Introduction 
The ubiquitous Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen able to infect a broad range of animals and plants hosts including humans.
In the course of infection, P. aeruginosa adapts to changing environmental conditions and coordinates the production of molecular determinants involved in host colonization and virulence [1].
Among these are pili and flagella, which are required for attachment and spreading on surfaces [2],[3].
Also necessary are protein secretion systems [4] and toxins required for cytotoxicity and survival within the host.
One powerful approach to dissect the interaction between pathogen and host is the use of simple infection models.
It has been demonstrated that the nematode Caenorhabditis elegans can be an effective model for studying virulence mechanisms used by a variety of bacterial pathogens [5],[6].
P. aeruginosa is capable of killing C. elegans in several distinct ways.
When the P. aeruginosa strain PA14 is cultured on a high-osmolarity peptone-glucose-sorbitol medium (PGS), worms succumb to intoxication termed "fast killing", as the exposed worms die within hours [7].
Nematodes exposed to PA14 grown on nematode growth media (NGM), succumb to "slow killing".
In this case the bacteria colonize the gut and the infected worms die over a number of days rather than hours [8].
The P. aeruginosa isolate PAO1 cultured on Brain Heart Infusion (BHI) agar induces worm paralysis and death within hours [9].
Finally, C. elegans death, called red death, is observed in response to PAO1 grown on phosphate-depleted medium in conjunction with physiological stress on the nematodes [10].
It is known that the P. aeruginosa isolates PA14 and PAO1 show genomic diversity.
Strains cultured in vitro for years are known to undergo changes in gene expression.
Individual genes and even entire genomic islands have been lost from laboratory isolates when compared to pathogenic strains [11],[12],[13].
In this study, we used the P. aeruginosa strain TBCF10839 (TB), which was isolated from a Cystic Fibrosis (CF) patient [14].
The TB strain belongs to an abundant clonal complex in the P. aeruginosa population [15].
It has a high resistance to detergents [16] and reactive oxygen intermediates [17] and is able to grow within polymorphonuclear leukocytes [18].
We developed a high-throughput killing assay using C. elegans as a host, which will be appropriate for a systematic screen of mutant libraries of any P. aeruginosa isolate of interest.
Increasing throughput in an assay such that an entire genome can readily be scanned in a short time period is an important advance.
Our protocol is based on a standard killing assay and makes use of a Biosorter to distribute nematodes into the wells of microtitre plates in a fully automated manner.
We screened a TB STM (signature tagged mutagenesis) mutant library of 2,200 non-redundant clones [19],[20] and selected a small group of mutants, significantly attenuated for virulence in C. elegans.
By testing these mutants in additional phenotypic assays, including adherence to epithelial cells and virulence in a mouse model, we identified a gene, cheB2, necessary for in vivo P. aeruginosa virulence.
The cheB2 gene belongs to the P. aeruginosa chemotaxis che2 gene cluster (cluster II, PA0180-PA0173) (Figure 1) [21].
P. aeruginosa has multiple copies of chemotaxis genes arranged in five clusters (Figure 1) [22].
The chemotaxis gene cluster I (PA1456-PA1464) and cluster V (PA3349-PA3348) have previously been shown to be essential for chemotaxis and flagellar mobility in P. aeruginosa [23],[24].
The wsp genes contained in cluster III (PA3708-PA3702) have been proposed to contribute to bacterial biofilm formation [25], whereas the cluster IV (Pil-Chp system, PA0408-PA0417) has been shown to be involved in twitching motility [26],[27].
The che2 genes from cluster II, and more particularly the cheB2 gene, were initially identified as required for an optimal chemotactic response in P. aeruginosa.
The CheB proteins are essential components of the chemotactic response and are responsible for demethylation of glutamate residues in methyl-accepting chemotaxis proteins (MCPs) and also deamidation of glutamine residues to form methyl-accepting glutamates [28].
MCPs sense the chemical stimuli that initiate the chemotactic activity [29].
In Escherichia coli, the loss of CheB leads decreased receptor sensitivity, due to an inability to reset the MCP's [30].
This modification leaves the chemotactic pathway in active states causing clockwise rotation of the flagella and continuous tumbling instead of runs and tumbles.
It has been reported previously in Salmonella typhimurium that a loss of proper chemotaxis control through a cheB mutation, leads to a 'tumbly' swimming phenotype and a strong reduction in the isolates ability to invade human epithelial HEP2 cells as well as a reduction in infectivity in a mouse ligated-loop model [31].
In our study we further compare the phenotypes of cheB2 and cheB1 mutants and propose that the cheB2 gene has a specific role during infection, which is essential for pathogenesis.
