Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.

The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the Helicobacter pylori ferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled H. pylori to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the pfr gene restricted growth of H. pylori under these conditions. The lowered total iron content in the pfr mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an H. pylori feoB mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of pfr transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant H. pylori Fur to the pfr promoter region. The functions of H. pylori Pfr in iron metabolism are essential for survival in the gastric mucosa, as the pfr mutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the pfr phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for H. pylori to survive in its hostile natural environment.

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To establish a system to analyze ATP synthesis by the archaeal A(1)A(o) ATP synthase and to address the nature of the coupling ion, the operon encoding the A(1)A(o) ATP synthase from the mesophile Methanosarcina mazei Gö1 was cloned in an expression vector and it was expressed in the F(1)F(o) ATP synthase-negative mutant Escherichia coli DK8. Western blot analyses revealed that each of the subunits was produced, and the subunits assembled to a functional, membrane-embedded ATP synthase/ATPase. ATP hydrolysis was inhibited by dicyclohexylcarbodiimide but also by tributyltin, which turned out to be the most efficient inhibitor of the A(o) domain of A(1)A(o) ATP synthase known to date. ATP hydrolysis was not dependent on the Na(+) concentration of the medium, and inhibition of the enzyme by dicyclohexylcarbodiimide could not be relieved by Na(+). The enzyme present in the cytoplasmic membrane of E. coli catalyzed ATP synthesis driven by an artificial DeltapH but not by DeltapNa or DeltamuNa(+).

The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes of Methanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques. Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied. The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria. In some of the proteolipids, the N-terminal methionine is excised. Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The molecular and biochemical data gave clear evidence that the mature proteolipid from M. jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F 1 F 0 ATPases and most archaeal A 1 A 0 ATPases. Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices. This finding puts in question the current view of the evolution of H ؉ ATPases and has important mechanistic consequences for the structure and function of H ؉ ATPases in general.Proton-pumping ATPases are found in all organisms with an overall applicable bipartite structure consisting of the membrane-extrinsic moiety (F 1 /V 1 /A 1 ), which synthesizes and/or hydrolyzes ATP, and the hydrophobic domain (F 0 /V 0 /A 0 ), which translocates ions across the membrane. Based on subunit composition and primary structures of the subunits, the archaeal A 1 A 0 ATPases and the eucaryal V 1 V 0 ATPases are closely related (1-5). However, they differ with respect to function. The V 1 V 0 ATPase exhibits only ATP hydrolase activity and serves to energize the membranes of certain organelles and cells. Methanogenic archaea are not fermentative but are strictly chemiosmotic, and the presence of an ATP synthase has been known for a long time (6, 7). Interestingly, the ATPases isolated from membranes of various methanogens were all classified as V 1 V 0 -like enzymes (now called A 1 A 0 ATPases), whereas F 1 F 0 -like enzymes have never been isolated (5). Inhibitor studies revealed that the A 1 A 0 ATPase from Methanosarcina mazei Gö1 is a ⌬ Hϩ -driven ATP synthase (8), which gave experimental evidence that methanogens synthesize ATP by means of the A 1 A 0 ATPase. Moreover, the genome of Methanococcus jannaschii harbors only genes encoding the A 1 A 0 ATPase but not the F 1 F 0 ATPase (9), which is clear evidence that this hyperthermophile also engages the A 1 A 0 ATPase for ATP synthesis.It was postulated that the diversion of the A 1 A 0 and V 1 V 0 ATPases took place by a duplication and subsequent fusion of the genes encoding the proteolipid, a very hydrophobic, membrane-integral subunit known to participate in transmembrane H ϩ transport, which in all hitherto known A 1 A 0 ATPases is 8 kDa but is 16 kDa in all V 1 V 0...