The Fur Regulon Directly Regulates Iron Metabolism In Yersinia Pestis

Plague, caused by Yersinia pestis, has ravaged human populations for many centuries and claimed hundred millions of lives in history. Iron is essential to virtually all organisms. Bacteria typically regulate their iron metabolism in response to iron availability. In Yersinia pestis this regulation is mediated by the ferric-uptake regulator protein (Fur) that directly controls the iron-dependent expression of more than 30 genes in Yersinia pestis strans.In this study,the fur gene in Y. pestis was replaced with kanamycin resistance cassette using the one-step disruption protocol. In our previous DNA microarray analysis, mRNA levels from wild-type (WT) Y. pestis cells treated with the iron chelator 2,2'-dipyridyl (DP) were compared with those supplemented with excessive iron, and then gene expression in the fur mutant was compared with that in the WT strain under iron rich condition. This analysis identified genes both directly and indirectly controlled by Fur, leading to the defining of iron-Fur modulon that was defined as a collection of genes whose transcription was affected by both the DP treatment (the iron starvation) and the fur mutation.Iron acquisition is critical for the survival of pathogenic bacteria during infection. In mammals, iron is bound to Fe3+-binding proteins (transferrin, lactoferrin and ferritin) and hemopoteins (hemoglobin, haptoglobin-hemoglobin, hemopexin and albumin, etc). The level of free iron (10–18 M) is too low to sustain bacterial growth. In light of the requirement for iron and the stiff competition for this nutrient, it is no surprise that successful pathogenic microorganisms have evolved miscellaneous strategies to scavenge iron from the iron or heme resources of mammalian hosts.In addition to the characterized iron acquisition systems (Ybt, Yfe, Yfu, Hmu, Has, Yfu, Yiu, Iuc and Fhu), there are still several other predicted iron assimilation (acquisition/storage) functions in the genome annotation of Y. pestis CO92, KIM and 91001. To give a full collection of iron assimilation systems in Y. pestis, all avalaible literatures about bacterial iron assimilation were reviewed, and the deriving protein sequences of reported bacterial iron assimilation genes were compared with the genomic sequence data of CO92, by using the BLASTP, to find their homologues in Y. pestis. In addition, the BLASTP analysis of all the iron-Fur modulon members of Y. pestis, as determined by our previous microarray expression analysis, was performed against the GenBank data to test whether each of them was homologous to a bacterial iron assimilation gene. Taken together, a total of 20 proven or putative iron assimilation-related genomic loci (80 genes) were found in Y. pestis.In the present work, a total of 34 putative operons (mainly responsible for the iron assimilation functions) were identified as the direct Fur targets, based on the combined use of real-time reverse-transcription (RT)-PCR, primer extension analysis, and electrophoretic mobility shift assay (EMSA). It was shown that Fur was a global regulator, both an activator and a repressor, governing a complex regulatory cascade in Y. pestis. The subsequent DNase I footprinting assay enabled the mapping of Fur-DNA interactions within the ybt locus, also known as the high pathogenicity island (HPI).The pgm locus is a 102-kb unstable DNA region (YPO1902-1967 on the chromosome of CO92) embedded between two IS100 elements in the same orientation . The recombination between the two IS100 elements will delete this locus. YPO1902-1917, containing the ybt locus, was referred to as the HPI. Mutants lacking the Ybt system (a well-characterized siderophore-based iron acquisition system) are completely avirulent to mice by subcutaneous infection, but are fully virulent via an intravenous route, suggesting that the Ybt is essential to iron acquisition at the site of flea bite, in the lymphatic system and/or the lymph nodes.In the present study, we confirmed that the Fur protein repressed the whole ybt locus in response to excess extracellular iron, and then defined the Fur sites within the ybt locus by using DNase I footprinting. In addition, the primer extension assay was used to determine the transcriptional start sites of Fur-dependent promoters and to help to localize the promoter -10 and -35 elements.In addition to the Fur protein, the YbtA has been shown to be another transcriptional regulator that functions as an activator of irp2-irp1-ybtUTE, ybtPQXS and fyuA, and as a repressor of its own transcription. The YbtA-DNA interactions within the HPI in Y. pestis has been elucidated recently.Taking all the above results together, we depicted the organization of Fur- and YbtA-dependent promoters, to construct a prototype Fur/YbtA regulatory network. Given the high conservation of HPI in the Yersinia spp., generally the above mechanisms of transcriptional regulation by YbtA should be shared by Y. pestis.