Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa

Abstract

Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrDeltaEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrDeltaEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrDeltaEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

FIG. 1.

Genetic organization of the vfr locus. (A) ORFs, putative rho-independent terminators, and the vfr transcriptional start site are represented by hollow arrows, lollipops, and a small filled arrow, respectively. The locations of transposon insertions are indicated by triangles. The filled triangle is the site of mTn 5-Tc insertion in 118C8. Subsequently, this insertion was transferred to P. aeruginosa PAK to create PAK vfr::mTn 5-Tc. The hollow triangle is the site of insertion of the tetracycline resistance cassette in PAO1 vfr:: Tc. Numbers below the solid line correspond to the P. aeruginosa PAO1 genome sequence. (B) Bold lines denote the region cloned from pMO011925 to create pUCP vfrA and pUCP vfrF and the region amplified by PCR from wild-type and PAK lasI-RT chromosomal DNAs and subsequently cloned to create pSB299.15A and pSB299.17A, respectively.

FIG. 2.

Subsurface twitching motility assay of wild-type P. aeruginosa PAK and pilA and vfr mutants. Bar, 1 cm.

FIG. 3.

Type IV pilus production by a vfr mutant. (A) ELISA of whole-cell samples of wild-type P. aeruginosa PAK and pilA and vfr mutants. PilA was detected with anti-PilA serum and is indicative of levels of surface pili in these strains. (B) Immunoblot assays of PilA in sheared surface pili. (C) PilA subunit remaining in whole-cell samples after surface pili have been sheared. The pilV mutant strain, which is defective in assembly of pili (3), was included in these assays to control for the contribution of the PilA subunit to surface samples as a result of cell lysis.

FIG. 4.

Amino acid sequence comparison of the cNMP-binding domains of P. aeruginosa Vfr and E. coli CRP. The residues forming the strongest interactions between E. coli CRP and cAMP are highlighted with an asterisk and shaded. The component of cAMP (ribose, cyclic phosphate, or the purine ring) targeted by CRP is indicated above the highlighted sequence. Numbers above and below the sequence indicate the corresponding residue positions in Vfr and CRP, respectively. Numbers in parentheses refer to the numbers of amino acids between the two aligned sequence blocks that are not shown but were taken into account in the CLUSTALW alignment. Lines below the sequence highlight the secondary-structure motifs (αC, α-helix C; β6 and β7, β strands 6 and 7, respectively) determined from the three-dimensional structure of CRP.

FIG. 5.

Homology-based structural models of the cNMP-binding domains of P. aeruginosa (Pa) Vfr and VfrΔEQERS based on E. coli (Ec) CRP, which served as a template. CRP, Vfr, and VfrΔEQERS are shown in stick form and colored in accordance with standard CPK. Only the six residues of CRP (and their corresponding residues in Vfr and VfrΔEQERS) that make the strongest contact with cAMP are shown. Hydrogen bonds (as determined from the three-dimensional structure of CRP) are represented by dotted green lines. To permit comparison, the residues of VfrΔEQERS are numbered in accordance with the scheme for Vfr (i.e., T132 and T133 of Vfr are actually T127 and T128 of VfrΔEQERS).