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The Pseudomonas Aeruginosa PilSR Two-Component System Regulates Both Twitching and Swimming Motilities


The Pseudomonas Aeruginosa PilSR Two-Component System Regulates Both Twitching and Swimming Motilities

Sara L N Kilmury et al. mBio.


Motility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times. The motility structures type IV pili and flagella are also involved in sensing surface contact, which modulates pathogenicity. In Pseudomonas aeruginosa, the PilS-PilR two-component system (TCS) regulates expression of the type IV pilus (T4P) major subunit PilA, while biosynthesis of the single polar flagellum is regulated by a hierarchical system that includes the FleSR TCS. Previous studies of Geobacter sulfurreducens and Dichelobacter nodosus implicated PilR in regulation of non-T4P-related genes, including some involved in flagellar biosynthesis. Here we used transcriptome sequencing (RNA-seq) analysis to identify genes in addition to pilA with changes in expression in the absence of pilR Among the genes identified were 10 genes whose transcription increased in the pilA mutant but decreased in the pilR mutant, despite both mutants lacking T4P and pilus-related phenotypes. The products of these inversely dysregulated genes, many of which were hypothetical, may be important for virulence and surface-associated behaviors, as mutants had altered swarming motility, biofilm formation, type VI secretion system expression, and pathogenicity in a nematode model. Further, the PilSR TCS positively regulated transcription of fleSR, and thus many genes in the FleSR regulon. As a result, pilSR deletion mutants had defects in swimming motility that were independent of the loss of PilA. Together, these data suggest that in addition to controlling T4P expression, PilSR could have a broader role in the regulation of P. aeruginosa motility and surface sensing behaviors.IMPORTANCE Surface appendages such as type IV pili and flagella are important for establishing surface attachment and infection in a host in response to appropriate cues. The PilSR regulatory system that controls type IV pilus expression in Pseudomonas aeruginosa has an established role in expression of the major pilin PilA. Here we provide evidence supporting a new role for PilSR in regulating flagellum-dependent swimming motility in addition to pilus-dependent twitching motility. Further, even though both pilA and pilR mutants lack PilA and pili, we identified sets of genes downregulated in the pilR mutant and upregulated in a pilA mutant as well as genes downregulated only in a pilR mutant, independent of pilus expression. This finding suggests that change in the inner membrane levels of PilA is only one of the cues to which PilR responds to modulate gene expression. Identification of PilR as a regulator of multiple motility pathways may make it an interesting therapeutic target for antivirulence compounds.

Keywords: cell surface; cystic fibrosis; flagellar gene regulation; two-component regulatory systems; type IV pili.


PA14 mutant homologs of inversely dysregulated genes affect motility phenotypes. (A to E) Available PA14 transposon mutant homologs of inversely dysregulated genes identified in strain PAK were tested for twitching (A), swarming (B), and swimming (C) motilities, biofilm formation (D), and pathogenicity toward Caenorhabditis elegans (E) to identify the functions of hypothetical proteins. All mutants displayed WT twitching, but only a subset had defects in swimming and/or swarming motility. Several mutants exhibited a hyperbiofilm phenotype, while two had defects in C. elegans killing (***, P < 0.005). For the motility assays, images representative of three independent experiments are shown.
Swimming motility is impaired in pilS and pilR mutants. Loss of pilS or pilR results in decreased swimming motility (53.7% ± 0.5% and 47% ± 1.4% of WT, respectively; P < 0.005) in a plate-based assay. pilA mutants swim comparably to the WT, indicating that the swimming defect is not PilA dependent. pilS and pilR mutants appear to acquire suppressors that overcome these defects resulting in asymmetrical flares. Reinoculation of swimming plates with cells from the interior of swimming zones—inside the white dotted circles of pilSR (inner) recapitulate the original phenotype, while cells taken from the flares (outer; from flares outside the white dotted circles, except for WT) swim to WT levels. Black asterisks denote the locations where cells were taken for the reinoculated swimming plates.
Loss of fleSR reduces twitching motility but increases pilA transcription. (A) Loss of fleSR reduces twitching motility by approximately 20%. Means plus standard errors (error bars) for six independent replicates are shown. Statistical significance was determined by one-way ANOVA (***, P < 0.005). (B) A lux-pilA luminescent reporter assay measuring pilA promoter activity indicated that pilA transcription is increased over 5 h. Means ± standard errors for four biological replicates are shown. RLU, relative luminescence units.
Model for pilSR-dependent regulation of fleSR and the fleSR regulon. Under conditions in which pilSR expression is decreased (low cAMP) or when PilSR is low (high intracellular PilA), fleSR transcription is decreased as is expression of the the fleSR regulon. Genes on a red background are those that had decreased expression in a pilR mutant in RNA-seq. Genes on a gray background had insufficient reads assigned to them from RNA-seq to accurately report differential expression. FleQ (blue) was not differentially expressed between WT or pilR, indicating that pilSR fits into the flagellar regulatory hierarchy after FleQ but before fleSR, as fleQ itself and most FleQ-dependent genes were unaffected by loss of pilR.

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    1. West AH, Stock AM. 2001. Histidine kinases and response regulator proteins in two-component signalling systems. Trends Biochem Sci 26:369–376. doi:10.1016/S0968-0004(01)01852-7. - DOI - PubMed
    1. Rodrigue A, Quentin Y, Lazdunski A, Méjean V, Foglino M. 2000. Two-component systems in Pseudomonas aeruginosa: why so many? Trends Microbiol 8:498–504. doi:10.1016/S0966-842X(00)01833-3. - DOI - PubMed
    1. Arora SK, Ritchings BW, Almira EC, Lory S, Ramphal R. 1997. A transcriptional activator FleQ regulates mucin adhesion and flagellar gene expression in Pseudomonas aeruginosa in a cascade manner. J Bacteriol 179:5574–5581. doi:10.1128/jb.179.17.5574-5581.1997. - DOI - PMC - PubMed
    1. Ritchings BW, Almira EC, Lory S, Ramphal R. 1995. Cloning and phenotypic characterization of fleS and fleR, new response regulators of Pseudomonas aeruginosa which regulate motility and adhesion to mucin. Infect Immun 63:4868–4876. - PMC - PubMed
    1. Jiménez-Fernández A, López-Sánchez A, Jiménez-Díaz L, Navarrete B, Calero P, Platero AI, Govantes F. 2016. Complex interplay between FleQ, cyclic diguanylate and multiple σ factors coordinately regulates flagellar motility and biofilm development in Pseudomonas putida. PLoS One 11:e0163142. doi:10.1371/journal.pone.0163142. - DOI - PMC - PubMed

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