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Journal of Bacteriology logoLink to Journal of Bacteriology
. 2006 Jun;188(11):4037–4050. doi: 10.1128/JB.02000-05

Acquisition and Evolution of the exoU Locus in Pseudomonas aeruginosa

Bridget R Kulasekara 1,, Hemantha D Kulasekara 1,, Matthew C Wolfgang 1,§, Lisa Stevens 1, Dara W Frank 2, Stephen Lory 1,*
PMCID: PMC1482899  PMID: 16707695

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 β- and γ-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.


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.

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 λ 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 1× 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 1× 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 0.5× 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.

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).

FIG. 1.

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.

TABLE 1.

Description of the features of ExoU island A

EXA ORF Annotation result(s) Orientation Start codon position Stop codon position Amino acid length G+C (%) Accession no. of closest homolog Conserved domain search result
tRNALys N/Aa 1 76 N/A N/A N/A N/A
TAGI repeat N/A 202 221 N/A 45 N/A N/A
1a Putative integrase 1729 446 427 61 AAP84129 None
1b Putative regulator of excisionase activity + 1113 1822 229 61 AAP94702 None
2 Conserved hypothetical protein 3657 1726 643 58 AAP84130 None
3 Conserved hypothetical protein 5855 3894 653 51 ABA73546 None
4 Hypothetical protein 6280 5891 129 49 AAN29662 None
5 Hypothetical protein 6530 6249 93 52 None None
6 UvrD/REP DNA helicase 9050 6768 760 59 EAR47340 COG0210
7 Hypothetical protein 10423 9239 394 58 ABC92876 None
8 DNA helicase-related protein 17105 10440 2,221 60 AAM41383 cd00221
IS407 left-terminal inverted repeat (67% identical) N/A 17512 17560 N/A N/A N/A N/A
9 IS407 transposase OrfA similar to PA0986 + 17580 17843 87 54 AAG04375 Pfam01527
IS407-associated repeat conserved with positions 53587-53974 (inverted) and 77997-78377 N/A 18358 18688 N/A 57 N/A N/A
10 N terminus of IS407 transposase OrfB similar to the N terminus of PA0987 + 17867 18388 173 61 ZP_00970131 None
11 C terminus of IS407 transposase OrfB similar to the C terminus of PA0987 + 18421 18717 98 55 ZP_00968899 None
12 Hypothetical protein 19245 19015 76 52 None None
13 Hypothetical protein with homology to the C terminus of colicin M + 19450 20319 289 47 AAO54114 None
14 Hypothetical protein + 20375 20803 142 42 None None
15 Putative plasmid stabilization factor 21214 20840 124 60 ZP_00971292 Pfam05016
16 Putative transcriptional regulator 21550 21218 110 59 AAP22603 COG3609
17 Conserved hypothetical protein 23472 21958 504 59 ZP_00965522 None
18 Hypothetical protein 23822 23469 117 61 ZP_00965523 None
19 Conserved hypothetical protein 25204 23822 460 64 AAP22580 None
20 Conserved hypothetical protein 26160 25222 312 64 ZP_00965525 None
21 Conserved hypothetical protein 26591 26160 143 61 AAP84145 None
22 Hypothetical protein similar to PA0980 + 26972 27256 94 46 AAP82951 None
23 Conserved hypothetical protein similar to PA0981 + 27287 27916 209 46 AAG04370 None
Repeat conserved with positions 34864-34902 (inverted) N/A 28466 28504 N/A 51 N/A N/A
24 Tn1721 transposase, truncated 31463 28500 987 65 CAG15097 Pfam01526
25 Hypothetical protein similar to PA2223 + 31697 32725 342 48 AAP84179 None
26 Hypothetical protein similar to PA2222 + 32718 33404 228 50 AAP84180 None
27 Hypothetical protein similar to PA2224 + 33436 34161 241 50 AAP84181 None
28 Tn1721 resolvase, C terminus 34470 34216 84 66 CAG15096 Pfam02796
29 Tn1721 resolvase, N terminus 34774 34505 89 63 CAG15096 None
Repeat conserved with positions 28466-28504 (inverted) N/A 34864 34902 None 54 None None
30 Hypothetical protein + 35174 35392 72 56 AAP82955 None
31 Conserved hypothetical protein similar to PA0982 36048 35389 219 61 AAP84147 None
32 Conserved hypothetical protein 36329 36045 94 58 ZP_00971301 None
33 Conserved hypothetical protein 39268 36326 980 63 AAP84149 None
34 Conserved hypothetical protein 39711 39268 147 64 AAP84150 None
35 Conserved hypothetical protein 41194 39689 501 63 ZP_00971305 None
36 Conserved hypothetical protein 42062 41178 294 67 AAP22570 None
37 Conserved hypothetical protein 42718 42059 219 60 AAP84153 None
38 Conserved hypothetical protein 43101 42715 128 66 AAP84154 None
39 Conserved hypothetical protein 43468 43112 118 59 AAP84155 None
40 Conserved hypothetical protein 43725 43486 79 65 AAP84156 None
41 Conserved hypothetical protein 44060 43722 112 67 AAP84157 None
42 Conserved hypothetical protein 44453 44154 99 57 ZP_00971311 None
43 Conserved hypothetical protein + 44626 44937 103 49 AAP84159 None
44 Hypothetical protein 46090 44981 369 47 AAP22563 None
45 Conserved hypothetical protein 47702 46221 493 59 AAP84161 None
46 Conserved hypothetical protein 48459 47713 248 62 AAP84172 None
47 TraG/TraD family protein 50690 48459 743 64 ZP_00965538 Pfam02534
48 Hypothetical protein 50963 50694 89 61 ZP_00965539 None
49 Conserved hypothetical protein 51472 50972 166 66 AAP22559 None
50 Conserved hypothetical protein 52050 51469 193 64 AAP84175 None
51 Conserved hypothetical protein 52790 52035 251 65 ZP_00965541 None
52 Conserved hypothetical protein 53481 52801 226 63 AAP84178 None
IS407-associated repeat (inverted) conserved with positions 18358-18688 and 77997-78377 N/A 53587 53974 None 56 None None
53 Transposase-derived hypothetical protein 54557 53610 315 58 CAI46956 None
54 Hypothetical protein + 54534 54707 57 56 AAO64283 None
55 KatA, catalase isozyme A + 55063 56511 482 61 AAGO7624 cd00328
56 Conserved hypothetical protein + 56582 56755 57 43 AAN67283 None
57 Alpha/beta hydrolase family protein 57751 56894 285 44 EAR24768 COG0596
58 Putative glutathione S-transferase + 57782 58393 203 44 EAN67479 COG0625
59 Pirin-related protein + 58761 59501 246 44 CAD17595 COG1741
60 Amidohydrolase family protein + 59588 60187 199 49 EAN17461 CD01012
61 Putative outer membrane channel lipoprotein + 60378 61859 493 46 CAD14996 COG1538
62 Putative multidrug resistance protein A + 61849 63057 402 45 ABC37909 COG1566
63 Putative multidrug resistance protein B + 63073 64593 506 42 AAQ58753 None
64 Putative LysR binding protein + 64991 65881 296 40 BAC51446 Pfam03466
65 Pseudogene, halogenase PltM 66762 66173 None 62 None None
66 Pseudogene, transcriptional regulator PltR 67497 66898 None 61 None None
5′ portion of PA0979 N/A 67616 67811 None 58 None None
67 Conserved hypothetical protein 69988 67913 691 64 ABB05606 None
68 HlyD/EmrA family membrane protein 71012 69933 359 68 ABB05605 COG1566
69 Hypothetical protein 71294 71025 89 62 ABB05604 None
70 Hypothetical protein + 71550 72785 411 64 ZP_00283098 None
71 Hypothetical protein with homology to the C terminus of PA0710 + 73307 73495 62 47 AAG04099 None
72 Pseudogene, helicase family protein 75860 73596 None 63 ZP_00965545
73 O-methyl transferase family protein 77404 75959 481 64 AAP84189 COG4123
74 Conserved hypothetical protein, C terminus 77646 77434 70 64 ZP_00965547 None
IS407 left-terminal inverted repeat (65% identical) N/A 77714 77762 None 51 None None
75 Transposase-derived hypothetical protein + 77667 78344 225 61 AAC16022 None
IS407-associated repeat conserved with positions 18358-18688 and 53587-53974 (inverted) N/A 77997 78377 None 59 None None
76 ExoU, type III secretion effector protein + 78481 80544 688 59 AAC16023 Pfam01734
77 SpcU, ExoU chaperone + 80541 80954 137 56 AAC16024 None
TAGI repeat N/A 81117 81136 N/A 45 N/A N/A
N/A PA0988 + 81347 N/A N/A N/A N/A N/A
a

N/A, not applicable.

FIG. 2.

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.

FIG. 3.

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 β- and γ-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).

FIG. 4.

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).

TABLE 2.

Description of the features of ExoU island B

EXB ORF Annotation result(s) Orientation Start codon position Stop codon position Amino acid length G+C (%) Accession no. of closest homolog Conserved domain search result
1 Hypothetical protein similar to PA0977 476 198 92 57 AAG04366 None
TAGI repeat N/Aa 200 219 N/A 45 N/A N/A
2 Hypothetical protein similar to the C terminus of an integrase 952 443 169 61 AAP84129 None
3 Transposase-derived hypothetical protein 1142 957 61 54 ZP_00971320 None
4 Putative IS30 family transposase, N terminus + 1498 2232 244 57 ZP_00463497 None
5 Putative IS30 family transposase, C terminus + 2266 2847 193 55 ZP_00463497 Pfam00665
6 Transposase-derived hypothetical protein 3187 2813 124 61 ZP_00971320 None
7 Transposase-derived hypothetical protein 3435 3187 82 66 AAV82054 None
8 Putative transposase 3731 3432 99 60 ZP_00971321 Pfam01527
9 Hypothetical protein similar to PA0980 + 3918 4202 94 47 AAP82951 None
10 Hypothetical protein similar to PA0981 + 4239 4862 207 46 AAG04370 None
11 Hypothetical protein similar to RS08 from PAPI-2 5288 4968 106 50 AAP82953 None
12 Hypothetical protein similar to RS09 from PAPI-2 + 5400 5603 67 57 AAP82954 None
13 Hypothetical protein similar to RL019 from PAPI-1 + 5647 5877 76 55 AAP84146 None
14 Hypothetical protein similar to the C terminus of PA0982 6047 5874 57 60 AAP22574 None
15 GNAT family acetyltransferase + 6091 6540 149 53 AAP82956 Pfam00583
16 ISPpu14 transposase Orf3, C terminus 7616 6543 357 59 NP_746094 Pfam03050
17 ISPpu14 transposase Orf3, N terminus 8122 7577 181 59 NP_746094 None
18 ISPpu14 transposase Orf2 8482 8142 111 64 NP_746095 Pfam05717
19 ISPpu14 transposase Orf1 8724 8479 81 58 NP_746109 None
20 Hypothetical protein similar to the N terminus of PA0982 9305 8859 178 61 AAG04371 None
21 Putative transposase similar to PA0983 + 9340 9627 95 57 AAG04372 Pfam01527
22 Colicin immunity protein similar to PA0984 + 10168 10494 108 41 AAP84212 None
23 Putative pyocin S5 similar to PA0985 12015 10519 498 46 AAP84213 None
24 Hypothetical protein + 12348 12752 134 58 None None
IS407 left-terminal inverted repeat (69% identical) N/A 13022 13070 N/A 53 N/A N/A
25 Hypothetical protein similar to IS407 transposase OrfB and PA0987 + 13134 13856 240 62 ZP_00138569 Pfam00665
26 ExoU, type III effector protein + 13981 16044 687 59 AAC16023 Pfam01734
27 SpcU, ExoU chaperone + 16041 16454 137 56 AAC16024 None
28 RNA polymerase sigma-70 factor, extracytoplasmic function family 17275 16691 194 59 EAP51975 COG1595
29 Hypothetical protein 17705 17583 40 47 None None
30 Hypothetical protein 18363 18157 68 54 None None
31 Hypothetical protein + 18670 19020 116 66 None None
32 Putative oxygen-independent coproporphyrinogen III oxidase + 19438 20862 474 63 EAP35559 COG0635
33 Putative nitric oxide reductase transcriptional regulator 22498 20864 544 68 NP_522520.1 COG3604
34 Putative quinol-dependent nitric oxide reductase + 22625 24901 758 65 ABC27611 COG3256
35 Transposase-derived hypothetical protein + 25262 25504 80 56 AAP82949 None
36 Conserved hypothetical protein 26346 25681 221 61 BAE50869 None
37 Putative regulator of mercury resistance proteins + 26375 26902 148 57 AAD40337 cd01108
38 Hypothetical protein 27321 27163 164 60 None None
39 Putative transcriptional regulator 27983 27558 141 49 EAP09715 COG1733
40 Hypothetical protein + 27996 28118 40 52 None None
41 NADH:flavin oxidoreductase family protein + 28326 29462 378 54 ZP_00136272 cd02933
TAGI repeat N/A 29794 29813 N/A 45 N/A N/A
42 PA0988 + 30024 N/A N/A N/A N/A N/A
a

N/A, not applicable.

FIG. 5.

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.

FIG. 6.

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.

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 β- and γ-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.

FIG. 7.

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.

FIG. 8.

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.

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.

REFERENCES

  • 1.Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. [DOI] [PubMed] [Google Scholar]
  • 2.Arai, H., Y. Igarashi, and T. Kodama. 1995. The structural genes for nitric oxide reductase from Pseudomonas aeruginosa. Biochim. Biophys. Acta 1261:279-284. [DOI] [PubMed] [Google Scholar]
  • 3.Bergogne-Berezin, E. 2003. Pseudomonads and miscellaneous gram-negative bacilli, p. 2203-2217. In J. Cohen and W. Powderly (ed.), Infectious diseases, 2nd ed. Mosby, New York, N.Y.
  • 4.Berthelot, P., I. Attree, P. Plesiat, J. Chabert, S. de Bentzmann, B. Pozzetto, and F. Grattard. 2003. Genotypic and phenotypic analysis of type III secretion system in a cohort of Pseudomonas aeruginosa bacteremia isolates: evidence for a possible association between O serotypes and exo genes. J. Infect. Dis. 188:512-518. [DOI] [PubMed] [Google Scholar]
  • 5.Busch, A., B. Friedrich, and R. Cramm. 2002. Characterization of the norB gene, encoding nitric oxide reductase, in the nondenitrifying cyanobacterium Synechocystis sp. strain PCC6803. Appl. Environ. Microbiol. 68:668-672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Deretic, V., M. J. Schurr, and H. Yu. 1995. Pseudomonas aeruginosa, mucoidy and the chronic infection phenotype in cystic fibrosis. Trends Microbiol. 3:351-356. [DOI] [PubMed] [Google Scholar]
  • 8.DeShazer, D., D. M. Waag, D. L. Fritz, and D. E. Woods. 2001. Identification of a Burkholderia mallei polysaccharide gene cluster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant. Microb. Pathog. 30:253-269. [DOI] [PubMed] [Google Scholar]
  • 9.Dobrindt, U., B. Hochhut, U. Hentschel, and J. Hacker. 2004. Genomic islands in pathogenic and environmental microorganisms. Nat. Rev. Microbiol. 2:414-424. [DOI] [PubMed] [Google Scholar]
  • 10.Ewing, B., and P. Green. 1998. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8:186-194. [PubMed] [Google Scholar]
  • 11.Ewing, B., L. Hillier, M. C. Wendl, and P. Green. 1998. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8:175-185. [DOI] [PubMed] [Google Scholar]
  • 12.Faure, K., D. Shimabukuro, T. Ajayi, L. R. Allmond, T. Sawa, and J. P. Wiener-Kronish. 2003. O-antigen serotypes and type III secretory toxins in clinical isolates of Pseudomonas aeruginosa. J. Clin. Microbiol. 41:2158-2160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Feltman, H., G. Schulert, S. Khan, M. Jain, L. Peterson, and A. R. Hauser. 2001. Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiology 147:2659-2669. [DOI] [PubMed] [Google Scholar]
  • 14.Finck-Barbancon, V., J. Goranson, L. Zhu, T. Sawa, J. P. Wiener-Kronish, S. M. Fleiszig, C. Wu, L. Mende-Mueller, and D. W. Frank. 1997. ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol. Microbiol. 25:547-557. [DOI] [PubMed] [Google Scholar]
  • 15.Finck-Barbancon, V., T. L. Yahr, and D. W. Frank. 1998. Identification and characterization of SpcU, a chaperone required for efficient secretion of the ExoU cytotoxin. J. Bacteriol. 180:6224-6231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Fraylick, J. E., J. R. La Rocque, T. S. Vincent, and J. C. Olson. 2001. Independent and coordinate effects of ADP-ribosyltransferase and GTPase-activating activities of exoenzyme S on HT-29 epithelial cell function. Infect. Immun. 69:5318-5328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Fraylick, J. E., M. J. Riese, T. S. Vincent, J. T. Barbieri, and J. C. Olson. 2002. ADP-ribosylation and functional effects of Pseudomonas exoenzyme S on cellular RalA. Biochemistry 41:9680-9687. [DOI] [PubMed] [Google Scholar]
  • 18.Fraylick, J. E., E. A. Rucks, D. M. Greene, T. S. Vincent, and J. C. Olson. 2002. Eukaryotic cell determination of ExoS ADP-ribosyltransferase substrate specificity. Biochem. Biophys. Res. Commun. 291:91-100. [DOI] [PubMed] [Google Scholar]
  • 19.Gordon, D., C. Abajian, and P. Green. 1998. Consed: a graphical tool for sequence finishing. Genome Res. 8:195-202. [DOI] [PubMed] [Google Scholar]
  • 20.Hacker, J., and J. B. Kaper. 2000. Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54:641-679. [DOI] [PubMed] [Google Scholar]
  • 21.Hauser, A. R., E. Cobb, M. Bodi, D. Mariscal, J. Valles, J. N. Engel, and J. Rello. 2002. Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit. Care Med. 30:521-528. [DOI] [PubMed] [Google Scholar]
  • 22.Hauser, A. R., P. J. Kang, and J. N. Engel. 1998. PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence. Mol. Microbiol. 27:807-818. [DOI] [PubMed] [Google Scholar]
  • 23.He, J., R. L. Baldini, E. Deziel, M. Saucier, Q. Zhang, N. T. Liberati, D. Lee, J. Urbach, H. M. Goodman, and L. G. Rahme. 2004. The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes. Proc. Natl. Acad. Sci. USA 101:2530-2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Hou, Y. M. 1999. Transfer RNAs and pathogenicity islands. Trends Biochem. Sci. 24:295-298. [DOI] [PubMed] [Google Scholar]
  • 25.Kiewitz, C., K. Larbig, J. Klockgether, C. Weinel, and B. Tummler. 2000. Monitoring genome evolution ex vivo: reversible chromosomal integration of a 106 kb plasmid at two tRNA(Lys) gene loci in sequential Pseudomonas aeruginosa airway isolates. Microbiology 146:2365-2373. [DOI] [PubMed] [Google Scholar]
  • 26.Klockgether, J., O. Reva, K. Larbig, and B. Tummler. 2004. Sequence analysis of the mobile genome island pKLC102 of Pseudomonas aeruginosa C. J. Bacteriol. 186:518-534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kresse, A. U., S. D. Dinesh, K. Larbig, and U. Romling. 2003. Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs. Mol. Microbiol. 47:145-158. [DOI] [PubMed] [Google Scholar]
  • 28.Kulasekara, H. D., I. Ventre, B. R. Kulasekara, A. Lazdunski, A. Filloux, and S. Lory. 2005. A novel two-component system controls the expression of Pseudomonas aeruginosa fimbrial cup genes. Mol. Microbiol. 55:368-380. [DOI] [PubMed] [Google Scholar]
  • 29.Kurahashi, K., O. Kajikawa, T. Sawa, M. Ohara, M. A. Gropper, D. W. Frank, T. R. Martin, and J. P. Wiener-Kronish. 1999. Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia. J. Clin. Investig. 104:743-750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Larbig, K. D., A. Christmann, A. Johann, J. Klockgether, T. Hartsch, R. Merkl, L. Wiehlmann, H. J. Fritz, and B. Tummler. 2002. Gene islands integrated into tRNA(Gly) genes confer genome diversity on a Pseudomonas aeruginosa clone. J. Bacteriol. 184:6665-6680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lomholt, J. A., K. Poulsen, and M. Kilian. 2001. Epidemic population structure of Pseudomonas aeruginosa: evidence for a clone that is pathogenic to the eye and that has a distinct combination of virulence factors. Infect. Immun. 69:6284-6295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lukashin, A. V., and M. Borodovsky. 1998. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res. 26:1107-1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Marchler-Bauer, A., A. R. Panchenko, B. A. Shoemaker, P. A. Thiessen, L. Y. Geer, and S. H. Bryant. 2002. CDD: a database of conserved domain alignments with links to domain three-dimensional structure. Nucleic Acids Res. 30:281-283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Mohd-Zain, Z., S. L. Turner, A. M. Cerdeno-Tarraga, A. K. Lilley, T. J. Inzana, A. J. Duncan, R. M. Harding, D. W. Hood, T. E. Peto, and D. W. Crook. 2004. Transferable antibiotic resistance elements in Haemophilus influenzae share a common evolutionary origin with a diverse family of syntenic genomic islands. J. Bacteriol. 186:8114-8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Nelson, K. E., C. Weinel, I. T. Paulsen, R. J. Dodson, H. Hilbert, V. A. Martins dos Santos, D. E. Fouts, S. R. Gill, M. Pop, M. Holmes, L. Brinkac, M. Beanan, R. T. DeBoy, S. Daugherty, J. Kolonay, R. Madupu, W. Nelson, O. White, J. Peterson, H. Khouri, I. Hance, P. Chris Lee, E. Holtzapple, D. Scanlan, K. Tran, A. Moazzez, T. Utterback, M. Rizzo, K. Lee, D. Kosack, D. Moestl, H. Wedler, J. Lauber, D. Stjepandic, J. Hoheisel, M. Straetz, S. Heim, C. Kiewitz, J. A. Eisen, K. N. Timmis, A. Dusterhoft, B. Tummler, and C. M. Fraser. 2002. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ. Microbiol. 4:799-808. [DOI] [PubMed] [Google Scholar]
  • 36.Nowak-Thompson, B., N. Chaney, J. S. Wing, S. J. Gould, and J. E. Loper. 1999. Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5. J. Bacteriol. 181:2166-2174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Pallen, M. J., R. R. Chaudhuri, and I. R. Henderson. 2003. Genomic analysis of secretion systems. Curr. Opin. Microbiol. 6:519-527. [DOI] [PubMed] [Google Scholar]
  • 38.Pohlmann, A., R. Cramm, K. Schmelz, and B. Friedrich. 2000. A novel NO-responding regulator controls the reduction of nitric oxide in Ralstonia eutropha. Mol. Microbiol. 38:626-638. [DOI] [PubMed] [Google Scholar]
  • 39.Raymond, C. K., E. H. Sims, A. Kas, D. H. Spencer, T. V. Kutyavin, R. G. Ivey, Y. Zhou, R. Kaul, J. B. Clendenning, and M. V. Olson. 2002. Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa. J. Bacteriol. 184:3614-3622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Romling, U., J. Greipel, and B. Tummler. 1995. Gradient of genomic diversity in the Pseudomonas aeruginosa chromosome. Mol. Microbiol. 17:323-332. [DOI] [PubMed] [Google Scholar]
  • 41.Roy-Burman, A., R. H. Savel, S. Racine, B. L. Swanson, N. S. Revadigar, J. Fujimoto, T. Sawa, D. W. Frank, and J. P. Wiener-Kronish. 2001. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J. Infect. Dis. 183:1767-1774. [DOI] [PubMed] [Google Scholar]
  • 42.Salzberg, S. L., A. L. Delcher, S. Kasif, and O. White. 1998. Microbial gene identification using interpolated Markov models. Nucleic Acids Res. 26:544-548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Sato, H., D. W. Frank, C. J. Hillard, J. B. Feix, R. R. Pankhaniya, K. Moriyama, V. Finck-Barbancon, A. Buchaklian, M. Lei, R. M. Long, J. Wiener-Kronish, and T. Sawa. 2003. The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU. EMBO J. 22:2959-2969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Schmidt, H., and M. Hensel. 2004. Pathogenicity islands in bacterial pathogenesis. Clin. Microbiol. Rev. 17:14-56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Schulert, G. S., H. Feltman, S. D. Rabin, C. G. Martin, S. E. Battle, J. Rello, and A. R. Hauser. 2003. Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia. J. Infect. Dis. 188:1695-1706. [DOI] [PubMed] [Google Scholar]
  • 46.Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959-964. [DOI] [PubMed] [Google Scholar]
  • 47.van der Meer, J. R., R. Ravatn, and V. Sentchilo. 2001. The clc element of Pseudomonas sp. strain B13 and other mobile degradative elements employing phage-like integrases. Arch. Microbiol. 175:79-85. [DOI] [PubMed] [Google Scholar]
  • 48.Wolfgang, M. C., B. R. Kulasekara, X. Liang, D. Boyd, K. Wu, Q. Yang, C. G. Miyada, and S. Lory. 2003. Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 100:8484-8489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Wong, G. K., J. Yu, E. C. Thayer, and M. V. Olson. 1997. Multiple-complete-digest restriction fragment mapping: generating sequence-ready maps for large-scale DNA sequencing. Proc. Natl. Acad. Sci. USA 94:5225-5230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Wong, S. M., and J. J. Mekalanos. 2000. Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 97:10191-10196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Wood, M. S., A. Byrne, and T. G. Lessie. 1991. IS406 and IS407, two gene-activating insertion sequences for Pseudomonas cepacia. Gene 105:101-105. [DOI] [PubMed] [Google Scholar]
  • 52.Ye, R. W., D. Haas, J. O. Ka, V. Krishnapillai, A. Zimmermann, C. Baird, and J. M. Tiedje. 1995. Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr. J. Bacteriol. 177:3606-3609. [DOI] [PMC free article] [PubMed] [Google Scholar]

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