Apoprotein isolation and activation, and vibrational structure of the Helicobacter mustelae iron urease.

The micro aerophilic pathogen Helicobacter mustelae synthesizes an oxygen-labile, iron-containing urease (UreA2B2) in addition to its standard nickel-containing enzyme (UreAB). An apoprotein form of the iron urease was prepared from ureA2B2-expressing recombinant Escherichia coli cells that were grown in minimal medium. Temperature-dependent circular dichroism measurements of holoprotein and apoprotein demonstrate an enhancement of thermal stability associated with the UreA2B2 metallocenter. In parallel to the situation reported for nickel activation of the standard urease apoprotein, incubation of UreA2B2 apoprotein with ferrous ions and bicarbonate generated urease activity in a portion of the nascent active sites. In addition, ferrous ions were shown to be capable of reductively activating the oxidized metallocenter. Resonance Raman spectra of the inactive, aerobically-purified UreA2B2 holoprotein exhibit vibrations at 495cm(-1) and 784cm(-1), consistent with ν(s) and ν(as) modes of an Fe(III)OFe(III) center; these modes undergo downshifts upon binding of urea and were unaffected by changes in pH. The low-frequency mode also exhibits an isotopic shift from 497 to 476cm(-1) upon (16)O/(18)O bulk water isotope substitution. Expression of subunits of the conventional nickel-containing Klebsiella aerogenes urease in cells grown in rich medium without nickel resulted in iron incorporation into a portion of the protein. The inactive iron-loaded species exhibited a UV-visible spectrum similar to oxidized UreA2B2 and was capable of being reductively activated under anoxic conditions. Results from these studies more clearly define the formation and unique properties of the iron urease metallocenter.

An ABC transporter and a TonB ortholog contribute to Helicobacter mustelae nickel and cobalt acquisition.

The genomes of Helicobacter species colonizing the mammalian gastric mucosa (like Helicobacter pylori) contain a large number of genes annotated as iron acquisition genes but only few nickel acquisition genes, which contrasts with the central position of nickel in the urease-mediated acid resistance of these gastric pathogens. In this study we have investigated the predicted iron and nickel acquisition systems of the ferret pathogen Helicobacter mustelae. The expression of the outer membrane protein-encoding frpB2 gene was iron and Fur repressed, whereas the expression of the ABC transporter genes fecD and ceuE was iron and Fur independent. The inactivation of the two tonB genes showed that TonB1 is required for heme utilization, whereas the absence of TonB2 only marginally affected iron-dependent growth but led to reduced cellular nickel content and urease activity. The inactivation of the fecD and ceuE ABC transporter genes did not affect iron levels but resulted in significantly reduced urease activity and cellular nickel content. Surprisingly, the inactivation of the nixA nickel transporter gene affected cellular nickel content and urease activity only when combined with the inactivation of other nickel acquisition genes, like fecD or ceuE. The FecDE ABC transporter is not specific for nickel, since an fecD mutant also showed reduced cellular cobalt levels and increased cobalt resistance. We conclude that the H. mustelae fecDE and ceuE genes encode an ABC transporter involved in nickel and cobalt acquisition, which works independently of the nickel transporter NixA, while TonB2 is required primarily for nickel acquisition, with TonB1 being required for heme utilization.

Comparative genomics and proteomics of Helicobacter mustelae, an ulcerogenic and carcinogenic gastric pathogen.

BACKGROUND: Helicobacter mustelae causes gastritis, ulcers and gastric cancer in ferrets and other mustelids. H. mustelae remains the only helicobacter other than H. pylori that causes gastric ulceration and cancer in its natural host. To improve understanding of H. mustelae pathogenesis, and the ulcerogenic and carcinogenic potential of helicobacters in general, we sequenced the H. mustelae genome, and identified 425 expressed proteins in the envelope and cytosolic proteome. RESULTS: The H. mustelae genome lacks orthologs of major H. pylori virulence factors including CagA, VacA, BabA, SabA and OipA. However, it encodes ten autotransporter surface proteins, seven of which were detected in the expressed proteome, and which, except for the Hsr protein, are of unknown function. There are 26 putative outer membrane proteins in H. mustelae, some of which are most similar to the Hof proteins of H. pylori. Although homologs of putative virulence determinants of H. pylori (NapA, plasminogen adhesin, collagenase) and Campylobacter jejuni (CiaB, Peb4a) are present in the H. mustelae genome, it also includes a distinct complement of virulence-related genes including a haemagglutinin/haemolysin protein, and a glycosyl transferase for producing blood group A/B on its lipopolysaccharide. The most highly expressed 264 proteins in the cytosolic proteome included many corresponding proteins from H. pylori, but the rank profile in H. mustelae was distinctive. Of 27 genes shown to be essential for H. pylori colonization of the gerbil, all but three had orthologs in H. mustelae, identifying a shared set of core proteins for gastric persistence. CONCLUSIONS: The determination of the genome sequence and expressed proteome of the ulcerogenic species H mustelae provides a comparative model for H. pylori to investigate bacterial gastric carcinogenesis in mammals, and to suggest ways whereby cag minus H. pylori strains might cause ulceration and cancer. The genome sequence was deposited in EMBL/GenBank/DDBJ under accession number FN555004.