Pseudomonas aeruginosa AlgR regulates type IV pilus biosynthesis by activating transcription of the fimU-pilVWXY1Y2E operon

Abstract

The response regulator AlgR is required for Pseudomonas aeruginosa type IV pilus-dependent twitching motility, a flagellum-independent mode of solid surface translocation. Prior work showed that AlgR is phosphorylated at aspartate 54, and cells expressing an AlgR variant that cannot undergo phosphorylation (AlgRD54N) lack twitching motility. However, the mechanism by which AlgR controls twitching motility is not completely understood. We hypothesized that AlgR functioned by activating genes within the prepilin fimU-pilVWXY1Y2E cluster that are necessary for type IV pilin biogenesis. Reverse transcriptase PCR analysis showed that the fimU-pilVWXY1Y2E genes are cotranscribed in an operon, which is under the control of AlgR. This supports prior transcriptional profiling studies of wild-type strains and algR mutants. Moreover, expression of the fimU-pilVWXY1Y2E operon was reduced in strains expressing AlgRD54N. DNase footprinting and electrophoretic mobility shift assays demonstrate that AlgR but not AlgRD54N bound with high affinity to two sites upstream of the fimU-pilVWXY1Y2E operon. Altogether, our findings indicate that AlgR is essential for proper pilin localization and that phosphorylation of AlgR results in direct activation of the fimU-pilVWXY1Y2E operon, which is required for the assembly and export of a functional type IV pilus.

FIG. 1.

The fimU-pilVWXY1Y2E gene cluster is an operon. (A) Map of the P. aeruginosa fimTU-pilVWXY1Y2E prepilin gene cluster. Numbers 1 to 7 represent the respective primer pairs spanning two adjacent genes used in RT-PCR analysis. Table 1 shows sequences for primers. (B) Agarose gel analyses of RT-PCRs. Lanes 1 to 7 are primer pairs as denoted in Fig. 1A. Lanes designated “R” represent the reactions where cDNA derived from PAO1 was amplified by the primer pairs 1 to 7, lanes G are controls with genomic DNA, lanes C are negative controls (RNA without RT or cDNA without primers, respectively), and lanes L are DNA ladders.

FIG. 2.

AlgR but not AlgRD54N binds directly to sequences upstream of fimU. (A) Gelcode blue stain of a sodium dodecyl sulfate-polyacrylamide gel with purified AlgR (lane 1) or AlgRD54N (lane 2). The arrow indicates the positions of these proteins (∼30 kDa). Lane L, protein ladder. (B) EMSA of DNA-protein complexes formed in the presence of AlgR and AlgRD54N and algD or NS DNA. Lanes 1 (algD) and 8 (NS DNA) represent free DNA (2.27 pM and 1.5 pM, respectively). Lanes 2 to 4 are incubated with 2.0, 4.27, or 6.0 pmol of AlgR, respectively; Lanes 5 to 7 are incubated with 2.0, 4.27, or 6.0 pmol of AlgRD54N, respectively. Lanes 9 and 10 have 2.0 pmol and 4.27 pmol of AlgR, respectively. (C) AlgR binding site (−45 CCGTTTGgC −36) localized with respect to fimU ATG. F1, F2, and F3 DNA fragments (−251 to +80, −117 to +23, and −228 to −117, respectively, with respect to fimU ATG). The asterisk denotes the γ-³²P label. (D) DNA-protein complexes formed in the presence of AlgR and AlgRD54N using DNA fragments illustrated in panel C. Lanes 1, 7, and 13 represent free DNA for F1 (1.5 pM), F3 (1.46 pM), and F2 (1.5 pM), respectively. Lanes 2 to 4, 8 to 10, and 14 to 16 are incubated with 1.0, 2.0, and 4.27 pmol of wild-type AlgR, respectively. Lanes 5 and 6, 11 and 12, and 17 and 18 are incubated with 2.0 and 4.27 pmol of AlgRD54N, respectively.

FIG. 3.

DNase I protection assays of the fimU promoter with AlgR and AlgRD54N. The binding reaction mixture contained the end-labeled DNA template strand of the F2 fragment (1.5 pM) illustrated in Fig. 2C. Lanes 3 to 6 contained 0.33, 0.83, 1.67, and 3.33 pmol of AlgR, respectively. Lanes 8 to 12 contained 0.33, 0.83, 1.67, 3.33, and 8.33 pmol of AlgRD54N, respectively. Lane 1 contains control reaction mixtures with free DNA lacking AlgR and DNase I; lanes 2 and 7 are DNase I-treated control reaction mixtures without protein. Molecular size markers (M) are indicated on the right in nucleotides. The brackets mark regions of DNA protected from DNase cleavage by AlgR and also containing the consensus AlgR binding sites: ABS2 (5′-CCGTTTGGC-3′) and ABS1 (5′-CCCGTTTGGC-3′).

FIG. 4.

Localization of the fimU-pilVWXY1Y2E promoter. (A) Positions of primers (A to G) spanning fimT and fimU used in RT-PCR analysis. The positions of these primers are relative to the fimT start codon. Table 1 shows the primer sequences. The minus and plus signs denote the absence or presence of RT-PCR product, respectively, using each primer indicated in combination with primer G. (B) Agarose gel analyses of RT-PCRs. Lanes denote primer pairs as indicated in panel A. Lanes designated “R” represent the cDNA reactions with the primer pairs indicated, lanes G are controls with genomic DNA, lanes C are negative controls (RNA without RT), and lane L is a DNA ladder. Sizes (bp) are indicated on the left. (C) Sequences of the 3′ end of fimT and the 5′ region of fimU (fimT stop codon and fimU start codons are indicated). The fimU promoter region, as identified by RT-PCR, is shaded. AlgR binding sites (ABS1 and ABS2) are underlined (5′-CCCTCGGGC-3′ and 5′-CCGTTTGGC-3′, respectively).