The Bordetella pertussis heme iron utilization system: Transport and heme-responsive gene regulation.

摘要

Iron is an essential nutrient for virtually all living cells, yet it is extremely limiting in most biological systems, including the human body. Pathogenic bacteria, such as the gram negative respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica, possess high-affinity iron uptake systems that allow them to scavenge free iron or obtain iron from host iron binding proteins. This study describes the identification and characterization of the Bordetella heme iron utilization system. Genes encoding a putative heme transport system (bhuRSTUV) were initially identified in the incomplete B. pertussis genome sequence (Wellcome Trust Sanger Centre) and their role in the utilization of heme and hemoprotein iron sources was confirmed by mutational, genetic complementation and phenotypic analyses. Immediately upstream of the bhu genes, two open reading frames ( hurIR) predicted to encode positive regulators similar to those found in other iron acquisition systems were identified. A hurI mutant strain, lacking a putative extracytoplasmic function σ factor, was defective in utilization of heme and hemoglobin as iron sources, consistent with a role for HurI in positive regulation of bhu genes. Regulation of bhu gene expression was monitored using transcriptional fusions with lacZ, and production of the BhuR outer membrane heme receptor protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These experiments revealed that the bhu genes are expressed maximally under iron starvation conditions in the presence of heme as an inducer. Heme-responsive bhu transcription was hurI-dependent, confirming that HurI is a positive regulator of bhu gene expression. Genetic and biochemical analyses identified two promoters within the hur-bhu gene cluster. The promoter upstream of hurI was found to be iron-regulated, hurI-independent and not responsive to heme, indicating that hurI expression is not autoregulated. Consistent with reporter fusion analyses, the bhuR promoter was hurI-dependent and heme-inducible. The data presented in this thesis, along with similarities between the hur-bhu system and other iron acquisition systems that are positively regulated by extracytoplasmic function a factors, led to a model for transcriptional activation of heme transport genes that is responsive to the presence of heme in the environment.