Identification of Genes Involved in Swarming Motility Using aPseudomonas aeruginosaPAO1 Mini-Tn5-luxMutant Library

Abstract

During a screening of a mini-Tn5-luxCDABE transposon mutant library of Pseudomonas aeruginosa PAO1 for alterations in swarming motility, 36 mutants were identified with Tn5 insertions in genes for the synthesis or function of flagellin and type IV pilus, in genes for the Xcp-related type II secretion system, and in regulatory, metabolic, chemosensory, and hypothetical genes with unknown functions. These mutants were differentially affected in swimming and twitching motility but in most cases had only a minor additional motility defect. Our data provide evidence that swarming is a more complex type of motility, since it is influenced by a large number of different genes in P. aeruginosa. Conversely, many of the swarming-negative mutants also showed an impairment in biofilm formation, indicating a strong relationship between these types of growth states.Pseudomonas aeruginosa is an opportunistic human pathogen that can cause nosocomial pneumonia, catheter and urinary tract infections, and sepsis in burn wound and immunocompromised patients (14,18,23,28,34). Moreover, P. aeruginosa is the most prevalent and significant pulmonary pathogen and the most common cause of eventually fatal lung disease in patients with cystic fibrosis (16,29). In addition to its intrinsic resistance to a broad spectrum of antimicrobial compounds, this organism is also noted for its metabolic diversity and extremely versatile lifestyle, which allows it to colonize a large number of different environments (33).Motility has been strongly implicated in the virulence of P. aeruginosa. It plays an important role in mobilization to and colonization of different environments, attachment of the bacteria to surfaces, and biofilm formation (27). P. aeruginosa is unusual in that it is capable of three different types of motility: flagellum-mediated swimming in aqueous environments and at low agar concentrations (Ͻ0.3% [wt/vol]); type IV pilus-mediated twitching on solid surfaces or interfaces; and, most recently observed, swarming on semisolid (viscous) media (0.5 to 0.7% [wt/vol] agar) (7,20,30). Swarming is described as a social phenomenon involving the coordinated and rapid movement of bacteria across a semisolid surface, often typified by a dendritic-like colonial appearance. It is widespread among flagellated bacteria, including Salmonella, Vibrio, Yersinia, Serratia, and Proteus (4,12,31).Recently, it was shown that swarming of P. aeruginosa is dependent on both flagella and type IV pili as well as the presence of rhamnolipids (2,7,20). It is induced under nitrogen limitation and in response to certain amino acids (e.g., glutamate, aspartate, histidine, or proline) when provided as the sole source of nitrogen (20). Like other swarming bacteria that differentiate from short, motile, vegetative swimmer cells into longer, hyperflagellated swarmer cells (10, 13), P. aeruginosa swarmer cells are elongated and can possess two polar flagella (20,30). In addition to these physical changes, swarmer differentiation can also be coupled to increased expres...

Swarming ofPseudomonas aeruginosaIs Controlled by a Broad Spectrum of Transcriptional Regulators, Including MetR

“…As a result, 35 transcriptional regulators were found to be involved in swarming, including a variety of two-component sensors and response regulators. Interestingly, in contrast to the results of a small study described previously (45), an inverse relationship between the ability of mutants to swarm and their ability to form biofilms was observed in this study. The defect in one swarming-deficient mutant, a metR mutant, was confirmed by complementation in trans with the cloned gene.…”

“…To investigate genes involved in swarming motility in P. aeruginosa, the Harvard PA14 transposon insertion mutant library was screened for swarming defects as described elsewhere (45). Briefly, mutants were grown overnight in Luria-Bertani (LB) broth, and 1 l of each culture was transferred using a custom 96-well pin device onto the surface of brain heart infusion agar plates containing 0.5% (wt/vol) Difco agar.…”

“…A recent screen of the 1,200 genes for which mutants are available in the P. aeruginosa PAO1 mini-Tn5-luxCDABE transposon mutant library (35) identified 36 mutants with altered swarming phenotypes (45). Moreover, a comparison of the gene expression profiles of swarmer cells and bacteria growing in the same medium under planktonic conditions showed that there was a Ͼ2-fold change in 7.5% of all P. aeruginosa genes.…”

“…The 417 genes that were differentially regulated in swarmer cells included no less than 18 predicted or known transcriptional regulator genes. Furthermore, differentially regulated genes were found to function in processes as diverse as transport, secretion, metabolism, and motility, suggesting that the regulation of swarming behavior is complex (45).…”

The sensor kinase PhoQ mediates virulence in Pseudomonas aeruginosa

“…Static microtitre biofilm assays were performed as previously described (Overhage et al, 2007). After 20 h incubation at 37 uC without shaking in LB medium, medium and non-adherent cells were discarded, and the wells were gently washed with deionized H 2 O. Surface-attached bacteria were then stained with 0.1 % (w/v) crystal violet for 20 min, followed by ethanol solubilization of crystal-violet-stained cells for quantification of absorbance at 600 nm using a microtitre plate reader (Bio-Tek Instruments).…”