Helicobacter pylori requires flagellar motility and chemotaxis to establish and maintain chronic infection of the human stomach. The pH gradient in the stomach mucus is essential for bacterial orientation and guides the bacterium toward a narrow layer of the mucus, suggesting that H. pylori is capable of energy sensing or taxis. In the present study, H. pylori wild-type behavior in a temporal swimming assay could be altered by electron transport inhibitors, indicating that a connection between metabolism and behavior exists. In order to elucidate mechanisms of behavioral responses of H. pylori related to energy sensing, we investigated the phenotypes of single and multiple mutants of the four proposed chemotaxis sensor proteins. All sensor mutants were motile, but they diverged in their behavior in media supporting different energy yields. One proposed intracellular sensor, TlpD, was crucial for behavioral responses of H. pylori in defined media which did not permit growth and led to reduced bacterial energy levels. Suboptimal energetic conditions and inhibition of electron transport induced an increased frequency of stops and direction changes in the wild type but not in tlpD mutants. Loss of metabolism-dependent behavior in tlpD mutants could be reversed by complementation but not by electron donors bypassing the activity of the electron transport chain, in contrast to the case for the wild type. TlpD, which apparently lacks transmembrane domains, was detected both in the bacterial cytoplasm and at the bacterial periphery. The proposed energy sensor TlpD was found to mediate a repellent tactic response away from conditions of reduced electron transport.Helicobacter pylori has the ability to survive and multiply in the mucous layer of the human stomach, where it colonizes persistently for many years. Gastric Helicobacter species need complete flagella and full motility to colonize their hosts persistently (5,13,23,42). Both for the first entry of H. pylori into the human gastric mucus and for chronic colonization, H. pylori probably exploits horizontal and vertical substance gradients to reach and stay in its optimal habitat. Motile H. pylori mutants deficient in chemotaxis by knockout mutagenesis of the chemotaxis histidine kinase gene cheA and other chemotaxis proteins have been found to be unable to colonize in mouse and gerbil colonization models (16,26). Analysis of nanobiopsies taken from the stomach mucus of anesthetized, Helicobacterinfected mice and Mongolian gerbils has shown that Helicobacter actively accumulates in a very narrow zone of the proximal stomach mucus, at a distance from the epithelial surface of between 0 and 15 m (45, 46). It is guided there by a vertical proton gradient within the gastric mucus. Changes in other chemical gradients in the mucus in vivo did not alter the horizontal distribution of H. pylori, suggesting that pH taxis may be dominant over other chemotactic responses (45). pH taxis, a manifestation of energy-dependent taxis, was therefore shown to be essential for persiste...

H. pylori is a gram-negative bacterium associated with gastric inflammation and peptic ulcer and considered a risk factor for gastric cancer in its natural habitat. However, the energy metabolism of H. pylori in the stomach remains to be clarified. H. pylori shows rather high respiratory activity with L-proline and significantly large amounts of L-proline are present in the gastric juice from H. pylori infected patients. We constructed a disrupted mutant of the put A gene, which encodes the proline utilization A (Put A) flavin-linked enzyme, in order to examine the role of put A in the gastric colonization of H. pylori. The put A disrupted mutant, ΔputA, was constructed by inserting a chloramphenicol resistant gene into put A. Δput A did not show respiratory activity using L-proline and could not incorporate L-proline into cells. Δput A also did not show motility in response to amino acids and did not display the swarming activity observed with the wild-type. Δput A had lost its ability to colonize the stomach of nude mice, an ability possessed by the wild-type. These findings indicate that put A may play an important role in H. pylori colonization on the gastric mucus layer.

Helicobacter pylori exhibits chemotactic responses to urea, flurofamide, acetohydroxamic acid, and sodium bicarbonate. In buffer, the chemotactic activities of a urease-positive strain were higher than those of the isogenic urease-negative strain. Moreover, the chemotactic activities of the urease-positive strain were increased in a viscous solution containing 3% polyvinylpyrrolidone, whereas those of the urease-negative mutant were not. These results are in accordance with the fact that the mutant strain did not show swarming in motility agar regardless of having flagella. Incubation of the wild-type strain with flurofamide resulted in partial inhibition of the chemotactic activities in the viscous solution. In addition, incubation with acetohydroxamic acid, a low-molecular-weight, diffusible urease inhibitor, resulted in complete loss of chemotactic activity in the viscous solution. The inhibition of the chemotactic activity by urease inhibitors paralleled the inhibition of urease. The chemotactic activity of H. pylori was also inhibited by the proton carrier carbonyl cyanide m-chlorophenylhydrazone, showing that H. pylori utilizes proton motive force for motility. These results indicate that cytoplasmic urease plays an important role in the chemotactic motility of H. pylori under a condition that mimics the ecological niche of the bacterium, the gastric mucous layer.

A 1.7 kb DNA fragment isolated from an E. coif genomic library was able to complement the thiamin requirement of strains carrying the thiM, thil and thiD mutations. The three genes encode hydroxyethylthiazole kinase, hydroxymethylpyrimidine (HMP) kinase and phosphomethylpyrimidine (HMP-P) kinase, respectively. Sequence analysis revealed that the 1.7 kb fragment contained two ORFs of 708 bp and 801 bp. The former ORF complemented the thiM mutation and the latter ORF both the thil and thiD mutations. The latter ORF was cloned into the expression vector pET3a, and the encoded protein was purified through three successive column chromatographies. The purified protein was able to convert HMP to its monophosphate and the monophosphate to its pyrophosphate. These results suggest that the two distinct enzyme activities, HMP kinase and HMP-P kinase, are indeed a bifunctional enzyme encoded by a single gene, designated thiDIJ.