General DNA manipulations.

Routine genetic manipulations were carried out using standard procedures (43). Primers were synthesized by AlphaDNA Inc. (Montreal, QC, Canada), and their sequences are available from us on request. Plasmid DNA was isolated using QIAprep spin miniprep kits (Qiagen Inc., Mississauga, ON, Canada), and agarose gel fragments were purified using a QIAquick gel extraction kit (Qiagen). Restriction endonucleases were from New England BioLabs (Mississauga, ON, Canada). DNA sequencing was carried out by the UBC NAPS unit.

Recombinant DNA manipulations.

For construction of a cbrA deletion mutant, an in-frame deletion of cbrA was obtained via splicing by an overlap extension PCR strategy (17). Briefly, primers were designed to amplify the gentamicin cassette of pPS858. Primers were also designed to amplify approximately 1-kb fragments located upstream and downstream of cbrA from PA14 genomic DNA with additional short sequences of overlap with the gentamicin cassette. Next, the three DNA fragments were fused together and the final product was boosted by a third PCR. The resulting fragment was cloned into pEX18Ap carrying a sacB sucrose sensitivity gene (16). This plasmid was transformed into E. coli S17-1 λpir and conjugated into P. aeruginosa PA14 to generate an in-frame deletion of the cbrA gene in the PA14 strain by allelic exchange. Selection for double recombinants was performed on plates containing gentamicin and 5% (wt/vol) sucrose. The deletion was confirmed by PCR and sequencing.

The suicide vector pME9673, obtained from Dieter Haas's laboratory, contained the deleted crcZ promoter and the 5′ region of crcZ (46). To construct a crcZ deletion mutant, a crcZ deletion was introduced from plasmid pME9673 into the chromosomal crcZ locus of P. aeruginosa strain PA14 by gene replacement as described previously (46). Briefly, the plasmid pME9673 was mobilized from E. coli DH5α into P. aeruginosa PA14 with the help of E. coli HB101/pRK2013. PA14 transconjugates carrying a chromosomally integrated copy of pME9673 were selected on tetracycline. Excision of the vector by a second crossover (i.e., tetracycline-sensitive derivatives) was subsequently obtained by enrichment with carbenicillin. The chromosomal crcZ deletion was confirmed by PCR and sequencing.

The PA14 cbrA deletion mutant and cbrB and crc transposon mutants were complemented by amplifying cbrAB, cbrB, or crc, including an upstream region of 400 base pairs, from PA14 genomic DNA by PCR and cloning each fragment into the broad-host-range vector pUCP18 or pUCP19 (50). The resulting hybrid plasmids, pUCP18::cbrAB⁺, pUCP19::cbrB⁺, and pUCP18::crc⁺, were transferred into the cbrA, cbrB, and crc mutants, respectively, by electroporation (3).

The crcZ deletion mutant was complemented by chromosomal insertion of a mini-Tn7 carrying the functional crcZ⁺ gene into the Tn7 attachment site of the mutant. The suicide plasmid, pME9818, which contained the cloned crcZ⁺ gene including the promoter region, was kindly provided by the laboratory of Dieter Haas (L. Abdou and D. Haas, unpublished data). Briefly, pME9818, carried by host E. coli S17-1, was transferred into the PA14 crcZ mutant by conjugation. Transposition of the mini-Tn7 carrying the crcZ⁺ gene into the chromosome of the crcZ mutant was facilitated by the E. coli SM10λpir helper carrying pUXBF-13. The P. aeruginosa strain carrying a crcZ⁺ insertion was selected with gentamicin and chloramphenicol and confirmed by PCR.

Motility experiments.

Swimming and twitching of P. aeruginosa PA14 wild type (WT) and mutants were examined on LB plates with 0.3% (wt/vol) agar and 1% (wt/vol) agar, respectively. Swarming for PA14 strains was examined on BM2-swarming plates containing BM2 with 0.1% (wt/vol) Casamino Acids substituted for 7 mM (NH₄)₂SO₄ and 0.5% (wt/vol) agar. To test the effects of the carbon source on the swarming motility of the cbrA and cbrB mutants, glucose was replaced with 0.4% (wt/vol) glycerol, 0.4% (wt/vol) mannitol, or 20 mM succinate. For iron complementation studies, Fe(II) sulfate was added to the swarming medium to a final concentration of 100 μM. Swimming and swarming motility were assayed by spotting 1 μl of mid-logarithmic-growth-phase cultures grown in LB broth or BM2-swarming medium onto the respective motility plates. Twitching motility was assessed by stab-inoculating a single colony grown overnight on LB broth at 37°C. The resultant diameters of the swim or twitch zones were measured after incubation for 20 h and 24 h at 37°C. The resultant swarming colonies were analyzed by measuring the surface coverage on agar plates after 20 h at 37°C. For each form of motility, 3 independent experiments were performed with 3 replicates for each mutant.

Biofilm assays and rapid attachment.

Biofilm formation was analyzed using an abiotic solid-surface assay as described elsewhere (9, 34). Overnight cultures were diluted in BM2-biofilm medium [62 mM potassium phosphate buffer, pH 7, 7 mM (NH₄)₂SO₄, 2 mM MgSO₄, 10 μM FeSO₄, 0.4% (wt/vol) glucose, 0.5% (wt/vol) Casamino Acids] in polystyrene microtiter plates (Falcon) and incubated at 8 h or 20 h at 37°C to study the initiation and mature biofilm formation, respectively. To test the effects of the carbon source on biofilm formation of the cbrA and cbrB mutants, glucose was replaced with 0.4% (wt/vol) glycerol, 0.4% (wt/vol) mannitol, or 20 mM succinate. For iron complementation studies, Fe(II) sulfate was added to the biofilm medium to a final concentration of 100 μM. Biofilms were stained with crystal violet, and absorbance was measured at 600 nm using a microtiter plate reader (Bio-Tek Instruments Inc.).

Rapid attachment was assayed as described previously with modifications (34). Overnight cultures were first diluted 1/100 into fresh BM2-biofilm medium and grown to an optical density at 600 nm (OD₆₀₀) of 0.5, and 100 μl was added to each well of a 96-well polystyrene microtiter plate. Cells were allowed to attach for 30 min at room temperature prior to staining with crystal violet as described above.

CR assay.

Congo red (CR) binding assays were performed as previously described (9). Briefly, tryptone-grown overnight cultures were diluted to OD₆₀₀s of 0.025 and 1, 5, and 10 μl were spotted onto CR plates (10 g/liter tryptone broth with 10 g/liter agar, 40 μg/ml Congo red, and 20 μg/ml Coomassie brilliant blue). The plates were incubated for 24 h at 37°C, followed by 48 h at room temperature to assess colony morphology.

MIC determination and killing experiments.

MICs were measured using standard broth microdilution procedures (26) in BM2-swarming medium. Growth was scored following 24 h of incubation at 37°C. For measuring MICs against polymyxin B, a modified assay was used to prevent artificially high MICs due to aggregation of the antibiotic and binding to polystyrene (52).

To perform killing experiments, cells of P. aeruginosa were grown to an OD₆₀₀ of 0.5 in BM2-swarming liquid medium or on BM2-swarming agar plates for 18 h (37). These cultures were diluted into 1× BM2-salts containing 1 μg/ml polymyxin B sulfate (Sigma). Samples were shaken at 37°C, and aliquots were taken at specified times, plated for survivors on LB agar, and grown overnight at 37°C. All experiments were repeated at least 3 times.

Cytotoxicity assays.

For the interaction assay, 16HBE14o- cells were seeded in 96-well plates (Corning Life Science, Corning, NY) at a density of 2 × 10⁴ cells/well and grown at 37°C with 5% CO₂ until 100% confluent (~48 h). Bacteria were grown in LB broth until mid-logarithmic phase, washed with phosphate-buffered saline, and resuspended and diluted in MEM containing 1% FBS and 2 mM l-glutamine. The interaction assay was performed at a multiplicity of infection (MOI) of 2 bacteria/cell in MEM containing 1% FBS and 2 mM l-glutamine, and the assay mixture was incubated at 37°C with 5% CO₂. At postinfection time points, medium was removed from the wells, placed in microtiter plates, and spun for 10 min at 3,000 rpm to pellet the bacteria and host cell debris. The level of lactate dehydrogenase (LDH) in the supernatant was then assayed in triplicate using a colorimetric cytotoxicity detection kit (Roche, Mannheim, Germany). As a positive control for maximum LDH release, cells were treated with 1% Triton X-100 (Sigma, Oakville, Canada), resulting in complete cell lysis, while untreated cells were used to assess background LDH release.

Growth curves.

P. aeruginosa mutants and wild type were grown overnight in LB medium, BM2-swarming medium, or BM2-biofilm medium. If necessary, cultures were diluted to obtain equal optical densities. Five-microliter portions of these cultures were added to 195 μl of fresh medium in 96-well microtiter plates. The growth of these cultures at 37°C under shaking conditions was monitored with a Tecan Spectrofluor Plus by determining the absorbance at 620 nm every 20 min for 24 h. Two independent experiments were performed with 3 replicates for each mutant.

Real-time quantitative PCR (RT-qPCR).

Total RNAs from the cbrA, cbrB, crcZ, and crc mutants were harvested under various conditions as follows: (i) for the swarming condition, cells were obtained from the leading edge of the dendritic swarm colonies of the PA14 wild type and the entire nonswarming colonies of the PA14 cbrA transposon mutant (ID33836), the cbrA deletion mutant, the cbrB transposon mutant, and the crcZ deletion mutant; (ii) for the polysaccharide synthesis-inducing condition, cells for RNA isolation were obtained by spotting 10 μl of diluted cultures grown in tryptone broth onto CR plates without Congo red or Coomassie brilliant blue; (iii) for the HBE cell infection condition, 16HBE14o- cells were seeded in tissue culture-treated petri dishes (Corning Life Science, Corning, NY) at a density of 2 × 10⁴ cells/well and grown at 37°C with 5% CO₂ until 100% confluent (~48 h). Bacteria were grown in LB medium until mid-logarithmic phase, washed with phosphate-buffered saline, and resuspended and diluted in MEM containing 1% FBS and 2 mM l-glutamine. The interaction assay was performed at an MOI of 100 bacteria/cell in MEM containing 1% FBS and 2 mM l-glutamine, and the assay mixture was incubated at 37°C with 5% CO₂. At 4 h postinfection, medium was removed from the dishes, placed in sterile Falcon tubes, and spun for 10 min at 3,000 rpm to pellet the bacteria. Subsequently, RNAs were isolated using RNeasy minicolumns (Qiagen) treated with DNase I (Invitrogen) to remove contaminating genomic DNA. Three micrograms of total RNA was combined with 0.5 μM deoxynucleoside triphosphates (dNTPs), 500 U SuperaseIN/ml (Ambion), and 10 μM dithiothreitol (DTT) in 1× reaction buffer and reverse transcribed with Superscript II reverse transcriptase (Invitrogen). The resultant cDNA was used as a template for qPCR. Analysis was carried out in the ABI Prism 7000 sequence detection system (Applied Biosystems) using the two-step RT-qPCR kit with SYBR green detection (Invitrogen). Fold change was determined using the comparative threshold cycle (C_T) method by comparison to the PA1544 housekeeping gene.

DNA microarray experiment.

Microarray experiments were performed on three independent cultures. The cbrA deletion mutant and wild-type PA14 were grown on a BM2-swarming plate containing 0.5% (wt/vol) agar for 18 h at 37°C. RNA was harvested from the leading edge of the dendritic swarm colonies of the PA14 wild type and of the entire nonswarming colonies of the cbrA mutant. As described previously (37), cells were resuspended in BM2-swarming medium supplemented with RNAprotect reagent (Qiagen, Germany). Harvesting of cells, RNA isolation, cDNA synthesis, hybridization to P. aeruginosa PAO1 DNA microarray slides (aminosilane coated) from the Institute for Genomic Research (TIGR) Pathogenic Functional Genomics Resource Center, analysis of microarray slides using ArrayPipe version 1.7, and RT-qPCR were performed as described previously.

Pyoverdine assay.

Bacterial strains were grown in CAA medium (5 g/liter low-iron Bacto Casamino Acids [Difco], 1.54 g/liter K₂HPO₄·3H₂O, 0.25 g/liter MgSO₄·7H₂O) at 37°C for 48 h. The supernatants were diluted 1/75 in 10 mM Tris-HCl, pH 7.5, and excited at 400 nm with a spectrofluorimeter (11).

The cbrA deletion mutant was impaired in swarming motility.

The abilities of the cbrA deletion mutant to swim, twitch, and swarm on 0.3%, 1%, and 0.5% agar, respectively, were examined. The cbrA deletion mutant exhibited minor defects in flagellum-mediated swimming and type IV pilus-mediated twitching motilities (data not shown). In contrast, this mutant's ability to swarm was completely abolished. The swarming-defective phenotype of this mutant could be restored to the wild-type level by introducing the wild-type cbrAB genes into the mutant (Fig. ​(Fig.1A).1A). We also investigated whether the inability of the mutant to swarm was influenced by differences in production of rhamnolipids. Using the rhamnolipid agar plate method (6, 7), no difference was observed between the diameters of the halos formed due to rhamnolipid produced from the cbrA mutant and the PA14 wild type (data not shown). To investigate whether the inability of the cbrA mutant to swarm was due to its poor ability to utilize glucose as the carbon source, we replaced glucose, in the swarming medium, with succinate, a carbon source that had been demonstrated, and confirmed in our growth studies, to sustain the wild-type growth of the P. aeruginosa cbrA mutant. Furthermore, swarming of the cbrA mutant was tested on other carbon sources, including glycerol and mannitol. Replacement of glucose with other carbon sources did not restore the ability of the cbrA mutant to swarm (data not shown).

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FIG. 1.

Swarming motility of the PA14 cbrA and cbrB mutants. Defective swarming motilities of the cbrA deletion mutant (ΔcbrA) and the cbrB transposon mutant (ΔcbrB) were restored to levels similar to that of the wild type, P. aeruginosa PA14 (WT), by transforming these mutants with plasmids containing the wild-type cbrAB genes or the wild-type cbrB gene.

The cbrA mutant exhibited enhanced biofilm formation.

P. aeruginosa forms biofilms on a number of surfaces, including tissues of the human host. Consequently, biofilm infections are virtually impossible to eradicate due to the biofilm's inherent resistance to conventional antibiotic therapies (28). Therefore, we investigated here the ability of the cbrA deletion mutant to form simple biofilms using static microtiter biofilm methods. These experiments demonstrated that the cbrA mutant showed a significant (P < 0.01 by Student's t test) but moderate (~40 to 60%) enhancement in biofilm formation as early as 8 h (Fig. ​(Fig.22 A and B). This biofilm phenotype could be successfully complemented by introducing the wild-type cbrAB operon into the mutant. To determine whether the biofilm formation phenotype occurred during initial attachment stage or later during biofilm development, a rapid (30-min) attachment assay was performed. No difference in early attachment was observed between the PA14 wild type and the cbrA mutant (data not shown).

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FIG. 2.

Biofilm formation of the PA14 cbrA and cbrB mutants. Cells were incubated in 96-well microtiter plates containing BM2-biofilm medium for 8 h (A) and 20 h (B) at 37°C for the cbrA mutant and 20 h at 37°C for the cbrB mutant (C). Surface-associated biofilm formation was analyzed by crystal violet staining of the adherent biofilm followed by ethanol solubilization of the crystal violet and quantification (A₆₀₀) of the stained well. Results shown are means with standard deviations for three biological experiments, each with eight technical repeats. **, statistically significant difference (P < 0.01) between the mutants and the wild type as determined by Student's t test.

The excess-biofilm-forming phenotype of the cbrA mutant led us to investigate further whether this mutant was altered for other biofilm-related functions. Chemical analyses of P. aeruginosa biofilms have suggested that the matrix is comprised of exopolysaccharides (EPS), DNA, RNA, proteins, and ions (47, 51). In P. aeruginosa, the psl and pel loci have been suggested to be involved in the production of the polysaccharide component of the matrix (9, 10). While alginate is a component of the extracellular matrix, studies have suggested that alginate is not a significant component of the extracellular polysaccharide present in the matrix of biofilms formed by P. aeruginosa under commonly used laboratory growth conditions (14, 54). Also, while P. aeruginosa strain PAO1 has both pel and psl loci, only the pel locus has been identified in the PA14 strain (9). To investigate whether the increased-biofilm-forming phenotype of this mutant was due to increased production of the pel-encoded extracellular matrix, we performed Congo red assays, as Congo red has been shown to bind the pel-encoded polysaccharide of P. aeruginosa PA14. The cbrA mutant showed substantially increased binding to Congo red compared to the PA14 wild type (Fig. ​(Fig.33).

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FIG. 3.

Congo red binding. Strains were spotted on Congo red and Coomassie brilliant blue plates and incubated for 24 h at 37°C, followed by incubation for 48 h at room temperature. Representative images of the colony morphology of P. aeruginosa PA14 (WT), the cbrA mutant (ΔcbrA), the cbrB mutant (ΔcbrB), the complemented cbrA/B⁺ strain, and the complemented cbrB⁺ strain are shown.

Furthermore, biofilm production of the cbrA mutant was assessed in different carbon sources, including succinate, mannitol, and glycerol. Interestingly, when glucose was replaced with succinate as the major carbon source, the cbrA mutant produced significantly less biofilm than in glucose at 20 h, while no such difference was observed for the wild type (Fig. ​(Fig.4).4). When mannitol or glycerol was provided as the carbon source, the cbrA mutant still produced significantly more biofilm than did the wild type (data not shown).

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FIG. 4.

Influence of carbon source on biofilm formation of the PA14 cbrA deletion mutant. Cells were incubated at 37°C for 20 h in 96-well microtiter plates containing BM2-biofilm medium supplemented with 0.4% (wt/vol) glucose (open bars) or 20 mM succinate (filled bars). Surface-associated biofilm formation was analyzed by crystal violet staining of the adherent biofilm followed by ethanol solubilization of the crystal violet and quantification (A₆₀₀) of the stained well. Results shown are means with standard deviations for three biological experiments, each with eight technical repeats. **, statistically significant difference (P < 0.01) between the mutants and the wild type as determined by Student's t test.

The cbrA mutant displayed enhanced cytotoxicity toward human bronchial epithelial cells.

P. aeruginosa possesses a large arsenal of virulence factors. One of the major virulence mechanisms employed by P. aeruginosa to intoxicate eukaryotic cells is the type III secretion system (T3SS). The T3SS, triggered by cell contact, injects toxins directly into the cytoplasm of the target cell. Moreover, the CbrAB system was previously demonstrated to be involved in the metabolic regulation of the T3SS and its effectors (41). Therefore, we examined the ability of the cbrA mutant to infect and destroy a monolayer of cultured 16HBE14o- epithelial cells. To measure the cytotoxic effects of the PA14 wild type and cbrA mutant on the epithelial cells, the amount of lactate dehydrogenase (LDH) released from the 16HBE14o- cells was quantified using an enzyme assay. The cbrA mutant displayed 3.5- and 2.2-fold-greater cytotoxicity than did the wild type at 4 h and 6 h postinfection, respectively (Fig. ​(Fig.5A).5A). Introducing the wild-type cbrAB operon into the cbrA mutant restored cytotoxicity to wild-type levels at both time points. Furthermore, to investigate whether the cbrA mutant was able to infect the epithelial cells better than the wild type was, due to an improved ability to adhere to the epithelial cells, adhesion assays were performed. No significant differences in adherence to 16HBE14o- cells at 1 h were observed between the wild type and the mutant (data not shown).

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FIG. 5.

In vitro cytotoxicity toward HBE cells. The abilities of the PA14 wild type (WT), the mutants (cbrA mutant [ΔcbrA] and cbrB mutant [ΔcbrB]), and the complemented strains (cbrA/B⁺ and cbrB⁺) to induce cell damage were determined by monitoring the release of intracellular lactate dehydrogenase (LDH) into the supernatant from HBE cells. Bacteria were cocultured with the cells, and LDH release was monitored at the time points indicated. Each result represents the mean of three independent biological repeats, each assayed in triplicate.

The cbrA mutant demonstrated increased resistance to polymyxins, aminoglycosides, and fluoroquinolones.

Recently, a microarray analysis indicated that among the many transcriptional changes, the cbrA gene was 2-fold downregulated (P < 0.05) in response to sublethal concentrations of tobramycin (Kindrachuk et al., submitted). Therefore, we were interested in investigating the role of CbrA in modulating resistance to a variety of clinical antibiotics, including aminoglycosides, cationic peptides, fluoroquinolones, cephalosporins, and carbapenems (Table ​(Table2).2). Intrinsic resistance of the mutant to these antibiotics was assessed by MIC assay in cells growing in BM2-swarming medium containing high (2 mM) Mg²⁺ to suppress the possibility of induction of genes by limiting Mg²⁺. Compared to the PA14 wild type, the cbrA mutant reproducibly exhibited a 2-fold-increased resistance to polymyxin B and colistin and 4-fold-increased resistance to ciprofloxacin and tobramycin. No difference was observed between the MIC values of the PA14 wild type and the mutant to piperacillin, tetracycline, cefepime, ceftazidime, and imipenem. Although the 2-fold change in MIC observed for the cbrA mutant to polymyxin B is often considered within the acceptable range of error of these assays, we confirmed the increased resistance of the cbrA mutant to polymyxin B by performing kill curve assays with cells taken from liquid swarm medium and from swarm plates (Fig. ​(Fig.6).6). The antibiotic susceptibility phenotype could be complemented to wild-type levels by introducing the wild-type cbrAB genes into the mutant.

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FIG. 6.

Polymyxin B resistance in the PA14 cbrA deletion mutant. Sensitivity to polymyxin B at 1 μg/ml was analyzed using cells from mid-log phase in swarm medium (A) or using cells directly off swarm plates and then plating diluted aliquots for survivors (B). For each condition, one representative experiment of four independent experiments that produced identical results is shown.

The moderate growth deficiency exhibited by the cbrA mutant was insufficient to explain its swarming, biofilm, or cytotoxicity phenotypes.

Previous studies identified CbrAB as an important regulatory element for the expression of several catabolic pathways and utilization of a variety of organic compounds as the sole carbon source (32). Li and Lu showed that a cbrAB mutant displayed weak growth when glucose was used as the sole carbon source, while growth on tricarboxylic acid (TCA) cycle intermediates was sustained (22). Therefore, we investigated whether any of the phenotypes observed for the cbrA mutant could be related to growth impairment when glucose was provided as the major carbon source in the swarming, MIC, and biofilm media. The growth of the cbrA mutant and the PA14 wild type was measured in the appropriate medium (BM2-swarm medium, LB medium, or BM2-biofilm medium) at 37°C using a Tecan Spectrofluor Plus to measure the absorbance at 620 nm every 20 min for 15 to 20 h under shaking conditions. The growth of the mutant and wild type was also determined by measuring the absorbance at 600 nm every 20 min during infection of 16HBE14o- cells. As shown in Fig. ​Fig.7A,7A, the cbrA mutant exhibited a very minor growth defect in LB medium. A moderate defect in growth under swarming conditions was observed for the mutant (Fig. ​(Fig.7B).7B). The moderate growth defect of the mutant in this medium (~50% change in growth rate), however, seemed insufficient to explain the complete abolition of swarming motility. The cbrA mutant also exhibited slight growth defects under biofilm-inducing (Fig. ​(Fig.7C)7C) and HBE infection conditions, but these growth defects also seemed insufficient to explain the increased biofilm production and cytotoxicity of the mutant. Furthermore, we investigated whether changing the carbon source from glucose to succinate would restore the ability of the mutant to swarm and form biofilm to the wild-type level. The cbrA mutant indeed showed wild-type growth when glucose was replaced with succinate in the BM2-swarming and BM2-biofilm media, as suggested from previous studies (22), but exhibited the same swarming and biofilm defects as in glucose media.

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FIG. 7.

Growth curves of P. aeruginosa PA14 wild type (WT) and cbrA deletion mutant. Cells were diluted and grown in LB broth (A), BM2-swarm medium (B), and BM2-biofilm medium (C). Growth was measured at 37°C using a Tecan Spectrofluor Plus.

Microarray analysis of the cbrA mutant.

To investigate how CbrA contributed to the various phenotypes observed, microarray studies were performed comparing the cbrA mutant to the PA14 wild type. For the microarray, RNAs from the PA14 wild type and the cbrA mutant were taken directly from BM2-swarm plates that had been incubated at 37°C for 20 h. The microarray revealed 236 genes that were differentially regulated by more than 2.0-fold (P ≤ 0.06) with 145 transcriptionally upregulated and 91 transcriptionally downregulated genes (see Table S1 in the supplemental material). A selection of these genes is presented in Table ​Table3.3. Of note, PAO1 DNA microarray slides were used to analyze gene expression of PA14 cbrA mutant and PA14 wild type, as there are no PA14-specific microarray slides available. The PA14 genome (6.5 Mbp) is slightly larger than that of PAO1 (6.3 Mbp), but the PA14 and PAO1 genomes are very similar, with greater than 92% of all genes in PA14 also present in PAO1. The additional genes in the PA14 genome that are absent in PAO1 have been suggested to contribute to its enhanced pathogenicity, as PA14 is more virulent than PAO1 (13).

TABLE 3.

Selected genes significantly dysregulated in cbrA mutant as determined using microarray

Category and gene IDa	Name	Fold changeb	P value	Function
Adaptation and protection
PA1159		−2.36	0.0353	Probable cold shock protein
PA2385	pvdQ	−3.01*	0.002	3-Oxo-C12-homoserine lactone acylase and pyoverdine biosynthesis
PA2386	pvdA	−2.39*	0.0011	l-Ornithine N5-oxygenase
PA2397	pvdE	−3.44	0.002	Pyoverdine biosynthesis protein
PA2399	pvdD	−2.24	0.0061	Pyoverdine synthetase D
PA2920		2.14	0.0068	Probable chemotaxis transducer
PA3349		2.15	0.0013	Probable chemotaxis protein
PA4223	pchH	−4.16*	8.59E−05	Probable ATP binding component of ABC transporter
PA4225	pchF	−2.53	0.0005	Pyochelin synthetase
PA4231	pchA	−2.48	0.0009	Salicylate biosynthesis isochorismate synthase
PA4356	xenB	2.27	0.0039	Xenobiotic reductase
PA4468	sodM	3.55	8.86E−05	Superoxide dismutase
PA4876	osmE	−2.00	0.0161	Osmotically inducible lipoprotein
Chemotaxis; cell wall/LPS/capsule
PA1423		2.55	0.0007	Probable chemotaxis transducer
PA2920		2.14	0.0068	Probable chemotaxis transducer
PA3157	wbpC	−2.3	0.0186	Probable acetyltransferase
PA3349		2.15	0.0013	Probable chemotaxis protein
PA3545	algG	2.08	0.0068	Alginate-C5-mannuronan-epimerase
Chaperones and heat shock proteins
PA1596	htpG	2.21	0.0016	Heat shock protein 90
PA2830	htpX	3.27	0.0001	Heat shock protein
PA3126	ibpA	3.26	6.23E−05	Heat shock protein
PA4352		2.65	0.0003	Putative universal stress protein
PA4761	dnaK	3.57*	5.33E−05	Molecular chaperone
PA4762	grpE	2.95	0.0004	Heat shock protein
PA5053	hslV	4.9	9.70E−05	ATP-dependent protease peptidase subunit
PA5054	hslU	3.07	0.0054	ATP-dependent protease ATP binding subunit
Antibiotic resistance and susceptibility
PA1178	oprH	9.36*	1.63E−06	PhoP/Q and low-Mg2+-inducible outer membrane protein H1 precursor
PA1170	phoP	4.16*	2.74E−05	Two-component response regulator
PA1797		10.37*	1.16E−06	Putative function in adaptive polymyxin resistance
PA2198		3.73	0.001	Putative antibiotic biosynthesis monooxygenase
PA3552	arnB	6.16*	6.84E−06	Hypothetical protein
PA3553	arnC	3.61	5.17E−05	Probable glycosyl transferase
PA3554	arnA	2.44	0.0021	Hypothetical protein
PA3555	arnD	4.49	1.57E−05	Hypothetical protein
PA3556	arnT	3.13	0.0001	Inner membrane l-Ara4N transferase
PA3557	arnE	4.51	2.97E−05	Hypothetical protein
PA3558	arnF	2.45	0.0028	Hypothetical protein

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^aID, identification. Information is according to the P. aeruginosa genome website (http://www.pseudomonas.com).

^bFold regulation of genes differentially expressed in cbrA mutant relative to PA14 wild type. A positive number indicates transcript upregulation and a negative number indicates transcript downregulation in the cbrA mutant. An asterisk indicates confirmation of gene regulation by RT-qPCR.

Analysis of the microarray data revealed dysregulation of genes involved in amino acid biosynthesis and metabolism, carbon compound catabolism, and central intermediary metabolism, consistent with the proposed role of CbrAB in the utilization of a variety of organic compounds as sole carbon source (22, 32). For example, hutU (urocanase), part of the hutUHIG operon involved in histidine catabolism, was moderately downregulated in the cbrA mutant (RT-qPCR revealed a fold change of −6.0 ± 0.6 relative to the wild type).

The cbrA microarray results were examined to identify genes that might influence antibiotic resistance of the mutant (since the microarray and MIC experiments utilized similar growth conditions). Moderate (3- to 10-fold) upregulation of the operons encompassing the two-component regulators oprH-phoPQ and pmrAB and of the downstream arnBCADTEF (lipopolysaccharide [LPS] modification) operon was observed in the microarray. To confirm the microarray results, RT-qPCRs were performed and revealed 9.3- ± 1.8-fold upregulation of oprH, 3.7- ± 0.5-fold upregulation of phoP, 3.1- ± 0.3-fold upregulation of phoQ, 10.2- ± 1.9-fold upregulation of arnB, and 4.0- ± 1.4-fold upregulation of pmrB. Activation of these operons is known to trigger bacterial resistance to cationic peptides and polymyxins in response to low-Mg²⁺ conditions by controlling the addition of aminoarabinose to lipid A, thereby reducing the net negative charge of LPS and limiting its interaction with polycationic peptides such as polymyxin B (29); however, their link to CbrAB had not been revealed previously. The involvement of the PhoPQ system in aminoglycoside resistance has also been defined (25, 38). Although the details of PhoPQ involvement in aminoglycoside resistance remain to be fully elucidated, PhoPQ appears to mediate resistance to aminoglycosides via a mechanism different from that involved with the polycationic peptides. The cbrA microarray also identified the upregulation of several heat shock protein genes, including htpG, ibpA, dnaK, grpE, hslV, and hslU. The upregulation of dnaK was confirmed by RT-qPCR, revealing 3.6- ± 0.2-fold upregulation in the mutant relative to the wild type. Recent studies have demonstrated that upregulation of heat shock genes prior to treatment with tobramycin led to increased resistance of P. aeruginosa to tobramycin (Kindrachuk et al., submitted). Although the cbrA microarray indicated upregulation of the mexX gene, known to be involved in involvement in aminoglycoside resistance (38), RT-qPCR failed to detect any significant changes in the transcriptional expression of this gene (fold change of 1.1 ± 0.3) in the cbrA mutant compared to the wild type.

Analysis of the microarray data also revealed moderate downregulation of several genes, PA0621, pvdD, PA3784, pchH, and pchF, which were possibly involved in the ciprofloxacin resistance phenotype observed in the cbrA mutant. A previous ciprofloxacin screen of the PA14 transposon mutant library by Breidenstein et al. showed that transposon mutants of PA0621, pvdD, PA3784, pchH, and pchF are more resistant than their wild-type parent strain toward ciprofloxacin (2). Flagella and type IV pili play important roles in biofilm and microcolony formation and are also required for swarming (19). Furthermore, studies have suggested that the flagellum secretion system plays a role in P. aeruginosa invasion of epithelial cells (8). However, the microarray did not reveal dysregulation in any flagellum- or type IV pilus-related genes. Furthermore, there was no dysregulation of the type III secretion apparatus or effector genes in the cbrA mutant.

This transcriptome analysis also highlighted several other interesting genes. These findings included moderate downregulation of the pyoverdine and pyochelin biosynthesis genes, including pvdA, pvdQ, and pchF. RT-qPCR confirmed downregulation of these genes in the mutant with values of −9.9 ± 1.3 for pvdA, −16.5 ± 2.4 for pvdQ, and −4.2 ± 0.6 for pchF. The downregulation of pvdQ in the cbrA microarray was of interest since a pvdQ mutant is swarming deficient (37) and has been suggested to play a role in biofilm formation and virulence of P. aeruginosa (18). Consistent with the qPCR data, we grew the cbrA mutant and the wild type in a low-iron medium to induce pyoverdine production and observed, using a spectrofluorimeter, that the mutant secreted less pyoverdine into the supernatant than did the wild type (Fig. ​(Fig.8).8). In a previous study, Jimenez et al. (18) showed that addition of iron in swarm plates restored the swarming of a swarming-impaired pvdQ mutant to the wild-type level. Therefore, we also tested whether addition of iron had any effect on swarming motility or the biofilm formation of the cbrA mutant. However, addition of iron did not restore swarming or biofilm formation of the cbrA mutant to the wild-type level (data not shown).

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FIG. 8.

Pyoverdine production of the PA14 cbrA deletion mutant. Cells grown in Casamino Acids medium for 48 h at 37°C were diluted, and the resultant sample was excited at 400 nm with a spectrofluorimeter.

Transcriptional analysis, by RT-qPCR, of the cbrA mutant under various growth conditions.

The microarray experiment of the cbrA mutant versus PA14 wild type did not reveal transcriptional changes in the expression of genes known to be involved in virulence (e.g., the type III secretion system) or genes involved in the production of exopolysaccharides in P. aeruginosa. This was not completely unexpected, since the growth conditions used for the microarray experiment and the conditions used to assay exopolysaccharide production or in vitro cytotoxicity experiments were different and it is known that CbrAB influences the utilization of many different carbon and nitrogen sources. Therefore, we isolated RNA from bacterial cells during in vitro infection of HBE cells and cells growing on Congo red plates. Consistent with the increased binding phenotype observed on the Congo red plates (Fig. ​(Fig.3),3), RT-qPCR revealed 2.5- to 5-fold upregulation of the exopolysaccharide pelD and pelF genes in the cbrA mutant compared to the PA14 wild type (Table ​(Table4).4). Bacterial cells obtained during HBE cell infection revealed moderate upregulation of the type III secretion apparatus and effectors (exoT, exoY, exoU, pcrV, exsA, and popD) and the type I secretion apparatus, aprD (Table ​(Table4),4), consistent with the enhanced cytotoxicity of this mutant.

TABLE 4.

Dysregulated genes in the cbrA, cbrB, crcZ, and crc mutants during human bronchial epithelial (HBE) cell line infection and growth on Congo red plates as determined by RT-qPCR

Expt and gene	Fold change relative to PA14 wild typea
	cbrA	cbrB	crcZ	crc
HBE cell infection
exoT	2.9 ± 0.3	3.4 ± 1.0	3.8 ± 0.9	−3.8 ± 1.2
exoY	2.7 ± 0.3	2.3 ± 0.6	2.1 ± 0.5	−4.3 ± 0.3
exoU	2.5 ± 0.4	2.7 ± 0.3	2.0 ± 0.4	−9.8 ± 1.0
pcrV	2.6 ± 0.3	2.2 ± 0.1	2.0 ± 0.3	−5.8 ± 0.9
exsA	2.2 ± 0.2	2.7 ± 0.5	1.7 ± 0.6	−2.7 ± 0.9
pcrD	1.4 ± 0.1	1.2 ± 0.5	1.2 ± 0.8	−1.4 ± 0.5
popD	2.9 ± 0.5	2.3 ± 0.5	2.4 ± 0.4	−6.6 ± 0.7
pscF	1.2 ± 0.1	1.4 ± 0.5	1.6 ± 0.9	−2.4 ± 0.3
pcrR	1.3 ± 0.3	1.8 ± 1.1	1.2 ± 0.9	−1.5 ± 0.5
lasB	1.2 ± 0.2	1.1 ± 0.6	1.5 ± 0.8	−1.2 ± 0.5
lipC	1.2 ± 0.1	−1.1 ± 0.3	1.1 ± 0.5	−1.3 ± 0.3
plcB	1.6 ± 0.5	1.3 ± 0.3	1.7 ± 0.6	−1.8 ± 0.6
PA4528	1.5 ± 0.3	1.3 ± 0.1	1.1 ± 0.3	−1.0 ± 0.5
aprD	2.5 ± 0.5	2.9 ± 0.4	2.4 ± 0.3	−6.2 ± 0.8
Congo red
pelD	2.6 ± 0.5	5.1 ± 0.5	2.7 ± 0.5	−4.1 ± 1.0
pelF	2.5 ± 0.4	4.5 ± 0.4	2.8 ± 0.5	−2.6 ± 0.8

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^aValues are represented as averages of at least 3 biological samples.

CbrA mediated regulation of swarming, biofilm formation, and cytotoxicity in conjunction with CbrB.

The CbrB response regulator has been identified to play a role along with its cognate sensor kinase CbrA in the global metabolic regulation of carbon and nitrogen utilization in P. aeruginosa (22). Therefore, we examined whether CbrB also interacted with CbrA to modulate virulence and virulence-related processes in P. aeruginosa. Similarly to the cbrA mutant, the cbrB mutant displayed impairment in swarming and excessive biofilm formation (Fig. ​(Fig.1B1B and ​and2C).2C). These phenotypes could also be complemented by introducing the wild-type cbrB allele into the cbrB mutant. Congo red assays revealed increased binding of Congo red to the cbrB mutant compared to the wild type (Fig. ​(Fig.3).3). Interestingly, on the Congo red plates, the cbrB mutant also showed wrinkled morphology while the cbrA mutant remained smooth like the wild type. Furthermore, the cbrB mutant exhibited 2-fold-enhanced in vitro cytotoxicity toward HBE cells (Fig. ​(Fig.5B).5B). Similarly to the cbrA mutant, the cbrB mutant also exhibited moderate growth defects in swarming/MIC and biofilm media (data not shown). However, while the cbrA mutant showed increased resistance to a variety of antibiotics, the cbrB mutant displayed wild-type MIC values for all antibiotics tested (Table ​(Table2).2). Based on the microarray results of the cbrA mutant, we examined the expression level of a variety of genes in the cbrB mutant under swarming conditions by RT-qPCR. As expected, RT-qPCR revealed no dysregulation in the genes that were thought to explain the altered susceptibility of the cbrA mutant; these findings included a lack of dysregulation of the oprH-phoPQ operon, the pmrAB operon, and the LPS modification operon (data not shown). Similarly to the cbrA mutant, upregulation of energy metabolism genes, such as nirS and norC, was observed in the cbrB mutant. The expression of pvdQ was also downregulated in the cbrB mutant, while other genes involved in pyoverdine biosynthesis, such as pvdA, were not dysregulated in the cbrB mutant (data not shown). However, similarly to the cbrA mutant, pyochelin biosynthesis genes, such as pchG, were downregulated in the cbrB mutant. In addition, RNA was isolated from the cbrB mutant from Congo red plates, and during HBE cell infection, RT-qPCRs were performed. The cbrB mutant revealed upregulation of the pel operon under the Congo red condition, but to a greater extent than that with the cbrA mutant (Table ​(Table4).4). This result was consistent with the wrinkled morphology observed only in the cbrB mutant and the increased biofilm formation of the cbrB mutant compared to the cbrA mutant. RT-qPCR analysis of the cbrB mutant during HBE cell infection also revealed upregulation of the type III secretion system during in vitro infection (Table ​(Table44).

We gratefully acknowledge financial support from the Canadian Institutes for Health Research (CIHR) and the Canadian Cystic Fibrosis Foundation (CCFF). A.T.Y.Y. received studentships from the CCFF and the Natural Sciences and Engineering Research Council of Canada (NSERC). R.E.W.H. holds a Canada Research Chair.

We thank Laetitia Abdou and Dieter Haas for providing the necessary plasmids and primers for construction of the PA14 crcZ deletion mutant and complementation of the mutant. We thank Shaan Gellatly for her help with the in vitro human bronchial epithelial cell infection experiments. We also thank the Institute for Genomic Research (TIGR) for providing the microarray slides.