Elevated exopolysaccharide levels in Pseudomonas aeruginosa flagellar mutants have implications for biofilm growth and chronic infections

Summary

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.

Keywords:

Phenotypes – Pseudomonas aeruginosa – Bacterial biofilms – Biofilms – Cystic fibrosis – Exopolysaccharides – Flagella – Deletion mutation

Introduction

One of the most important CF pathogens is Pseudomonas aeruginosa [1]. During chronic infection, it predictably acquires a number of adaptive mutations that are believed to aid in long-term persistence [2–4]. These adaptations include alginate overproduction [5], amino acid auxotrophy [6], loss of lipopolysaccharide O-antigens [7] and loss of flagellar motility [8]. Although the fitness benefits that these different mutations confer is uncertain, several seem intuitive. For example, amino acid auxotrophy may relieve the metabolic burden of amino acid biosynthesis in the amino acid rich environment of CF airway secretions. Another is the loss of motility functions, which is thought to assist in the evasion of the host immune system by eliminating production of a key antigen and pathogen-associated molecular pattern, flagellin.

Several similar genetic adaptations are observed in laboratory biofilms. This is potentially important because CF airway infections are thought to have biofilm etiology [9, 10]. One P. aeruginosa phenotype that is selected for in laboratory biofilms is the rugose small colony variant (RSCV) [11, 12] (Fig 1A). These variants have also been isolated from CF sputum [12–15]. RSCVs make profuse amounts of the extracellular polysaccharides (EPS) Pel and Psl as well as the extracellular adhesin CdrA [16, 17]. Expression of Pel and Psl is associated with tolerance to some clinically-relevant antibiotics [18, 19], and it safeguards P. aeruginosa against complement-mediated opsonization and neutrophil phagocytosis [20]. Pel, Psl and CdrA are also known to play a role in biofilm aggregate formation [17, 18, 21], which is associated with protection from antimicrobials [22–24] and host defenses [16, 25]. It is not surprising, therefore, that prior reports have associated the appearance of RSCVs in CF sputum to poor patient outcomes [26, 27].

Expression of the RSCV morphotype is linked to elevated levels of the second messenger cyclic diguanylate (c-di-GMP) [28–32]. Several loci have been linked to the RSCV phenotype, including the wsp operon [29], the tpb (also denoted aws or yfi) loci [28, 33, 34], amrZ [35, 36], dsbA [37], genes of the rsm signalling pathway [21, 38] and pvrR [30]. Previous work has estimated that RSCVs represent approximately 5% of laboratory grown biofilm populations and that wsp mutations account for a majority of these [16]. RSCVs harbouring mutations in the wsp and yfi loci have also been observed among CF isolates [2, 39].

In this study, we found that the frequency of RSCVs in laboratory biofilms and CF isolates has been grossly underestimated. Using transposon mutagenesis, we sought to identify novel genetic elements linked to the phenotype. We found 22 genes in our screen that we grouped into four classes. Perhaps the most surprising class were mutations in genes encoding flagellum structure and function. Unlike the other classes of RSCVs, flagellar mutants overexpressed Pel and Psl in a surface-contact-dependent fashion. We also found that flagellar mutations are selected for at high frequency in laboratory biofilms, and that increased Pel and Psl expression provides the primary fitness benefit. Suppressor mutagenesis revealed that Psl overexpression by flagellar mutants requires the mot genes as well as components of the Pil-Chp surface-sensing system. Finally, we show that flagellar mutant RSCVs are also found among CF clinical isolates. We propose that gain-of-function phenotypes (Pel and Psl overexpression) offer a new interpretation of the benefits of flagellar mutations in isolates from laboratory biofilms and CF infections.

Results and discussion

P. aeruginosa RSCVs are abundant in laboratory biofilms and CF isolates

P. aeruginosa biofilms grown in vitro rapidly undergo mutation and selection [11]. A manifestation of this is the appearance of diverse colony morphotypes when biofilm bacteria are plated onto solid growth media. Using a growth medium optimized for RSCV detection (Vogel-Bonner minimal medium supplemented with the dyes Congo Red and Coomasie brilliant blue), we found that RSCVs are observed ~9-fold more frequently than on traditional lysogeny broth (LB) agar (Fig 1A and 1B). Many isolates displaying the RSCV phenotype on VBMM had a wild type smooth colony morphotype when re-streaked on LB agar.

A key question for these isolates is whether RSCV-linked traits (elevated Pel and Psl production) are only expressed on VBMM-based agar compared to LB. To test this, Psl and PelC expression were measured for strains that exhibited the RSCV phenotype on VBMM but were smooth when grown on LB. We observed that all these strains still overexpressed PelC and/or Psl on both VBMM and LB (S1 Fig). These data indicate that elevated EPS expression is a feature of these strains regardless of the growth medium or colony morphotype.

We next asked if RSCVs are more common than previously thought among CF sputum isolates (Fig 1C). We examined 416 P. aeruginosa isolates cultured from 46 pediatric CF patients enrolled in a two-year study of respiratory bacterial epidemiology at Seattle Children's Hospital [40]. Only a single isolate had an RSCV phenotype on LB agar. By contrast, 56 (13.5%, Fig 1C) of these isolates (from 40% of the patients) displayed the RSCV phenotype on VBMM-based agar medium. By comparison, perhaps the best-known CF-related P. aeruginosa adaptive colony morphology, mucoidy [41], was observed in 80 (19.2%, Fig 1C) of the isolates (from 49% of the patients).

Identification of genes that are linked to the RSCV phenotype

Work by Starkey and colleagues [16] indicated that wspF mutations account for 70% of the RSCVs that are isolated on LB agar from biofilms. We randomly isolated 25 RSCVs on VBMM agar from biofilms (i.e. 5 RSCVs from each of 5 different drip-flow biofilm reactors in order to increase the likelihood that these RSCVs were non-isogenic) and attempted to complement these mutants with a wild type copy of the wspF gene; however, in only a single instance did this restore the wild type colony morphology of these biofilm isolates. Analogously, transformation of 10 RSCVs (from 10 different patients) recovered on VBMM-based agar from CF sputum with a plasmid bearing wspF restored smooth colony morphology in only 3 of 10 isolates. These data suggest that the majority of RSCVs isolated from biofilms and from CF sputum may be wsp-independent.

To identify wsp-independent genes linked to the RSCV phenotype, we carried out near-saturation transposon mutagenesis of P. aeruginosa PAO1 harboring an engineered deletion of wspR, the diguanylate cyclase of the wsp system [29]. This allowed us to eliminate the isolation of wsp-related mutations that might confer the RSCV phenotype. Here we used the transposon miniTn5Pro, which carries the repressor araC as well as an outward-facing, arabinose-inducible promoter [42]. Approximately 35,000 miniTn5Pro mutants were scored for colony morphology on selective VBMM agar with and without arabinose. This random gene-disruption-gene-activation strategy produced 57 genetically distinct RSCVs in which the transposon was mapped to 22 different genes from 17 different operons (Table 1). Collectively, the identified genetic elements could be placed into four groups based on known and predicted functions: 1) the regulator of secondary metabolism (rsm) signalling pathway, 2) the periplasmic thiol-disulfide interchange protein (dsbA), 3) flagellum biosynthesis, and 4) diguanylate cyclases and phosphodiesterases that synthesize and degrade c-di-GMP, respectively. We also placed oligoribonuclease (orn) in this last category. The orn gene encodes a 3ʹ→5ʹ exoribonuclease that degrades pGpG, an intermediate product produced by EAL-domain phosphodiesterases in the two-step pathway for c-di-GMP degradation [43, 44]. Only a single mutant displayed an arabinose inducible RSCV phenotype, and in this case the transposon insertion point was mapped upstream of the diguanylate cyclase siaD.

A limitation of transposon mutagenesis is that it is difficult to predict whether transposon insertions cause polar effects that disrupt the expression of adjacent genes in operons. Therefore, unmarked deletion alleles were constructed for genes chosen to represent each of the four groups of RSCV-linked mutations. As expected, introducing these deletion alleles into the wild type PAO1 (Fig 2) and ΔwspR strains (S2A Fig) duplicated the RSCV phenotype of transposon mutants. Each of these mutants had smooth, and in some instances small, colony morphology on LB agar (S2B Fig). Each mutant strain was then complemented using miniTn7 to insert a single copy of the deleted gene and its native promoter elsewhere on the chromosome (Fig 2). Additionally, precise, in-frame deletion mutations were constructed in ten flagellar genes. Genes were chosen to represent operons from each of the known tiers (Table 1) of the four-tier flagellar gene regulation hierarchy that orders flagellum assembly in P. aeruginosa [45]. Our results show that some but not all disruptions in flagellar operons may result in rugose colony morphology on VBMM (S3 Fig). Moreover, with the exception of ΔfleQ (which is at the first tier of flagellar gene regulation), we noted that RSCV-linked mutations can occur in operons at every tier of the flagellar gene regulation hierarchy. Taken together, these data suggest that the assembly status of the P. aeruginosa flagellum regulates EPS production, which is similar to observations previously reported for other bacterial species [46, 47].

Flagellar mutants overproduce EPS in a surface contact-dependent fashion

Transcriptional profiling has identified a role for the master flagellar regulator FleQ in repressing the pel and psl operons [45]. However, there is little evidence that other flagellar mutations increase the expression of EPS genes. Here, we assessed molecular markers of EPS production for four flagellar mutants that expressed the RSCV phenotype on VBMM (ΔflhA, ΔfliM, and ΔflgL and ΔfliC) and for three that did not (ΔfleQ, ΔflgB and ΔmotABCD). When grown in shaking liquid culture, none of these mutants displayed increased expression of Psl (Fig 3A and 3B). Moreover, apart from ΔfleQ, none showed overexpression of PelC (S4 Fig). By contrast, nearly all these mutants showed elevated Psl when grown on solid medium (Fig 3C and 3D). Except for the ΔmotABCD strain, nearly all these mutants also overexpressed PelC when grown on VBMM agar (S4 Fig). These data suggest that surface contact might be a stimulus for flagellar mutants to express biofilm matrix polysaccharides. These observations are consistent with reports that the flagellum has a role in mechanosensation that is similar to other bacteria [48], including V. cholerae [49], Vibrio paraheamolyticus [50], Caulobacter crescentus [51], Proteus mirabilis [52] and Bacillus subtilis [53].

Suppressor mutagenesis reveals a role for the flagellum stator and the Pil-Chp surface-sensing system in regulating EPS production in flagellar mutants

To reveal the regulatory pathway that links flagellar mutations to EPS overexpression, we carried out transposon mutagenesis of the ΔfliC strain. We again utilized minTn5Pro to enable random gene disruption and activation, but this time sought ΔfliC transconjugates that exhibited a smooth colony morphology. Approximately 21,000 ΔfliC miniTn5Pro mutants were evaluated on VBMM agar with or without arabinose, and this analysis yielded 115 genetically distinct mutants in which the transposon was mapped to 70 genes. A subset of 58 candidate second-site suppressor mutations, none of which were arabinose-responsive, mapped to only 26 genes in 17 operons (Table 2).

To further investigate these findings, in-frame deletion mutations were constructed in genes representing key suppressor mutations: gacS, fleQ, fleN, motAB, motABCD, sadB, sadC, siaD, pilA, and PA1769. Introducing these deletions into the ΔfliC background in all cases abolished the RSCV-phenotype on VBMM (Fig 4A). To further distinguish suppression of EPS expression from changes in colony morphology, we used an enzyme-linked immunosorbent assay (ELISA) to quantify the amount of Psl produced by each of these double, triple and quintuple mutants. Mutations in gacS, fleQ, sadB, motAB and motABCD signifcantly reduced Psl production by the ΔfliC strain (Fig 4B). Together, motAB or motCD encode a stator complex that provides flagellar torque [54, 55]. Here, we conclude that EPS overexpression by flagellar mutants involves a regulatory pathway that not only requires SadB [56–58], but also involves the flagellum stator proteins. These observations are consistent with a recent report from Baker and colleagues [59] in which the MotCD stator was observed to interact with the diguanylate cyclase SadC to stimulate c-di-GMP production under conditions not permissive to motility. SadC was also identified in our suppressor screen. In addition, we found a number of type IV pilus genes linked to the Pil-Chp surface sensing system, including four separate insertions in pilY1 (Table 2), which encodes a pilus tip protein that has a putative mechanosensory domain [60]. We also found four separate insertions in the pilMONPQ operon (Table 2), which encodes the pilus alignment complex that is thought to transduce stimuli from PilY1 [61]. Because a ΔpilA ΔfliC double mutant did not have decreased levels of Psl production (Fig 4), one interpretation of these data is that type IV pilus-dependent motility might be important for the RSCV phenotype of flagellar mutants; however, another possibility is that the stator-dependent pathway of surface-sensing requires some parts of the Pil-Chp surface-sensing system to stimulate the RSCV phenotype on agar surfaces. While a complete mechanism is not yet evident, a teleological explanation is that altered function of flagellum motor components engages a signal transduction pathway that leads to biofilm formation. These observations might be informative to our understanding of the early stages of biofilm formation.

These observations are not without precedent: flagellin (flaA) mutants of Vibrio cholerae O139 also display a rugose colony morphology [47]. In V. cholerae, this gain-of-function phenotype depends on the Vibrio polysaccharide (vps) genes, the master regulator of the vps gene cluster (VpsR) and the sodium-driven flagellar motor (MotX) [62]. Work by Wu and colleagues [46] has also identified a stator-dependent signal transduction process that regulates V. cholerae EPS gene expression by modulating intracellular c-di-GMP. This flagellum-dependent biofilm response (FDBR) depends on the activities of at least three different diguanylate cyclases [46]. Under certain conditions and similar to P. aeruginosa [63, 64], V. cholerae O139 flagellar mutants grown on glass coverslips can form thick biofilm monolayers or microcolonies with architectures that are distinct from wild type [47]. Additionally, EPS-producing V. cholerae has an advantage in biofilm competition against isogenic, EPS-deficient strains; however, a cost of EPS-production is an impaired ability to disperse to new locations [65].

Flagellar mutants predictably and reproducibly evolve in laboratory biofilms

One context in which EPS-overproducing flagellar mutants might have an advantage is in biofilms. EPS-mediated adhesion may allow bacteria to better occupy space on the substratum [66] and polymer production may provide better access to oxygen and nutrients as biofilms grow [67]. To test the hypothesis that flagellar mutants might account for RSCVs isolated from in vitro grown biofilms (Fig 1), we began by inoculating five independent reactors with a founding population of wild type PAO1 taken from a single, shaken overnight culture. After 5 d, a random sample of RSCVs was isolated on VBMM agar from each reactor. Genome sequencing revealed that RSCV-linked mutations were found in each isolate that were previously identified by transposon mutagenesis (Table 3). Subsequently, the identified mutant alleles were introduced into the ancestral PAO1 strain, producing the RSCV phenotype (S5 Fig). All these genotypes overproduced PelC (S6A Fig) and Psl (S6B Fig). The most frequent RSCV-linked mutations occurred in genes encoding flagellum biosynthesis and function. Mutants bearing these alleles had a frequency from 23% to 63% in each of the replicate biofilm populations (Table 3). Altogether, these observations indicate that EPS-overproducing flagellar mutants reproducibly evolve during P. aeruginosa biofilm growth.

EPS overproduction provides flagellar mutants with a biofilm fitness advantage

Competitive co-culture was used to assess the fitness of precisely defined genotypes in laboratory-grown biofilms. Here we used a starting ratio of 1 mutant or complemented cell to 1000 ancestral cells. Viable cell counting was enabled by genetically tagging bacteria with an antibiotic resistance gene (aacC1). The aacC1 gene is not selectively neutral, as the wild type PAO1 strain labelled in this manner decreased in frequency approximately 6-fold during competition with its antibiotic-sensitive ancestor (Fig 5A). To account for this change in fitness, all changes in frequency of aacC1-labelled mutant cell lines were calculated relative to the frequency of the aacC1-labelled wild type strain. Correlating with previous reports and with only a single exception (ΔPA5295), we found that acquisition of an RSCV-linked mutation was strongly associated with an increase in biofilm fitness (Fig 5A and 5B).

Subsequently, we focused on flagellar mutants in order to disentangle the roles of EPS production and loss of flagellar function to biofilm fitness. During the course of co-culture, the ΔfliC mutant increased in frequency 150-fold and genetic complementation eliminated the increased biofilm fitness of this genotype (Fig 5B). In contrast to ΔfliC, a ΔfliCpelFpslD triple mutant had greatly diminished biofilm fitness, increasing in frequency only 2.5-fold relative to the aacC1-labelled control (Fig 5B). Genetic complementation of this triple mutant with single copies of pelF and pslD expressed from their native promoters restored the biofilm fitness of this strain, which increased ~410-fold in frequency during co-culture (Fig 5B). These data suggest that Pel and/or Psl are essential for the biofilm fitness of a flagellar mutant. To further test this interpretation, we directly tested for the loss of swimming motility during experimental evolution of wild type and ΔpelFpslD strains in drip-flow reactors (Fig 5C). After 5 days, 35.4% of the biofilm isolates from wild type and 14.9% from the ΔpelFpslD strain were negative for swimming motility. By contrast, repeating experimental evolution with the ΔpelFpslD mutant that had been complemented with pelF and pslD yielded a population where 39.8% of biofilm isolates were negative for swimming motility. Overall, these results suggest that EPS expression is a gain-of-function phenotype that significantly contributes to the biofilm fitness of flagellar mutants.

Flagellar mutants and other classes of RSCVs described here are found among CF isolates

P. aeruginosa that have lost flagellar motility have been reported among CF isolates [8, 68]. Here we examined clonally related isolates from previous studies of P. aeruginosa genetic diversity in CF respiratory infections [2, 40, 69]. Initially, we identified eight closely related pairs of isolates (Fig 6A and S1 Table, each pair recovered from a different, individual CF patient) in which one isolate had smooth colony morphology and the other displayed an RSCV phenotype on VBMM (not on LB; S7 Fig). An additional two pairs of isolates were identified among early and late isolates from prior [2] genome sequencing projects. All the RSCVs displayed upregulation of PelC relative to their related strains with smooth colony morphology (Fig 6A). Data from directed [2] as well as de novo genome sequencing was used to identify putative RSCV-linked mutations. In five cases we successfully identified mutations (retS_2078C>A, wspF_{474_477ΔTTCGinsCAGAC}, wspF_{635_636ΔCG}, morA_3430C>T, and fleQ_364C>T), that when introduced into PAO1, caused the RSCV phenotype (Fig 6B). We were unsure as to why the wspF mutant allele was smooth on LB for two of these clinical isolates. We postulate that this difference could be due to an epistatic interaction in the genetic background of the CF isolates. Regardless, these findings indicate that CF RSCV isolates are associated with similar gain-of-function phenotypes (EPS overexpression), notably including a flagellar mutant.

A new way to interpret the fitness benefits of flagellar mutations

Because an accurate and established small animal model for CF respiratory infection is not available, it is difficult to evaluate what selective forces are at work in vivo. Several factors could lead to bacterial diversification in CF airways, and these pressures might include immune functions, fluctuations in antibiotics, O₂ tension or nutrient availability, among others. P. aeruginosa non-mucoid isolates that have lost their flagella have been reported for at least thirty-five [8, 68] years and there are two classic interpretations for the benefits of this adaptation. First, loss of the flagellum confers immune evasion, which could allow P. aeruginosa flagellar mutants to escape negative selection by host immune cells [70]. Second, mutation of flagellar genes could afford a metabolic benefit to mutant cells by eliminating the energetic cost of synthesizing the flagellum, which could provide flagellar mutants with an increased growth rate. Our discovery that flagellar mutants have a gain-of-function phenotype–Pel and Psl expression–offers another perspective on the selective pressures that amplify flagellar gene mutations (Fig 7). One possibility is that positive selection for aggregation and biofilm growth could favor loss of the flagellum; alternatively, immune selection or an increased growth rate could lead to the evolution of non-flagellated bacteria that are also particularly successful at forming biofilms. Because there are multiple parameters that may drive expression of the RSCV phenotype on VBMM agar but not on LB, which might include, for example, nutrition-dependent modulation of c-di-GMP signaling enzymes [71, 72], we have yet to decipher the molecular mechanism for this phenomenon. However, we observed that flagellar mutants display an RSCV phenotype and overexpress PelC and Psl on synthetic cystic fibrosis sputum medium (SCFM) agar (S8 Fig). Staudinger and colleagues have also observed that flagellar mutants aggregate in mucus-based gels and that aggregation decreases the susceptibility of P. aeruginosa flagellar mutants to antibiotics [73]. We remark that our interpretation for how these gain-of-function phenotypes might provide benefits during infection may not be exclusive of other classic interpretations for the benefits of the loss of flagella. Nevertheless, because Pel and Psl production are associated with biofilm fitness, we propose that loss of the flagellum may lead to biofilm growth that contributes to increased bacterial persistence during infection.

Materials and methods

Bacterial strains and isolates, plasmids and growth conditions

Bacterial strains or isolates and plasmids are listed in S1 Table and S2 Table, respectively. Collection of all clinical isolates was approved by the Seattle Children’s and/or University of Washington Hospital Institutional Review Boards (IRBs). P. aeruginosa was grown at 37°C in lysogeny broth (LB, 10 g L^-1 tryptone, 5.0 g L^-1 yeast extract, 5.0 g L^-1 NaCl) or Vogel-Bonner Minimal Media (VBMM; 0.2 g L^-1 MgSO₄•7H₂O, 2.0 g L^-1 citric acid, 3.5 g L^-1 NaNH₄HPO₄•4H₂O, 10 g L^-1 K₂HPO₄, pH 7.0) [74] with 10 mM citrate. VBMM with Congo red (CR), brilliant blue R (BB) and 1.0 g L^-1 casamino acids (VCBA) was used to grow clinical isolates. Semi-solid plate media were prepared by adding 1.5% w/v Bacto agar to LB or 1.0% noble agar to VBMM. Additional details of growth conditions, antibiotic selection and plasmid construction are described in S1 Text.

Digital photography

An Olympus SZX-ILLK100 stereomicroscope equipped with a C-7070 wide zoom digital camera was used to photograph colonies growing on agar media. Pictures of Petri plates were captured using a Pentax Optio W10 digital camera with tripod. Digital images were adjusted for contrast and brightness using Photoshop CS6 (Adobe).

Transposon mutagenesis

Established protocols were used for mutagenesis of P. aeruginosa with the transposon miniTn5-Pro [42]. Transconjugants were selected on VBMM plates containing 100 μg/ml gentamicin (Gm) + CR/BB. This selection was repeated on plates that additionally contained 0.2% arabinose. Transposon insertion sites were identified by plasmid rescue of the flanking genomic regions followed by DNA sequencing (see S1 Text).

Construction of deletion and site-directed mutants

Deletion alleles were assembled in vitro by removing an in-frame fragment of coding sequence from each gene. This was done by joining PCR products amplified from the adjacent regions of the chromosome by splicing by overlapping extension (SOE) PCR. These DNA fragments were then cloned by restriction into the suicide vector pEX18Gm. Alternatively, cloning of deletion alleles was carried out using Gateway recombination with the suicide vectors pDONRPEX18Gm or pEX18GmGW as previously described [75] (see S1 Text). Alleles containing point mutations that evolved in either biofilm or clinical isolates were directly cloned by PCR using primers specific for the target open reading frames (ORFs) and then introduced into allelic exchange vectors using Gateway technology (see S1 Text). Unmarked deletion and site-directed mutations were then introduced into the P. aeruginosa chromosome by two-step allelic exchange using established procedures [75].

Complementation analysis

PCR primers (S3 Table) targeting wild type alleles and their native promoters were tailed with attB sequences and these PCR products were then cloned by Gateway BP recombination into pDONR223. The plasmid inserts were sequenced using universal forward and reverse M13 primers (S3 Table) and then transferred by LR recombination into pUC18-miniTn7T-Gm-GW [76]. For the purpose of complementation analysis with two ORFs, we used the plasmid pUC18-miniTn7T2.1-Gm-GW [77] and two divergently transcribed ORFs were assembled in multiple steps using SOE-PCR and Multisite Gateway cloning technology (see S1 Text). The miniTn7 constructs were then introduced into the P. aeruginosa chromosome via electroporation with the helper plasmid pTNS1 [78]. Insertion at the neutral attTn7 adjacent to P. aeruginosa glmS was confirmed by PCR using the primers PTn7L, PTn7R, Gm-up and Gm-down (S3 Table) as previously described [76].

Drip-flow biofilm reactors

P. aeruginosa biofilms were grown in drip-flow reactors using 1% tryptic soy broth according to standard protocols [79]. Biofilms were recovered using a cell scraper to dislodge biomass into sterile PBS and these were then disrupted using a tissue homogenizer. Biofilm cells were serially diluted ten-fold in PBS and then plated onto either LB or VBMM + CR/BB agar.

Genome sequencing and bioinformatics

Illumina sequencing technology was used to collect deep-coverage genome sequence data. The procedures for library construction, sequencing, genome assembly and SNP analysis are detailed in S1 Text. Sequencing data have been deposited with links to BioProject accession number PRJNA625996 in the National Center for Biotechnology (NCBI) BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/).

Immunoblots

Chemiluminescent Western and dot blots for PelC and Psl, respectively, were carried out by established methods [80] with slight modification (see S1 Text). Horse-radish peroxidase linked secondary antibodies were used in conjunction with the Pierce SuperSignal West Pico ECL reagent (Thermo Scientific) to visualize proteins bound by primary antibodies and Chemiluminescence was captured and quantified using a FluorChem Q (Alpha Innotech).

ELISA assays

ELISA assays were carried out according to the method of Byrd and colleagues [80] with slight modification using a human monoclonal anti-Psl antibody, Cam-003 (MedImmune) [81] (see S1 Text).

Motility assays

The swimming medium was VBMM that contained 0.3% w/v bacto-agar (Difco). Swim plates were stab inoculated with bacteria from an LB or VBMM agar culture grown overnight at 37°C using a sterile toothpick. Plates were then incubated at 37°C for 16–18 h and photographed.

Supporting information

S1 Text [pdf]
Supplementary materials and methods and supplementary references.

S1 Table [pdf]
Bacterial strains.

S2 Table [pdf]
Plasmids.

S3 Table [pdf]
Primers and sequencing adapters.

S1 Fig [a]
. strains with nutrition-dependent RSCV-linked genotypes overexpress PelC and/or Psl even when grown under conditions where the RSCV phenotype is not expressed.

S2 Fig [a]
Nutrition-dependent RSCVs do not require WspR and have smooth, and in some instances small, colony morphology on LB agar.

S3 Fig [tif]
Only some deletion mutations in flagellar operons are linked to the . RSCV phenotype.

S4 Fig [tif]
Overexpression of PelC by some flagellar mutants is surface-contact dependent.

S5 Fig [top]
Mutant alleles that evolved during biofilm growth are linked to the RSCV phenotype.

S6 Fig [a]
Biofilm-evolved flagellar mutant genotypes overexpress PelC and Psl.

S7 Fig [a]
Nutrition-dependent colony morphology of closely related pairs of CF isolates.

S8 Fig [a]
Flagellum mutants overexpress extracellular polysaccharides and display the RSCV phenotype on synthetic cystic fibrosis sputum medium (SCFM) agar.