Heme Utilization in Bordetella avium Is Regulated by RhuI, a Heme-Responsive Extracytoplasmic Function Sigma Factor

Abstract

Efficient utilization of heme as an iron (Fe) source by Bordetella avium requires bhuR , an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that rhuI encoded a potential in- trans regulator of the heme receptor gene in trans . Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR . Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuI was correlated with a dramatically increased expression of BhuR. A putative rhuI -dependent bhuR promoter was identified in the 199-bp region located proximal to bhuR . When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced into Escherichia coli , promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E . coli demonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the rhuI -dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.