Characterization of the NifU and NifS Fe-S cluster formation proteins essential for viability in Helicobacter pylori

The Fe-S cluster formation proteins NifU and NifS are essential for viabili ty in the ulcer causing human pathogen Helicobacter pylori. Obtaining viabl e H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS wa s unsuccessful even by growing the potential transformants under many diffe rent conditions including low O-2 atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, t his second copy of the gene could be disrupted at high frequency. This indi cates that the procedures used for transformation were capable of nifU muta genesis, so that the failure to recover mutants is solely due to the requir ement of nifU for H. pylori viability. H. pylori NifU and NifS were express ed in Escherichia coli and purified to near homogeneity, and the proteins w ere characterized. Purified NifU is a red protein that contains similar to1 .5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S](2+,+) cluster by characteristic UV-visible, EPR, a nd MCD spectra. The primary structure of NifU also contains the three conse rved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein. Purifi ed NifS has a yellow color and UV-visible spectra characteristic of a pyrid oxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters. Th e results here indicate that the NifU type of Fe-S cluster formation protei ns is not specific for maturation of the nitrogenase proteins and, as H. py lori lacks other Fe-S cluster assembly proteins, that the H. pylori NifS an d NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clus ters.