Regulation of the phototrophic iron oxidation (pio) genes in Rhodopseudomonas palustris TIE-1 is mediated by the global regulator, FixK

The pioABC operon is required for phototrophic iron oxidative (photoferrotrophic) growth by the alpha-proteobacterium Rhodopseudomonas palustris TIE-1. Expression analysis of this operon showed that it was transcribed and translated during anaerobic growth, upregulation being observed only under photoferrotrophic conditions. Very low levels of transcription were observed during aerobic growth, suggesting expression was induced by anoxia. The presence of two canonical FixK boxes upstream of the identified pioABC transcription start site implicated FixK as a likely regulator. To test this possibility, a Delta fixK mutant of R. palustris TIE-1 was assessed for pioABC expression. pioABC expression decreased dramatically in Delta fixK versus WT during photoferrotrophic growth, implying that FixK positively regulates its expression; coincidently, the onset of iron oxidation was prolonged in this mutant. In contrast, pioABC expression increased in Delta fixK under all non-photoferrotrophic conditions tested, suggesting the presence of additional levels of regulation. Purified FixK directly bound only the proximal FixK box in gel mobility-shift assays. Mutant expression analysis revealed that FixK regulates anaerobic phototrophic expression of other target genes with FixK binding sites in their promoters. This study shows that FixK regulates key iron metabolism genes in an alpha-proteobacterium, pointing to a departure from the canonical Fur/Irr mode of regulation.