espA espB tir pathotypes among the 12 STEC strains and described the new espAbetav variant. As previously observed, the O157 serogroup is associated with eaegamma, O26 is associated with eaebeta, and O103 is associated with eaeepsilon. However, the unexpected eaezeta allele was detected in 5 of the 12 isolates. PCR amplification and pulsed-field gel electrophoresis using the I-CeuI endonuclease followed by Southern hybridization indicated that the LEE was inserted in the vicinity of the selC (three isolates), pheU (two isolates), or pheV (six isolates) tRNA gene. Six isolates harbored two or three of these tRNA loci altered by the insertion of integrase genes (CP4-int and/or int-phe), suggesting the insertion of additional foreign DNA fragments at these sites. In spite of great genetic diversity of LEE pathotypes and LEE insertion sites, bovine strains harbor alleles of LEE genes that are frequently found in clinical STEC strains isolated from outbreaks and sporadic cases around the world, underscoring the potential risk of the bovine strains on human health.     

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.     

Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1Tyr)

virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.     

EilA, a HilA-like regulator in enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a diarrhoeal pathogen in developing and industrialized countries. Most EAEC virulence factors thus far described are encoded on virulence plasmid pAA, yet recent completion of the EAEC genome has suggested the presence of additional factors encoded on chromosomal islands. Previous reports have recognized the presence of a type III secretion system (T3SS), designated ETT2, at the glyU locus of prototype EAEC strain 042, along with possible T3SS effectors at the selC locus. The selC locus was also noted to harbour homologues of Salmonella enterica regulator HilA and of invasin from Yersinia spp., yet previous publications suggested that these loci may be silent. Here, we show that the genes of the selC locus are present inconsistently among a collection of well-characterized EAEC strains. Notably, however, there was perfect correlation between the presence of hilA-homologue eilA and predicted Yersinia invasin homologue gene eaeX. We hypothesized that if expressed, the putative gene product EilA would contribute to EAEC virulence in part by activation of the T3SS and its effectors. An eilA mutant was constructed in EAEC strain 042, and complementation was achieved by cloning the eilA gene under control of an arabinose-dependent promoter. In this system, we observed expression of at least seven genes to be affected by expression of eilA, either directly or indirectly: selC locus genes eipB, eipC, eipD, eicA and eaeX (renamed here air), as well as glyU ETT2 genes eivF and eivA. Notably, the eilA mutant was shown to be less adherent to epithelial cells in culture and to form less abundant biofilms than the isogenic parent. These effects were recapitulated in the air mutant, suggesting that the predicted outer membrane protein product of the air gene is involved as an accessory adhesin and aggregin of EAEC, coexpressed with the T3SS. Our data suggest that the T3SS of EAEC and presumed effectors located on different chromosomal islands may be coordinately activated by EilA, which also activates the genetically linked high molecular weight bacterial surface protein Air. Contributions of this new putative virulence-related regulon in EAEC may include adherence, aggregation, and as yet uncharacterized roles for the T3SS.     

The ETT2 gene cluster, encoding a second type III secretion system from Escherichia coli, is present in the majority of strains but has undergone widespread mutational attrition

ETT2 is a second cryptic type III secretion system in Escherichia coli which was first discovered through the analysis of genome sequences of enterohemorrhagic E. coli O157:H7. Comparative analyses of Escherichia and Shigella genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3 Salmonella pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized E. coli reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of E. coli strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic E. coli O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion system-associated locus that likely encodes the ETT2 translocation apparatus was found in some E. coli strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative E. coli strain O42.     

A pseudo-tRNA modulates antibiotic resistance in Bacillus cereus

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNA(Other), which does not function in translation. Deletion of tRNA(Other) led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNA(Other). This suggested that tRNA(Other) might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3' and 5'-ends of tRNA(Other). tRNA(Other) expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5' leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNA(Other) may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell.     

Sip,an integrase protein with excision,circularization and integration activities,defines a new family of mobile Staphylococcus aureus pathogenicity islands

We report the complete sequence of Staphylococcal pathogenicity island bovine 2 (SaPIbov2), encoding the biofilm-associated protein Bap. SaPIbov2 contains 24 open reading frames, including sip, which encodes a functional staphylococcal integrase protein. SaPIbov2 is bordered by 18 bp direct repeats. The integration site into the chromosome lies at the 3' end of a gene encoding GMP synthase. SaPIbov2 has extensive similarity to previously described pathogenicity islands of Staphylococcus aureus. The principal difference is that toxin genes present in the other pathogenicity islands are exchanged for a transposon-like element that carries the bap gene and genes encoding an ABC transporter and a transposase. Also, SaPIbov2 can be excised to form a circular element and can integrate site-specifically and RecA-independently at a chromosomal att site in a Sip-dependent manner. This was demonstrated both in S. aureus and with plasmid substrates ectopically in Escherichia coli. Thus, SaPIbov2 encodes a functional recombinase of the integrase family that promotes element excision and insertion/integration. In addition, we demonstrated that the presence of SaPIbov2 facilitated the persistence of S. aureus in an intramammary gland infection model. Finally, different bovine isolates of S. aureus were found to carry islands related to SaPIbov2, suggesting the existence of a family of related pathogenicity islands.     

Occurrence and functional compatibility within Enterobacteriaceae of a tRNA species which inserts selenocysteine into protein.

The selC gene from E. coli codes for a tRNA species (tRNA(UCASer] which is aminoacylated with L-serine and which cotranslationally inserts selenocysteine into selenoproteins. By means of Southern hybridization it was demonstrated that this gene occurs in all enterobacteria tested. To assess whether the unique primary and secondary structural features of the E. coli selC gene product are conserved in that of other organisms, the selC homologue from Proteus vulgaris was cloned and sequenced. It was found that the Proteus selC gene differs from the E. coli counterpart in only six nucleotides, that it displays the same unique properties and that it is expressed and functions in E. coli. This indicates that the unique mechanism of selenocysteine incorporation is not restricted to E. coli but has been conserved as a uniform biochemical process.