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J Bacteriol. Aug 2009; 191(15): 5013–5019.
Published online May 29, 2009. doi:  10.1128/JB.00473-09
PMCID: PMC2715730

Intraspecies Signaling Involving the Diffusible Signal Factor BDSF (cis-2-Dodecenoic Acid) Influences Virulence in Burkholderia cenocepacia[down-pointing small open triangle] §

Abstract

Burkholderia cenocepacia produces a diffusible fatty acid signal molecule, cis-2-dodecenoic acid (BDSF), that has been implicated in interspecies and interkingdom communication. Here, we show that BDSF also acts as an intraspecies signal in B. cenocepacia to control factors contributing to virulence of this major opportunistic pathogen.

Many bacteria use cell-cell communication systems involving the synthesis and perception of diffusible signal molecules to monitor aspects of their environment such as cell density or confinement to niches and to modulate their behavior accordingly (reviewed in references 3, 7, 16, 26, and 27). The signal molecules produced by bacteria are structurally diverse. Many gram-negative bacteria use N-acyl homoserine lactones (N-AHLs) as signals, although other fatty acid derivatives, including cis-unsaturated fatty acids, are also found. The first example of this latter class of molecule to be described was the diffusible signal factor DSF from the plant pathogen Xanthomonas campestris pv. campestris, which is cis-11-methyl-2-dodecenoic acid (1, 25). Until recently, it was thought that signal molecules of the DSF family were restricted to the xanthomonads, which do not synthesize N-AHLs (4, 11). However, work by Boon and colleagues (2) has demonstrated that Burkholderia cenocepacia, the opportunistic pathogen of cystic fibrosis patients and immunocompromised individuals, produces the DSF-like molecule cis-2-dodecenoic acid (BDSF). This molecule is able to activate DSF-dependent responses in X. campestris pv. campestris and to inhibit germ tube formation in Candida albicans, suggesting a role in interspecies and interkingdom communication. B. cenocepacia produces N-AHLs to regulate a wide range of functions that include virulence, biofilm formation, and motility (9, 23). This raises the question as to whether BDSF has any role as an intraspecies signal in B. cenocepacia J2315, an issue that we address here.

BDSF synthesis requires BCAM0581, which is related to the DSF synthase RpfF of X. campestris pv. campestris (1, 2). We have examined the role of BDSF in signaling by comparing the phenotypes of wild-type B. cenocepacia J2315, an rpfF mutant with a deletion in bcam0581, and a complemented mutant strain (Table (Table1).1). A strain carrying an in-frame deletion of rpfF was constructed by allelic exchange. DNA fragments comprising the upstream and downstream regions flanking rpfF in the genome were amplified by PCR, using B. cenocepacia genomic DNA as a template and the primer sets RPFFUF/RPFFUR and RPFFDF/RPFFDR, respectively (Table (Table1).1). The amplicons were mixed, diluted, and used as a template for PCR with RPFFUF and RPFFDR as the primers. To facilitate construction, restriction sites for BamHI and HindIII were engineered into these primer sequences. The amplified DNA fragment was cut with BamHI and HindIII and ligated into the allelic exchange vector pEX18Tc to give pEXRPFF. The construction was verified by DNA sequencing. The construct was introduced into B. cenocepacia J2315 by electroporation. The resulting mutants were selected on LB agar plates containing tetracycline (100 μg ml−1). Colonies harboring second crossover events were selected on LB agar containing 10% sucrose. The rpfF deletion mutant was identified by colony PCR, using the primer pair RPFFCOMF and RPFFCOMR (Table (Table1).1). To construct a complementing clone, the rpfF gene (bcam0581) with its promoter was amplified using B. cenocepacia J2315 chromosomal DNA as a template and the primers RPFFCOMF and RPFFCOMR (Table (Table1).1). The amplified fragment was cloned into the TOPO vector (Invitrogen, CA) and sequenced. A 2.0-kb HindIII-BamHI fragment carrying rpfF with its promoter was excised and ligated into pBBR1MCS for complementation analysis. This construct was introduced into B. cenocepacia J2315 rpfF by electroporation, and the complemented strain was selected on LB agar plates containing chloramphenicol (20 μg ml−1). Strains of Burkholderia cenocepacia were routinely grown at 37°C in Luria-Bertani (LB) medium or minimal M9 medium (20). No difference was observed in the growth rates of the strains. The wild type, the rpfF strain, and the complemented rpfF strain were tested for the production of BDSF by using an X. campestris pv. campestris bioassay (1). For these experiments, BDSF was extracted from culture supernatants with ethyl acetate as described previously (1). As expected from the work of Boon et al. (2), DSF-like activity was detected in the wild-type strain and the complemented rpfF mutant but not in the rpfF strain.

TABLE 1.
Strains, plasmids and primers used in this study

The role of BDSF in regulation in B. cenocepacia was initially investigated by comparative proteomic studies of the wild type and the rpfF mutant that were grown in M9 medium (20) to an optical density at 600 nm of 0.8. Whole-cell protein extraction, two-dimensional (2D) electrophoresis, 2D gel imaging and analysis using PDQuest, in-gel tryptic digestion of spots, and mass spectrometry of tryptic peptides were carried out as described previously (15). Representative 2D gels are shown in the supplemental material. Mutation of rpfF led to a reduced abundance of several identifiable proteins in each of three replicate experiments. These proteins included the chaperone, DnaK; CblD, which is implicated in cable pilus production; FliJ, a soluble component of the type III flagellar protein export system; and BCAM0791, a protein of the major facilitator superfamily (Table (Table2).2). Three hypothetical proteins (BCAM0242, BCAM0114, BCAM1600) showed increased abundance in the rpfF mutant. The results from this proteomic analysis were extended by measurement by reverse transcription-PCR (RT-PCR) of the transcript levels of selected genes, using the primers given in Table Table1.1. RNA was isolated from cultures of the wild-type B. cenocepacia J2315 strain, the rpfF mutant, the complemented rpfF mutant, and the rpfF mutant supplemented with 50 μM BDSF, grown to an optical density at 600 nm of either 0.8 or 1.5 in M9 medium, and converted to cDNA by using avian myeloblastosis virus reverse transcriptase (Promega), using conditions specified by the manufacturer. The quantitative PCR was carried out using Platinum SYBR green quantitative PCR SuperMix-UDG (Invitrogen). The primers used to amplify the genes are listed in Table Table1.1. As a control, RT-PCR was similarly applied to analyze expression of the 16S rRNA gene. The reverse transcription and PCR protocols were as described by Sambrook et al. (20). The amplified products were subjected to electrophoresis on 2.0% agarose gels and stained with ethidium bromide. RT-PCR analysis was repeated twice for each gene tested.

TABLE 2.
Proteomic analysis of the effects of mutation of rpfF on Burkholderia cenocepacia J2315

Mutation of rpfF did not lead to significant alterations in transcript levels for fliJ, dnaK, or bcam0791 (Fig. (Fig.1A).1A). However, transcript levels for cblD were lower in the rpfF mutant than those in the wild type (Fig. (Fig.1A).1A). The cblD gene is transcribed as part of the cbl operon, which comprises cblBACDS (19, 24). This operon is adjacent to the separately transcribed genes—cblR, which encodes a two-component regulator, and cblT, which encodes a sensor histidine kinase. CblT and CblR together with CblS, which is a second sensor histidine kinase, control cable pilus expression at the level of transcription of the cblBACDS operon (18). Relative transcript levels of these different cbl genes in the wild type and the rpfF mutant were established (Fig. (Fig.1B).1B). Mutation of rpfF led to lower transcript levels for cblR but no significant change for cblT. Transcript levels of cblBACS showed the same pattern of accumulation as did those of cblD, being lower in the rpfF mutant than in the wild type, which was expected from the organization of these genes within an operon.

FIG. 1.
Effect of mutation of rpfF on the expression of selected genes in B. cenocepacia J2315. (A) Effect of rpfF mutation on the expression of genes encoding proteins with altered abundance as determined by proteomic analysis. (B) Effect of mutation of rpfF ...

Complementation with the rpfF gene or addition of exogenous 50 μM BDSF restored cblD transcript levels to wild-type levels (Fig. (Fig.1C).1C). Similar observations were seen for other genes in the operon; results for cblA and cblS are also shown in Fig. Fig.1C.1C. These findings indicated that the effects of BDSF signaling on the abundance of the CblD protein, revealed by proteomic analysis, was exerted at least in part at the level of gene transcription. The mechanisms by which the levels of FliJ and BCAM0791 are regulated remain obscure.

The effect of mutation of rpfF on the abundance of CblD and FliJ suggested that BDSF signaling may be involved in regulation of bacterial motility and biofilm formation (to which flagella can contribute) or adhesion to host cell surface molecules such as mucin (to which cable pili can contribute). Consequently, the effect of mutation of rpfF on these phenotypes was tested and compared with the effects of disruption of each gene within the cblBACDS operon and fliJ. These mutant strains were created by directed insertional inactivation using pEX18Tc carrying an internal fragment of each gene. An internal fragment (approximately 500 bp) of each of the fliJ, cblA, cblS, and cblD genes was amplified using the primers detailed in Table Table1,1, cloned into the TOPO vector (Invitrogen), and sequenced. These fragments were then excised with EcoRI and ligated into the suicide plasmid pEX18Tc. These constructs were introduced into B. cenocepacia J2315 by electroporation. The mating mixture was plated onto LB agar containing tetracycline (100 μg ml−1) to select for insertion mutants. Candidate strains were analyzed by colony PCR using the primers detailed in Table Table11 to confirm disruption of the genes by directed insertion of the suicide vector.

The rpfF mutant showed reduced motility, which was assayed on 0.6% Eiken agar plates as described previously (5, 10). In contrast, mutation of the cbl genes did not influence motility in this assay, and mutation of fliJ had only a small effect (data not shown). The rpfF mutant showed reduced adherence to porcine mucin assayed using bacteria suspended in phosphate-buffered saline to an optical density at 600 nm of 1.0 and mucin-coated microtiter plates as previously described by Sajjan et al. (17, 18) (Fig. (Fig.2B).2B). These effects were more pronounced than those seen after mutation of the cbl genes, which gave an approximately 10% reduction in numbers of adhering bacteria, and mutation of fliJ, which had no effect. The rpfF mutant showed reduced biofilm formation after 24 h growth in 24-well microtiter dishes in LB medium, as quantified by crystal violet staining (14). In comparison, mutation of the cbl genes and fliJ had no effect on biofilm formation under these conditions. Complementation of the rpfF mutation with the wild-type rpfF gene restored motility, adherence to mucin, and biofilm formation to wild-type levels (Fig. 2A to C). Furthermore, addition of exogenous 50 μM BDSF restored motility and biofilm formation of the rpfF mutant to wild-type levels (data not shown). Taken together, these findings suggested that BDSF signaling has a pleiotropic regulatory influence in B. cenocepacia J2315, affecting motility, adherence to mucin, and biofilm formation. These effects cannot, however, be explained solely through regulation of the levels of FliJ or Cbl proteins.

FIG. 2.
Loss of BDSF signaling through mutation of rpfF has multiple phenotypic effects in B. cenocepacia J2315. The rpfF mutant showed reduced motility in 0.6% Eiken agar (A), reduced adherence to porcine mucin (B), and reduced biofilm formation (C), ...

The role of BDSF signaling in regulation of factors such as motility and biofilm formation, known to contribute to virulence in B. cenocepacia, prompted us to examine the effects of rpfF mutation on virulence, using the Galleria mellonella larva infection model (12, 21, 22). Larvae were inoculated with 10 μl of bacterial suspensions containing 1 × 106 CFU made in phosphate-buffered saline using a 10-μl Hamilton syringe. Each trial consisted of 10 larvae, and three independent trials were carried out for each strain. Following injection, larvae were placed in a static incubator in the dark at 37°C and were monitored for survival at 24 h postinfection. The results (Fig. (Fig.3)3) showed that the rpfF mutant killed a smaller number of larvae than did the wild type, within 24 h. Complementation of the rpfF mutant with the wild-type rpfF gene restored killing to wild-type levels (Fig. (Fig.3).3). This reduction in virulence seen in the rpfF mutant was more pronounced than that seen after mutation of either fliJ or the different cbl genes (Fig. (Fig.3),3), which may reflect a broad regulatory influence of BDSF signaling on virulence functions in B. cenocepacia J2315.

FIG. 3.
Virulence of Burkholderia cenocepacia strains in the Galleria mellonella larva infection model. Larvae were injected with 106 bacteria and monitored for survival 24 h postinfection. Each bar is representative of the results for five independent trials ...

Overall, the findings suggest that in addition to having a role in interkingdom signaling, BDSF is an intraspecies signal for B. cenocepacia. Several lines of evidence indicate that this signaling function is independent of N-AHL signaling, which is also known to regulate virulence as well as other functions in Burkholderia spp. (23). Mutation of rpfF does not influence the levels of N-AHLs produced by B. cenocepacia J2315, and mutation of cepI or cciI, which encode enzymes for the synthesis of N-AHLs, does not give rise to the same phenotypic alterations as does mutation of rpfF (R.P. Ryan, unpublished data). Although BDSF and DSF are structurally similar molecules, bioinformatic analysis results suggest that the signal transduction mechanism associated with BDSF signaling in B. cenocepacia is different from that established for DSF in xanthomonads. In the latter case, the two-component system RpfC/RpfG couples DSF perception to alterations in the cellular level of the second messenger cyclic di-GMP (16). The genome of B. cenocepacia J2315 does not, however, encode a histidine kinase with a sensory input domain related to that of RpfC. Although the mechanism by which the BDSF signal is transduced is currently unknown, the definition of specific transcriptional responses of the rpfF mutant to exogenous BDSF should allow appropriate genetic screens to be devised.

Supplementary Material

[Supplemental material]

Acknowledgments

The work in the BIOMERIT Research Centre is supported by a Principal Investigator Award from the Science Foundation of Ireland to J.M.D.

We thank T. Tolker-Nielsen and L. Yang for supplying reagents.

Footnotes

[down-pointing small open triangle]Published ahead of print on 29 May 2009.

§Supplemental material for this article may be found at http://jb.asm.org/.

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