| Iron (Fe) is an essential element for nearly all pathogenic bacteria. Within vertebrate hosts, Fe is sequestered in high affinity complexes, effectively maintaining the free Fe levels at <10−18 M. Although successful pathogens have developed elaborate systems to acquire Fe from host Fe complexes, the Fe-stress encountered in the host serves as a regulatory signal for the bacterium. Generally, this regulation is achieved through the F&barbelow;e u&barbelow;ptake r&barbelow;egulator (Fur), which acts as a transcriptional repressor under high Fe conditions. Previous work identified the Fur-regulated Bhu heme uptake system in Bordetella avium which is required for utilization of heme and hemoproteins as sole Fe sources. Monitoring the presence of the heme receptor BhuR in the outer membrane as a measure of expression, it was found that the system is responsive to both Fe and heme. This study characterized three genes required for heme induction: (1) rhuI, a putative extracytoplasmic function (ECF) sigma factor, (2) rhuR, a transmembrane regulator necessary for RhuI activity; and (3) bhuR, an outer membrane heme receptor. RhuI-dependent promoter sequences (P bhuR) identified in the 100 by non-coding region preceding bhuR are activated in low Fe, high heme conditions in B. avium. Unlike many sigma factors which are negatively regulated, the transcriptional activity of RhuI requires the presence of RhuR, which is cotranscribed with RhuI and is localized to the inner membrane of the cell. Protein fusion studies showed that the activation domain of RhuR is separable from the remainder of the protein. The N-terminal region of the protein, alone or as a fusion to BlaM, constitutively activates RhuI, consistent with a cytoplasmic localization for this portion of the protein. The C-terminal region is predicted to be periplasmic and to interact with BhuR. Expression of the three component induction system in Fe-stressed conditions is dependent upon the upstream rhuIR promoter (PruhIR), which is RhuI-independent and Fur-regulated. At a low frequency, a transcript originating from the rhuIR operon continues into bhuR. This readthrough transcription is predicted to he essential to produce RhuI, RhuR and BhuR in Fe-stress to allow for induction upon the introduction of heme or hemoproteins. |
