Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori.
The nickel-containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)- and 200 (HypB:kan)-fold lower than for the wild-type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H(2)-oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease-containing fractions of the mutant strains contained about four (HypA:kan)- and five (HypB:kan)-fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.
Olson
J W
JW
Department of Microbiology, 813 Biological Sciences Building, University of Georgia, Athens, GA 30602, USA.
Mehta
N S
NS
Maier
R J
RJ
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000201:enzymology*; D006864:Hydrogenase / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009154:Mutation; D009532:Nickel / Q000378:metabolism*; D014510:Urease / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; C089637:HypB protein, bacteria; D008667:Metalloproteins; D009532:Nickel; D006864:Hydrogenase; D014510:Urease; D019204:GTP-Binding Proteins