| Iron starvation is used by many pathogens as a signal that they are in a host environment and results in the expression of virulence factors that are transcriptionally regulated through the ferric uptake regulator protein, Fur. A Fur homolog has been identified in pathogenic Neisseria; however a detailed analysis of Fur on Neisserial gene expression has previously not been conducted. My hypothesis is that the Fur protein, controls the expression of numerous genes, in addition to those involved in iron acquisition, that are required for the virulence of the pathogenic Neisseria. In this study we have demonstrated the in vitro binding of Neisseria gonorrhoeae Fur (Gc-Fur) to the promoter/operator regions of well-defined iron transport genes as well as to genes involved in major catabolic, secretory and recombination pathways of gonococci. The Gc-Fur protein was expressed as a recombinant protein and purified by ion-exchange chromatography. Gc-Fur was shown to bind specifically to the promoter/operator region of the gene encoding a previously identified Fur regulated periplasmic binding protein (FbpA) in a metal-ion dependent fashion, demonstrating that purified repressor is functional. In silico analysis of the partially completed gonococcal genome (FA1090) identified Fur boxes in the promoters of several genes including tonB, fur, recN, sect, sodB, hemO, hmbR, fumC, a hypothetical gene (Fe-S homolog) and the opa family of genes. We demonstrated binding of Fur to the operator regions of these genes including all eleven opa genes (A through K) by electrophoretic mobility shift assay. In contrast, we did not detect binding of purified Escherichia coli Fur to the regulatory regions of 8 of the 11 opa members. These results indicate that target DNA sequence specificities exist between these two closely related regulatory proteins. Furthermore, we observed differences in the relative strengths of binding of Gc-Fur to these genes suggesting the differential control of these gene targets. To further define the gene repertoire that is regulated by iron and Fur in Neisseria, we utilized DNA microarray technology to monitor simultaneous expression of the entire gene repertoire of N. meningitidis MC58 in response to iron limitation. These results suggest that iron regulates the expression of at least 235 genes in N. meningitidis, some of which may play an important role in pathogenesis. An in silico search of the iron-regulated genes for the presence of a Fur binding sequence revealed that 44% of these genes have the potential to be regulated by Fur. Neisseria Fur was demonstrated to bind to the promoter/operator DNA fragments of representative genes from both the iron-repressed and iron-activated gene clusters which contain highly conserved Fur binding sequences. Our results demonstrate that this repressor protein binds to the regulatory regions of a broad array of genes and indicates that the Neisseria Fur regulon is likely to be larger than originally proposed. |
