Multiple haem-utilization loci in Helicobacter pylori

To identify genes responsible for the utilization of haem as an iron source in Helicobacter pylori. a siderophore synthesis mutant of Escherichia coli was transformed with an ordered cosmid library of H. pylori NCTC 11638. Fo ur independent cosmids were found that were able to complement this mutant on iron-restrictive solid media containing different haem compounds as the sole source of iron. Hybridization experiments revealed that the four cosmi ds contained unrelated DNA fragments. No major differences were observed in the growth of the four transformants on iron-restrictive solid media to wh ich different haem compounds had been added. None of the cosmids could conf er the ability to use haem as an iron source to an E. coli aroB tonB mutant , which means that transport of iron and/or haem across the outer membrane requires a functional TonB protein. Further characterization of the cosmids revealed that one of them was also able to complement E. coli aroB hemA, i ndicating that the haem molecule is taken up as a whole by this haem-biosyn thesis mutant. Expression of this haem-uptake system could not be repressed by excess iron. Another cosmid expressed two polypeptides in E. coli which were specifically immunoreactive with a polyclonal antiserum raised agains t whole cells of H. pylori. The production of these proteins appeared to be iron repressible. One of these proteins has the same molecular mass as a p reviously described 77 kDa haem-binding iron-repressible outer-membrane pro tein (IROMP) of H. pylori.