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Journal of Bacteriology, June 2006, p. 4037-4050, Vol. 188, No. 11
0021-9193/06/$08.00+0     doi:10.1128/JB.02000-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Acquisition and Evolution of the exoU Locus in Pseudomonas aeruginosa

Bridget R. Kulasekara,1,{dagger} Hemantha D. Kulasekara,1,{ddagger} Matthew C. Wolfgang,1,§ Lisa Stevens,1 Dara W. Frank,2 and Stephen Lory1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115,1 Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, Wisconsin 532262

Received 31 December 2005/ Accepted 22 March 2006


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ABSTRACT
 
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 tRNALys 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 ß- and {gamma}-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNALys-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 tRNALys 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.


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INTRODUCTION
 
Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium with the ability to survive in diverse ecological niches. It is also an opportunistic pathogen that causes a wide variety of acute and chronic infections in individuals with compromised host defenses and those with cystic fibrosis (7). Infections by P. aeruginosa are important in a variety of clinical settings and account for 10% to 20% of nosocomial infections. The outcome of hospital-acquired pneumonia caused by P. aeruginosa is especially severe as it has a high mortality rate (3). The genome of this organism encodes a variety of secreted virulence factors utilizing dedicated secretion systems (46). Those secreted by the type III secretion system (TTSS) appear to play a major role in the outcome of infection, particularly for acute pneumonias (21, 41). The TTSS is a specialized protein-targeting system whereby the bacterial secreted factors (the so-called effectors) are directly introduced into the host cell though an injection-like apparatus. A number of gram-negative bacterial pathogens use this secretion mechanism, and based on the conserved sequences of the components of the secretion machineries, they appear to function using a conserved mechanism (37). The consequences of the action of the TTSS are determined by the specific biochemical activities of the individual effectors on the host cell. Most strains of P. aeruginosa carry genes encoding a functional TTSS and a combination of three effectors (13, 48). Among these, genes encoding exoenzyme T (ExoT) and exoenzyme Y (ExoY) are conserved in the genomes of nearly all P. aeruginosa strains regardless of their origin, while most strains carry the gene for either ExoS or ExoU but not both (4, 13, 48). The carriage of exoS is more prevalent among P. aeruginosa isolates. ExoS is a bifunctional cytotoxic protein, with the carboxy-terminal half specifying an ADP-ribosyltransferase activity targeting the Ras family of small G proteins that causes cell death (17, 18). The amino-terminal domain is a GTPase-activating protein directed toward the Rho family of low-molecular-weight GTPase proteins (16). The exoU gene has been shown to occur in a significant fraction of strains responsible for hospital-acquired pneumonia, keratitis, and ear infections (21, 31, 45). ExoU is a potent cytototoxin with phospholipase A2 activity (43). Although the precise role of ExoU in the various infections by P. aeruginosa is not known, this effector has been associated with the development of septic shock in an animal model (29).

The basis for the incompatibility of exoU and exoS within the same P. aeruginosa genome is not known. Interestingly, exoU and exoS do not reside at the same locus (14, 22, 46). Several lines of evidence have indicated that the determinants for expression and secretion of ExoU (the exoU gene and the spcU gene encoding its cognate chaperone [15]) are located within a genomic island. Genomic islands are segments of bacterial genomes that have been acquired through horizontal gene transfer. They often are integrated at sites adjacent to tRNA genes, are flanked by direct repeats, display a percent G+C and codon usage that are different from those of the core genome, and contain mobile genetic elements (9, 44). Genomic islands are subject to rearrangements, and over time, many of the genes originally present may be absent or mutated. Rearrangements may also result in translocation of a portion of an island to a different location on the chromosome (26).

Southern blot analysis of exoU-containing strains suggested that the exoU/spcU locus is linked to a highly polymorphic region (14). This observation implied that the exoU and spcU genes are located within a region of the chromosome associated with genomic plasticity, a conjecture that was also supported by microarray analysis of genomic differences between various P. aeruginosa strains (48). The percent G+C of exoU/spcU is 58.8, well below 66.7, the mean percent G+C of the PAO1 genome. Finally, in some isolates not carrying ExoU, the region of the core genome exoU/spcU borders (adjacent to the PA0988 homolog) is separated by less than 1 kbp from a tRNA gene (25).

Association of horizontally transferred DNA with tRNA or tmRNA genes is believed to be a result of the activities of specific integrases that carry out site-specific recombination between attP of the acquired genetic element and the 3' end of a tRNA gene (attB). As a result, the newly integrated genetic element is flanked by a tRNA gene (attL) and the 3' end of a tRNA gene (attR) (24). In P. aeruginosa, two identical tRNALys genes adjacent to PAO1 homologs PA0976 and PA4541 can be sites for plasmid integration and are often associated with genomic islands (23, 25, 26). In strain PAO1, the 3' region adjacent to the open reading frame (ORF) PA0976 contains an 8.9-kbp genomic island (from PA0977 to PA0987) encoding nine hypothetical unknown proteins, a colicin protein, and a neighboring immunity protein (25, 46). We have previously used yeast recombinational cloning to demonstrate that in two unrelated P. aeruginosa strains, the exoU/spcU pair is found on an 81-kbp DNA segment flanked by tRNALys and homologs of P. aeruginosa PAO1 genes PA0976 and PA0988. In strain PA14, this region is occupied by a 14-kbp genomic (pathogenicity) island called PAPI-2, containing the coding sequences for ExoU and SpcU (23).

In certain isolates of P. aeruginosa, this tRNALys site proximal to the PA0976 homolog serves as a site for the reversible integration of plasmids. For example, in strains of lineage K (a common clone found in Europe), plasmid pKLK106 can be integrated either at this tRNALys site or at an identical tRNALys gene located adjacent to the PA4541 homolog. In P. aeruginosa clone C strains (another common clone found in Europe), this tRNALys site proximal to the PA0976 homolog is occupied by the genomic island PAGI-4. However, it was found that in these same strains, plasmid pKLC102, closely related to pKLK106, can integrate into the tRNALys gene adjacent to the PA4541 homolog (26). The integration event of pKLK106 or pKLC102 in all strains is accompanied by a direct repeat flanking the integrated plasmids, consisting of the 3' end of the tRNALys gene (25).

In this paper, we have probed the chromosomal regions that exoU and spcU occupy in four strains isolated from geographically distant locations. Using yeast recombinational cloning, four additional unique exoU/spcU-containing genomic islands were identified, and three were sequenced and annotated. This allowed us to deduce the evolutionary history of these islands that may have originated from a single ancestral mobile genetic element.


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MATERIALS AND METHODS
 
Strains and growth conditions. The source of each P. aeruginosa strain, containing exoU, is as follows: strains 6077 and 19660 are from ocular infections from Berkeley, CA; strain X13273 is a blood isolate from Seattle, WA; strains S54485, JJ692, and U2504 are urinary tract isolates from Seattle, Washington, Minneapolis, Minnesota, and Gainesville, Florida, respectively. Antibiotic concentrations used for Escherichia coli were 10 µg/ml for gentamicin, 5 µg/ml for tetracycline, and 50 µg/ml for streptomycin. For P. aeruginosa, gentamicin was used at a concentration of 30 µg/ml and tetracycline was used at a concentration of 60 µg/ml. Luria broth (LB) and LB agar were used if the medium used is not indicated.

Yeast recombinational cloning. The recombinational cloning vector pLLX13 and techniques used have been described by Wolfgang et al. (48). pLLX13 was modified to contain the flanking targeting sequences of the tRNALys-associated hypervariable region, including PA0975 and PA0989, and is called p0975-0989capture (48). Following successful recombinational cloning, the vectors were referred to by the name of the strain used for cloning, followed by "pCap0976.1."

Generation of transposon insertions in pCap0976.1:6077 and introduction of mutated genes into the JJ692 chromosome. The mariner-based transposon, on plasmid pBT20, was used to generate transposon insertions (28). A library of random transposon insertions in pCap0976.1:6077 was generated by combining Escherichia coli GeneHogs/pCap0976.1:6077 in phosphate-buffered saline (PBS) at an optical density at 600 nm (OD600) of 20, with E. coli SM10 {lambda} pir/pBT20 at an OD600 of 40. Five 50-µl amounts were incubated on dry LB agar plates at 37°C for 2 h. Spots were resuspended in 1 ml of 1x PBS, and 200 µl was plated onto LB plates containing streptomycin and gentamicin. A total of 40,000 colonies were recovered and used for purification of plasmid DNA. The resulting plasmids were reelectroporated into E. coli GeneHogs, and transformants were plated on gentamicin- and tetracycline-containing media to select for pCap0976.1:6077-containing transposon insertions. Thirty-two E. coli GeneHogs p0976.1:6077::BT20 isolates were individually mated into P. aeruginosa JJ692/pSW(I-SceI) (50) and resuspended in 1x PBS. Dilutions were plated onto minimal medium A with gentamicin (30 µg/ml) agar plates (6) in order to isolate single colonies. Double recombination events were verified by loss of tetracycline resistance present on the vector. Insertions were mapped using semirandom PCR (28) and sequencing of each amplicon.

Sequencing of the ExoU islands. ExoU island B was sequenced using randomly subcloned pUC19 libraries, generated from the P. aeruginosa-specific portion of pCap0976.1:19660. Sequence gaps were closed using custom primers. To sequence ExoU island A, a cosmid library of 6,077 genomic DNAs was first constructed, consisting of ~40-kbp inserts in the sCos-DBI vector (49). Cosmids were screened for the presence of exoU and EXA24 by using colony hybridization of PCR products specific for these two chromosomal regions. Two cosmids hybridizing to the probes were identified that spanned from 31,460 bp to 80,000 bp of ExoU island A. Two random M13 libraries were constructed from these two cosmids and were subsequently sequenced. The remaining portion was sequenced by creating a pUC19 library using the DNA from pCap0976.1:6077. As before, sequencing gaps were closed using custom primers. The sequence of ExoU island C was generated by sequencing EcoRI and PstI fragments of pCap0976.1:X13273 subcloned into pUC19 and by using custom primers. Sequencing reads were assembled and viewed using Phred, Crossmatch, Phrap, and Consed (10, 11, 19).

ORF prediction and annotation. Putative ORFs were predicted using, first, Glimmer (42) trained on the P. aeruginosa PAO1 complete genome sequence (46) and, subsequently, GeneMark (32). The minimum number of amino acids used for a predicted coding sequence was 30. The first possible start codon was used for all ORFs except when an alternate start codon was used by homologous coding sequences. Predicted protein sequences were annotated using the BLAST algorithm (1) to search the complete NCBI database on 5 March 2006. CDD (33) was used to find putative domains. Only conserved domains producing alignments greater than 65% are included. If more than one significant alignment was produced per protein sequence, then the domain with the smallest E value (expected number of high-scoring segmented pairs with a score greater than or equal to 5 [1]) is listed.

PFGE. P. aeruginosa strains were grown to an OD600 of 0.5 to 1.5 in LB. Cultures were resuspended in an equal volume of phosphate-buffered saline and mixed with equal volumes of 1% low-melting-point agarose dissolved in phosphate-buffered saline. Eighty-microliter volumes were added to individual plug molds. Once solidified, the plugs were lysed as described by Romling et al. (40), after which the lysis solution was replaced with 10 mM Tris-Cl and 10 mM EDTA. The plugs were washed once in water and three times in SpeI digestion buffer (New England Biolabs) at 55°C for 1 h each, after which 30 units of SpeI was added to the plug combined with 300 µl of SpeI buffer and incubated overnight. Half of a plug was used for pulsed-field gel electrophoresis (PFGE). The marker used was the Lambda Ladder PFG marker from New England Biolabs. Conditions for PFGE were 1% agar and 0.5x Tris-borate-EDTA buffer at 14°C and 6 V per cm. The initial switch time was 0.22 s, and the final switch time was 54.17 s, with an angle of 120° and a total time of 25.6 h.

Nucleotide sequence accession numbers. The nucleotide sequences of ExoU islands A, B, and C have been deposited in the GenBank database and assigned the accession numbers DQ437742, DQ437743, and DQ437744, respectively.


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RESULTS
 
Genomic variability among ExoU/SpcU-containing P. aeruginosa isolates. Six P. aeruginosa strains, 19660 (strain 1), X13273 (strain 2), S54485 (strain 3), 6077 (strain 4), JJ692 (strain 5), and U2504 (strain 6), were chosen for analysis of their ExoU/SpcU-encoding loci. These six strains were the ExoU-containing representatives from an 18-member panel, analyzed previously for genome variability by hybridization of genomic DNA to a P. aeruginosa PAO1 whole-genome DNA microarray (48). The phylogenetic relationships of the 18 strains used in this study showed a limited clonal association between the exoU/spcU-containing isolates and the rest of the strains carrying the exoS gene. Five of the six exoU/spcU-containing strains grouped within a distinct cluster, and three strains within this branch (strains 4 to 6) belonged to a separate cluster (with strains 4 and 6 being the most closely related). To further assess the relatedness of these strains, we employed PFGE of chromosomal DNA digested with the restriction endonuclease SpeI (Fig. 1A). Based on this analysis, strains 4 and 6 appear to be clonal variants as the majority of the restriction fragments are conserved. This result is thus in close agreement with the earlier microarray analysis (48).


Figure 1
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  		FIG. 1. Molecular analysis of the genome content of six ExoU-encoding strains. (A) Pulsed-field gel electrophoresis of SpeI-treated chromosomal DNA from the six strains. The assigned number of each strain is listed above the respective lane. (B) NcoI fingerprints of the recombinational cloning vector containing the captured regions between PA0975 and PA0989 homologs from six strains. Lanes are labeled with the assigned number of the strain that was used for cloning. The bands from vector DNA are indicated with an asterisk.

Capture of the exoU/spcU-containing hypervariable segments by yeast recombinational cloning. We have previously shown that in strains 4 and 5, the 81-kbp DNA segment, designated ExoU island A, occupies the PAO1 PA0977-to-PA0987 chromosomal region.

We employed yeast recombinational cloning (39, 48) with a capture vector carrying 1-kbp targeting sequences from PA0975 to PA0976 and PA0988 to PA0989 to isolate the exoU/spcU locus from four P. aeruginosa strains (strains 1, 2, 3, and 6). Restriction endonuclease (NcoI) fingerprinting revealed that strain 6 (in addition to strains 4 and 5) contains ExoU island A.

These data, together with those of the earlier hybridization study, indicate that strains 4 to 6 may have evolved from the same ancestral strain in spite of their origins from three geographically distant locations within the United States. The remaining three strains did not carry the 81-kbp ExoU island A and instead carried insertions of 60 kbp (strain 3), 32 kbp (strain 1), and 6 kbp (strain 2).

Sequence analysis of ExoU island A. We sequenced and annotated ExoU island A from strain 4. The 81.17-kbp segment is predicted to encode 77 open reading frames (Table 1 and Fig. 2). As with other genomic islands, the percent G+C (57.0) differed from that of the core genome (PAO1, 66.7). Another feature of ExoU island A which is common in genomic islands that are integrated next to a tRNA gene is the presence of a gene carrying an integrase (int), presumed to be responsible for incorporation of genetic elements into the tRNALys gene (47). The ExoU island A int gene is almost identical to the integrase gene that was identified at the same location on the reversibly integrating plasmids pKLC102 and pKLK106 (25, 26). Almost identical int genes also exist at the same location on genomic islands PAPI-1 and PAPI-2, both integrated at tRNALys genes in PA14 (23). A genetic pattern common to the tRNA-associated genomic islands of P. aeruginosa is created following integration of pKLC102 into the tRNALys gene in clone C strains. On the episomal form of pKLC102, attP is located between xerC (int gene) and the gene encoding a homolog of the chromosomal partitioning factor Soj. Following incorporation, xerC borders the end of the integrated plasmid proximal to the tRNA gene (attL), and soj is located at the opposite end, bordered by a duplicated 3' end of the tRNA gene (attR). This genetic organization is preserved in the highly homologous PAPI-1 and, additionally, in PAGI-2 and PAGI-3 (23, 26, 30). In ExoU island A, a duplication of the 3' end of the tRNALys gene at the opposite border and soj are absent, suggesting that the ancestral form of ExoU island A had undergone a deletion event following its chromosomal integration, resulting in the loss of the partial tRNA gene and soj. Deletion of these features may have had a stabilizing effect on the ancestral ExoU island A, fixing it permanently into the chromosome. The absence of a repeated partial tRNA gene would make excision from the chromosome, dependent on the partial tRNA duplication, impossible (47). Additionally, soj is homologous with a chromosomal partitioning gene that is likely important for the maintenance of a large self-replicating DNA element, such as the ancestral ExoU island plasmid.


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  		TABLE 1. Description of the features of ExoU island A


Figure 2
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  		FIG. 2. A schematic representation of the features of ExoU island A. Predicted genes and their relationships to horizontally transmissible genetic elements discussed in the text are depicted by arrows, pointing in the direction of transcription. Patterns and colors are assigned based on homology. Percent G+C is shown, based on a sliding 75-bp window. The gray shaded area indicates sequence conservation of the island borders with the core genome.

A unique 20-bp sequence flanks ExoU island A (Fig. 3). This sequence is 125 bp downstream of the tRNALys gene next to the PA0976 homolog (Fig. 2) and, on the opposite end of the island, is 172 bp downstream of spcU. A similar flanking sequence is observed in numerous genomic islands associated with the tRNALys gene. A sequence 90% identical to this repeat flanks PAPI-1 and pKLC102. In both cases, it is approximately 125 bp downstream from tRNALys (attL) and, on the opposite end, is only 29 bp downstream from attR. A sequence highly similar to the above-mentioned repeats also flanks the PAO1 tRNALys-associated genomic island, where it is also approximately 125 bp downstream from tRNALys (attL) but is 123 bp downstream from attR. A similar sequence is present 123 bp downstream of an unoccupied tRNALys (attB) that is proximal to the PA0976 homolog of strain K2 and that has served as an integration site for pKLK106 (accession number AF285422). This sequence also overlaps with 1 bp of the 3' portion of tRNALys proximal to PA4541 in strain PAO1. Given the ubiquitous presence of this repeat in tRNALys-associated genomic islands, we have named it the tRNALys-associated genomic island (TAGI) repeat. We speculate that the TAGI repeat sequence may be an accessory element necessary for integration and/or excision of these islands.


Figure 3
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  		FIG. 3. Alignment of the TAGI repeats with repeats flanking exoS and the sequence marking the exoS deletion in exoU-carrying strains. *, K2 attB is located next to the PA0976 homolog. **, In PAO1, this putative attB site is proximal to PA4541. Gray shading denotes nucleotides that are not conserved with the consensus sequence. The boxed region indicates the repeated sequence associated with exoS. Sequences for PAPI-1 were taken from a sequence with accession number AY273869. The coordinates for the left repeat are from 925 to 944, and the coordinates for the right repeat are from 108,786 to 108,805. The sequence for the left repeat of pKLC102 was taken from the sequence with accession number AF285426, and the coordinates are from 256 to 275. The sequence for the right repeat of pKLC102 was taken from the sequence with accession number AF285425, and the coordinates are from 507 to 526. The sequence from the PAO1 attB site was taken from the sequence with accession number AE004868 and the coordinates are from 117 to 98 (reverse and complement). The sequences from the sites flanking exoS in PAO1 were taken from accession number AE004801. For the left repeat, the coordinates are from 8,661 to 8,642 (reverse and complement), and for the right repeat, the coordinates are from 7,210 to 7,191 (reverse and complement). The remaining PAO1 sequences from the tRNALys-associated genomic island were taken from accession number AE004531. The coordinates for the left repeat are 1,812 to 1,831, and the coordinates for the right repeat are from 10,759 to 10,778. The sequence from PA14 was taken from ABQ07000001 and had coordinates from 162,446 to 162,475.

The relationship between ExoU island A and other P. aeruginosa tRNALys-associated genomic islands is further verified by examining ORFs encoded by ExoU island A. Homology searches of predicted protein sequences encoded by ExoU island A reveal a number of encoded proteins with unknown function, specifically EXA32 to EXA40, EXA43 to EXA44, and EXA46 to EXA48, that share both homology and synteny with a subset of genes located on the P. aeruginosa plasmid pKLC102 (Fig. 4) and genomic islands PAPI-1, PAGI-2, and PAGI-3 (23, 26, 30). Moreover, this set of genes is widely conserved in at least 10 species belonging to ß- and {gamma}-Proteobacteria, ranging from Ralstonia metallidurans to Haemophilus influenzae, and belongs to a larger set, comprised of 33 ORFs found among this group of Proteobacteria (34). Additionally, these conserved coding sequences are often located on genomic islands that in most cases share other characteristic features, such as association with a tRNA gene and the presence of an integrase. It has been postulated that this set of 33 ORFs may be involved in plasmid maintenance or horizontal gene transfer (26). Additional proteins encoded by ExoU island A are clearly associated with plasmid maintenance and transmission, based on their sequence similarity to proteins with known plasmid-associated functions. These include a putative plasmid stabilization factor (EXA15), several putative helicases (EXA3, EXA6, EXA8, EXA45, and EXA72), and a TraG/TraD family protein (EXA47).


Figure 4
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  		FIG. 4. An illustration of the homology and synteny between PAPI-1, pKLC102, and ExoU island A. Homologies were determined using the Pustell Matrix feature of MacVector, using a similarity score of 70% and a window size of 60 or 70 bp.

In addition to the absence of an attR site and soj, the presence of pseudogenes and partial components of operons also suggest that rearrangements and deletions have occurred. For example, two genes in ExoU island A are homologs of the Pseudomonas fluorescens biosynthesis operon for the antifungal compound pyoluteorin (36). As in P. fluorescens, these two genes are located next to each other and are transcribed in the same direction; however, the eight remaining ORFs present in P. fluorescens are absent in ExoU island A.

Genomic islands often encode several transposons and insertion sequences. Duplicated insertion sequences facilitate rearrangements and deletions in genomic islands (20) and have been shown to cause large-scale chromosomal inversions in P. aeruginosa (27). Several genes with homology to transposons or insertion sequences were identified in ExoU island A. Three ORFs, EXA9, EXA10, and EXA11, are homologous to the two genes comprising the IS407 element whose sequences in the PAO1 genome are represented by PA0986 and PA0987. EXA9 is homologous to PA0986, EXA10 is homologous to the 5' portion of PA0987, and EXA11 is homologous to the 3' portion of PA0987. IS407 has been identified in other species, such as Burkholderia cepacia (51) and Burkholderia mallei (8), in addition to being found in locations such as P. aeruginosa O-antigen biosynthetic clusters (39, 46). A 392-bp sequence, inclusive of the segment homologous to the 3' portion of PA0987, is repeated three times within this island (Fig. 2). The sequence is present as two direct repeats, inclusive of EXA11 and EXA75, and one inverted repeat, inclusive of EXA53. The three repeated sequences together share 74% nucleotide identity. Additionally, a 6.4-kbp sequence that apparently contains a remnant of a transposon (EXA24 to EXA29) is present in ExoU island A and is flanked by a 39-bp, 90%-conserved, inverted repeat. The region between the repeats includes a truncated transposase gene, EXA24, and two ORFs with homology to the 5' and 3' portions of a resolvase, EXA28 and EXA29.

Sequence analysis of ExoU island B. To determine whether exoU is at a conserved location, we used PCR to amplify the region that spans exoU and PA0988. All six exoU-carrying strains, with the exception of strain 1, gave identically sized products (data not shown). The absence of an amplicon suggests that in strain 1, the exoU gene is located at a unique site. We therefore sequenced the captured island from this strain and named it ExoU island B. Sequencing of ExoU island B showed that the 29.85-kbp segment contains 41 predicted ORFs and has a mean percent G+C of 56.8 (Table 2 and Fig. 5).


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  		TABLE 2. Description of the features of ExoU island B


Figure 5
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  		FIG. 5. An illustration of the features of ExoU island B and ExoU island C. The organization of this figure is the same as that described in the legend to Fig. 2.

In strain 1, the exoU/spcU pair resides in a different location relative to the PA0988 homolog from that of most exoU-containing strains, and exoU and spcU are located at approximately equal distances from either junction with PAO1 homologous DNA. Comparison of ExoU island B to the other ExoU-encoding islands reveals that ExoU island B is the most similar to PAPI-2, as the two islands contain blocks of homologous DNA segments. As with PAPI-2 and ExoU island A, ExoU island B contains many genes that are homologous to the region between PA0976 and PA0988 in PAO1. ExoU island B contains a truncated homologue of PA0977, EXB1, and homologues of PA0980 through PA0985 (EXB9, EXB10, EXB14, and EXB20-23). The homologue of PA0982 has apparently been disrupted by an ISPpu14-type transposon common in Pseudomonas putida (35), resulting in EXB14 and EXB20. Also, less conserved is a portion of IS407, homologous to PA0987, that is present in ExoU island A.

ExoU island B encodes the C-terminal end of an integrase (EXB2) highly similar to the integrase located on other tRNALys-associated genomic islands, pKLC102, pKLK106, PAPI-2, and ExoU island A. This portion of an integrase is approximately 30 amino acids longer than the C-terminal portion of the integrase located on the analogous PAO1 genomic island. Other genes that have DNA-associated or plasmid-related functions or are conserved hypothetical genes located on other P. aeruginosa tRNA-associated genomic islands are not located on this island. However, the unique 20-bp TAGI repeat that flanks ExoU island A, PAPI-1, and pKLC102 also flanks ExoU island B.

ExoU island B encodes several proteins that share sequence similarity to proteins of known function. A putative nitric oxide reductase, NorB (EXB34), dependent on quinol for the passage of electrons, and its regulator NorR (EXB33) are encoded by ExoU island B. A nitric oxide reductase is encoded within the core genome; however, this enzyme is dependent on cytochrome c for the passage of electrons (2, 46). Cytochrome c-dependent NorB is part of the denitrification pathway that sequentially reduces nitrate to diatomic nitrogen and is used by P. aeruginosa during growth under anaerobic conditions (52). A quinol-dependent nitric oxide reductase in P. aeruginosa has not been previously identified. Expression of quinol-dependent NorB is regulated by the nitric oxide-activated response regulator NorR in Ralstonia eutropha (38). Quinol-dependent nitric oxide reductase is encoded by the genomes of some pathogenic bacteria that do not have denitrification ability, such as Neisseria gonorrhoeae and Staphylococcus aureus (5).

Sequence analysis of ExoU island (islet) C. The sequence encoding ExoU/SpcU from strain 2 consists of a 3.89-kbp segment located between genes homologous to PA0976 and PA0988 (Fig. 5). This region contains exoU, spcU, and an additional ORF and is very similar to the extremities of ExoU island A. The island is 85.2% identical to ExoU island A for the first 256 bp. The remaining nucleotide sequence is 99.8% identical to the sequence from the right border of ExoU island A, upstream of the PA0988 homolog. Most likely, a repeat similar to the 392-bp repeat present in ExoU island A associated with IS407 facilitated a deletion in an ancestral island in strain 2, as this repeated sequence in ExoU island A forms the junction between the two different sequence homologies. The 20-bp TAGI repeat mentioned above also flanks ExoU island C.

A host-vector system for functional analysis of genomic islands. We have incorporated several features into the design of the vector p0975-0989capture used for recombinational cloning to allow targeted insertional mutagenesis of defined regions of a bacterial chromosome and facilitate generating libraries of mutants in a specific genomic island. The outline of this approach is shown schematically in Fig. 6A. Following capture of a genomic island by yeast recombinational cloning, the recombinant plasmid is propagated in E. coli, where it is subjected to transposon mutagenesis. The library of transposon insertions is then transferred into P. aeruginosa by conjugation. The P. aeruginosa strain carries a plasmid, expressing the I-SceI restriction endonuclease, which efficiently excises the insert DNA from the plasmid by cleavage at the two flanking I-SceI sites included in the capture vector. The linear DNA is then capable of integrating into the chromosome by double reciprocal recombination (50). Following selection for the resistance determinant carried by the transposon, a library of mutants is obtained which carries insertions limited to the genomic island. A number of different P. aeruginosa recipients can be used, including the strain which was the source of the captured genomic island and a different strain which carries an identical island.


Figure 6
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  		FIG. 6. (A) A schematic description of the technique used to introduce captured segment-specific mariner transposon insertions into the bacterial chromosome. TS1 and TS2 are targeting sequences used to capture a specific chromosomal segment by yeast recombinational cloning, bla is the carbenicillin resistance determinant, tetR is the tetracycline resistance determinant, cyh is the cycloheximide resistance determinant, oriT is the origin of transfer, and pI-SceI is the plasmid carrying the gene for the I-SceI restriction endonuclease. (B) Illustration of the mariner insertions in ExoU island A marked by arrows.

We have tested the utility of this system by using the captured ExoU island A from strain 4. We have generated a library of mariner transposon insertions in pCap0976.1:6077 that was subsequently introduced into P. aeruginosa strain 5 carrying plasmid pSW(I-SceI). Following selection on gentamicin-containing medium, individual clones were screened for the loss of tetracycline determinant, which indicated insertion of the island into the chromosome by double reciprocal recombination following excision of the entire insert fragment by I-SceI. Fifty to 90 percent of gentamicin-resistant colonies were sensitive to tetracycline. We isolated 38 clones and successfully amplified 31 inserts, which were sequenced to determine the junctions between the transposon and genomic DNA. The distribution of mariner insertions is shown in Fig. 6B. The relatively random distribution of the transposon insertions is the result of low target specificity of the mariner element. This strategy could be used as a general method for functional analysis of proteins encoded within genomic islands.


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DISCUSSION
 
Acquisition of genes encoding virulence traits through horizontal gene transfer is an important mechanism in the evolution of pathogenic bacteria. The cytotoxin ExoU and its cognate chaperone SpcU are encoded by the genomes of a significant fraction of P. aeruginosa isolates, particularly those associated with acute pneumonia and ocular infections. We have investigated the composition of the genomic environment associated with the exoU/spcU gene pair. We used yeast recombinational cloning to examine the exoU/spcU locus from four isolates and sequenced three of the four captured islands. A comparison of the ExoU island A, B, and C sequences to published sequences provided the basis for a conclusion that exoU and spcU were acquired through a mobile genetic element. A comparison of the relatedness and synteny of the genes in the largest ExoU-encoding island with plasmid pKLC102 and pathogenicity island PAPI-1 indicates a close evolutionary relationship. A schematic illustrating the homologous regions shared between ExoU island A, PAPI-I, and pKLC102 is shown in Fig. 4. Because conserved hypothetical gene clusters in ExoU island A appear to be a subset of those found in pKLC102, we hypothesize that ExoU island A was derived from an integrative plasmid related to pKLC102.

Sequence analysis indicates that the ancestral exoU/spcU-containing plasmid acquired several insertion sequences and transposons. Following chromosomal integration, the ancestral element also underwent deletions, resulting in elimination of a number of genes required for transmissibility and episomal maintenance. Moreover, the number of conserved segments in the ExoU islands varies greatly, yet each strain retains full-length exoU and spcU genes, suggesting that these strains were subjected to environmental pressures selecting for the ability to secrete functional ExoU. Since the strains selected for this study were previously identified as exoU carriers, we cannot exclude the possibility that in strains lacking exoU, islands or plasmids related to ExoU island A may carry other genes that are maintained in the chromosome due to different environmental selective pressures. Indeed, in strain PAO1, the location occupied by the various ExoU islands contains a segment apparently derived from the same ancestral plasmid element. Moreover, the pathogenicity island PAP1-1, although integrated at a different tRNALys locus, appears to be derived from the same ancestral plasmid related to plasmid pKLC102.

A model describing the evolutionary history of the various ExoU islands and their relationship to the ancestral integrative plasmid is shown in Fig. 7. The ancestral plasmid, related to pKLC102, initially acquired exoU/spcU likely through a horizontal gene transfer event. The invariant association of exoU/spcU with IS407 suggests that this set of genes may have transposed as part of a mobile element. Insertion of exoU/spcU into the pKLC102-like ancestral plasmid may have occurred in a different bacterial species belonging to ß- and {gamma}-Proteobacteria, followed by a subsequent transfer to a P. aeruginosa recipient, where it integrated into the tRNALys site. Certain strains of P. aeruginosa, such as PAO1, probably acquired a form of the ancestral pKLC102 lacking exoU/spcU that integrated into one of the two tRNALys genes. This explains the existence of two different lineages of genomic islands among different P. aeruginosa strains differing in the presence or absence of exoU. Once these elements were integrated, recombination events could have deleted portions of the plasmid, eliminating factors needed for autonomous plasmid maintenance, thus fixing them as permanent genomic islands in the chromosome. Although this model predicts acquisition of exoU and spcU through transposition onto the plasmid element, we cannot exclude the possibility that they were acquired after integration of the pKLC102-like plasmid into the P. aeruginosa chromosome, possibly as part of a mobile genetic element with IS407 as depicted in Fig. 7.


Figure 7
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  		FIG. 7. A model depicting the evolution of the ExoU islands in different P. aeruginosa strains. An ancestral transmissible plasmid acquires exoU and is transmitted into a recipient, where it undergoes various alterations, including deletions, inversions, and acquisition of additional insertion sequences and transposons, leading to the forms as they are found in strains analyzed in this work. A similar plasmid lacking exoU leads to a different lineage of P. aeruginosa exemplified by strain PAO1. In the inset box is an alternative route for acquiring the exoU determinant, with IS407, following integration of the ancestral plasmid into the recipient's genome.

Sequence comparison of the tRNALys-associated genomic islands also provided evidence enabling us to speculate on the absence of exoS in exoU/spcU-containing strains. We hypothesize that prior to acquisition of exoU/spcU, the recipient strains contained exoS, which was subsequently deleted due to genetic determinants encoded by the ancestral ExoU island A. The exoS gene appears to have been acquired by the P. aeruginosa genome prior to exoU/spcU because of its lack of association with a variable portion of the chromosome and relatively typical percent G+C content. Currently, less than 1% of P. aeruginosa isolates lack both exoS and exoU/spcU (13), leading us to believe that recipient strains, prior to acquisition of exoU/spcU, contained exoS due to evolutionary pressures. Because exoS currently shows no association with features of a genomic island, it appears that at the time of exoU/spcU acquisition, plasmid incompatibility factors could not have served as a basis for mutual exclusion of exoS and exoU/spcU. Thus, it appears that exoU/spcU-containing strains lack exoS due to a targeted deletion event caused by a product of a gene linked to exoU/spcU at the time of acquisition. We compared the 10-bp direct repeat flanking exoS that also marks the site of deletion in strains lacking exoS (data not shown) with the TAGI repeat and noticed a slight homology between the two sets of repeated sequences (Fig. 3). Because the TAGI repeat may be involved in site-specific recombinase-mediated excision and integration of tRNALys islands, we postulate that the recombinase acting on the TAGI repeat may also play a role in the excision of exoS.

We have compared the distributions of single nucleotide polymorphisms in exoU and spcU (Fig. 8). Although the sequences of these genes are highly conserved, a specific distribution of nucleotide polymorphisms can be identified. Clearly, exoU/spcU in ExoU island A and island C represents a clonal variant, with only one nucleotide difference. In contrast, the same gene pair in PAPI-2 and ExoU island B is more polymorphic, with numerous substitutions at different locations within the coding sequence representing a more evolved lineage of the ancestral strain.


Figure 8
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  		FIG. 8. Single nucleotide polymorphisms of exoU/spcU.

The ExoU/SpcU pair is encoded by a genomic island that is hypothesized to have evolved from an ancestral plasmid similar to pKLC102. The integrase gene on this plasmid is 93% identical to this gene on ExoU island A and has been demonstrated to have an ability to integrate into both tRNALys genes adjacent to PA0976 or PA4541 homologs. However, exoU/spcU has not been found on genomic islands integrated at the tRNALys gene next to the PA4541 homolog. Possibly, in strains that had the ability to act as recipients of the ancestral ExoU island A, this site was not available for integration. It is also equally likely that acquisition of the determinants of ExoU/SpcU expression may have been an extremely rare event, meaning that the majority of approximately 25% of strains containing this gene may be the result of clonal expansion from the original recipient strain of the ancestral plasmid. Transfer of exoU as a rare event supports the idea that the ancestral island was rapidly rendered irreversibly integrated into the chromosome, as this would prevent further transmission of the exoU-carrying element. ExoU/SpcU-carrying strains show many signs of being clonally related. Strains carrying ExoU have been shown to have a relatively conserved genetic repertoire, as they are usually serotypes O1, O10, and O11 (4, 12). We have shown that strains 4, 5, and 6, isolated from geographically diverse locations, are very closely related and have most likely diverged from the same ancestral isolate. In addition, strains 2 to 6 were clustered as being more closely related to each other than to other ExoS-containing isolates, and at least one strain, strain 2, has an exoU/spcU locus that appears to have had a large-scale deletion stemming from an ancestral island that appeared to be very similar to ExoU island A.


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ACKNOWLEDGMENTS
 
This work was supported by NIH grant GM068516 to S.L., and work in D.F.'s laboratory was supported by NIH grant AI49577.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. Phone: (617) 432-5099. Fax: (617) 738-7664. E-mail: stephen_lory{at}hms.harvard.edu. Back

{dagger} Present address: Seattle Biomedical Research Institute, 307 Westlake Avenue N, Seattle, WA 98109. Back

{ddagger} Present address: Department of Genome Sciences, University of Washington, 1705 NE Pacific, Seattle, WA 98195. Back

§ Present address: Department of Microbiology and Immunology and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599. Back


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Journal of Bacteriology, June 2006, p. 4037-4050, Vol. 188, No. 11
0021-9193/06/$08.00+0     doi:10.1128/JB.02000-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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