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We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.

The ferric-uptake regulator (Fur) protein is an Fe(2+)-dependent transcriptional repressor. To clarify the global regulation of Helicobacter pylori proteins by Fur according to the growth phase, we compared the proteome profiles of H. pylori 26695 and its isogenic fur mutant, harvested during in vitro culture. Clustering analysis of the proteome profiles of the two strains revealed that the growth-phase-dependent protein regulation in the wild-type strain was largely altered in the fur mutant. Reverse transcriptase-PCR analysis of several H. pylori genes showed that a major switch in transcription occurred 12 h earlier than in the wild type, indicating that the fur mutation induced an earlier transcriptional switch from log to stationary phase. Several H. pylori proteins also showed changes in their patterns of protein post-translational modification (PTM). In particular, the HydB protein, which was detected as four spots on 2-dimensional electrophoresis gels, underwent two types of PTM, which were under different kinds of regulation. These data demonstrate that a fur mutation affects the growth-phase-dependent regulation of proteins and mRNAs, suggesting a role for Fur in controlling the global regulation of cellular processes in response to changing growth environments.