A ΔbifA mutant does not swarm, likely due to high cellular c-di-GMP pools.
In previous studies, we reported that the bifA
) encodes a phosphodiesterase (PDE) that is able to degrade c-di-GMP and influence surface-associated behaviors of Pseudomonas aeruginosa
). Our studies showed that deletion of the bifA
gene leads to an increase in cellular c-di-GMP pools and that the ΔbifA
mutant exhibits a hyperbiofilm phenotype as well as a complete loss of swarming motility (27
We predicted that the nonswarming phenotype of the ΔbifA
mutant results from the enhanced production of the pel
-derived polysaccharide. However, a pelA
mutation in the ΔbifA
mutant background resulted in only a minor improvement in the swarming defect, indicating that excess Pel polysaccharide production is not the primary contributor to the ΔbifA
swarming impairment (Fig. A). This finding is consistent with a previous report from our group (27
). Thus, we postulated that high levels of c-di-GMP inhibit swarming motility of the ΔbifA
mutant via one or more Pel-independent factors.
FIG. 1. Genetic analyses of the ΔbifA swarming defect. (A) Impact of Pel polysaccharide on the ΔbifA swarming impairment. Representative images of swarms formed by the WT, the ΔbifA mutant, the ΔbifA ΔpelA double mutant, (more ...)
To first confirm that the accumulation of excess c-di-GMP does, in fact, inhibit swarming motility in the ΔbifA
mutant, we tested whether expression of a nonendogenous PDE in the ΔbifA
mutant could rescue the swarming defect. We expressed the Pseudomonas fluorescens rapA
gene, encoding a PDE previously shown to possess c-di-GMP-degrading activity (38
), under the control of the arabinose-inducible pBAD promoter. As shown in Fig. , expression of pRapA in the ΔbifA
mutant background is able to rescue the swarming defect of the ΔbifA
mutant. These results indicate that degradation of c-di-GMP in the ΔbifA
mutant is able to restore swarming motility and supports the hypothesis that excess c-di-GMP inhibits swarming motility in the ΔbifA
Identification of suppressors of the ΔbifA swarming motility defect.
To better understand how c-di-GMP negatively affects swarming motility, we sought to identify those factors required for swarming inhibition in the ΔbifA mutant. To this end, we performed mariner transposon mutagenesis of the ΔbifA mutant and screened for restoration of swarming motility. We screened approximately 5,500 mariner transposon mutants in the ΔbifA mutant background on swarm agar plates (Fig. , top panel) and isolated suppressor strains with mutations in four different genes.
We mapped these transposon mutations in the suppressors to sadA
, and sadB
(Fig. ). The swarm phenotypes of three representative ΔbifA
suppressor mutants are shown in Fig. , bottom panel. The ΔbifA sadA
mutant exhibits a pattern of swarming motility distinct from that of the WT, with shorter and more numerous tendrils. We isolated five independent pvrS
mutants as suppressors of the ΔbifA
swarming defect (Fig. ). For the representative ΔbifA pvrS
mutant (58A9) shown in Fig. , the extent of swarming of the mutant is greater than that of the WT. The remaining four pvrS
mutants suppress the ΔbifA
mutant phenotype to various degrees, which likely reflects the different positions of the transposon insertion in each mutant (see below). The three independent ΔbifA pilY1
suppressor mutants isolated exhibit an unusual swarm morphology relative to the WT in that the mutants lack distinct tendril formation and instead show a more uniform swarming pattern radiating from the origin of inoculation (Fig. ). The fourth gene, sadB
, was previously shown to be required for inhibition of swarming by the ΔbifA
). Given that mutations in the sadB
gene completely suppress the swarming defects of the ΔbifA
mutant, the isolation of sadB
mutants served to validate the screen.
Enhanced expression of genes encoding putative PDEs suppresses the ΔbifA swarming defect.
Of the genes identified as suppressors in this screen, two genes, sadA
, are located directly adjacent to genes that code for proteins containing EAL motifs and thus are predicted to encode c-di-GMP phosphodiesterases (sadR
, respectively) (Fig. ). The sadA
gene (also known as rocA
) encodes a response regulator that has been previously shown, together with the adjacent genes, sadR
, to be involved in biofilm formation as well as regulation of Cup fimbrial gene expression (28
). The pvrS
gene encodes a sensor histidine kinase and is adjacent to the response regulator gene pvrR
, which has been shown to influence the frequency of formation of antibiotic-resistant small-colony variants of P. aeruginosa
Given the proximity of the transposon insertions in sadA
to a PDE-encoding gene and the fact that overexpression of the RapA PDE in the ΔbifA
mutant is able to rescue the swarming defect, we hypothesized that the phenotypic suppression of the ΔbifA
swarming defect observed for the ΔbifA sadA
and ΔbifA pvrS
mutants was due to enhanced expression of sadR
rather than to inactivation of either sadA
. This also seemed a reasonable prediction based on the fact that the mariner
transposon used in these studies harbors an outward-directed Ptac promoter and has been shown in previous studies to enhance expression of genes adjacent to transposon insertion sites (30
). Furthermore, in a recent study, overexpression of pvrR
led to a reduction in intracellular c-di-GMP levels, with a corresponding impact on c-di-GMP-sensitive phenotypes (36
As shown in Fig. , the Ptac promoter for several of the transposon insertion alleles is oriented toward the adjacent PDE-encoding gene. In cases where the opposite orientation is found, it is possible that the promoter driving expression of the aaC1 gene, encoding resistance to gentamicin, is able to suitably activate transcription of adjacent genes. To address the possibility that sadR and pvrR are overexpressed in these mutants, we performed qRT-PCR to examine expression of the pvrR gene in several of the ΔbifA pvrS::Mar mutants, as well as expression of the sadR gene in the ΔbifA sadA::Mar 51G2 mutant (Fig. ). The results show that expression of the pvrR gene is elevated in each of the ΔbifA pvrS::Mar mutants, ranging from 8-fold to 17-fold higher than that in the ΔbifA mutant (Fig. , top panel). Likewise, expression of the sadR gene in the ΔbifA sadA::Mar 51G2 mutant is elevated approximately 30-fold relative to that in the ΔbifA single mutant (Fig. , bottom panel).
To further confirm that inactivation of either the sadA or pvrS gene is not responsible for suppression of the ΔbifA swarming impairment, we tested the ΔbifA ΔsadA double mutant and a ΔbifA mutant containing a single crossover insertion in the pvrS gene and found that they are indistinguishable from the ΔbifA single mutant in swarming motility, CR binding, and biofilm formation assays (data not shown). Taking these results together with the data from the RT-PCR experiments, we infer that suppression of the swarming motility defect exhibited by the ΔbifA sadA::Mar and ΔbifA pvrS::Mar mutants occurs via overexpression of the adjacent PDE-encoding genes, sadR and pvrR, respectively. These data are consistent with our observation that overexpression of a heterologous PDE (i.e., RapA) also restores swarming to a ΔbifA mutant (Fig. ).
Mutations in the pilY1 gene suppress multiple ΔbifA mutant defects.
We isolated three independent pilY1
mutants as suppressors of the ΔbifA
swarming defect (Fig. ). The pilY1
gene encodes a protein involved in type IV pilus biogenesis and twitching motility, a form of surface-associated motility of P. aeruginosa
that is distinct from swarming motility. While studies from our laboratory and others have previously shown that pili are not absolutely required for swarming motility by this bacterium (49
), it has been reported that pili participate in the patterning of swarming motility (31
To better understand the potential role of the pilY1 gene in c-di-GMP-mediated swarming suppression, we further characterized the ability of ΔbifA pilY1::Mar suppressors to affect each of the ΔbifA mutant phenotypes resulting from the accumulation of c-di-GMP. We observed that ΔbifA pilY1::Mar mutants show markedly decreased biofilm formation (Fig. , bottom row, and B) and CR binding (a measure of Pel polysaccharide production) relative to those of the ΔbifA single mutant (Fig. , middle row). Furthermore, we generated an in-frame deletion of the pilY1 gene and observed the same suppression of these ΔbifA mutant phenotypes as we observed for the mariner suppressor mutants (Fig. , fourth column).
FIG. 2. Phenotypes of the bifA suppressor mutants. (A) pilY1 mutations suppress multiple defects of the ΔbifA mutant. Top panel, typical swarm images for (left to right) the WT, the ΔbifA mutant, a representative ΔbifA pilY1::Mar mutant (more ...)
Given that the pilY1
gene maps to a region containing other pilus-related genes and has been shown to be cotranscribed as a unit with members of this locus (fimU-pilVWXY1Y2E
) (Fig. ), we assessed whether suppression of the ΔbifA
mutant defects was due to inactivation of pilY1
alone, or possibly due to polar effects of the ΔpilY1
mutation, by generating a His-tagged version of the pilY1
gene on a multicopy plasmid under the control of the pBAD promoter. Introduction of this construct (pPilY1His) into the ΔbifA
mutant background restored the hyperbiofilm and hyper-CR binding phenotypes as well as swarming inhibition to the same extent observed for the ΔbifA
single mutant (Fig. ). This and all complementation studies with pPilY1His were performed in the absence of added arabinose and thus rely upon the basal expression of the pBAD promoter to drive PilY1 production. These results indicate that mutation of pilY1
alone is responsible for the observed suppression of the ΔbifA
Finally, we also constructed a ΔpilY1 single mutant strain. This strain is defective for twitching motility (not shown), has reduced CR binding, and shows both increased swarming motility (a 2-fold increase in percent surface coverage of the swarm plate) compared to the WT and an altered swarming pattern. Thus, the ΔpilY1 single mutant does have a small but reproducible positive impact on swarming motility, consistent with a role for PilY1 in repressing swarming motility.
Loss of type IV pili is not sufficient for full suppression of ΔbifA defects.
Both the ΔpilY1 and ΔbifA ΔpilY1 mutants fail to twitch in twitching motility assays, and we can complement these twitching defects using the pilY1 complementation construct described above (data not shown). Given that loss of pilY1 function leads to disruption of type IV pilus biogenesis, it is formally possible that suppression of the ΔbifA mutant defects is due to loss of surface piliation and/or twitching motility. To test this possibility, we introduced a deletion mutation of the pilA gene, which encodes the major pilin subunit of P. aeruginosa, into the ΔbifA mutant background and assessed the suppression of each of the ΔbifA mutant phenotypes in this ΔbifA ΔpilA double mutant compared to the ΔbifA ΔpilY1 double mutant (Fig. ).
The data show that elimination of pilA function in the ΔbifA mutant background does lead to partial suppression of the swarming, CR binding, and biofilm formation phenotypes. However, the suppression observed for the ΔbifA ΔpilA mutant is not nearly as robust as that observed for the ΔbifA ΔpilY1 double mutant in each of the assays tested. These results indicate that it is not loss of surface pili per se that leads to suppression of the ΔbifA mutant defects but that loss of PilY1 function in particular is responsible for full suppression of the ΔbifA mutant defects.
Although the ΔbifA ΔpilY1 and the ΔbifA ΔpilA double mutants both lack functional surface pili (as determined by the twitching motility assay), they differ in that the ΔbifA ΔpilY1 mutant produces pilin whereas the ΔbifA ΔpilA mutant does not (Fig. ). To control for the possibility that a potential buildup of pilin in cells of the ΔbifA ΔpilY1 mutant contributes to suppression of the swarming defect, we constructed a ΔbifA ΔpilY1 ΔpilA triple mutant and found no difference in the swarming, biofilm, and CR binding phenotypes compared with those of the ΔbifA ΔpilY1 mutant (Fig. ). Together, these data indicate that it is loss of PilY1 function and not loss of pili that suppresses the ΔbifA swarming defect.
Global c-di-GMP pools are not detectably altered by deletion of the pilY1 gene.
Deletion of the pilY1
gene in the ΔbifA
mutant background suppresses all of the c-di-GMP-related phenotypes of the ΔbifA
mutant, suggesting that such a mutation might lead to a decrease in the cellular pools of c-di-GMP in the ΔbifA
mutant. To test this notion, we examined levels of c-di-GMP in ΔbifA
mutant cells grown in the presence of radiolabeled inorganic phosphate, followed by nucleotide extraction, analysis by 2D TLC, and autoradiography (22
Inspection of the two-dimensional TLC autoradiographs revealed no detectable difference in the levels of c-di-GMP between the two strains (Fig. ). Quantification of c-di-GMP and normalization to total 32Pi incorporation (for which there is no significant difference between the two strains [P = 0.55, n = 3]) showed no significant difference between the ΔbifA single mutant and ΔbifA ΔpilY1 double mutant (P > 0.05) (Fig. ). These data suggest that the pilY1 mutation has little impact on the global levels of c-di-GMP in cells of the ΔbifA mutant.
FIG. 3. Quantification of global c-di-GMP pools. (A) Shown are autoradiographs of representative two-dimensional TLC plates used to separate [32P]orthophosphate-labeled, acid-extracted whole-cell extracts prepared from the ΔbifA mutant and the Δ (more ...)
To further investigate c-di-GMP cellular pools, we used liquid chromatography-mass spectrometry (LC-MS) to quantify the levels of c-di-GMP in these strains. Strains were grown to OD600
of ≈0.6 prior to acid extraction of nucleotides, followed by LC-MS analysis. Consistent with our previous studies, the ΔbifA
mutant accumulates approximately 3-fold more c-di-GMP than the WT (Fig. ) (27
). Consistent with our observations using the whole-cell labeling method described above, mutation of pilY1
has no detectable impact on c-di-GMP levels in either the WT or the ΔbifA
Expression of the pel polysaccharide genes is not altered by deletion of the pilY1 gene.
Mutation of the pilY1
gene in the ΔbifA
mutant background suppresses the hyper-CR binding phenotype of the ΔbifA
mutant and thus abrogates the increased production of Pel polysaccharide. It is plausible that pilY1
affects Pel polysaccharide production by influencing pel
gene expression, and if so, we would predict that pel
gene expression is reduced in the ΔbifA
mutant relative to the ΔbifA
single mutant. Thus, we assessed pel
gene expression in the WT, ΔbifA
mutant, and ΔbifA
mutant backgrounds. Consistent with our previous studies (27
), we observe no statistically significant difference (using the criterion of a P
value of <0.05 [n
= 3]) in pelA
expression between the WT (0.012 ± 0.001 pg input cDNA) and the ΔbifA
mutant (0.013 ± 0.004 pg input cDNA). Furthermore, our data show that the pilY1
mutation confers no significant change in pelA
gene expression in the ΔbifA
(0.01 ± 0.002 pg input cDNA) mutant relative to the ΔbifA
single mutant. These data indicate that excess Pel polysaccharide production in the ΔbifA
mutant is likely not due to a pilY1
-mediated increase in pel
Enhanced expression of pilY1 represses swarming in a pilus-independent manner.
The identification of the pilY1 gene as a suppressor of the ΔbifA swarming defect indicates that PilY1 is important for inhibition of swarming motility in the ΔbifA mutant. Additionally, a pilY1 single mutant is a hyperswarmer relative to the WT (Fig. ), suggesting that the pilY1 gene can repress swarming in the WT. Based on these observations, we assessed whether the pilY1 gene could act as a repressor of swarming when expressed from a multicopy plasmid in the WT background.
As shown in Fig. (left panel), arabinose-induced expression of the pPilY1His plasmid was indeed able to repress swarming motility by the WT. Based on the swarm suppression results with the ΔbifA ΔpilA mutant above indicating that the pilus might affect pilY1 function, we next asked whether pilY1-mediated repression of swarming required functional pili. As shown in Fig. (right panel), overexpression of pilY1 in the ΔpilA mutant was still able to repress swarming motility. These results further confirm that the pilY1 gene is able to function as a repressor of swarming motility and indicate that pilin production is not required for this effect.
FIG. 4. Increased expression of PilY1 represses swarming motility. (A) Enhanced expression of pilY1 represses swarming in a pilus-independent manner. Shown are representative images of the WT (left panel) and the ΔpilA mutant (right panel) carrying either (more ...) Expression of pilY1 in a ΔbifA mutant.
Our data thus far suggest that PilY1 functions to repress swarming motility. Thus, one possibility is that levels of c-di-GMP modulate pilY1 gene expression and that the high levels of c-di-GMP in a ΔbifA mutant could enhance pilY1 expression, thus providing an explanation for the loss of swarming motility upon loss of BifA function. To address this hypothesis, we examined the levels of pilY1 gene expression in the WT and ΔbifA mutant backgrounds by qRT-PCR. Expression of the pilY1 gene is not different in the ΔbifA mutant and the WT, suggesting that elevated levels of c-di-GMP do not affect pilY1 transcription (Fig. ).
To address whether c-di-GMP affects pilY1 expression in a posttranscriptional manner, we examined whether levels of PilY1 protein are altered in the ΔbifA mutant relative to the WT using a polyclonal PilY1 antibody. As shown on a representative Western blot in Fig. (top panel), the levels of PilY1 protein in the WT (lane 1) and the ΔbifA mutant (lane 2) are comparable. Quantification of PilY1 protein levels on a Western blot reveals no significant difference between the WT and ΔbifA mutant backgrounds (P = 0.78, n = 3 replicates each), indicating that c-di-GMP exerts no detectable posttranscriptional effects on PilY1 levels.
To further assess whether the levels of PilY1 in the ΔbifA background would be sufficient to repress swarming, we utilized the pPilY1 arabinose-inducible pMQ80 construct to titrate the levels of PilY1 in the WT and determine the impact on swarming motility. When the pilY1 gene is expressed from the pPilY1His plasmid in the WT strain (WT/pPilY1His) in the absence of arabinose (Fig. , lane 3), we observe a 13-fold increase in PilY1 protein levels relative to PilY1 levels in the ΔbifA mutant (lane 2), as detected using the PilY1 antibody (top panel). Importantly, we do not observe an impact on swarming motility (bottom left panel) under these conditions, indicating that this increase in PilY1 above the levels in the ΔbifA mutant is not sufficient to repress swarming motility of the WT.
For robust repression of WT swarming motility, arabinose was added to a final concentration of 0.2%. Under these conditions, a substantial induction of PilY1 protein expression occurs (Fig. , lane 7) and swarming motility of the WT strain is repressed (bottom right panel). Using intermediate arabinose concentrations of between 0 and 0.2%, we can estimate a minimum level of PilY1 protein that is sufficient to repress swarming in the WT. At 0.01% arabinose, we still do not reliably observe swarming repression at levels of PilY1 that are ~15-fold above that in the ΔbifA mutant. However, we begin to observe swarming repression with the addition of 0.05% arabinose, which results in PilY1 levels that are ~30-fold higher than those in the ΔbifA mutant. Together, these data argue that swarming repression in the ΔbifA mutant is not due primarily to increased expression of the PilY1 protein.
PilY1 functions genetically upstream of the DGC SadC and specifically requires SadC, but not other DGCs, for repression of swarming motility.
Previous work in our laboratory has shown that the SadC protein, a diguanylate cyclase (DGC), functions in a genetic pathway with the BifA protein to modulate levels of c-di-GMP in the cell and thereby influence both biofilm formation and swarming motility (37
). As shown in those studies, mutating the sadC
gene in the ΔbifA
mutant background led to suppression of the ΔbifA
mutant swarming motility defect, as well as partial suppression of the hyper-biofilm formation and CR binding phenotypes (37
), results that are similar to those observed for the ΔbifA
double mutant. Furthermore, overexpression of the sadC
gene in the WT leads to inhibition of swarming motility as well as stimulation of biofilm formation and CR binding (37
). Given the similarities between the effects of the sadC
genes on surface-associated behaviors, we wondered whether these two genes might function together in a pathway to regulate biofilm formation and swarming motility. To determine the potential epistatic relationship between the pilY1
genes, we first asked whether the sadC
gene is required for PilY1-mediated repression of swarming. When overexpressed in the ΔsadC
mutant, pPilY1His fails to repress swarming, indicating that sadC
functions downstream of pilY1
in the repression of swarming (Fig. , second row).
FIG. 5. PilY1 functions genetically upstream of the SadC diguanylate cyclase. (A) Shown are representative swarms for the WT and the ΔsadC, ΔwspR, and ΔPA1107 DGC mutants carrying either the empty vector (left column) or the pPilY1His (more ...)
Given that there are 16 predicted DGCs encoded by the P. aeruginosa
PA14 genome, we assessed whether this requirement by pilY1
for a downstream DGC was specific for SadC or whether other DGCs might also fulfill this function. In particular, we tested two additional DGC mutants for their impacts on pilY1
-mediated swarming repression. The wspR
gene encodes a CheY-like response regulatory component of a chemosensory signal transduction pathway (16
). The WspR protein contains a GGDEF domain and was shown to possess DGC activity and to be required for the hyperbiofilm phenotype of a wspF
). The PA1107
gene also encodes a GGDEF domain-containing protein and was shown previously to possess DGC activity and to stimulate hyperbiofilm formation upon overexpression (29
). As shown in Fig. , enhanced expression of pPilY1His is still able to repress swarming in both the ΔwspR
mutant backgrounds, indicating a specific interaction between the pilY1
gene and sadC
-encoded DGC in the repression of swarming.
To further confirm that the pilY1 gene functions upstream of the sadC gene in a genetic pathway, we performed the reciprocal experiments to those described above. That is, we overexpressed the sadC gene in the ΔpilY1 mutant to determine whether pilY1 function is required for repression of swarming by sadC. Using an arabinose-inducible hemagglutinin (HA)-tagged version of the sadC gene (pSadC), we found that sadC overexpression was still able to repress swarming to the same extent in the ΔpilY1 mutant background as in the WT (Fig. ), indicating that pilY1 likely functions upstream of sadC in swarm repression.
Cellular localization of PilY1.
According to the signal peptide prediction program SignalP, the PilY1 protein harbors an N-terminal signal peptide resembling a Sec secretion system signal peptide for transport across the inner membrane. The bacterial localization prediction tool PSORTB predicts that the PilY1 protein localizes to the outer membrane and also is secreted to the extracellular environment. Consistent with these predictions, previous studies using the P. aeruginosa
PAK strain showed that PilY1 was found in the insoluble membrane fraction as well as the pilus-associated extracellular shear fraction (3
). Results from a phoA
fusion screen to identify exported proteins in P. aeruginosa
PAO1 indicated that PilY1 is transported across the cytoplasmic membrane (32
To determine the cellular localization of PilY1 in P. aeruginosa strain PA14, we performed cellular fractionations of WT cells and detected PilY1 using a polyclonal PilY1 antibody. In WT cells, we observe PilY1 in the whole-cell (WC) lysate and in the cytosolic (Cyt) fraction as well as in the total membrane (TM) and inner membrane (IM) fractions (Fig. ). We do not detect PilY1 in the outer membrane (OM) fraction. Given predictions that PilY1 is a secreted protein, we tested whether PilY1 was present in the supernatant (Sup) fraction and the so-called cell-associated (CA) fraction, which refers to proteins weakly bound to the cell surface (see Materials and Methods for details). We can detect PilY1 in the supernatant after concentrating these samples approximately 300-fold. However, we are unable to detect PilY1 in the CA fractions.
FIG. 6. PilY1 cellular localization. Cellular fractions of the WT, ΔpilY1 mutant, and ΔpilA mutant were separated by SDS-PAGE. Fractions are indicated as supernatant (Sup), cell-associated (CA), whole-cell (WC), soluble cytoplasmic (Cyt), total (more ...)
The integrity of the inner and outer membrane fractions was confirmed by Western blotting using antibodies to SecY, an inner membrane protein (1
), and OprF, an outer membrane protein (7
Based on our findings indicating that loss of pilin production by mutation of pilA partially suppresses the ΔbifA swarming defect, we hypothesized that pilin production and/or pilus formation might affect PilY1 cellular localization. Thus, we tested whether PilY1 localization is altered in a ΔpilA mutant relative to the WT. As shown in Fig. (compare lane 3 to lane 1), we observe that the supernatant fraction of the ΔpilA mutant lacks detectable PilY1 relative to the WT. The remaining cellular fractions contain levels of PilY1 that are comparable in the ΔpilA mutant and the WT. These data suggest that loss of pilin (and/or pilus formation) affects localization of PilY1 to the supernatant fraction. Furthermore, it is plausible that the absence of PilY1 from the supernatant fraction of the ΔpilA mutant contributes to the partial suppression of swarming observed in the ΔbifA ΔpilA double mutant. However, we cannot exclude the possibility that loss of surface pili alone contributes to the partial swarming restoration observed for the ΔbifA ΔpilA double mutant.
Structure/function analysis of PilY1.
To better understand the potential mechanism by which PilY1 represses swarming motility, we undertook a structure/function analysis of the protein. A schematic diagram of the predicted PilY1 structure is shown in Fig. . At the N terminus, the PilY1 protein has a Sec secretion signal for transport across the inner membrane. Besides other PilY1 homologs from various species, the remainder of the N-terminal region bears little resemblance to other proteins and lacks any obvious protein motifs. However, a 200-amino-acid region within the N terminus shows weak similarity to the von Willebrand factor A (VWA) domain. Most VWA domains that have been well characterized to date belong to extracellular eukaryotic proteins, including cell adhesion and extracellular matrix (ECM) proteins, and are involved in protein-protein interactions (14
). However, many intracellular eukaryotic proteins with a diverse array of functions, including transcription, DNA repair, and proteolysis, also contain VWA domains (62
). Recently, VWA domains in prokaryotic proteins have also been identified (45
), although the precise role of this domain in these proteins has not yet been established, with the exception of a recent study showing a role for the VWA domain of the Streptococcus agalactiae
PilA protein, a pilus-associated adhesin, in adherence of this organism to epithelial cells (26
FIG. 7. Structure/function analysis of PilY1. (A) A schematic diagram of the PilY1 protein. Numbers indicate amino acids, and predicted regions of PilY1 are denoted as follows: signal sequence (SS), N terminus (NT), von Willebrand factor A (VWA), and the domain (more ...)
The most obvious domain of PilY1 lies within the C-terminal half of this protein, which shares similarity (24% identity and 46% similarity) with the C terminus of the PilC proteins of pathogenic Neisseria
species. PilC of Neisseria
has been shown to associate with the bacterial cell surface, including localization to the tip of the pilus, and is required for bacterial adherence to epithelial cells (39
). A recent crystal structure-based analysis of the PilC-like domain of PilY1 has shown this domain to be essential for regulating pilus extension and retraction mechanisms involved in twitching motility in a calcium-dependent manner (41
To test the functions of these various PilY1 domains in swarming inhibition as well as twitching motility, we generated deletion constructs of the pilY1 gene (Fig. ) and expressed the His-tagged PilY1 variants from each of these constructs in the WT background. However, all of the deletion constructs except PilY1ΔPilC produced proteins that appeared to be unstable relative to full-length PilY1His protein as assessed by Western blotting (not shown), making it difficult to draw any firm conclusions regarding the functions of these domains.
Thus, we proceeded to characterize the PilY1ΔPilC construct. The results show that the PilY1ΔPilC protein is able to inhibit swarming motility as well as the full-length PilY1 protein, indicating that the PilC-like region of the PilY1 protein is dispensable for swarming inhibition (Fig. ). Expression of the PilY1ΔPilC protein in the WT background has no impact on twitching motility. In complementation assays, expression of the PilY1ΔPilC protein is unable to complement the twitching defect of the ΔpilY1
mutant (not shown), a result consistent with recent findings regarding the essential role of this domain in twitching motility (41
Given that the N-terminal half of the PilY1 protein is sufficient to inhibit swarming motility, we focused on this region for further mutagenesis experiments. We first undertook a random PCR mutagenesis approach by mutagenizing the PilY1ΔPilC construct and screening for loss of swarming inhibition when expressed in the WT background. We screened approximately 1,000 strains expressing mutant variants of PilY1, and about 30% of those mutants recovered swarming motility. However, upon closer investigation using Western dot blot analysis, we found that only 3% of these swarmer strains produced a detectable PilY1ΔPilC protein, indicating that the vast majority of the mutations were likely causing PilY1 protein instability. Furthermore, when those constructs making apparently stable PilY1ΔPilC protein were retransformed into WT P. aeruginosa, the swarming phenotypes were not reproducible, indicating that the mutation in the original mutant strains was not linked to the plasmid. Thus, we chose to take a more directed mutagenesis approach.
Alignment of the PilY1 protein across pseudomonads as well as other bacterial species using the ClustalW2 sequence alignment tool indicated there were a number of highly conserved amino acid residues throughout the N terminus of the protein, and we used this alignment as a guide to generate site-directed mutations. A subset of the highly conserved amino acids found in the alignment fall within the putative VWA-like region of PilY1. The alignment of PilY1 proteins spanning two of the conserved sites within the VWA-like region is shown in Fig. .
To determine whether either of these conserved regions is important for PilY1 function in swarming repression, we changed the threonine in the conserved “TPL site” to an alanine in one construct and changed the aspartic acid and glycine in the conserved “MTDG site” to alanine residues in a second construct. We then overexpressed these His-tagged mutant PilY1 proteins (PilY1-APL and PilY1-MTAA) in the WT background and assessed swarming motility. As shown in Fig. (right panel), expression of the PilY1-MTAA mutant protein led to only partial swarming inhibition relative to the complete inhibition observed for the WT protein (left panel), whereas the PilY1-APL mutant protein behaved like the WT in regard to swarming inhibition (data not shown). The stability of the PilY1-MTAA protein was comparable to that of the WT PilY1 protein as assessed by Western blotting (Fig. ). These data indicate a potential role for the conserved aspartic acid and glycine amino acids in swarming inhibition by PilY1. Furthermore, these data are consistent with our observations showing that the PilY1-MTAA construct only partially complements the swarming phenotype of the ΔbifA ΔpilY1 double mutant (Fig. ). Similarly, the PilY1-MTAA construct only partially complements the twitching phenotype of the ΔpilY1 mutant (not shown).
Our observations above indicate that the PilY1-MTAA mutant protein shows only partial function in swarming inhibition. To further investigate this, we determined whether the cellular localization of PilY1-MTAA differs from that of WT PilY1 when overexpressed in the WT background. When expression is induced with arabinose, we observe elevated levels of WT PilY1 in all of the cellular fractions relative to the WT carrying vector alone (Fig. ). In contrast, the levels of PilY1-MTAA in the Sup and CA fractions are greatly reduced relative to those fractions containing WT PilY1. In all other cellular fractions, the levels of PilY1-MTAA are comparable to those of the WT PilY1. These data indicate that localization of the PilY1-MTAA mutant protein is altered in both the Sup and CA fractions and suggest that the mutated amino acids may affect proper extracellular localization of the PilY1-MTAA protein. Taken together with the results showing an absence of PilY1 in the ΔpilA mutant Sup fraction relative to the WT, these data support the notion that extracellular localization of PilY1 may contribute to its function in swarming inhibition.