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Journal of Bacteriology, August 2005, p. 5278-5291, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5278-5291.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Suppression-Subtractive Hybridization To Identify Genes in the Burkholderia cepacia Complex That Are Unique to Burkholderia cenocepacia

Steve P. Bernier and Pamela A. Sokol*

Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta T2N 4N1, Canada

Received 24 February 2005/ Accepted 11 May 2005


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ABSTRACT
 
We have previously shown differences in virulence between species of the Burkholderia cepacia complex using the alfalfa infection model and the rat agar bead chronic infection model. Burkholderia cenocepacia strains were more virulent in these two infection models than Burkholderia multivorans and Burkholderia stabilis strains. In order to identify genes that may account for the increased virulence of B. cenocepacia, suppression-subtractive hybridization was performed between B. cenocepacia K56-2 and B. multivorans C5393 and between B. cenocepacia K56-2 and B. stabilis LMG14294 Genes identified included DNA modification/phage-related/insertion sequences and genes involved in cell membrane/surface structures, resistance, transport, metabolism, regulation, secretion systems, as well as genes of unknown function. Several of these genes were present in the ET12 lineage of B. cenocepacia but not in other members of the B. cepacia complex. Virulence studies in a chronic lung infection model determined that the hypothetical YfjI protein, which is unique to the ET12 clone, contributes to lung pathology. Other genes specific to B. cenocepacia and/or the ET12 lineage were shown to play a role in biofilm formation and swarming or swimming motility.


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INTRODUCTION
 
Cystic fibrosis (CF) is the most common life-threatening autosomal recessive disease in the Caucasian population, affecting more than 50,000 individuals worldwide (25) with an incidence of 1 in 3,200 live births (51). Despite a better understanding of CF and improved therapies and life expectancy over the last decades, 90% of the morbidity and mortality involving this genetic disorder is caused by progressive loss of lung function (12, 25, 37, 51). Respiratory failure in CF lungs is related to severe host inflammatory responses caused by chronic infection leading to progressive airway obstruction, long-term tissue damage, and early death (15, 37, 45). The most common pathogens affecting CF lungs are Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and the Burkholderia cepacia complex (Bcc) (53). Despite the fact that P. aeruginosa remains the most prevalent CF pathogen and that P. aeruginosa infection is the most common cause of mortality in CF patients, several studies report that clinical outcomes of CF patients infected with Bcc are often worse than those infected with P. aeruginosa (32). Bcc infection can result in "cepacia syndrome," which is characterized by a rapid pulmonary decline with necrotizing pneumonia, fever, and occasional septicemia (58).

Bcc is a group of closely related bacterial species recognized to be important opportunistic pathogens in immunocompromised patients, including those with CF and chronic granulomatous disease (10, 24). To date, there are nine Bcc species that have been isolated from sputum of CF patients (8, 9, 35). Burkholderia cenocepacia (formerly genomovars III) and Burkholderia multivorans (formerly genomovars II) are the two species most commonly recovered from CF sputum, but their proportions vary geographically. In Canada, 80% of the Bcc CF isolates are B. cenocepacia compared to 9% B. multivorans (62); however, in the United States, B. multivorans is recovered from 38% of CF patients colonized with Bcc compared to 50% for B. cenocepacia (35). Highly transmissible B. cenocepacia clones, such as the ET12, Midwest, and PHDC (Philadelphia-DC) lineages, have been identified in outbreaks in Europe and North America (6, 39).

Strains belonging to the B. cenocepacia ET12 lineage contain both the B. cepacia epidemic strain marker (BCESM) (39) and the cable pili gene (cblA) (63). ET12 strains were shown to be responsible for outbreaks in Canada and the United Kingdom and were linked to patient-to-patient transmission (62, 63). The PHDC clone has also been implicated in outbreaks, yet it lacks these two putative virulence markers (6, 8), demonstrating that the BCESM and cable pili are not necessary for transmissibility. The BCESM has recently been shown to be part of a genomic island, the B. cenocepacia island (cci), which is involved in pathogenicity and metabolism (2).

Virulence factors known to play a significant role in the pathogenesis of B. cenocepacia ET12 strains include two quorum-sensing systems, CepIR (61) and CciIR (2), iron acquisition via siderophore production (60, 66), a protease (14), a type III secretion system (65), and a type IV secretion system (TFSS) (19). Invasion and survival in epithelial cells (4, 7, 56), macrophages (42), amoebae (41), and acanthamoeba (33) have also been demonstrated. Analysis of a signature-tagged mutagenesis (STM) library in a chronic pulmonary infection model identified several B. cenocepacia genes directly involved in host survival, including genes involved in cellular metabolism, regulation, DNA replication and repair, cell surface proteins, and polysaccharide production (31).

Recently, we have shown differences in virulence between species of the Bcc using alfalfa and rat agar bead infection models (3). For most strains tested there was a correlation in virulence between the two models, and B. cenocepacia was one of the most virulent species in these two infection models (3). This observation is consistent with the clinical profile since CF patients infected with B. cenocepacia frequently experience worse outcomes than patients infected with other Bcc species (32, 40). Burkholderia stabilis (formerly genomovars IV) and B. multivorans were generally avirulent in the alfalfa infection model. In the rat agar bead chronic infection model, lung pathology changes were significantly less for B. multivorans strains than B. cenocepacia, and B. stabilis strains did not persist in the lung as well as other species (3).

The objectives of this study were to identify genes that may account for the differences in virulence between B. cenocepacia, B. multivorans, and B. stabilis in CF patients, plants (alfalfa), and animals (rats). Suppression-subtractive hybridization (SSH), which has previously been used to identify virulent determinants between closely related Burkholderia species (17, 52), was performed independently between B. cenocepacia K56-2 and B. multivorans C5393 and B. stabilis LMG 14294, respectively. Select unique B. cenocepacia K56-2 genes were evaluated for their distribution within the Bcc, their implication in virulence using a chronic lung infection model, as well as their role in biofilm formation and swarming and swimming motility.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are described in Table 1. Cultures were grown in Luria broth (LB) (Invitrogen, Burlington, Ontario, Canada) or on 1.5% LB agar plates supplemented with antibiotics when required and incubated at 37°C for 24 h. When appropriate, antibiotics were used at the following concentrations: 50 µg/ml of kanamycin (Km) and 1,200 µg/ml of trimethoprim (Tp) for Escherichia coli and 100 µg/ml of Tp for B. cenocepacia. Plasmids were purified from overnight cultures in LB medium supplemented with appropriate antibiotic by using a QIAprep Spin Miniprep kit (QIAGEN, Mississauga, Ontario, Canada). For animal studies, overnight cultures were grown in TSB-DC medium (46). For swarming and swimming motility assays, cultures were grown in nutrient broth (NB) (Difco) supplemented with 0.5% glucose. All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada).


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  		TABLE 1. Bacterial strains and plasmids used in this study

Construction of suppression-subtractive hybridization libraries. Genomic DNA was isolated using a Wizard genomic DNA purification kit (Promega, Madison, Wisconsin). Suppression-subtractive hybridization was carried out using a PCR-Select bacterial genome subtraction kit (Clontech, Palo Alto, CA) as recommended by the supplier except that two hybridization temperatures of 73°C and 85°C were used in separate experiments due to the high G+C content of B. cenocepacia. Two suppression-subtractive hybridization libraries were constructed using B. cenocepacia K56-2 genomic DNA as the tester in both libraries and B. multivorans C5393 and B. stabilis LMG 14294 genomic DNA as the driver in their respective library. PCR products generated in each library were cloned into pCR2.1-TOPO (Invitrogen) using a TOPO TA cloning kit (Invitrogen) and transformed into TOP10 cells (Invitrogen). Resulting transformants were individually grown overnight in LB medium with kanamycin (50 µg/ml) at 37°C in 96-well microtiter plates and stored in 15% glycerol at –70°C.

Screening of B. cenocepacia K56-2-specific clones in each library. Subtracted libraries were screened for tester-specific fragments using a modified dot blot hybridization previously described (50, 72). Plasmid DNA from individual clones was isolated using a QIAprep Spin Miniprep kit (QIAGEN). Approximately 200 ng of plasmid DNA was spotted in duplicate onto wet GeneScreen Plus hybridization transfer membranes (Perkinelmer Life Sciences, Boston, MA) using a BIO-DOT SF blotting apparatus (Bio-Rad Laboratories, Richmond, CA) in a 96-well format. Denatured DNA was hybridized with approximately 1 µg of RsaI-digested genomic DNA from tester and driver DNA separately, previously end-labeled with [{alpha}-32P]dCTP (GE Healthcare).

Distribution of B. cenocepacia K56-2-specific genes within the B. cepacia complex. Genomic DNA from 43 strains belonging to different species of the Bcc (11, 38) was isolated using Chelex 100 as previously described (68). Dot blot hybridization was carried out as described above except that DNA was spotted onto nitrocellulose membranes and probes used were obtained by PCR amplification from clones with primers M13F and M13R (Invitrogen), purified by QIAquick PCR Purification kit (QIAGEN), and labeled with [{alpha}-32P]dCTP (GE Healthcare). For PCR analysis, primers were designed to amplify internal regions specific to genes unique to B. cenocepacia K56-2.

DNA manipulations. Molecular biology techniques were performed as generally described (57). Restriction enzymes and T4 DNA polymerase were purchased from Invitrogen. T4 DNA ligase was purchased from New England Biolabs (Mississauga, Ontario, Canada). Oligonucleotide primers were purchased from University of Calgary Core DNA and Protein Services. DNA fragments used in cloning procedures were purified with a QIAquick gel extraction kit (QIAGEN), and recombinant plasmids were electroporated into E. coli by using a Gene Pulser (Bio-Rad Laboratories) according to the manufacturer's recommendations.

DNA sequencing and analysis. DNA sequencing reactions were performed by Macrogen Inc. (Seoul, Korea) with an ABI 3730XL automatic DNA sequencer. Fragments identified by SSH were analyzed by using the unpublished annotation from the B. cenocepacia J2315 sequencing project at the Sanger Institute (http://www.sanger.ac.uk/projects/B_cenocepacia/) using Artemis software (55). Each open reading frame (ORF) predicted by the Sanger Institute was used to identify homologous sequences with the BLASTX (http://www.ncbi.nlm.nih.gov/BLAST/) program.

Construction of different B. cenocepacia K56-2 mutants by allelic exchange. Construction of deletion mutants in B. cenocepacia K56-2 was performed as follows. Two sets of PCR primers were designed for each gene targeted for mutagenesis. Each set of primers amplified two discontinuous parts of the gene in order to create a deletion. Each primer contained a restriction site at the 5' end in order to facilitate cloning into pEX18Tc (29). The fragment amplified with the first primer set was cloned into pEX18Tc or pBluescript for SRC2, followed by the cloning of the fragment generated by the second set of primers. A Tp resistance cassette obtained from p34E-Tp (18) was then inserted between the two fragments of the targeted gene into the restriction site generated by the primers used to clone the two fragments. This plasmid was transferred from E. coli to B. cenocepacia K56-2 by triparental mating using the mobilizing plasmid pRK2013 (22). Tpr transconjugants were plated onto 5% sucrose to select for excision of the plasmid. Confirmation of the mutant genotype was preformed by PCR.

Biofilm assay. Biofilm assays were performed as previously described (48) with minor modifications. Biofilms were formed on polystyrene pegs by placing a 96-peg lid (Nunc, Roskilne, Denmark) in a 96-well microtiter plate (Nunc) containing 5 µl of a 0.4 optical density at 600 nm suspension diluted in 145 µl of LB broth (Invitrogen). The plate was incubated for 24 h at 37°C on a rocking platform. The lid was removed, air dried for 10 min, stained with 200 µl of 1% crystal violet (Sigma) in a 96-well plate for 1 min, and rinsed three times in separate plates containing double-distilled water. The stained pegs were decolorized with 175 µl of 95% ethanol in a microtiter plate for 1 min. The quantity of crystal violet removed was measured using a Wallec Victor2 model 1420 multilabel counter (PerkinElmer Life Sciences) set to measure absorbance at 600 nm.

Swarming and swimming motility assays. Motility assays were performed as previously described (34). Briefly, 1 microliter of an overnight culture was spotted in the middle of a swarm plate (NB, 0.5% glucose, 0.5% agar) or a swim plate (NB, 0.5% glucose, 0.25% agar), allowed to dry for 1 h at room temperature, and incubated for 24 h for the swarming and 12 h for swimming assays at 37°C. Diameters of swarming and swimming zones were measured.

Animal studies. Sprague-Dawley rats (150 to 175 g) (Charles River Canada, Inc.) were tracheostomized under anesthesia and inoculated with approximately 104 CFU of the appropriate strain embedded in agar beads as previously described (5). At 14 days postinfection, the lungs from four to five animals from each group were removed aseptically and homogenized (Polytron Homogenizer; Brinkman Instruments, Westbury, N.Y.) in 3 ml of phosphate-buffered saline (10 mM sodium phosphate, 150 mM NaCl, pH 7.5). The homogenates were serially diluted in phosphate-buffered saline and plated on trypticase soy agar (Difco) and B. cepacia isolation agar (27) with 100 µg/ml of trimethoprim when required. The lungs of four to five additional animals from each group were removed en bloc, fixed in 10% formalin, and examined for quantitative pathological changes. Infiltration of the lung with inflammatory cells and exudate was measured as previously described (3).

Alfalfa infection assay. Virulence studies in the alfalfa infection model were performed as previously described (3).

Statistical analysis. Analysis of variance (ANOVA) was performed with INSTAT software (GraphPad Software, San Diego, Calif.). A P value of <0.05 was considered statistically significant.


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RESULTS
 
Construction of suppression-subtractive hybridization libraries and screening for B. cenocepacia K56-2-specific clones. SSH was carried out between B. cenocepacia K56-2 and B. multivorans C5393 and B. stabilis LMG 14294 in order to identify genomic regions specific to B. cenocepacia K56-2 that could potentially be linked to its increased virulence. Genomic DNA from the tester DNA (B. cenocepacia K56-2) and the driver DNA (B. multivorans C5393 or B. stabilis LMG 14294) were digested with the restriction enzyme RsaI, ligated with two different adaptors (Clontech), and subjected to two rounds of hybridization. Hybridization temperatures of 73°C and 85°C were used in separate experiments. After two rounds of hybridization, the unhybridized tester-specific fragments were subsequently amplified twice by PCR using adaptor-specific primers (Clontech) and cloned into pCR2.1-TOPO (Invitrogen). The four subtracted libraries were screened by dot blot hybridization with both tester and driver genomic DNA to identify clones that hybridized with K56-2 but not C5393 or LMG 14294. The K56-2-specific clones were sequenced and compared to those from the B. cenocepacia J2315 sequencing project at the Sanger Institute (http://www.sanger.ac.uk/projects/B_cenocepacia/). B. cenocepacia strains J2315 and K56-2 belong to the same clonal group (38). Characteristics of the clones generated from each of the four SSH libraries were similar, with an average size of approximately 500 bp and a G+C content of 54%, with the exception of the library generated using B. multivorans C5393 as driver DNA with a hybridization temperature of 85°C, in which the average size of each insert was 1,240 bp and the G+C content was 65%.

Identification of sequences present in B. cenocepacia K56-2 and absent from B. multivorans C5393 and/or B. stabilis LMG 14294. Plasmid DNA from 384 clones from the B. multivorans C5393 subtracted libraries and 301 clones from the B. stabilis LMG 14294 subtracted libraries were analyzed, and 205 were shown to be specific to B. cenocepacia K56-2 by dot blot hybridization for an efficiency of approximately 30%. Of these 205 clones, 102 were from the B. multivorans C5393 subtracted libraries and 103 from the B. stabilis LMG 14294 subtracted libraries. Sequencing of these clones led to the identification of 89 different ORFs. Genes identified from these 205 unique clones are shown in Tables 2 and 3. These ORFs were assigned to one of nine classes: DNA modification/phage-related/insertion sequences, cell membrane/surface structures, resistance, transport, metabolism, regulation, secretion systems, and unknown functions. Of the 89 different genes identified by SSH, 82 were present in a single copy (Table 2) and seven had multiple copies (Table 3) within the B. cenocepacia J2315 genome. All of the multiple copy genes coded for insertion sequence (IS) elements and were identified in both subtraction libraries (Table 3). Seven different IS elements were 95 to 100% identical at the nucleotide level to the B. cenocepacia J2315 genome and were located in 153 different locations (Table 3). For example, IS407A and transposase and inactivated derivatives (COG2801) are present in at least 13 copies each in the genome (Table 3).


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  		TABLE 2. B. cenocepacia K56-2 sequences identified by SSH libraries that are present in single copy in the B. cenocepacia J2315 genome


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  		TABLE 3. B. cenocepacia K56-2 sequences identified by SSH that are present in multiple copies in the B. cenocepacia J2315 genome

Although fewer genes were identified by using B. stabilis LMG 14294 as driver DNA than B. multivorans C5393, the distribution in classes of genes between the two libraries was almost identical (Table 4). More than half of the genes identified were DNA modification/phage-related/IS elements. With the exception of a few enzymes related to DNA replication and repair (Table 2), most of the genes in that category were phage-related genes (Table 2) and multicopy IS elements (Table 3). These genes were all located in low G+C regions, demonstrating the preference of SSH in identification of A+T rich regions.


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  		TABLE 4. Distribution of genes identified by suppression-subtractive hybridization between B. cenocepacia and B. multivorans or B. stabilis

Several genomic regions were identified containing multiple SSH clones within the same gene cluster (Table 2). For example, three clones were identified within a 7.7-kb low G+C content cluster on chromosome 1 including a bacteriophage P4 integrase (BCAL1118), a hypothetical yfjI (BCAL1122), and an IS402-like element (BCAL1125). Two clones were identified in a fusaric acid resistance cluster containing three genes: BCAL1176, BCAL1177, and BCAL1178. An outer membrane receptor (BCAL1783) gene and a biopolymer transport gene (BCAL1787) were located in a putative iron transport cluster. Two different type IV secretion systems, one on the chromosome 2 and one on the resident plasmid (19) were identified in multiple clones. A lipopolysaccharide (LPS) synthesis cluster as well as a putative capsular polysaccharide cluster previously described (50) were identified by the isolation of three clones containing portions of genes in each of these clusters.

Distribution of selected B. cenocepacia K56-2 clones identified by SSH across the B. cepacia complex. Twenty of the 89 different clones identified in B. cenocepacia K56-2 by the four SSH libraries were selected to determine their distribution within nine species of the Bcc (11, 38). Criteria chosen for the selection of these genes were based on their clustering in the genome mapping and their possible roles in virulence. One of the clones contained the cblA pili gene which has previously been shown to be present primarily in the B. cenocepacia ET12 lineage (38); therefore, we used it as a positive control for the dot blot hybridization approach. PCR was used to validate the dot blot hybridization data for yfjI, the Vgr-related protein gene, and the phospholipase-like gene.

Most of the genes analyzed were unique to the ET12 clone of B. cenocepacia (Table 5). Genes that were present only in the ET12 clone included yfjI (BCAL1122), the fusaric acid genes (BCAL1176 and BCAL1177), and the autotransporter adhesin genes (BCAM0224 and BCAM0225). The genes identified within the putative capsule polysaccharide cluster (BCAL3229, BCAL3234, BCAL3235, BCAL3241, and BCAL3242) were present in all ET12 strains and possibly one strain of B. cepacia, which hybridized weakly compared to the ET12 strains. Genes present on the resident plasmid were present in the ET12 clone and one strain of Burkholderia pyrrocinia only. A type IV secretion system gene, virB4, was shown to be present in 9 out of 11 strains of B. cenocepacia and absent from the other species of the Bcc. A Vgr-related protein gene and a phospholipase-like gene were detected in six of the B. cenocepacia strains including the ET12 strains. A putative membrane protein (BCAL1535) associated with or within a fimbriae operon was identified, and the adjacent cpaC (BCAL1528) gene that has homology to a pilus assembly protein was determined to be present only in B. cenocepacia. Interestingly, genes within a LPS cluster did not have the same distribution pattern; wbiF was widely distributed in the Bcc compared to the ABC-transporter (wzm) and rmlD which were present in the B. cenocepacia ET12 clone, and one strain of Burkholderia vietnamiensis and Burkholderia dolosa, respectively. Two of the multicopy IS elements were widely distributed, whereas the putative transposition helper protein, the transposase, and inactivated derivatives were present in the ET12 strains and in few other species (Table 5).


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  		TABLE 5. Distribution of selected B. cenocepacia K56-2 genes in the B. cepacia complex

Construction and characterization of mutations in genes unique to B. cenocepacia. Deletion mutants were constructed in selected genes including the hypothetical yfjI (BCAL1122), the Vgr-related protein gene (BCAM0148), the phospholipase-like gene (BCAM0149), an autotransporter adhesin (BCAM0223), cpaC (BCAL1528) of the fimbriae cluster, and the virB1-virB2 genes of the chromosomal type IV secretion system (Table 1). These genes were selected based on their uniqueness to B. cenocepacia and/or to the ET12 clone (Table 5) and their possible roles in virulence.

Virulence studies were conducted using the rat agar bead model to determine if these genes contributed to lung damage and/or to the persistence of chronic infections. Rats infected with SB002 (yfjI) had a significant decrease in lung pathology compared to rats infected with K56-2 (P < 0.001) despite no significant difference in the number of bacteria being recovered from the lungs, suggesting an important role for the hypothetical YfjI protein in inflammation in this model (Fig. 1). Rats infected with SB004 (phospholipase-like gene), SB006 (autotransporter adhesin), and SRC2 (chromosomal type IV secretion system) did not have significant differences in either lung pathology or bacterial persistence (Fig. 1). One of the animals infected with wild-type B. cenocepacia K56-2 had cleared the bacteria from its lungs. Although the number of bacteria recovered from lungs infected with K56-2 were less than the mutant strains, the differences were not statistically significant (Fig. 1A). None of these mutants, including SB003 (Vgr-related protein) and SB007 (cpaC) (Table 1), were attenuated in virulence in the alfalfa infection model (data not shown).



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  		FIG. 1. Virulence studies in a chronic lung infection model. A. Means ± standard deviations of CFU recovered 14 days postinfection from the lungs of four to five rats per bacterial strain. B. Means ± standard deviations of the percent of the lung with inflammatory exudates. Four or five rats were analyzed per group at 14 day postinfection. SB002 is significantly different than K56-2 (P < 0.001) by ANOVA.

Using SSH, we identified genes that were previously shown to be required for survival in the rat agar bead model using a STM approach including genes involved in LPS synthesis and synthesis of a putative polysaccharide capsule (31). B. cenocepacia K56-2 mutant strains with transposon insertions in LPS or capsule genes (Table 1) were used together with our deletion mutants to identify phenotypic properties which might be important for survival and/or the increased virulence of B. cenocepacia compared to both B. multivorans C5393 and B. stabilis LMG 14294.

B. cenocepacia has previously been shown to form biofilms on abiotic surfaces (13, 30). Five transposon mutants with insertions in genes of the LPS and the putative capsule polysaccharide clusters, 32D2 (wbiG) and 34D8 (rmlD) from the LPS cluster and 10F1 (hypothetical protein), 17D8 (fkbH), and 36B4 (cpxA) from the capsule cluster (31) (Table 1), and six mutants with deletions in genes unique to B. cenocepacia and/or the ET12 clone (SB002, SB003, SB004, SB006, SB007, and SRC2) (Table 1) were tested for their ability to form biofilms. Strains SB003 (Vgr-related protein), SB006 (autotransporter adhesin), as well as the LPS mutants 32D2 and 34D8 produced significantly more biofilm compared to the wild-type strain K56-2 (P < 0.001) as shown in Fig. 2. Strain 36B4, with a mutation in the capsule cluster, produced slightly more biofilm compared to its parent strain (P < 0.05). None of the mutants tested were defective in biofilm formation in the conditions used.



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  		FIG. 2. Biofilm formation. Strains were grown in LB broth for 24 h at 37°C on a rocking platform in a 96-well polystyrene microtiter plate with a pegged lid. Biofilms formed on polystyrene pegs were stained with 1% crystal violet. Values shown are the means ± standard deviations for four pegs. This experiment was repeated at least three times with similar results. 36B4 is significantly different than K56-2 (*, P < 0.05) by ANOVA. SB002, SB006, 32D2, and 34D8 are significantly different than K56-2 (**, P < 0.001) by ANOVA.

B. cenocepacia was previously shown to differentiate into swarmer cells and migrate over agar (30, 34). Swarming and swimming phenotypes of selected mutants are displayed in Table 6. B. cenocepacia K56-2 was able to swarm over an agar surface, and differentiation from vegetative cells into typical elongated swarmer cells (23) was observed by microscopy (data not shown). Mutants SB003, SB006, SB007, 10F1, and 34D8 exhibited major swarming defects as demonstrated by much smaller zones of swarming on agar surface compared to the wild-type strain B. cenocepacia K56-2 (Table 6). With the exception of 36B4, which was slightly reduced in swarming motility, the other mutants were similar to the wild type (Table 6).


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  		TABLE 6. Comparison of swarming and swimming motility between mutants and wild-type B. cenocepacia K56-2

The swimming motility phenotype of these mutants was also determined. SB003, SB007, 10F1, and 17D8 were defective in swimming compared to the parent strain using soft agar plates (Table 6). Swimming motility from overnight cultures was analyzed by microscopy, and only SB003 was nonmotile (Table 6), which could explain its defect in both swarming and swimming motility using swarm and swim agar plates.


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DISCUSSION
 
SSH has been used successfully in the identification of virulence determinants and genetic diversity of several bacterial pathogens (reviewed by Winstanley) (71). A capsule polysaccharide cluster was identified in both Burkholderia pseudomallei (52) and Burkholderia mallei (17) by SSH with the nonpathogenic Burkholderia thailandensis and was demonstrated to be an important virulence determinant of both pathogens in animal infection models (17, 52). Genomic diversity was also observed between clinical isolates of B. pseudomallei with the identification of a B. mallei-specific prophage in B. pseudomallei 1026b by comparison to the sequenced B. pseudomallei K96243 (16). SSH has been used previously to analyze differences between strains of B. cenocepacia (36, 50). IS1363 identified by SSH between a B. cenocepacia PHDC strain and a non-PHDC strain of B. cenocepacia is present almost exclusively in two B. cenocepacia epidemic clones, ET12 and PHDC, which were implicated in outbreaks within the CF population (36). B. cenocepacia is divided into four groups based on the recA sequence, and a putative capsule polysaccharide cluster, a Vgr-related protein gene, and a phospholipase-like gene were previously identified by SSH between a highly transmissible B. cenocepacia recA type A and a nontransmissible recA type B (50).

Previous studies using SSH in different Burkholderia species used a hybridization temperature of 73°C (16, 17, 50, 52). In the present study, these conditions resulted in isolation of fragments with a G+C content averaging 54%, which is similar to previous SSH studies involving Burkholderia species (16, 17, 50, 52). Increasing the hybridization temperature to 85°C when using B. multivorans C5393 as driver DNA made it possible to identify genes with an average G+C content corresponding to that of the genome; although this temperature did not result in a SSH library with an increased G+C content with B. stabilis LMG 14294 as the driver.

Although we may have been conservative in our screening of the libraries by dot blot hybridization with labeled genomic DNA, the 20 clones chosen for distribution studies within the Bcc were always absent from either or both driver strains (Table 5), demonstrating the efficiency of our approach. Although not confirmed by hybridization or PCR, the remaining genes identified in Tables 2 and 3 are highly likely to be absent from either C5393 or LMG 14294. Several independent clones contained genes localized within the same cluster or operon, which demonstrates the effectiveness of the SSH screening approach used. Not all differences between the compared species were identified, however, since some genes known to be unique to B. cenocepacia ET12 including the genes of the cci genomic island (2) and IS1363 (36) were not identified. Because of the extent of variation between these species, it would be very unlikely that this method could ever approach identification of all differences.

Many ET12-specific genes or clusters identified in this study had previously been shown to be implicated in B. cenocepacia virulence, including genes that were required for survival in the rat agar bead model using a STM approach (31). Our study showed that genes within the capsule polysaccharide cluster and the wzm-rmlD genes of the LPS cluster were not widely distributed within the Bcc (Table 5).

Virulence studies in the rat agar bead model were performed only on a selected number of our B. cenocepacia K56-2 deletion mutants (Fig. 1). Natural colonization steps are bypassed in this infection model, therefore mutation of genes involved in adherence is unlikely to alter virulence. cpaC is predicted to be part of an operon encoding for a fimbriae structure. Therefore, strain SB007 with a mutation in cpaC was not tested in the agar bead model. Strain SB003 (Vgr-related protein) was also not tested in this model since the phospholipase-like gene mutation (SB004) is located 3 bp downstream of the Vgr-related protein gene and probably in the same operon.

Although there is no clinical evidence that Bcc forms biofilms in CF lungs, there is evidence that P. aeruginosa forms biofilms in chronically infected CF lungs (49, 59). Flagella-driven motility like swarming was demonstrated to facilitate colonization of the urinary tract by Proteus mirabilis (1) and to upregulate virulence gene products such as hemolysin, urease, and protease (23). Phenotypes such as biofilm formation and swarming and swimming motility may correlate with virulence, and therefore we determined that mutations in selected genes identified by SSH influenced these phenotypes.

Genes wbiF and rmlD, which are part of an O-antigen synthesis cluster, were identified in both studies. Mutations in rmlD and wbiG, which is downstream of wbiF, affect the lipid A core and sensitivity to human serum (47). Interestingly, we have shown that these genes also affect biofilm production, suggesting that the LPS phenotype influences biofilm formation. The swarming motility phenotype was also reduced for the rmlD mutant (34D8) (Table 6), which is consistent with some observations made in Salmonella enterica serovar Typhimurium, where O-antigen mutants were rescued by surfactin for swarming, indicating that LPS O antigen improves surface fluidity for colonies to swarm (64). Surprisingly, the distribution of genes within this O-antigen cluster in the Bcc varies depending on their location in the cluster. wbiF is present in most of the Bcc species, whereas wzm-rmlD are present in the ET12 clone of B. cenocepacia and one strain of both B. vietnamiensis and B. dolosa each (Table 5). Ortega et al. (47) showed that this O antigen cluster has several transcriptional units and wbiF and wzm-rmlD are located in two different transcriptional units. Further, the G+C content seems to change at the limits of these two transcriptional units, suggesting that the genes rmlBACD-wzm-wzt-vioA-wbxFED (47) may have been acquired at different times in evolution.

We identified three clones within a putative capsule polysaccharide cluster as well as IS elements, ISRS011 that flanks one end of the cluster and IS407 elements that are present within the cluster. One gene of this putative capsule polysaccharide cluster, cpxA (wzt), had previously been identified by SSH (50). Using wcbB and cpxA as probes, Parsons et al. (50) reported that the cluster was present in B. cenocepacia ET12 strains, B. cepacia Cep509 and ATCC25416, and B. multivorans CF-A1-1. PCR analysis could only confirm the presence of both wcbB and cpxA in B. cenocepacia ET12. Amplification of only one of these two genes could be performed in B. cepacia strains while none were amplified in B. multivorans, suggesting that there were either sequence differences or possibly other genes that cross-hybridized (50). In our study, although there was weak hybridization with B. cepacia Cep509 for all three probes used, we have shown that this cluster is primarily found in the ET12 clone by dot blot hybridization using probes for fkbH (BCAL3229), glycosyltransferase (BCAL3234), UDP-galactopyranose mutase (BCAL3235), ctrC (BCAL3241), and wcbD (BCAL3242) (Table 5).

As previously reported by Parsons et al. (50), these genes have homology to the capsule polysaccharide genes of B. mallei (17) and B. pseudomallei (52). Although there are similarities between the clusters of the three species, there are several differences including insertions and deletions of several genes. Although the interruption of wcbO by IS407-like elements likely inactivates the wcbOPQRS operon, this inactivation was only observed in B. cenocepacia J2315 (50). Hunt et al. (31) identified three STM mutants with transposons inserted in this polysaccharide cluster (mutants 10F1, 17D8, and 36B4), indicating that these genes are important for survival in the lungs of a chronic lung infection model and suggesting a possible role in host resistance.

The hypothetical yfjI gene identified by SSH was also previously found to be important for survival (31). The presence of a bacteriophage P4 gene adjacent to yfjI and genes in a low G+C content region suggests that this region was acquired by horizontal transfer since phages have been implicated in the movement of virulence factors between bacterial species (67). Although yfjI homologues are present in other bacterial species, the role of this gene is unknown. It is predicted to encode a cytoplasmic protein (PSORTb of 8.96). Its contribution to lung pathology (Fig. 1A) and its uniqueness to B. cenocepacia ET12 warrant further studies on its role in virulence of B. cenocepacia ET12 and possibly other species with yfjI homologues.

Both our study and that of Parsons et al. (50) identified a cluster containing a Vgr-related protein and a phospholipase-like gene. Vgr-related genes are present in several gram-negative bacteria and contain repeated dipeptide motifs (valine-glycine repeats) that are often associated with Rhs elements (rearrangement hot-spots) in E. coli (69, 70). P. aeruginosa PAO1 contains 10 Vgr homologues (70), and six of them were found to be associated with genomic islands (20). Although their functions are yet to be defined, Vgr-related proteins are often associated with ligand-binding proteins at the bacterial surface or are secreted (69). In this study, we showed that the Vgr-related protein gene was involved in biofilm formation and swarming and swimming motility (Fig. 2 and Table 6), suggesting that a mutation in a Vgr-related protein gene affects phenotypes that are surface related. In P. aeruginosa, competition studies between a phospholipase D (PLD) mutant and its parent strain in the rat agar bead model demonstrated that PLD contributed to the ability of P. aeruginosa to persist in the lungs (70). In our study, the rats infected with the PLD mutant had slightly less bacteria recovered from the lungs although the difference was not significant. It is possible that a greater difference might have been observed in a competition assay with the wild-type strain. PLDs are widely distributed and found in both eukaryotic and prokaryotic cells (21). They have been associated in bacterial pathogenesis with the murine toxin of Yersinia pestis (54) as well as in the virulence of Corynebacterium ovis (44). PLD is a member of a superfamily that includes prokaryotic and eukaryotic PLDs, cardiolipin synthase, phosphatidylserine synthase, poxvirus envelope proteins, bacterial endonuclease, and helicase. All members of the superfamily have one or two copies of the HKD motif, which is a conserved active site [HxK(x)4D or HxK(x)4D(x)6GSxN] (21, 43). The phospholipase-like gene in B. cenocepacia contains these two distinct HKD motifs.

Engledow et al. (19) demonstrated the presence of two different TFSSs in B. cenocepacia K56-2, one located on chromosome 2 and one on the resident plasmid. In this study, we also identified these two TFSSs (Table 2) and showed that the chromosomal one was unique to B. cenocepacia (Table 5). Some pathogenic bacteria use TFSS to translocate virulence factors, which are DNA or protein macromolecules, to a large array of target cells (26). The plasmid-encoded TFSS of B. cenocepacia K56-2, which is primarily found in B. cenocepacia ET12 (Table 5), is directly involved in plant pathology as demonstrated by plant tissue watersoaking symptoms. The chromosomal TFSS does not appear to be involved in plant virulence as demonstrated by our studies with the alfalfa model (data not shown) and those of Engledow et al. (19).

More than half of the genes identified were IS elements (Table 4). Most of the IS elements were present in multiple copies in the B. cenocepacia J2315 genome, and seven of these multiple copy IS elements were found in 153 locations. IS elements in B. cenocepacia ET12 suggest the presence of several genomic islands other than the described cci (2), since these islands are often characterized by regions of low G+C containing IS elements.

Although this study has not identified all the differences that may account for the increased prevalence and virulence of the B. cenocepacia ET12 clone versus the other species of the Bcc, we have demonstrated that genes unique to B. cenocepacia and/or ET12 may play a role in the increased virulence of the ET12 clone. We have also determined that the previously uncharacterized yfjI is important for virulence, and further studies are in progress to determine the function of this gene.


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ACKNOWLEDGMENTS
 
This study was supported by the Canadian Institutes of Health Research (CIHR). S.P.B is the recipient of a Studentship from the Canadian Cystic Fibrosis Foundation (CCFF).

We thank B. Pohorelic, R. Chen, and C. Kooi for experimental assistance and D. E. Woods for histopathology analysis. We thank J. Parkhill and M. Holden at the Welcome Trust Sanger Institute for access to the annotation data of the B. cenocepacia J2315 genome sequence prior to publication.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, 3330 Hospital Dr. N. W., Calgary, Alberta, Canada T2N 4N1. Phone: (403) 220-6037. Fax: (403) 270-2772. E-mail: psokol{at}ucalgary.ca. Back


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Journal of Bacteriology, August 2005, p. 5278-5291, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5278-5291.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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