Deficient motility is one of the characteristic hallmarks observed in Pseudomonas aeruginosa strains that chronically colonize the lungs of cystic fibrosis (CF) patients. Pseudomonas aeruginosa TB is a nonpiliated CF isolate known to be defective in twitching motility. Complementation confirmed a direct link of this phenotype to an intragenic out-of-frame deletion in pilQ (PA5040). Sequence alignment of pilQ derived from TB vs. PAO1 suggests that close direct repeats framing the deletion site may have triggered this mutation. This type of mutation could play a role in the emergence of pathoadaptive mutations of P. aeruginosa in the CF lung habitat.
Pseudomonas aeruginosa is an opportunistic pathogen that can cause chronic infection in the lungs of cystic fibrosis (CF) patients (Armstrong, 2006). During the initial phase of infection, different modes of motility and adhesion to host epithelial cell surface structures can be mediated by cell appendages such as flagella and type IV pili (Tfp).
Tfp are polarly localized, filamentous surface appendages produced by most Gram-negative bacteria and have been most extensively studied in Neisseriae species. In P. aeruginosa, Tfp are involved in adhesion to biotic and abiotic surfaces (Giltner et al., 2006), and in biofilm formation at later stages of infection (Klausen et al., 2003). To date, over 40 genes from the Pseudomonas genome database are known to encode structural proteins of this machinery (Whitchurch, 2006). These genes include two-component systems that regulate the two-dimensional mode of translocation termed twitching motility. This type of motility is mediated by assembly and retraction of the pilus filament powered by two antagonistic nucleotide-binding proteins PilB and PilT, respectively. The pilus filaments consist of thousands of copies of PilA pilin subunits that are cleaved and N-methylated by an endopeptidase PilD prior assembly (Kaiser, 2000). The dodecameric transmembrane protein PilQ plays a central role in extruding the pilus fibre through the bacterial outer membrane and thereby facilitating the presence and functionality of extracellular pili (Whitchurch, 2006).
In P. aeruginosa, PilQ (77 kDa; ORF PA5040) is encoded by the last gene of the polycistronic operon pilMNOPQ and belongs to the superfamily of secretins known to form transmembrane pores (Collins et al., 2001). Structurally, this pilus assembly apparatus is reminiscent of the type II protein secretion system (Bitter et al., 1998; Peabody et al., 2003) with PilQ sharing high homology with XcpQ (Bitter et al., 1998). Both systems also use the same endopeptidase PilD (also referred to as XcpA) for protein processing (Bally et al., 1992). In fact, pseudo-pili formation has been reported – among others – for PulG pilins from the Klebsiella oxytoca pullulanase (Pul) secreton (Sauvonnet et al., 2000), for GspG pilins excreted through the Gsp secreton in Escherichia coli (Vignon et al., 2003) and for XcpT pilins from XcpQ in P. aeruginosa (Durand et al., 2003).
Although Tfp are prominent virulence factors during acute infection, P. aeruginosa is known to undergo genetic adaptational processes during chronic colonization in the CF lungs, resulting in nonpiliation. Two traits were described of how loss of pili had occurred during clonal evolution in the CF lung. First, loss of motility was associated with the inactivation of the alternative sigma factor rpoN (Mahenthiralingam et al., 1994). Second, large chromosomal inversions led to the disruption of pilB, which encodes the NTP powering assembly of Tfp (Kresse et al., 2003). Here, another target and type of mutation that led to the loss of pili in a P. aeruginosa CF isolate is reported.
Materials and methods
Bacterial strains and growth conditions
The P. aeruginosa strains and the plasmids used in this study are listed in Table 1. The panel of 75 P. aeruginosa strains of diverse geographic origin and habitats and of unrelated SpeI genotypes has been described previously (Morales et al., 2004). All bacterial strains were grown in Luria–Bertani (LB) broth at 37°C. Tetracycline 200 μg mL−1 was added to maintain the pME6010 construct in P. aeruginosa TB. The mutation frequency of strain TB after exposure to rifampicin and streptomycin was determined by serial dilutions according to the assay described by Oliver et al. (2002).
Table 1. Bacterial strains, plasmids and primers used in this study
pME6010 containing the 2322 bp BglII/EcoRI PCR product bearing the pilQ gene from PAO1
DNA manipulation and PCR sequencing
A 2322 bp DNA fragment containing pilQ (PA5040) corresponding to the position 5 677 980–5 675 659 of the PAO1 genome sequence (Stover et al., 2000) was amplified from the genomic DNA of P. aeruginosa PAO1 and TB and ligated into the BglII and EcoRI site of the shuttle vector pME6010 (Heeb et al., 2000). Introduction of the plasmid construct into strain TB was carried out by electroporation. Twitching-positive revertants were selected on LB agar containing 200 μg mL−1 tetracycline. Sequencing of the PCR product was performed by MWG Biotech.
Twitching motility assay
Twitching motility was assayed according to the protocol by Alm & Mattick (1995). Briefly, bacteria from liquid culture were subsurfacely inoculated on LB agar allowing bacterial locomotion at 37°C. After 24 h, the twitching zone on the petridish was visualized by Coomassie staining.
Southern blot analysis of the pilQ deletion site
Genomic DNA of the 75 strain panel was extracted as described by Chen & Kuo (1993). Gel-separated XhoI-restricted genomic DNA was transferred onto a nylon N+ membrane (Hybond N+, Amersham Bioscience), fixed and hybridized with a PCR-generated probe corresponding to the intragenic deletion site of pilQ in TB (pos. 5677703–5677436 of the PAO1 genome). Probe-reactive bands were visualized by chemiluminescence with CDP Star as the substrate (Allefs et al., 1990).
Immunoblot analysis of extracellular pili
Equal amounts of late stationary stage liquid culture from P. aeruginosa PAO1 and TB were sedimented at 6000 g. Proteins from whole-cell lysates and extracellular protein supernatant precipitates (20 000 g, 30 min) were separated on 15% SDS-PAGE and blotted onto a nitrocellulose membrane. Detection of PilA with anti-PilA antibodies (1 : 1000) raised against linear epitopes of PilA in PAO1 (pos. 134–149, CKSTQDPMFTPKGCDN) and in TB (pos. 60–74, GKKLVSNDSPKNDEY) (Eurogentec) was carried out using the ECL Western Blot detection kit according to the manufacturer's protocol (Amersham).
The nontwitching P. aeruginosa TB strain harbours a deletion in the pilQ gene
The CF isolate P. aeruginosa TB that is known to survive within professional phagocytes (Tümmler, 1987) is a nontwitching strain (Fig. 1). Whereas the genetic reference strain PAO1 exhibited a twitching zone of 4.5 cm after a 24-h subsurface incubation on LB agar, P. aeruginosa TB was strongly impaired in twitching motility (twitching zone <1 cm). Consistent with this finding, extracellular Tfp was not detected from the protein precipitates of the culture supernatant using polyclonal anti-PilA antibodies, although pilin subunits were abundantly expressed in both strains PAO1 and TB (Fig. 2). Hence, it was suspected that strain TB is deficient in one or more elements of the pilus biogenesis apparatus. By screening a panel of transposon mutants that are inactivated in genes of the pil operons, twitching could be restored in a pilQ mutant by complementation in trans with the pilQ gene of strain PAO1 (Fig. 1), but not with that of strain TB.
Sequencing of the PCR-amplified pilQ gene of strain TB revealed a 281 bp out-of-frame deletion stretching from position 145 to 426 downstream of the ATG start codon. This frame shift mutation should lead to a truncated nonfunctional PilQ protein that should not able to form the multimeric PilQ pore in the outer membrane that is necessary for pilus translocation. The deletion is flanked by a direct repeat AGCCCG.
This mutation does not reflect an increased mutability of the TB strain, because the global mutation frequency was determined to be 1 × 10−7 in the same range as that of 1 × 10−8 for the nonmutator reference strain PAO1.
Prevalence of the intragenic deletion in pilQ is low in the P. aeruginosa population
Nonpiliation of P. aeruginosa is one of the hallmarks observed for many CF isolates, but is uncommon for isolates from other habitats (Mahenthiralingam et al., 1994). Hence, it was aimed to investigate the prevalence of the TB-specific intragenic pilQ deletion (i) among other variants of the TB clone defined by more than 75% SpeI fragment identity (Kiewitz & Tümmler, 2000) and (ii) among unrelated P. aeruginosa strains isolated from different habitats including CF lungs (Morales et al., 2004).
Genomic DNA from the clonal variants of TB (892, 63 741 and VA24437; see Table 1) served as templates to amplify pilQ from these strains. All strains but TB carried the full-length pilQ gene (Fig. 3). Interestingly, the CF isolate 892 that is closely related to strain TB could not twitch, although it harboured a wild-type pilQ gene. This finding underscores the multifactorial aetiology of nonmotility in CF isolates.
To investigate the prevalence of the TB-specific pilQ genotype among P. aeruginosa strains, a DNA probe encompassing the pilQ sequence that is deleted in TB was hybridized onto Southern blots of XhoI-restricted genomic DNA of 75 P. aeruginosa isolates of diverse origin and genotype (Morales et al., 2004). Ralstonia solanacearum and three Burkholderia multivorans strains were included as negative controls, whereas strain PAO1 served as the positive control. Apart from TB and VA24437, hybridization signals were present in all P. aeruginosa strains, indicating that the intragenic deletion mutation in pilQ is TB-specific. The negative hybridization signal for strain VA24437 was attributed to large divergence from the PAO1 pilQ sequence because a pilQ gene of expected size could be amplified by PCR and the strain was proficient in twitching (Fig. 3). In summary, the intragenic deletion was solely identified in the original index case, strain TB.
Intragenetic deletion flanked by direct repeats as a pathoadaptive mutation mechanism in P. aeruginosa CF isolates
Most nonmotile strains lack either Tfp or flagella that are immunogenic, and the absence of such cellular appendices can thus be beneficial for these strains in evading host immune defences. Loss-of-motility mutants have been reported to be either affected by mutations in rpoN (Mahenthiralingam et al., 1994), frame shift mutations in a polyG tract of algR (Beatson et al., 2002) or large chromosomal inversions interrupting the pilB gene in P. aeruginosa clone C (Kresse et al., 2003). The out-of-frame deletion in the pilQ gene of the CF isolate TB is a further, yet undescribed mechanism that leads to loss of twitching motility. Small deletions have already been reported for the mucA (reviewed in Bragonzi et al., 2006) and the mutS genes (Oliver et al., 2002) of CF isolates. Moreover, large deletions of 119 or 189 kbp were detected in isolates from two CF patients (Ernst et al., 2003). The intragenic deletion in the pilQ gene of strain TB was probably triggered by close direct repeats and represents a further mechanism that contributes to the molecular evolution of P. aeruginosa, particularly during the phenotypic conversion in the chronically colonized CF lungs. According to the in silico analysis of free-living fully sequenced proteobacteria, the illegitimate recombination between close repeats may allow bacterial populations to cope with novel or infrequent kinds of stresses (Rocha et al., 2002) and to adapt to unusual niches as in the CF lungs for P. aeruginosa.
The authors thank Nina Cramer for the determination of the mutation frequencies. Y.S.T.C. and J.K. have been members of the European Research Training Group ‘Pseudomonas: Pathogenicity and Biotechnology’ sponsored by the Deutsche Forschungsgemeinschaft. Further financial support by the Deutsche Forschungsgemeinschaft (SFB 587, A9) is gratefully acknowledged.