Phosphorylation of thePseudomonas aeruginosaResponse Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility

Abstract

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.The bacterium Pseudomonas aeruginosa is a serious and life-threatening pathogen of immune-compromised patients, such as those suffering from AIDS or severe burns or who are undergoing cancer chemotherapy or organ transplantation, and is one of the most common causes of hospital-acquired infections. P. aeruginosa is also responsible for Ͼ95% of the mortality of cystic fibrosis (CF) patients (13, 31). Chronic infection of the lower respiratory tract of CF patients results in the emergence of mucoid strains of P. aeruginosa which overproduce the highly viscous exopolysaccharide alginate. Alginate production plays an important role in CF infections by serving an adherence function and by conferring protection to the bacterium from both antibiotics and the host immune system (13).Colonization and establishment of infection by P. aeruginosa are dependent on the production of a number of virulence factors, including extracellular toxins, proteases, lipases, siderophores, and polar filamentous structures called type IV fimbriae. These fimbriae are essential for the attachment of the pathogen to host epithelial tissues and mediate a form of surface translocation known as twitching motility, which is implicated in the spread of infection (23) or the aggregation into microcolonies during biofilm formation (30). Mutants that either lack type IV fimbriae or produce nonfunctional fimbriae show reduced infectivity (18,23). Twitching motility has been shown to occur by fimbrial extension and retraction, probably involving the assembly and disassembly of the fimbrial strand (25, 38).Unlike P. aeruginosa strains commonly isolated fr...

Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila

“…Two independent insertions that had the same consequences for the interaction with D. melanogaster have been found in algR. In P. aeruginosa, AlgR regulates a number of processes including fimbrial biogenesis, biofilm formation and cyanide production 25,26 . Altogether, genetic analysis indicates that GacA is a master regulator of the interaction and that PrtR and AlgR regulators, seem to play secondary roles in the infection process.…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…The surface-dependent induction of algC expression correlated with stability of attachment, with cells that did not undergo algC upregulation demonstrating reduced ability to remain attached to the surface relative to that of cells with activated expression (33). While these findings suggested increased alginate production upon attachment, with alginate production contributing to biofilm resistance and being inversely linked to both flagellum-driven motility and twitching motility (48,139,165), it is now apparent that alginate is neither the major matrix polysaccharide nor required for biofilm development by nonmucoid P. aeruginosa strains, which are the first to colonize CF patients (121,144,167). Instead, recent chemical and genetic studies have demonstrated that the major polysaccharides produced by P. aeruginosa strains PAO1 and PA14 are Psl and Pel, with the roles of these two polysaccharides differing with respect to attachment and biofilm formation in a strain-specific manner.…”

Identification of two new genes involved in twitching motility in Pseudomonas aeruginosa

“…Type IV pili are flexible surface filaments about 6 nm in diameter produced at the poles of the bacterial cell (Beatson et al, 2002;; they are essential for the attachment of the pathogen to host epithelial tissues and also mediate a form of surface translocation known as twitching motility (Bradley, 1980;. Twitching motility has been shown to be required for the initial attachment and development of a biofilm by P. aeruginosa (O'Toole & Kolter, 1998;Costerton et al, 1999;Mcbride, 2001;Whiteley et al, 2001;Whitchurch et al, 2002). Once a biofilm is developed, cells growing in the biofilm can become 10-1000 times more resistant to the effects of antibiotics than their planktonic counterparts (Mah & O'Toole, 2001).…”

“…Biofilm bacteria embedded in an extracellular polymeric matrix cannot be eradicated even with the most aggressive antibiotics. Mutants that either lack type IV pili or are twitchingmotility deficient show loss of ability for biofilm initiation (O'Toole & Kolter, 1998) and reduced infectivity (Kang et al, 1997;Whitchurch et al, 2002).…”