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J Bacteriol. Apr 2008; 190(8): 2671–2679.
Published online Feb 1, 2008. doi:  10.1128/JB.01659-07
PMCID: PMC2293252

Swarming of Pseudomonas aeruginosa Is a Complex Adaptation Leading to Increased Production of Virulence Factors and Antibiotic Resistance[down-pointing small open triangle]

Abstract

In addition to exhibiting swimming and twitching motility, Pseudomonas aeruginosa is able to swarm on semisolid (viscous) surfaces. Recent studies have indicated that swarming is a more complex type of motility influenced by a large number of different genes. To investigate the adaptation process involved in swarming motility, gene expression profiles were analyzed by performing microarrays on bacteria from the leading edge of a swarm zone compared to bacteria growing in identical medium under swimming conditions. Major shifts in gene expression patterns were observed under swarming conditions, including, among others, the overexpression of a large number of virulence-related genes such as those encoding the type III secretion system and its effectors, those encoding extracellular proteases, and those associated with iron transport. In addition, swarming cells exhibited adaptive antibiotic resistance against polymyxin B, gentamicin, and ciprofloxacin compared to what was seen for their planktonic (swimming) counterparts. By analyzing a large subset of up-regulated genes, we were able to show that two virulence genes, lasB and pvdQ, were required for swarming motility. These results clearly favored the conclusion that swarming of P. aeruginosa is a complex adaptation process in response to a viscous environment resulting in a substantial change in virulence gene expression and antibiotic resistance.

Swarming motility is a multicellular phenomenon involving the coordinated and rapid movement of a bacterial population across a semisolid surface (14). It is widespread among flagellated bacteria, including Salmonella, Vibrio, Yersinia, Serratia, and Proteus (9, 18). Swarming is highly dependent on bacterial cell density, nutrient growth medium, and surface condition moistness (53). In addition to physical changes such as an increase in the number of flagella or cell elongation, swarmer cell differentiation results in substantial alterations in metabolic bias and gene expression, indicating that swarming represents a complex lifestyle adaptation in response to particular medium conditions rather than merely a form of locomotion (18, 45).

Swarming of Pseudomonas aeruginosa is often typified by a dendritic colonial appearance. This gram-negative bacterium is a major cause of hospital-acquired bacterial infections and the most significant pulmonary pathogen in cystic fibrosis patients (17, 20, 44). It is one of the most difficult infections to treat due to its high natural (intrinsic) antibiotic resistance. It possesses three types of movement depending on medium viscosity, namely, swimming in aqueous environments, twitching on solid surfaces or interfaces, and swarming on semisolid, viscous media, such as those containing 0.4 to 0.7% (wt/vol) agar. It has been previously shown that swarming of P. aeruginosa is dependent on both flagella and type IV pili, which mediate actual movement, as well as on rhamnolipids, which are proposed to enable swarming cells to overcome the strong surface tension of the water surrounding swarming cells and thus modulate the swarming motility pattern (5, 10, 25, 35, 41). P. aeruginosa swarming is increased under nitrogen limitation and in response to certain amino acids (25).

Recently, we showed that swarming of P. aeruginosa represents an adaptation that is influenced by a large number of cooperating genes (41). Using a Tn5-lux mutant library (29), we were able to isolate more than three dozen swarming-negative mutants with transposon insertions in regulatory, metabolic, chemosensory, type II secretion system, hypothetical, motility, and quorum-sensing-related genes (41). Moreover, it was demonstrated that many mutants with altered swarming motility were also defective in biofilm formation, indicating that it may play a key role in early biofilm development (41, 48).

To understand the nature of the complex adaptation that accompanies swarming motility, we investigated here the influence of swarming motility on gene expression by performing microarrays of bacteria from the leading edge of a swarm zone compared to bacteria growing in identical medium under swimming conditions. Major shifts in gene expression patterns were observed under swarming conditions, including, among others, the up-regulation of a large number of virulence-related genes. Also, actively swarming cells exhibited adaptive antibiotic resistance against all tested antibiotics compared to what was seen for their planktonic counterparts. By analyzing a large subset of the up-regulated genes, we were able to show that two virulence genes, lasB and pvdQ, were required for swarming motility. These results clearly favor the conclusion that swarming of P. aeruginosa is a complex adaptation process in response to a viscous environment, resulting in a substantial change in virulence gene expression and antibiotic resistance.

MATERIALS AND METHODS

Bacterial strains, primers, and growth conditions.

Bacteria included P. aeruginosa PAO1 (50) and P. aeruginosa PA14 (43), as well as a large number of derived Tn5 mutants from the P. aeruginosa mutant libraries (29, 30) as described below. Escherichia coli XL1-Blue (4) and plasmids pUCP18 (47) and pUCP18-mini-Tn7-Gm (6) were used for cloning. The sequences of DNA primers (AlphaDNA, Montreal, Canada) used in these studies are available from the authors upon request. Cultures were routinely grown in Luria-Bertani broth, BM2 minimal medium [62 mM potassium phosphate buffer, pH 7, 7 mM (NH4)2SO4, 2 mM MgSO4, 10 μM FeSO4, 0.4% (wt/vol) glucose] or BM2-swarming medium, comprising BM2 with 0.1 or 0.5% (wt/vol) Casamino Acids substituted for 7 mM (NH4)2SO4. Solid medium contained either 0.3% (wt/vol) agar for assessing swimming motility, 0.5% agar for swarming assessments, or 1% agar for twitching motility assessments (25, 41). When required for plasmid or transposon selection or maintenance, tetracycline, gentamicin, and carbenicillin were added to final concentrations of 50, 15, and 300 μg/ml, respectively. Routine genetic manipulations were carried out using standard procedures (32).

Swarming motility and biofilm assays.

Except as otherwise noted, swarming of P. aeruginosa PAO1 was examined on BM2-swarming plates containing 0.5% (wt/vol) agar (Difco) and 0.5% (wt/vol) Casamino Acids as described previously (41), since these conditions result in a more circular swarm zone, permitting easier determination of swarm colony diameter. Since P. aeruginosa PA14 does not swarm consistently under these conditions, BM2-swarming plates supplemented with 0.1% (wt/vol) Casamino Acids were utilized, resulting in dendritic colonies that were analyzed by measuring the agar plate coverage as previously described (5). All swarm experiments were repeated five times independently. Bacteria were inoculated on swarm agar plates as 1- to 2-μl aliquots of an overnight culture grown in BM2 glucose, and plates were incubated for 18 h at 37°C.

Abiotic solid surface assay biofilm formation was analyzed in 96-well polystyrene microtiter plates after 20 h of incubation at 37°C as described previously (15, 40). After crystal violet staining, the absorbance was measured at 595 nm using a microtiter plate reader (Bio-Tek Instruments Inc.).

DNA microarray experiments.

Microarray experiments were performed on four independent cultures. P. aeruginosa PAO1 was grown for 6 h to the late logarithmic growth phase (optical density at 600 nm, 0.9 to 1.0) in liquid BM2-swarming medium under shaking (swimming) conditions or for 18 h at 37°C on a BM2-swarming plate containing 0.5% (wt/vol) agar and 0.1% (wt/vol) Casamino Acids (to obtain dendritic swarm colony growth enabling actively swarming cells to be more easily harvested from the leading edge of the swarm colony [within 2 to 3 mm of the swarm zone edge]). Cells were harvested either by centrifugation (swimming) or by a sterile inoculating loop (swarming, within 2 to 3 mm of the swarm zone edge) and were resuspended in BM2-swarming media supplemented with RNAprotect reagent (Qiagen, Germany). RNA isolation, cDNA synthesis, and hybridization to microarray slides (The Institute for Genomic Research [TIGR] Pathogenic Functional Genomics Resource Center) along with analysis of DNA microarray slides using ArrayPipe version 1.7 and quantitative reverse transcription-PCRs (RT-PCRs) were performed as described previously (38). P. aeruginosa microarray slides from TIGR consist of 18,432 elements with 5,552 oligonucleotides and 3 oligonucleotide replicates (for more information, visit http://pfgrc.tigr.org/slide_html/array_descriptions/P_aeruginosa.shtml). Only genes that exhibited changes, compared to the swimming control, of twofold or more and P values of ≤0.05 were considered in this study.

Mutant complementation.

For complementation of the P. aeruginosa PA14 pvdQ mutant ID27758 from the corresponding P. aeruginosa PA14 mutant library (30), the pvdQ gene was amplified by PCR and cloned as a HindIII fragment into the broad-host-range vector pUCP18. The resulting hybrid plasmid, pUCP18::pvdQ, was transferred into the P. aeruginosa PA14 pvdQ mutant ID27758 by electroporation (6), resulting in strain P. aeruginosa PA14 ID27758(pUCP18::pvdQ).

For complementation of the P. aeruginosa PAO1 lasB mutant 23_F8 from the corresponding P. aeruginosa PAO1 mutant library (29), the lasB gene including the promoter region was amplified by PCR and cloned as a BamHI fragment into plasmid pUC18-mini-Tn7-Gm (6). The resulting hybrid plasmid, pUCP18-mini- Tn7-Gm::lasB, was coelectroporated with pTNS2 into the P. aeruginosa PAO1 lasB mutant 23_F8 by electroporation (6), resulting in P. aeruginosa PAO1 strain 23_F8(lasB+) with a chromosomally integrated lasB+ gene.

Comparison of antibiotic resistance levels.

P. aeruginosa PAO1 cultures were grown for 6 h in BM2 medium with 0.3% agar under swimming conditions or on BM2-swarming agar plates with 0.5% agar and 0.5% Casamino Acids for 18 h under swarming conditions. Swim plates containing BM2 medium were supplemented with different concentrations of antibiotics and inoculated with 1 μl of culture grown under swimming conditions (inoculum of 1 × 104 to 1 × 105 cells). Swarm agar plates containing BM2-swarming medium supplemented with different concentrations of antibiotics were inoculated with cells taken from the outside of a swarming colony (within 2 to 3 mm from the swarm zone edge) by use of a sterile toothpick (inoculum of 1 × 104 to 1 × 105 cells). After 18 h of incubation at 37°C, growth was determined by measuring the swimming or swarming colony diameter as previously described (41).

MIC.

MICs were measured using standard broth microdilution procedures in BM2 and BM2-swarming medium (31). Growth was scored following 24 h and 48 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 (55).

Killing experiments.

Cells of P. aeruginosa were grown to an optical density at 600 nm of 0.4 to 0.6 in BM2 or BM2-swarming liquid medium (i.e., supplemented with 0.1% Casamino Acids) or on BM2-swarming agar plates containing 0.1% (wt/vol) Casamino Acids for 18 h. These cultures were then diluted 1:100 into prewarmed 10 mM sodium phosphate buffer, pH 7.5, containing 2 μg/ml polymyxin B sulfate (Sigma). Samples were shaken at 37°C and aliquots were withdrawn at specified times, plated for survivors on LB agar, and grown overnight at 37°C. All experiments were repeated three times.

Microarray accession number.

The ArrayExpress accession number is E-FPM1-10.

RESULTS

Microarray analyses of P. aeruginosa swarmer cells.

Based on mutant studies, it would appear likely that P. aeruginosa swarming is a complex adaptation to movement in a viscous environment in that it is influenced by a substantial number of cooperating genes and exhibits a strong interrelation with biofilm formation (41, 48). To further understand swarming motility, gene regulation events associated with swarming were investigated, comparing bacteria at the leading edge of a swarm zone (within 2 to 3 mm of the swarm zone edge) to those grown under swimming conditions. Analysis of four independent experiments demonstrated the statistically significant (P < 0.05 by Student's t test) greater-than-twofold change in expression levels of 417 genes (7.5% of all P. aeruginosa genes) (see Table S1 in the supplemental material; also see Table Table11 for selected examples). Of these, 309 were up-regulated and 108 down-regulated, including 40% hypothetical or conserved hypothetical genes with no currently defined function. RT-PCR experiments confirmed the microarray data for genes PA0044 (exoT), PA1706 (pcrV), PA3478 (rhlB), PA3841 (exoS), and PA4225 (pchF) (data not shown).

TABLE 1.
Selected P. aeruginosa genes that were up-regulated under swarming motility conditions cf. swimming conditions

The 243 annotated genes were analyzed by functional category according to the Pseudomonas genome database V2 (www.pseudomonas.com). Under swarming conditions, differential expression of genes from 21 different functional classes was observed, demonstrating the complex differences accompanying growth under swarming compared to swimming conditions (Fig. (Fig.1).1). Most interestingly, we observed under swarming conditions the up-regulation, by 2- to 13-fold, of a large number of genes associated with virulence factor synthesis and export. Among these were genes coding for the type III secretion system (TTSS) and related secreted factors exoS, exoT, and exoY; genes aprA, lasB, and pIV, encoding the extracellular proteases alkaline protease, elastase, and protease IV, respectively; the rhamnosyltransferase gene rhlB; and genes encoding the biosynthesis of the siderophores pyochelin and pyoverdine as well as redox-active phenazine compounds (Table (Table11).

FIG. 1.
Functional classification of nonhypothetical genes with a change in expression level of greater than twofold.

Although we and others have shown recently that swarming of P. aeruginosa PAO1 is dependent on both flagella and type IV pili (25, 41) and swarm cells of P. aeruginosa are described to be hyperflagellated and to possess two polar flagella (25), genes related to flagella and type IV pili biosynthesis and function were not up-regulated under swarming conditions. In contrast, genes fimU, pilW, pilX, pilY1, and pilY2, comprising one of the gene clusters involved in type IV pilus assembly and biosynthesis, were down-regulated by three- to eightfold.

Interestingly, with more than 70 differentially regulated genes relating to various types of bacterial metabolism, including cofactor biosynthesis, energy, carbon compounding, and central intermediary, fatty acid, and amino acid metabolism (see Table S1 in the supplemental material), actively swarming cells exhibited a striking difference in the gene expression profile of metabolic genes compared to what was seen for swimming cells. For example, various genes associated with glucose metabolism and uptake, including those for the carbohydrate-selective sugar porin OprB and other carbohydrate transporters, were down-regulated, whereas genes involved in fatty acid metabolism and nitrite reduction were up-regulated in swarmer cells. In this context it is worth mentioning that nitrite, which is present in the cystic fibrosis lung, promotes swarming and rhamnolipid production (49) and that mutants of P. aeruginosa with defects in nitrite reduction exhibit a swarming-negative phenotype (51).

Furthermore, no fewer than 18 predicted or known transcriptional regulatory genes were observed to be differentially regulated under swarming conditions, indicating that a complex regulatory network was involved in the swarmer differentiation process. These included pcrR, pcrG, pcrH, exsB, exsA, ptxS, gltR, rhlR, ntrB, ybbL, rnk, glnK, lasR, PA0610, PA2897, PA4288, PA4843, and PA5116, several of which are known to have a role in the regulation of virulence. As anticipated for a social-behavior-like swarming, these included the two major quorum-sensing regulators lasR and rhlR, with two- and sixfold up-regulated gene expression levels, respectively. This up-regulation of lasR and rhlR under swarming conditions is consistent with the concept that swarming motility is a social bacterial phenomenon and explains the overlaps of the swarming transcriptome with the LasR and RhlR transcription profiles that were described previously (46).

To demonstrate that changes in gene expression are specific to swarming and do not reflect a response to surface growth under nonswarming conditions, RT-PCR was performed for a subset of genes by use of RNA from actively swarming cells in comparison to RNA isolated from cells grown on BM2-swarming media for 5 h under nonswarming conditions (BM2 plus 1.5% agar). As shown in Table Table2,2, all analyzed genes showed differential gene expression, with an up-regulation in the range of 2- to 15-fold under swarming conditions in comparison to what was seen for nonswarming surface growth. These findings underline the swarming-specific gene profile obtained by the microarray experiment described above.

TABLE 2.
Gene expression of selected P. aeruginosa genes under swarming conditions cf. nonswarming conditions

Elastase LasB and PvdQ are essential for normal swarming behavior.

Since such a large number of virulence-related genes were up-regulated under swarming conditions by more than twofold, we were interested in whether these virulence-associated genes were required for swarming motility. Using the opportunity to analyze gene functions afforded by two P. aeruginosa mutant libraries, namely, the PAO1 lux fusion library (29) and the PA14 mutant library (30), which comprise nearly every nonessential gene, we tested strains with mutations in up-regulated, virulence-associated genes for their swarming abilities (Table (Table3).3). No swarming defects relative to the parental wild-type strains were observed for tested mutants with transposon insertions in the TTSS or effectors exoT or exoY, alkaline protease secretion apparatus, or pyochelin, phenazine, and pyoverdine biosynthesis pathways. However, a known virulence-related gene, lasB, was shown to play an important role in swarming motility. Four independently derived lasB mutants from both transposon mutant libraries (Table (Table33 and Fig. Fig.2A)2A) showed strong swarming defects but normal swimming and twitching motility phenotypes compared to their corresponding wild-type strains. Moreover, all four tested lasB mutants also showed a strong impairment in biofilm formation with about 50% less biomass in the respective microtiter plate assays (Fig. (Fig.2B).2B). The swarming as well as the biofilm phenotype could be completely restored by the expression of the chromosomally integrated lasB+ gene (Fig. (Fig.22).

FIG. 2.
Swarming (A) and biofilm formation (B) of wild-type P. aeruginosa PAO1, P. aeruginosa PAO1 lasB mutant 23_F8, and the complemented lasB mutant. (A) Shown is a representative swarm plate and quantification (five plates) after 18 h of incubation. (B) Biofilm ...
TABLE 3.
Swarming motility of selected P. aeruginosa mutants

Since high cell density and quorum sensing are described to play an important role in swarming motility, we analyzed the pvdQ gene (PA2385) in more detail. In addition to its involvement in pyoverdine biosynthesis (26), PvdQ exhibits an acyl-homoserine lactone (HSL) acyclase activity and is known to degrade the quorum-sensing signal 3-oxododecanoyl-HSL (3OC12HSL), among other long-chain acyl-HSLs (19). The pvdQ gene was up-regulated threefold under swarming conditions (Table (Table1).1). We tested the respective pvdQ mutant, P. aeruginosa PA14 ID27758 from the PA14 mutant library, for its swarming ability and demonstrated a strong decrease in swarming motility of about 75% compared to what was seen for wild-type P. aeruginosa PA14 (Table (Table33 and Fig. Fig.3).3). In contrast, this pvdQ mutant revealed normal swimming and twitching motility as well as normal biofilm formation (data not shown). The swarming phenotype could be completely restored by expression of the cloned pvdQ+ gene (Fig. (Fig.33).

FIG. 3.
The pvdQ mutant exhibits a strong swarming motility defect. Swarming levels of wild-type P. aeruginosa PA14, the pvdQ mutant, and the complemented pvdQ mutant PA14 ID27758(pUCP18::pvdQ) were assayed on swarm plates after 18 h of incubation, and agar plate ...

Since the genome of P. aeruginosa reveals three open reading frames that exhibit homologies to pvdQ, namely, PA0305, PA1032, and PA1893, all corresponding mutants from the PA14 and PAO1 mutant libraries were analyzed for their swarming phenotype. However, none of the tested mutants showed any swarming defect compared to the wild-type strains (data not shown). Thus, these homologs do not seem to be necessary for normal swarming behavior under the tested conditions.

Swarmer cells exhibit enhanced antibiotic resistance.

Recently, it was demonstrated that swarm-cell differentiation in Salmonella enterica serovar Typhimurium results in elevated resistance to various antibiotics (22). To test whether P. aeruginosa also reveals altered antibiotic susceptibility under swarming conditions, we compared the antibiotic resistance of actively swarming cells with that of swimming cells by use of media solidified with appropriate agar concentrations.

As shown in Fig. Fig.4,4, swarmer cells of P. aeruginosa PAO1 grown on BM2-swarming agar plates supplemented with 0.5% Casamino Acids and 0.5% agar exhibited a 4- to 16-fold increase in antibiotic resistance toward polymyxin B, gentamicin, and ciprofloxacin compared to what was seen for swimming cells on BM2 medium with 0.3% agar. To analyze if the determined antibiotic resistance is due to the slightly different medium compositions for swarm and swim agar plates as opposed to the swarming growth state, we performed MIC experiments in liquid cultures as described previously (37). Identical MICs of 0.5, 2, and 0.1 μg/ml for polymyxin B, gentamicin, and ciprofloxacin, respectively, were recorded for BM2 medium with 0.5% Casamino Acids and BM2-swarming medium supplemented with 0.5% (wt/vol) Casamino Acids, using either swarming or swimming cells as precultures. This experiment demonstrated clearly that the observed antibiotic resistance is a result of swarmer differentiation of P. aeruginosa cells and is not due to a medium effect.

FIG. 4.
Swarmer cells of P. aeruginosa exhibit enhanced antibiotic resistance. Cells of P. aeruginosa were spot inoculated on swim and swarm agar plates containing different concentrations of polymyxin B, gentamicin, and ciprofloxacin. Growth was determined by ...

To verify these results, kill curves were performed. Cells of P. aeruginosa PAO1 were examined for sensitivity to killing by 2 μg/ml polymyxin B after growth under swimming conditions in BM2 or BM2-swarming liquid medium in comparison to what was seen for swarmer cells grown on BM2-swarming agar plates. Very high susceptibility to polymyxin B, with 5-log orders of killing occurring in 5 min, was observed with cells grown under swimming conditions in BM2 medium, whereas cells grown under swarming conditions showed high polymyxin resistance, with less than a 1-log order of killing after 10 min.

Subinhibitory concentrations of aminoglycosides do not influence swarming motility.

It has been shown previously by our group that subinhibitory concentrations of aminoglycoside antibiotics induce the gene expression of various motility genes, including genes for the biosynthesis and function of flagella and pili (33). With this in mind, we examined the swarming motility of P. aeruginosa PAO1 in the presence of subinhibitory concentrations of aminoglycosides. Using BM2-swarming medium supplemented with 0.5% Casamino Acids and subinhibitory concentrations of gentamicin in the range of 0.1 to 0.5 μg/ml, we determined swarm zones after 18 h of inoculation. No statistically significant change in swarming motility was detectable using subinhibitory concentrations of gentamicin or tobramycin, demonstrating that subinhibitory concentrations of aminoglycosides do not increase swarming motility under the tested conditions.

DISCUSSION

Microbiological and functional genomic studies have led to the concept that P. aeruginosa can adapt its lifestyle to various different circumstances through the up-regulation of large subsets of genes located around the genome. Indeed, its prominence as an opportunistic pathogen and widespread environmental isolate is likely to reflect this capability in part. Known complex adaptations in P. aeruginosa include the formation of organized structured communities termed biofilms (16, 24), the adaptation to nutrient starvation by iron, phosphate, and sulfur (39), the switch to anaerobic growth (13), and a phenomenon known as quorum sensing (52). In this study, we present the first global approach to investigating the P. aeruginosa swarming phenotype, which reflects life in viscous environments such as mucosal surfaces, and present evidence that confirms that it represents another type of complex adaptation of P. aeruginosa.

Our microarray data clearly revealed that P. aeruginosa gene expression during swarming is substantially different from that observed during growth under lower viscosities (swimming/planktonic conditions). More than 400 genes, approximately 7.5% of the genome, were altered in expression. In addition to a large number of hypothetical genes with unknown functions, there was a broad distribution of known genes with altered expression into 21 functional classes, including various metabolic pathways, transport and secretion, regulation, adaptation, and motility among others. The differential expression of such a large and diverse set of genes indicates that there was a striking physiological diversity between planktonic growth and growth on a viscous, semisolid surface. Conceptually similar results were recently made with Salmonella enterica serovar Typhimurium by use of genomic and proteomic approaches (23, 53), although the details differed from those observed here.

This diversity in the transcriptomes of these two physiological states of a P. aeruginosa cell is remarkable given that another complex adaptation, biofilm formation, was associated with only 73 differentially regulated genes compared to planktonic cells (54). These genes were largely related to phages, motility, and translation, while genes associated with metabolic functions were hardly affected at all. Thus, despite the recently reported relationship between swarming motility and biofilm formation (41, 48), a comparison of our microarray data with those published for biofilm formation strongly indicate that the physiological states of cells adapted to swarming motility and to biofilm formation differ substantially.

Keeping in mind that a viscous or semisolid environment is likely to reflect bacterial life in the mucous blanket covering epithelial surfaces or the viscous hyperabundant mucous in the cystic fibrosis lung, one of the most interesting observations of our studies is the up-regulated expression of various virulence factors during swarming. Besides genes coding for extracellular proteases or biosynthesis of the siderophores pyoverdine and pyochelin, which are involved in iron acquisition in the host (42), genes for the complete TTSS apparatus, including those for the related effector proteins required for colonization and dissemination (28), were up-regulated in swarming cells. Furthermore, genes coding for the biosynthesis of the redox-active compound phenazine, which plays an important role in pulmonary tissue damage and other effects in chronic lung infections by P. aeruginosa (36), as well as the multidrug efflux pump MexGHI-OpmD, which confers antimicrobial resistance (1, 2), were up-regulated in swarming cells. This phenomenon of increased virulence factor expression in swarming cells is consistent with observations with other bacteria such as Proteus and Salmonella (53) but has not to date been described for P. aeruginosa. The up-regulated expression of a variety of virulence factors would benefit the population of bacterial cells in the process of colonizing new environments, including host mucosal surfaces.

Since our microarray data revealed virulence expression as a dominant phenotype of P. aeruginosa swarm cells, we analyzed to what extent these virulence factors are necessary for normal swarming behavior. Although the swarm cell invests a lot of energy in virulence factor synthesis, most of these processes do not appear to play obligatory roles in swarming motility. No mutants affected either in iron acquisition or the biosynthesis of phenazine or in the TTSS apparatus and effectors were found to be deficient in swarming motility. However, two genes associated with virulence, pvdQ and lasB, were newly identified here to play important roles in swarming behavior.

The protein produced by the pvdQ gene, which is involved in pyoverdine biosynthesis (26), also exhibits acyl-HSL acylase activity (19). Since other mutants with transposon insertions in genes necessary for pyoverdine biosynthesis did not reveal a swarming defect, the acylase activity rather than its involvement in pyoverdine synthesis seems likely to be the important function influencing the swarming defect. One of the preferred substrates of the PvdQ protein is one of two major quorum-sensing signals, 3OC12HSL, which affects both the LasR and QscR systems (7). This observation is consistent with previous conclusions that the 3OC12HSL signal is necessary for normal swarming and mutants and that defects in the synthesis of this signal lead to deficiencies in swarming motility (25). Since it was demonstrated that the pvdQ mutant with defects in signal degradation also exhibited swarming defects, it seems likely that a specific concentration of signal is important for this type of multicellular behavior. Given our previous demonstration that an rhlR mutant influencing the expression of the second signal, butanoyl-HSL, was also defective in swarming, a specific balance of these signals might be important or conversely a degradation product induced by pvdQ might act as a signal during swarmer differentiation. Regardless, we hypothesize that quorum regulation of swarming is uniquely regulated through degradation rather than synthesis of quorum-sensing signals.

The extracellular elastase LasB, encoded by the lasB gene, is one of the most studied virulence factors of P. aeruginosa. This protease exhibits tissue-damaging activity, is capable of degrading various plasma proteins among other proteins, and is highly expressed during tissue colonization and infection, particularly in the lungs (27, 34). Interestingly, lasB mutants of P. aeruginosa also showed a strong defect in biofilm formation. Our results are consistent with the secretion of LasB by the type II secretion system, since mutants with defects in this secretion apparatus also exhibit strong swarming and biofilm defects (41). The involvement of extracellular proteases in swarming motility has been also demonstrated for strains of Bacillus subtilis and Vibrio vulnificus (8, 11, 12, 21). However, the functions of these processes are still unknown.

The diversity between swarmer and planktonic cells was also reflected in differential susceptibility to antimicrobial compounds. Our studies revealed that swarmer cells of P. aeruginosa are up to 16 times more resistant to the tested antibiotics than are planktonic cells. The mechanisms for this elevated antibiotic resistance were not investigated in detail here but are likely to be complex. For example, altered expression of the ATP-dependent protease Lon might be responsible in part for ciprofloxacin resistance (3), while the decreased expression of outer membrane components, including certain porins, the increased expression of efflux pump systems, such as the multidrug efflux pump MexGHI-OpmD (2), and the differential expression of genes associated with lipopolysaccharide (see Table S1 in the supplemental material) are all likely to have influenced susceptibility. Similarly, elevated resistance to multiple antibiotics was previously ascribed to complex changes in cell physiology and a more positively charged lipopolysaccharide (23, 53).

Overall, our studies clearly demonstrate that swarmer cells of P. aeruginosa respond and adapt to a viscous, semisolid environment through altered gene expression, resulting in a unique physiological state (complex adaptation) that differs from both planktonic and biofilm growth. The substantial overexpression of numerous virulence factors benefits the swarmer population in the process of environmental and/or host colonization and infection. Finally, our data indicate two new possibilities for cell-to-cell signaling involved in swarming motility of P. aeruginosa, which will be analyzed in more detail in the future.

Supplementary Material

[Supplemental material]

Acknowledgments

We acknowledge funding from the Canadian Institutes of Health Research. J.O. was supported by postdoctoral fellowships from the Alexander von Humboldt Foundation and Canadian Cystic Fibrosis Foundation. R.E.W.H. holds a Canada Research Chair.

Footnotes

[down-pointing small open triangle]Published ahead of print on 1 February 2008.

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

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