Journal of Bacteriology, July 2002, p . 3598-3604, Vol . 184, No . 13

Like many bacteria, P . aeruginosa has a quorum-sensing system by which it polls cell density and regulates the expression of many genes at high cell densities, including those encoding extracellular proteases, toxins, surfactants, and others (11, 14, 20, 50) . Such systems usually consist of a transcriptional activator and a biosynthetic enzyme, the latter of which is responsible for producing a small diffusible molecule, termed an autoinducer (normally an acyl-homoserine lactone), that forms a complex with the former at high threshold levels to induce transcriptional activation of other genes (11, 14, 50) . In P . aeruginosa, there are two well known quorum-sensing systems termed las and rhl . The lasR and rhlR genes encode transcriptional activators, and lasI and rhlI encode acyl-homoserine synthases that are responsible for the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, respectively . LasR complexed with N-(3-oxododecanoyl)-L-homoserine lactone regulates transcription of lasI, rhlR, lasA, lasB, apr, and toxA, the latter four of which encode key virulence factors . RhlR complexed with N-butyryl-L-homoserine lactone regulates transcription of rhlI, rhlA, lasB, and phzA, which also encode key virulence factors (for recent reviews, see references 11, 14, and 50) . Therefore, a hierarchy exists, with the las quorum-sensing system apparently situated above the rhl system .

However, it is becoming clear that the hierarchical quorum-sensing system is a great deal more complex than initially anticipated . Several additional regulatory proteins have been shown to exert control over the las or rhl system, including RpoS (19), GacA (31), RelA (41), and Vfr (1) . There are also a number of conflicting reports in the literature, for example, that RpoS regulates rhlI (51), not vice versa, as previously reported (19) . In fact, it has recently been shown that a secondary-site mutation in the P . aeruginosa wild-type strain used to construct a lasR knockout mutant accounts for at least some of this confusion (17) .

Recently, it was reported that the las and rhl quorum-sensing systems are required for twitching motility in P . aeruginosa (16) . We have been studying the genetic control of the biogenesis and function of type IV pili in this bacterium for many years and, with others, have characterized a large number of genes, now totaling more than 35, that are involved in this process, including the genes encoding the main structural subunit (pilA or pilin), the leader peptidase (pilD), other proteins involved in pilus assembly and twitching motility (many of which are homologous to other proteins required for type II protein secretion and DNA uptake in P . aeruginosa and other bacteria), and at least three regulatory systems, including the two-component sensor regulator systems pilSR and algR/fimS and a complex chemosensory system (pilGHIJK, chpABCDE) (2) . Here we show that twitching motility is unaffected by mutations in the genes encoding the las and rhl quorum-sensing systems but that such mutations can lead to specific secondary mutations in other key regulatory genes that do affect twitching motility and that apparently represent compensatory mutations induced by distortions in the regulatory architecture of the cell .

 
Isolation of genes and construction of isogenic mutants. Allelic-exchange mutations were constructed in the genes lasR, lasI, rhlR, rhlI, and pheC by using the sucrose selection system described previously (4, 33) . All reference sequence data for these genes, as well as algR and vfr, and their local restriction site topography were taken from the P . aeruginosa genome database (http://pseudomonas.bit.uq.edu.au) (9) . Comparison of amplimer sequences with the P . aeruginosa genome database provides information about the exact nature of the cloned gene sequences .

lasI, rhlI, lasRI, rhlRI, and pheC loci were amplified from chromosomal DNA by using flanking amplimers (designed against the 5' upstream and 3' downstream regions adjacent to the relevant coding regions), whose sequences are available on request . The lasRI amplicon was cloned into the XhoI-BamHI sites of pOK12 (41), whereas the rhlRI and pheC amplicons were cloned into the NotI-XhoI sites of pOK12 (note that the rhlRI amplicon was cloned as a NotI-SalI fragment due to an internal XhoI site) . All plasmids were sequence verified . Single-knockout mutations in these genes were made by insertion of the gentamicin resistance cassette (Gm) from pX1918GT (34) or the tetracycline resistance cassette (Tc) from pSM-TET (25) into restriction sites within lasI (EcoRI), lasR (SalI), rhlI (EcoRI), and rhlR (BamHI) . The disrupted genes were then isolated by digestion with SpeI and inserted into the corresponding site of the suicide vector pRIC380 (4) . This vector carries the genes sacBR, which promote sensitivity to sucrose, and oriT, which enables conjugal transfer . The constructs were then used to transform E . coli donor strain S17-1 in preparation for mating with P . aeruginosa . Following conjugation, the transconjugants were plated on 5% sucrose medium containing tetracycline or gentamicin, as appropriate, to select for colonies in which the plasmid had been excised while leaving the homologously recombined mutated gene in the chromosome . Mutants were confirmed by Southern blot analysis .

The double mutations lasI::Gm rhlI::Tc and lasR::Gm rhlR::Tc were made by sequential antibiotic resistance insertions and allelic replacements by using the strategy outlined above . The quadruple mutation lasRI::Gm rhlRI::Tc was made by sequential replacement of SalI-EcoRI and BamHI-EcoRI fragments, which span lasRI and rhlRI, with gentamicin and tetracycline resistance cassettes, respectively . The lasRI::Gm knockout also removes a small open reading frame termed rsaL between lasR and lasI (10) .

The pheC knockout was made similarly by insertion of the tetracycline resistance cassette into its BamHI site, and the rhlI::Gm pheC::Tc double mutation was made by subsequent insertion of the gentamicin cassette into the EcoRI site of rhlI .

PAKlasI::Tc and PAKrhlI::Tc were constructed by using allelic-exchange plasmids that differed from those used to construct the respective PAO1 mutants . Specifically, lasI and rhlI were amplified from chromosomal DNA by using flanking amplimers (sequences are available on request) . The resulting amplicons were inserted as BamHI-XbaI fragments into pOK12 . Single-knockout mutations in these genes were made by insertion of the Tc-encoding sequence from pSM-TET (25) into EcoRI within lasI and into KpnI within rhlI . The disrupted genes were then isolated by using SpeI and inserted into the corresponding site of the suicide vector pRIC380 (4) . Isogenic knockout mutants of P . aeruginosa PAK were made as described previously . For complementation studies, pUCPlasI and pUCPrhlI were constructed by excising lasI and rhlI from the corresponding pOK12 plasmids as XhoI-XbaI fragments and inserting them into the E . coli-P . aeruginosa shuttle expression vector pUCPKS (44) (with lac) .

All knockout mutations were confirmed by Southern analysis with indicative restriction digests and relevant probes .

Wild-type and mutated vfr and algR alleles were PCR amplified from chromosomal DNA by using specific amplimers (sequences available on request) . PCR products were inserted against lac as KpnI-HindIII or BamHI-HindIII fragments, respectively, into the E . coli-P . aeruginosa shuttle expression vector pUCPSK (44) for sequence analysis and complementation studies .

DNA sequencing. PCR-amplified genes were subcloned into pUCPSK (44) as described above . Plasmid DNA was isolated by alkaline lysis minipreps, and cloned inserts were sequenced (both strands) by using universal primers and internal primers with dye terminator chemistry on an ABI 377 DNA sequencer by the Australian Genome Research Facility, University of Queensland, Brisbane, Queensland, Australia . DNA sequences were compared with reference sequences from the P . aeruginosa genome (http://pseudomonas.bit.uq.edu.au) .

Twitching motility assay. Twitching motility was assayed by the subsurface agar method described previously (3, 35) . Briefly, the P . aeruginosa strain to be tested was stab inoculated through a 1% agar plate, and after overnight growth at 37°C, the zone of twitching motility elaborated at the interstitial surface between the agar and the petri dish was visualized by staining with Coomassie brilliant blue R250 .

Mutation assays. Wild-type PAK, PAKlasI::Gm, and PAKrhlI::Tc were recovered from frozen stocks and streaked to single colonies on LB agar . To compare the frequencies of spontaneous twitching defects and streptomycin resistance among wild-type PAK and the PAKlasI::Tc and PAKrhlI::Tc mutants, dilutions of 2 ml of LB broth cultures (inoculated from a single colony and incubated for 24 h at 37°C in 10-ml polystyrene tubes) were diluted and spread plated on LB agar with or without 500 µg of streptomycin (Sigma) ml^-1 . Twitching-defective colonies were identified after 24 h of incubation at 37°C by colony morphology and confirmed by twitching stab assay . Streptomycin-resistant colonies were scored on antibiotic-containing plates after 32 h of incubation at 37°C and patched onto fresh antibiotic-containing plates to confirm their phenotype .

Serial subcultures were performed by inoculating a fresh tube containing 2 ml of LB broth with 20 µl of a 10^-4 dilution of the previous day's culture for 4 consecutive days . Broths were incubated at 37°C with agitation overnight, and dilutions were plated on LB agar plates . Each day for 4 days, at least 1,000 CFU from each experiment were scored for twitching motility by colony morphology and the phenotype of up to 50 twitching-defective colonies from each experiment was confirmed by twitching stab assay .

Competitive 1:1 growth assays were carried out by coinoculating fresh tubes containing 2 ml of LB broth with 20 µl of a 10^-3 dilution of an overnight culture of each test strain . Similarly 1:100 or 100:1 competitive assays used 20 µl of a 10^-3 dilution and 10^-1 dilution of an overnight culture of each test strain or vice versa, respectively . Competitions were incubated overnight as described above, and the relative fitness of each strain was determined by plate counts using colony morphology to distinguish strains .

 
We examined several avenues to try to resolve the discrepancy between our observations and those reported by Glessner et al . (16) . Those authors reported that the level of twitching motility in strain JP1 (PAO1lasI) was reduced to 30% of the wild-type level and that strains PDO100 (PAO1rhlI) and JP2 (PAO1rhlIlasI) exhibited complete loss of twitching motility . These strains have been widely used to study quorum sensing in P . aeruginosa (11) . We considered the possibility that our quorum-sensing mutants were somehow defective; however, the integrity of all of the mutants was confirmed by Southern analysis and the elastase- and rhamnolipid-deficient phenotypes of our lasI and rhlI mutants, respectively, were consistent with those reported in the literature (8, 15, 17) (data not shown) .

The PDO100 and JP2 strains that were studied by Glessner et al . (16) contain an 5-kb deletion that knocks out not only rhlI but also the adjacent pheC gene (8) . PheC is homologous to FliY of E . coli, which has been implicated in cell density sensing and appears to be a periplasmic binding protein predicted to feed into chemosensory systems (26), raising the possibility that loss of pheC was responsible for the twitching motility defects observed in these strains . We therefore constructed an isogenic pheC knockout, as well as an rhlI::Gm pheC::Tc double mutant, but again found no effect on twitching motility .

Twitching-defective variants spontaneously arise among lasI and rhlI mutants. During the course of these experiments, however, we noticed that spontaneous defects in twitching motility, similar to those reported by Glessner et al . (16), commonly appeared in the PAKlasI and PAKrhlI quorum-sensing mutants . Specifically, nontwitching variants of PAKrhlI arose frequently during the allelic-exchange procedure and reduced-twitching variants of PAKlasI arose frequently after recovery from frozen stock culture . We isolated the stable twitching-defective mutants from these PAKlasI and PAKrhlI cultures . These mutants were nontwitching in the case of the PAKrhlI strain and showed reduced twitching in the case of the PAKlasI strain (hereinafter referred to as PAKrhlI-NT and PAKlasI-RT, respectively) (Fig . 1), phenotypes that were indistinguishable from those described for PDO100 and JP1, respectively (16) (see above) . We confirmed that PAKrhlI-NT and PAKlasI-RT were bona fide variants of the PAKrhlI::Tc and PAKlasI::Tc strains, respectively, by Southern analysis (data not shown) . Furthermore, we were unable to complement the twitching phenotypes of PAKrhlI-NT and PAKlasI-RT with cloned wild-type rhlI and lasI, respectively (data not shown) . Therefore, the likeliest explanation was the occurrence of a spontaneous secondary-site mutation within a gene required for normal twitching motility .

Careful examination of the phenotypes of the PAKrhlI-NT and PAKlasI-RT strains and comparison of these with the known twitching-defective mutants in our collection (on the basis of such indices as the nature of the twitching zones, the micromorphology of the colony edge under twitching conditions, the susceptibility of these strains to pilus-specific bacteriophage, and the level of expression of the structural subunit PilA and its subcellular location [data not shown]) indicated that the phenotype of the PAKrhlI-NT strain was similar to that of algR mutants and the phenotype of the lasI-RT strain was similar to that of vfr mutants . AlgR is an atypical regulator that was first identified as being required for alginate production in mucoid strains of P . aeruginosa (12) and later shown to be also required for twitching motility, along with its cognate sensor FimS (49) . Vfr is a homolog of the E . coli catabolite repressor protein CRP (6, 18, 48) and has been shown to regulate exotoxin A production, regA transcription, and quorum sensing, apparently via control of lasR transcription (1, 47, 48) . We have recently identified Vfr as being required for normal twitching motility by screening of a transposon library of twitching-defective mutants (5) . In agreement with the phenotypic similarities between these mutants, we found that twitching motility could be restored to PAKrhlI-NT and PAKlasI-RT by complementation with wild-type algR and wild-type vfr, respectively (Fig . 1) . Conversely, PAKrhlI-NT and PAKlasI-RT were not complemented by wild-type vfr and algR, respectively .

Mutations in algR and vfr cause the twitching defect in PAKrhlI-NT and PAKlasI-RT, respectively. We then isolated and sequenced the algR gene from wild-type strain PAK and the PAKlasI::Tc, PAKlasI-RT, PAKrhlI::Tc, and PAKrhlI-NT mutant strains . The algR sequence was identical to that of the wild type in all of the strains except PAKrhlI-NT, which had a GGGGTGGGGG transversion causing a V241G substitution . Homopolymeric runs of nucleotides are common sites for mutations . We also found that PAKrhlI-NT mutants spontaneously reverted to a normal-twitching phenotype at low frequencies when cultured on solid medium . Reversion was characterized by the emergence of a kidney-shaped flare of twitching cells at the edge of an otherwise smooth, nontwitching colony and was typically observed in approximately 1% of PAKrhlI-NT colonies after 3 or 4 days of incubation . The algR alleles from two such revertants were also sequenced, and both were found to have recovered the wild-type algR sequence (data not shown) .

We also isolated and sequenced the vfr gene from wild-type PAK and the PAKrhlI::Tc, PAKrhlI-NT, PAKlasI::Tc, and PAKlasI-RT mutant strains . The vfr sequence was identical to that of the wild type in all of the strains except PAKlasI-RT, which had a very precise 15-nucleotide in-frame microdeletion in the second half of an imperfect tandem repeat situated in the region of Vfr that closely matches the CRP cyclic AMP-binding domain (46), the net result of which is the loss of five residues (EQERS) . Interestingly, and perhaps importantly, this allele (VfrEQERS) is able to restore elastolytic activity but not twitching motility when supplied in trans to vfr-null mutants (5) .

The PAKlasI-RT (VfrEQERS) mutants were also found to revert spontaneously to a normal-twitching phenotype at low frequencies . vfr alleles from two such revertants were cloned and sequenced . Both vfr sequences contained the EQERS deletion, but each sequence contained a different point mutation elsewhere in vfr that caused an amino acid substitution (S194R and D36A) . These vfr alleles were also able to complement vfr knockout mutants for twitching motility (data not shown)—therefore, these mutations appear to overcome the effect of the EQERS deletion . Interestingly, the revertant carrying the D36A mutation did not recover twitching motility completely and the corresponding vfr allele could only complement vfr knockout mutants to 75% of wild-type twitching motility . This indicates that the effect of the EQERS deletion is only partly overcome by the D36A mutation . Analysis of specific compensatory mutations in the vfr alleles of further lasI-RT revertants should yield significant insights into the structure and function of Vfr itself .

Twitching-defective variants predominate in liquid culture of quorum mutants. We were interested in further characterizing the emergence of the spontaneous mutations among lasI and rhlI mutants . Our findings suggested either that these secondary mutations were occurring at high frequencies as a result of a hypermutator phenotype (due to down-regulation of systems that are involved in DNA repair or that protect cells from oxidative damage) or that such mutations had some selective advantage in this background . However, the frequency at which twitching defects arose among lasI and rhlI cells plated on solid medium was found to be similar to that in the wild type, between 10^-4 and 10^-6 in three independent trials (data not shown), which is consistent with the normal rate of spontaneous mutation given that a large number of genes are known to be required for twitching motility (representing approximately 0.1% of the genome) . In addition, there was no significant difference in the frequency of spontaneous streptomycin resistance mutations in these strains (data not shown), which frequency was consistent with previous studies (27) .

When serial subcultures in liquid medium (with shaking) were carried out, both PAKrhlI::Tc and PAKlasI::Tc cultures, but not wild-type PAK cultures, developed twitching defects (as measured by plating of dilutions after every subculture) that appeared to predominate as subculturing continued (Table 2) . These experiments were repeated several times . The majority of these twitching-defective variants exhibited phenotypes that appeared to be identical to those of the S1 and S2 variants previously described by Deziel et al . in an environmental isolate of P . aeruginosa (strain 57RP) (13) . Our S1- and S2-like mutants were defective in both twitching motility and swimming motility and formed a pellicle at the air-broth interface when cultured as a static broth but rapidly reverted to a normal twitching-swimming state, apparently by phase variation (data not shown), as had been previously observed by Deziel et al . (13) . We did not find such variants among the wild-type cultures, suggesting that these as-yet-unidentified phenotypic variants, which were observed otherwise only when P . aeruginosa cells were grown as a biofilm or in static broth culture, also have a selective growth advantage in quorum-sensing mutants when they are grown in a shaken broth culture . Perhaps importantly, Deziel et al . noted that, apart from strain 57RP, S variants also arose in P . aeruginosa strains that demonstrated a long lag phase before growth on hexadecane (13) . It has been reported that rhl mutants grow poorly in hexadecane (28) . Taken together, these observations add support to the idea that the appearance of S variants may be quorum sensing related .

 
Interestingly, Deziel et al . also observed that S variants show decreased elastase and increased pyocyanin production (13) . However, it was difficult to measure the pyocyanin phenotypes of our S variants due to the fact that strain PAK only produces very low levels of pyocyanin and because of the rapidity of reversion of such variants to a wild-type phenotype . Furthermore, as noted earlier, our lasI and rhlI mutants demonstrated elastase and rhamnolipid deficiencies that were consistent with published reports (8, 15, 17) . This precluded the measurement of quorum-sensing-related phenotypes in the S variants of the rhlI and lasI mutants . Nevertheless, the range of phenotypes affected in S variants suggests that the apparent phase variation occurs within or affects the expression of a gene encoding a global regulator . However, neither our S1 nor our S2 variants could be complemented by pUCPvfr or pUCPalgR and the vfr and algR alleles from representative S variants were identical in sequence to the wild type (data not shown), indicating that vfr and algR are not mutated in these S variants .

Interestingly, all of the nontwitching variants isolated from serial broth subcultures of our PAKrhlI mutants were found to resemble either S1- or S2-type variants (Table 2) but not the original PAKrhlI-NT strains . Therefore, we have yet to identify the precise conditions (if any exist) that selected for the algR mutation in rhlI mutants that occurred at high frequencies during the allelic-exchange procedure . In contrast, after four consecutive subcultures, two of six PAKlasI cultures contained reduced-twitching mutants that resembled the original PAKlasI-RT variant and were subsequently complemented with pUCPvfr . Significantly, these independent isolates both contained precisely the same mutation in vfr as previously observed in PAKlasI-RT, suggesting that in an agitated liquid culture, there is a selective pressure for this particular mutation in P . aeruginosa lasI mutants .

To detect whether there may be a selective advantage of the VfrEQERS mutation, we performed a number of direct competitions . Unlike the S1 and S2 mutants, which always outcompete the parent strain (or the wild type), PAKlasI-RT mutants harboring the VfrEQERS mutation do not appear to have a significant growth advantage over normal-twitching PAKlasI mutants in overnight 1:1 or 1:100 competitive assays (data not shown) . Therefore, it is possible that the selective advantage of this mutation is more subtle than any that can be detected by a simple competitive assay . Consistent with this hypothesis are our findings that vfr mutations generally arise after three or four consecutive subcultures and do not numerically predominate, whereas S1 or S2 variants usually arise after one or two subcultures and often become the most predominant type after four subcultures (Table 2) . We found no difference in the overall unselected rate of mutation (as measured by the frequency of streptomycin resistance development), suggesting that a generally higher incidence of mutation in the PAKlasI mutant could not be the cause of these VfrEQERS mutations . However, we cannot rule out the possibility that a specific type of mutation (one that favors deletion of the 15-nucleotide sequence within vfr but has no effect on the genes for streptomycin sensitivity) occurs more often in lasI mutants .

Quorum-sensing mutants and spontaneous mutation of regulatory genes. The frequent occurrence of nontwitching variants of PAKrhlI::Tc that harbor mutations in algR and that appear to be phenotypically similar to that described by Glessner et al . (16) indicates the possibility of a specific compensatory regulatory mutation occurring in algR in the rhlI background . Similarly, the fact that three independent twitching-defective variants of lasI mutants all contained the same precise mutation in vfr and are phenotypically similar to that reported for lasI mutants by Glessner et al . (16) suggests that this mutation has a specific advantage in the lasI background, probably as a compensatory mechanism by which to relieve stresses on the regulatory circuitry imposed by the lack of the lasI quorum-sensing signal . Interestingly, the twitching defects reported in the lasI mutant JP1 and the rhlI mutant PDO100 were not reported to be complemented by wild-type lasI or rhlI provided in trans (16) . Furthermore, our findings that S1 and S2 phase variants that affect a variety of phenotypes appear commonly in quorum-sensing mutants but not wild-type cells grown in normal broth culture also suggests that spontaneous mutation is selected for by distortions in the quorum-sensing regulatory system . Taken together, these results suggest that mutations in the lasI and rhlI quorum-sensing systems create conditions that can select for secondary mutations in other regulatory genes, presumably because the latter relieve regulatory and/or physiological stresses on the cells caused by the primary mutations . These secondary mutations may themselves also cause new stresses and selective pressures that may induce flow-on genetic changes during subculturing of such mutants, many of which have been grown and studied in (different) laboratories for many years and that may have gone unnoticed in the past . Indeed, if it had not been for the fact that twitching motility is an easily scored phenotype in P . aeruginosa colonies, this problem would not have been recognized at all, and there may well be other genes that are also subject to selection pressure in such regulatory mutants but whose incidence is hidden . These unanticipated changes may also account for much of the confusion and contradictory results in the literature . This has been demonstrated recently by a study showing that a pre-existing mutation in a wild-type strain used to create a defined lasR mutant is the reason behind contradictory reports of the role of lasR in the control of rhamnolipid production (17) . In the light of this finding, it appears that other reports of starvation selection for the elastase and rhamnolipid phenotypes in lasR mutants (42) and the related study showing dksA inhibition of quorum sensing (7) might have been similarly affected by this unexpected mutation .

At the minimum, our findings indicate that reports of the phenotypic effects of mutations in the las and rhl quorum-sensing systems in P . aeruginosa (and probably other bacteria) need to be confirmed by complementation of the mutants, as certain phenotypic characteristics might be due to secondary mutations in regulators such as AlgR and Vfr and, possibly, other global regulatory proteins . Clearly, any quorum-sensing mutant exhibiting a twitching motility defect harbors a secondary-site mutation and should be appropriately checked . The fact that the vfr mutations involve a precise deletion in a tandem repeat that affects some signaling pathways but not others suggests that this may be an important site for phase variation, as does the incidence of mutations in a poly-G tract in algR . Similar influences may be at work during the evolution of P . aeruginosa communities in the cystic fibrosis lung, where it is known that there are mutations in genes affecting transcriptional regulation and that there are different ratios of lactones than those observed in vitro (37) . More broadly, it is clear that caution is required when investigating regulatory mutations, as such mutations may cause stresses in the cell that create conditions for the emergence of compensating mutations in other genes .

This work was supported by the National Health and Medical Research Council of Australia and the Australian Research Council .

Present address: Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom .

Present address: Department of Medicine, University of California San Francisco, San Francisco, CA 94143-0654 .

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