ABSTRACT
FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through activation of the adenylate cyclase, CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV’s cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, required both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds - lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH, or expressing constitutively-active CyaB - pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV-dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV’s role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.
IMPORTANCE Pseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility, and are involved in surface sensing which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements – including the PilSR two-component system – to cell poles for optimal function.
INTRODUCTION
Type IV pili (T4P) are polar filamentous surface appendages made by a broad range of bacteria and archaea (1, 2). They can be divided into two sub-families, type IVa (T4aP) and type IVb (T4bP), which – though clearly related – differ in their pilin subunits and assembly system architectures (2). T4aP are involved in several processes including DNA uptake, surface attachment, and twitching motility (3-5). During twitching motility, T4aP undergo repeated cycles of assembly and disassembly, acting as molecular grappling hooks to pull the cells along surfaces. Well-studied T4aP model species include Neisseria spp., Myxococcus xanthus, and Pseudomonas aeruginosa (6, 7). Although core structural components of the T4aP assembly machinery and pilus fibre are shared, each species has unique regulatory elements that control the function of the T4aP machinery in response to their specific environmental requirements. Without these regulatory proteins, the bacteria make non-functional T4aP systems (8-11).
The P. aeruginosa Chp system is a putative chemosensory system that controls both twitching motility and intracellular levels of the second messenger, cyclic adenosine monophosphate (cAMP) (12-14). It resembles the well-studied Che system of E. coli, but lacks a CheZ-like phosphatase. Rather, similar to Sinorhizobium meliloti (15), it has two CheY-like response regulators, PilG and PilH (14, 16). PilG is proposed to regulate activation of CyaB and pilus extension (17), while PilH has been proposed to be either a phosphate sink that limits downstream signalling through PilG in lieu of a phosphatase (17, 18), or a separate response regulator controlling function of the T4aP retraction ATPase, PilT (12).
The Chp system positively regulates intracellular levels of cAMP by activating the major adenylate cyclase, CyaB (17). Deletion of pilG results in decreased cAMP, surface piliation, and twitching motility, while pilH mutants have increased cAMP and surface piliation but decreased twitching relative to wild type (17). Supplementation of a pilG mutant with exogenous cAMP restored surface piliation but not twitching motility (17), suggesting that PilG regulates pilus biogenesis and function by at least two pathways. A recent study (18) showed that of the two proteins, PilH is the preferred target of ChpA phosphorylation, consistent with its proposed role as a phosphate sink. Decreased twitching motility in the pilH background may reflect hyperphosphorylation of PilG, perturbation of the chemotactic response, and uncoordinated movement.
Important for P. aeruginosa virulence is its ability to switch from a planktonic to sessile state when cells contact surfaces (19, 20). T4aP-mediated surface interaction is proposed to lead to signalling through the Chp system, upregulating surface-associated virulence phenotypes by increasing intracellular levels of cAMP (20). Vfr (virulence factor regulator) binds cAMP and modulates the expression of >200 genes, including the type II secretion system (T2SS) and its effectors, and T4aP assembly components including the motor ATPases PilBTU, the alignment subcomplex PilMNOP, the secretin PilQ, and the PilSR two-component system that regulates PilA levels (21). This regulatory circuitry allows for just-in-time expression of components required for a surface-associated lifestyle in response to surface contact.
FimV is also required for T4aP function and CyaB activation (17), and is proposed to link into the Chp system via the cytoplasmic protein, FimL (22). FimV is a 97 kDa inner-membrane protein with one transmembrane segment. Its periplasmic domain contains a lysin (LysM) motif that binds peptidoglycan (PG) (23), and its cytoplasmic domain contains three discontinuous tetratricopeptide repeat (TPR) motifs involved in protein-protein interactions (24). FimV homologs have been identified in other T4P-producing bacteria (10) although their overall sequence identity is low, with the most conserved features being the LysM motif (COG3170), the single transmembrane segment, and a highly conserved cytoplasmic “FimV C-terminal domain” – TIGR03504 – encompassing a single TPR repeat and capping helix (25).
FimV homologs have been characterized in several species (8-11, 26–28), but their functions are not necessarily conserved. Deletion of FimV in Legionella pneumophila resulted in loss of twitching motility and cell elongation, while deletion of the Neisseria meningitidis FimV homolog TspA led to decreased host cell adhesion but no effect on twitching motility or surface piliation. The Vibrio cholerae homolog, HubP, functions as a protein interaction hub, although its role is not limited to T4P localization. Deletion of HubP altered the cellular distribution of the chemotactic and flagellar machinery, and the chromosomal origin, oriCI (28). HubP from Shewenella putrifaciens is responsible for localization of the chemotactic machinery, but not the flagellar system (27). Yamaichi et al. (28) showed that polar localization of V. cholerae HubP was dependent on the conserved LysM motif (25). Wehbi et al. (29) showed that P. aeruginosa fimV mutants have decreased levels of the T4aP alignment subcomplex proteins, PilMNOP, while inframe deletion of FimV’s LysM motif resulted in fewer PilQ multimers, suggesting that PG binding is important for optimal secretin formation. A recent study (30) confirmed that FimV participates in localization of PilMNOPQ to sites of future cell division, ultimately placing T4aP assembly systems at both poles of newly divided cells.
T4aP-mediated twitching motility requires both cAMP-dependent and independent inputs (17). For example, provision of exogenous cAMP to mutants lacking PilG restored piliation but not motility, and a mutant expressing a constitutively active form of CyaB but lacking FimV failed to twitch (25). FimL was proposed to be a scaffold protein linking PilG to the C-terminal TPR motif of FimV, leading to CyaB activation, and FimV localized both FimL and PilG to cell poles (22, 25). However, of these three proteins, only FimL is dispensable for twitching motility in cAMP-replete conditions. Thus, the FimV-FimL-PilG model fails to explain the cAMP-independent roles of FimV and PilG in twitching.
Here we provide evidence supporting different cAMP-independent roles for FimV and PilG in regulation of twitching motility. We show that in addition to polar localization of FimL, PilG, and PilMNOPQ (22, 30), FimV is responsible for polar localization of PilS, the membrane-bound sensor kinase that controls pilA transcription. These data show that FimV plays a central role in control of twitching motility that overlaps with – but is distinct from – that of the Chp system.
RESULTS
FimV is required for Chp activation of CyaB
FimL, FimV, and PilG are all required for activation of CyaB, with FimL proposed to link FimV to the Chp system through PilG (17, 22, 31, 32). However, while phenotypes associated with fimL deletion could be rescued by deletion of cpdA or by increasing intracellular cAMP levels in other ways (22, 32, 33), provision of exogenous cAMP failed to restore motility in a pilG mutant (17). We investigated whether the cAMP-independent function of PilG also required FimV by comparing PilU levels – a proxy for intracellular cAMP levels (21, 25) – twitching, and piliation in fimL, fimV, and pilG single mutants or in double mutants also lacking cpdA to prevent degradation of endogenous cAMP (32) (Figure 1). To confirm that FimV and FimL were epistatic to PilG, we also examined pilH fimL and pilH fimV double mutants. In the absence of PilH, cells are predicted to have hyper-phosphorylated PilG, consistent with the high levels of cAMP observed in a pilH background (17, 18).
PilU levels were decreased in fimL, fimV, and pilG, to 28%, 10% and 22% of wild type, respectively, consistent with roles in regulating cAMP synthesis (10, 12, 17, 32), (Figure 1). Both pilG and fimV were twitching deficient, while fimL twitching resembled that of wild type, as reported previously (32, 34). The cpdA mutant had high levels of PilU, surface piliation, and wild type twitching, consistent with high cAMP levels (17, 32). Deletion of cpdA in fimL, fimV, or pilG increased PilU levels relative to the corresponding single mutants, to at least wild-type levels (Figure 1), showing that CyaB retains residual activity in those backgrounds. However, only the cpdA fimL mutant was motile, confirming that both PilG and FimV have cAMP-independent roles in T4aP function. Also consistent with previous reports (17), a pilH mutant assembled surface pili but was twitching impaired (~39% of wild type). The pilH fimV and pilH fimL double mutants had PilU levels similar to those of fimV and fimL single mutants, suggesting that despite its hyper-phosphorylation in the absence of pilH (18), PilG was unable to activate CyaB without FimV or FimL, confirming that all three are required for the Chp system to stimulate cAMP synthesis.
Decreased levels of PilMNOPQ in fimV are due to decreased cAMP
Wehbi et al. (29) showed previously that fimV mutants had reduced levels of PilMNOP, and that a fimV∆LysM mutant with an in-frame deletion of the LysM motif had fewer PilQ secretins. However, transcription of the pilMNOPQ operon is Vfr-and thus cAMP-dependent (21). To determine if any of these phenotypes were independent of cAMP, we examined levels of PilMNOPQ and PilU, and twitching motility in fimV, fimV∆LysM, a mutant encoding only the cytoplasmic domain of FimV (fimV1194), and in a fimV cyaB-R456L double mutant that expresses constitutively active CyaB (25, 35).
The fimV mutant had low levels of PilU (~23% of wild type), reflecting low cAMP levels, while the fimV cyaB-R456L double mutant had wild type levels of PilU (Figure 2). The fimV1194 mutant had ~29% of wild-type PilU, suggesting that even though its protein partners PilG and FimL are cytoplasmic, expression of FimV’s cytoplasmic domain alone was insufficient to activate CyaB. Complementation of fimV and fimV1194 in trans with a construct expressing full-length FimV increased PilU levels to ~58% and ~65% of wild type, respectively. Surprisingly, the fimVLysM mutant had ~89% of wild type PilU, suggesting that the LysM motif and thus PG binding was dispensable for CyaB activation.
The fimV mutant had decreased levels of PilMNOP, and few detectable PilQ multimers, and all were restored to wild type with full-length FimV. Supporting the hypothesis that their levels were dependent on Vfr and cAMP, the fimV cyaB-R456L double mutant had wild type levels of PilMNOPQ. Despite this, the fimV cyaB-R456L double mutant had no recoverable surface pili (Figure 2) and could not twitch, confirming a cAMP-independent role(s) for FimV in pilus assembly and twitching motility. The fimV1194 mutant had low levels of PilMNOP and no detectable PilQ multimers, but these could be rescued by complementation with full length FimV. fimV∆LysM had essentially wild type PilMNOPQ levels, and could assemble surface pili (Figure 2); however, twitching was ~37% of wild type. The motility defect in fimV∆LysM suggests that PG binding is important for FimV’s cAMP-independent function(s).
Wehbi et al. (29) showed that the fimV1194 mutant (which expresses the cytoplasmic domain of FimV) was unable to twitch, but motility could be rescued by complementation with a plasmid expressing only the periplasmic domain of FimV (residues 1-507, pFimV507), suggesting that together, the two FimV fragments could restore function without being physically connected. A fimV deletion mutant complemented with empty vector or pFimV507 had similar PilU levels, suggesting that the periplasmic domain alone is not sufficient to activate CyaB (Figure 3A). Unexpectedly, despite its ability to restore motility in the fimV1194 background (Figure 3B), pFimV507 did not significantly increase PilU levels in that background, suggesting that the cytoplasmic and periplasmic domains cannot activate CyaB efficiently when they are not covalently linked. However, CyaB activation is not strictly required for twitching, as both fimL (Figure 1) and cyaAB mutants have low cAMP levels and piliation, but near wild-type motility (17, 32). We next tested if FimV’s periplasmic domain played a cAMP-independent role in twitching by complementing the fimV cyaB-R456L mutant with pFimV507. pFimV507 failed to restore twitching in fimV cyaB-R456L (Figure 3B), suggesting that the cytoplasmic region of FimV plays a cAMP-independent role in motility. Taken together, the data show that the cAMP-independent role(s) of FimV requires both domains.
FimV is required for PilS localization
The V. cholerae homolog of FimV, HubP, interacts with multiple proteins and has broad regulatory function (28). FimV is required for bipolar localization of PilG and FimL and the T4aP structural proteins PilMNOPQ, but not the Chp methyl-accepting chemotaxis protein (MCP) PilJ (22, 30). To determine if FimV was required for localization of other T4aP regulators, we examined its effects on localization of PilS (36, 37), the histidine sensor kinase component of the PilRS two-component system that regulates pilA transcription in response to changes in PilA levels in the inner membrane (38).
In wild-type cells, PilS-YFP was localized to both poles (Figure 4) as reported previously (36), while in the absence of FimV, PilS-YFP was diffuse in the inner membrane. Interestingly, the localization pattern of PilS-YFP in fimV∆LysM was similar to wild type, suggesting that PG-binding was not critical for PilS localization. However, PilS-YFP was mislocalized in fimV1194, suggesting that the cytoplasmic domain is insufficient for PilS localization. Finally, because PilG also has cAMP-independent effects on motility (17) (Figure 1), we examined PilS-YFP localization in a pilG mutant. Localization was similar to wild type, suggesting that PilG and FimV have distinct cAMP-independent roles in motility (Figure 4). These data also suggest that PilS localization is independent of intracellular cAMP concentration.
FimV deletion does not affect swimming motility
As the V. cholerae and S. putrefaciens homologs of FimV modulate swimming motility (27, 28), we tested whether loss of fimV impaired swimming in P. aeruginosa. We saw no effect of FimV deletion on swimming motility (Figure 5), suggesting that it is not essential for flagellar function in P. aeruginosa. Consistent with reports that swimming is negatively regulated by high cAMP (21), the cpdA mutant was swimming impaired (~53% relative to wild type). Deletion of cpdA in the fimV (~77%), fimL (~90%), and pilG (~85%) backgrounds reduced swimming relative to the single mutants, potentially due to increased cAMP levels (Figure 1). Suprisingly, despite having very high cAMP levels (17), and levels of piliation similar to the cpdA mutant (Figure 1), pilH had ~93% swimming motility relative to wild type (Figure 5). This finding suggests that high levels of cAMP and piliation do not necessarily inhibit swimming motility.
DISCUSSION
PilG, FimL, and FimV were recently proposed to be components of a surface-sensing pathway that activates CyaB (22), with FimL acting as a scaffold protein connecting PilG to FimV. Consistent with this model, we saw that increasing the level of PilG phosphorylation through deletion of pilH (18) failed to increase levels of PilU if either FimV or FimL was missing (Figure 1). However, only the fimL mutant twitched (Figure 1) following introduction of compensatory mutations that increased cAMP levels (17, 32), confirming that both PilG and FimV have cAMP-independent roles in twitching (17, 25). Thus, FimL's role in twitching is limited to its ability to connect PilG and FimV, leading to CyaB activation via an as-yet unknown mechanism. Interestingly, Nolan et al. (33) identified other suppressors of fimL that mapped outside the cyaA, cyaB, pilG, pilH, vfr, and cpdA loci. How those uncharacterized loci fit into the FimV-FimL-PilG signalling axis remains to be determined.
Since FimL's role is limited to the cAMP-dependent pathway (22, 32) and twitching motility in the fimL background is essentially wild type (Figure 1), PilG and FimV both function – together or independently of one another – in its absence. PilG polar localization is dependent on FimV, but PilG remains localized to the poles when FimL is absent (22). These data imply that PilG interacts with FimV directly, or indirectly via another, as-yet unidentified adaptor protein. That component is unlikely to be part of the Chp system; the MCP PilJ localizes to the poles independently of FimV, and PilG retains bipolar localization in the absence of both PilJ (Figure S1) and the Chp system kinase, ChpA (22). Identifying this interaction partner – potentially among the list of proteins recovered in a recent PilG pulldown/mass spectrometry study (22) could help to clarify how PilG contributes to cAMP-independent regulation of twitching.
Because restoration of cAMP levels in a pilG mutant by supplying exogenenous cAMP (17), constitutively activating CyaB (35), or deleting cpdA (Figure 1A) restores piliation but not twitching motility, the cAMP-independent role of PilG may be the coordination of pilus retraction to permit directional movement. In M. xanthus, the Chp-like Frz system controls the asymmetric subcellular distribution of the PilB and PilT motor ATPases in cells undergoing T4aP-mediated S-motility, to coordinate movement (39). It is likely that asymmetric T4aP retraction similarly occurs in P. aeruginosa, as pilus retraction at both poles simultaneously would result in zero net movement.
How PilG might regulate pilus retraction remains unclear. CheY interacts with FliM at the E. coli flagellar switch complex to control the direction of flagellum rotation (40), but the T4Ap system lacks an obvious FliM equivalent. However, the T4aP system was recently discovered to have a rotary motor (41–43). A hexameric PilB or PilT ATPase docks into the PilM ring at the base of the apparatus and encircles the cytoplasmic domains of the PilC platform protein (7), rotating it clockwise or counterclockwise, respectively, to insert or extract pilin subunits from the pilus in a stepwise manner (43). Transient interactions of phospho-PilG with PilM, PilC, or the motor ATPases might dictate which ATPase is docked at the leading versus lagging pole. In pilH mutants, hyperactivation of PilG may dysregulate asymmetric pilus retraction, leading to hyperpiliation and impaired motility (Figure 1A).
Interestingly, the cAMP-independent role of PilG appears dependent on, but distinct from, that of FimV. In addition to FimL and PilG (22), FimV is required for polar localization of the structural components PilMNOPQ (30) and the PilSR two-component system (Figure 4). However, unlike its homologues in V. cholerae and S. putrefaciens (27, 28), its deletion does not affect swimming (Figure 5). FimV and its homologs are emerging as protein interaction hubs that bind to septal PG via their LysM motif to target their partners to the septum during division, ensuring the correct placement of polar and partitioning systems during and after separation of daughter cells (28, 30). Although studies of L pneumophila FimV and N. meningitidis TspA (9, 11) did not address the role of the LysM motif or localization in function, the phenotypes of mutants lacking these proteins could reflect consequent mislocalization of motility or adhesion systems.
Septal PG binding by FimV was dispensable for CyaB activation (Figure 2) even though FimL, PilG, and CyaB are located at the cell poles (22, 32). It is possible that deletion of the LysM motif alone does not completely mislocalize FimV, as it likely has other interaction partners that help to confine it to the cell poles; attempts to test this hypothesis using a FimV∆LysM-YFP fusion have not been successful to date. Alternatively, FimV∆LysM may be present at the cell pole due to diffusion in the inner membrane in sufficient quantities to promote CyaB activity. Consistent with only partial mislocalization of FimV∆LysM, PilS-YFP remained mostly localized to the poles in that background, while deletion of FimV’s entire periplasmic region led to PilS delocalization (Figure 4). Transmembrane domains 5 and 6 of PilS (44), and the membrane-embedded MASE2 domain of CyaB (35) are sufficient for their polar localization. It is possible that they interact with FimV via its transmembrane segment, or are integrated into the FimV hub through as-yet unidentified intermediaries.
In summary, this work helps to resolve the cAMP-dependent and independent regulation of P. aeruginosa twitching motility by FimV and PilG. The cAMP-independent role of FimV is likely coordinate localization of multiple T4aP structural and regulatory components to the cell poles, while that of PilG may be to control pilus retraction in a way that allows for directional movement; experiments to test this idea are underway. Characterization of the FimV protein-interaction network will identify its full repertoire of direct and indirect interaction partners, and clarify the links between polar localization and function.
MATERIALS AND METHODS
Bacterial growth and culture conditions
Bacterial strains and plasmids are listed in Table 1. Unless otherwise stated, untransformed P. aeruginosa strains and all E. coli strains were grown on LB agar at 37 °C. Antibiotic selection was as follows unless stated otherwise: gentamicin, 15μg/ml for E. coli and 30μg/ml for Ρ. aeruginosa; kanamycin, 50μg/ml for E. coli; ampicillin, 100mg/ml for E. coli. All P. aeruginosa strains containing a FimV complementation construct were grown on media supplemented with 0.1% (w/v) arabinose.
Mutant generation
Mutants were made as previously described (17). Deletion constructs for the generation of fimL, cpdA, and fimV mutants were designed to include 100 nucleotides upstream and downstream of the gene to be deleted. The pilH construct was designed to include the first 12 nucleotides and last 30 nucleotides of the gene. The upstream and downstream fragments were amplified from the PAK chromosome using the primer sets described in Table 2. Inserts were cloned into the pEX18Gm suicide vector at the Sacl and HinDIII sites (pEX18Gm::fimL), Kpnl and EcoRI sites (pEX18Gm::cpdA), and HinDIII and Kpnl sites (pEX18Gm::pilH). Suicide vectors were verified by DNA sequencing.
After verification, plasmids (pEX18GM::fimL, pEX18Gm::cpdA, pEX18Gm::pilH, and pEX18Gm::fimVLysM) were transformed into E. coli SM10 cells. Plasmids were transferred to P. aeruginosa by conjugation at a ratio of 6:1 (E. coli to P. aeruginosa). 100 μl of the 6:1 mixed culture were spotted onto LB 1.5% agar (w/v) and incubated overnight at 37°C. The mating mixture was resuspended in 5 ml of LB and 100 μl was plated onto Pseudomonas isolation agar supplemented with Gm 100 μg/ml and grown overnight at 37°C. Single colonies were resuspended in 1 ml LB and plated onto LB 1.5% agar lacking sodium chloride and supplemented with 8% (w/v) sucrose, and grown overnight at 30°C. Resulting single colonies were replica plated onto LB and LB supplemented with Gm 30 μg/ml. Mutants were verified by PCR, and the pilH mutant was screened by western blotting with anti-PilH antiserum. Double mutants were generated in the same manner.
Plasmid construction
The coding region of the first 507 residues of FimV was amplified by PCR using pBADGr::fimV as a template, and cloned into pBADGr. The PCR amplified DNA was digested with, purified, and ligated into pBADGr at the Kpnl and Xbal sites with T4 DNA ligase according to manufacturer’s instructions (Thermo Scientific).
A version of piIS lacking its stop codon was amplified from the PAK chromosome and cloned into pBADGr::yfp in-frame with yfp at the EcoRI and Smal sites. In-frame ligation was confirmed by DNA sequencing.
A version of PilG lacking its stop codon was amplified from the PAO1 chromosome and cloned into the pMarkiC vector, which encodes (Gly3-Ser)3-YFP at the HinDIII site of pUCP20Gm. PilG was ligated into the EcoRI and Xbal sites. In-frame ligation was confirmed by DNA sequencing.
Immunoblotting
Western blotting of whole cell lysates was performed as previously described (45). In brief, whole cell lysates were prepared from strains were grown overnight on LB 1.5% agar, or in the case of plasmid transformed strains, LB 1.5% agar supplemented with 0.1% (w/v) arabinose. Cell growth was then resuspended in 1X PBS and normalized to an OD600 of 0.6. Cells were pelleted by centrifugation at 2,300 ×g for 5 min. Pellets were then resuspended in 175 μl of 1X SDS-PAGE loading dye. Cell lysates were resolved on 15% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked in 5% skim milk dissolved in PBS (pH 7.4) for 1 h, washed in PBS, and incubated with PBS-diluted antisera raised against the FimV periplasmic domain (1:1000), PilU (1:5000), PilM (1:1000), PilN (1:1000), PilO (1:1000), PilP (1:1000), or PilQ (1:1000), or polyclonal anti-GFP antibody (Novus Biologicals; 1:5000) for 1 h, washed, incubated with alkaline phosphatase-conjugated goat-anti-rabbit secondary antibody (1:3000, Bio-Rad) for 1 h, and washed. Blots were developed using 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitro blue tetrazolium (NBT). Data are representative of n = 3 independent experiments.
Sheared surface protein preparation
Surface pili were analyzed as previously described (46). In brief, strains of interest were streaked in a grid-like pattern onto LB 1.5% agar, or in the case of plasmid-transformed strains, LB 1.5% agar supplemented with 0.1% (w/v) arabinose and grown overnight at 37 °C. Cells were gently scraped from the plates using a sterile coverslip and resuspended in 4.5 ml PBS (pH 7.4). Surface appendages were sheared by vortexing the cells for 30 s. The OD600 for each strain was measured, and an amount of cells equivalent to 4.5 ml of the sample with the lowest OD600 was pelleted by centrifugation at 16,100 × g for 5 min. When necessary, PBS was added to samples to a final volume of 4.5 ml prior to centrifugation. Supernatants were removed and centrifuged again at 16,100 × g for 20 min to remove remaining cells. Supernatants were collected and mixed with 5 M NaCl and 30% (w/v) polyethylene glycol (Sigma; molecular weight range ~8000) to a final concentration of 0.5 M NaCl and 3% (w/v) polyethylene glycol, and incubated on ice for 30 min. Precipitated surface proteins were collected by centrifugation at 16,100 x g for 30 min. Supernatants were discarded and samples were centrifuged again at 16,100 x g for 2 min. Pellets were resuspended in 150 μl of 1X SDS-PAGE sample buffer (80 mM Tris, pH 6.8, 5.3% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, 0.02% (w/v) bromophenol blue, 2% (w/v) SDS). Samples were boiled for 10 min and resolved by 15% SDS-PAGE. Bands were visualized by staining with Coomassie brilliant blue (Sigma). Data are representative of n = 3 independent experiments.
Twitching assay
Twitching motility was tested as previously described (46). In brief, cells from an overnight culture were stab inoculated to the interface between LB 1% agar, or in the case of plasmid-transformed strains, LB 1% agar supplemented with 0.1% (w/v) arabinose and the underlying tissue culture-treated polystyrene petri dish, and incubated at 37 °C for 16 h (Thermo Fisher). Twitching zones were visualized by removing the agar and staining cells on the petri dish with 1% (w/v) crystal violet and washing with water to remove unbound dye. Twitching zones were measured by analyzing the diameter of each twitching zone in pixels using ImageJ software (NIH). Twitching zones were normalized to the twitching diameter of wild type PAK in each individual experiment. Data are representative of n = 3 independent experiments.
Fluorescence microscopy
P. aeruginosa strains transformed with pBADGr::FimV-eYFP were grown overnight. Microscopy was performed using 8-well 1.0 borosilicate chambered coverglass (LabTek). Chamber slides were prepared by adding LB 1% agar supplemented with 0.1% (w/v) arabinose to create an agar layer ~3mm in thickness and covering the bottom of the chamber. Agar was allowed to solidify with the lid off to prevent condensation. Bacteria were stab inoculated to the interface between the agar and coverglass. Slides were wrapped in foil to prevent photobleaching, and incubated at 37°C for 1h in the dark. Cells were then imaged using an EVOS FL Auto microscope, with a monochrome camera for brightfield imaging and a YFP LED light cube for fluorescence imaging, through a 60X oil immersion objective at room temperature. Representative fields were cropped from larger images and enlarged using ImageJ software (NIH) (47).
Fluorescence images were quantified using the MicrobeJ plugin for ImageJ (48). Brightfield and fluorescence images were arranged into a stack on ImageJ. Regions of interest corresponding to the bacteria were selected based on the brightfield image, and thresholding particles based on length (0.5μm-5μm), width (0.2μm-1.5μm), and area (0.75μm2-max), and fit to rod-shaped bacteria. Pixel intensity profiles were generated by MicrobeJ using the profile option on the fluorescence image, using 1μm width and 0.5μm extensions. Intensity profiles were plotted along a Y-axis of range 0-140, and the X-axis was partitioned into 50 bins. Pixel intensity profiles were generated for the YFP channel. Data are representative of at least 3 independent trials.
Swimming assay
Cells from overnight cultures were resuspended in sterile PBS and standardized to OD600 0.6. Two μl of cell suspension were spotted onto LB 0.3% agar and allowed to dry onto the surface of the agar. Plates were incubated at 30°C for 16h with the agar side down. Data are representative of n=3 independent experiments.