A fixed-time diffusion analysis method determines that the three cheV genes of Helicobacter pylori differentially affect motility Helicobacter pylori is a chemotactic bacterium that has three CheV proteins in its predicted chemotaxis signal transduction system. CheV proteins contain both CheW-and responseregulator-like domains. To determine the function of these proteins, we developed a fixed-time diffusion method that would quantify bacterial direction change without needing to define particular behaviours, to deal with the many behaviours that swimming H. pylori exhibit. We then analysed mutants that had each cheV gene deleted individually and found that the behaviour of each mutant differed substantially from wild-type and the other mutants. cheV1 and cheV2 mutants displayed smooth swimming behaviour, consistent with decreased cellular CheY-P, similar to a cheW mutant. In contrast, the cheV3 mutation had the opposite effect and the mutant cells appeared to change direction frequently. Additional analysis showed that the cheV mutants displayed aberrant behaviour as compared to the wild-type in the soft-agar chemotaxis assay. The soft-agar assay phenotype was less extreme compared to that seen in the fixed-time diffusion model, suggesting that the cheV mutants are able to partially compensate for their defects under some conditions. Each cheV mutant furthermore had defects in mouse colonization that ranged from severe to modest, consistent with a role in chemotaxis. These studies thus show that the H. pylori CheV proteins each differently affect swimming behaviour. INTRODUCTIONMany micro-organisms move in a directed fashion in response to their environment. A common mechanism for movement is via rotary motor organelles called flagella. These motors are regulated by the chemotaxis signal transduction system, which transduces environmental cues into a swimming response. The core of this signal transduction system consists of chemoreceptors, a kinase (CheA), a receptor-kinase coupler (CheW) and a phosphorylatable response regulator (CheY) that controls flagellar rotation (Blair, 1995;Szurmant & Ordal, 2004).There are additional proteins that modulate the amount of phosphorylated CheY (CheY-P). Accessory proteins responsible for adaptation and other functions abound, such as methylation of the chemoreceptors by CheR. As more and more prokaryotic genomes have been sequenced, it is becoming clear that motile microbes each have the core signal transduction proteins with a somewhat unique set of these modulator proteins, although it is not yet apparent why particular microbes have particular sets (Blair, 1995;Szurmant & Ordal, 2004). One such microbe is Helicobacter pylori, a bacterium that uses flagellar motility and chemotaxis to fully colonize animal stomachs (Eaton et al., 1992(Eaton et al., , 1996 Foynes et al., 2000;Ottemann & Lowenthal, 2002;Terry et al., 2005). Understanding some of the less-usual attributes of this microbe's chemotactic signal transduction system was the goal of this study.H. pylori contains h...