| Plague has ravaged human populations for many centuries and claimed hundred millions of lives in history. Plague normally cycles between fleas and rodent hosts, with humans providing an unproductive alternative. Yersinia pestis is the etiologic agent of plague. Since Y. pestis undergoes various types of environmental changes during transmission and infection, a wide range of rapid, adaptive responses to the changing niches are required. There are specific regulators responsing to the environmentally specific signals and controlling the expression of their specific target genes (including virulence genes). Finally, the complex regulating network of virulence genes is composed of them, and the viability and pathgenicity of Yersinia pestis is controlled by this virulence-related regulating network.Pathogen responses to signal stimulation, and expresses virulence-related regulators. These regulators play irreplaceable role during this process. The global or specific regulators control the expression of virulence genes. Additionally, the products that are expressed by some virulence regulated genes build up a complex system. The system in charge of virulence regulating functions is refered to virulence regulated system, which is always taken as research hotspot in pathogen bacteriology, incluing quorum sensing (QS) system, two-component system and Fur system etc.These systems are divided into many subsystems to control expression of different type of virulence genes and play the regulation role at different virulence-related stage of bacteria.Zur belongs to the Fur family and regulates the transport of zinc ions. Zinc is an essential component of protein and has function in all living cells. The two-component system PhoP-PhoQ can govern bacterial virulence, helps bacteria to adapt to Mg2+-limiting environments, survival and proliferation in macrophage. For further indentifing target genes of Zur and PhoP, especially the directly regulated target genes, and investigating the mechanism of Zur or PhoP regulation, we employed gene knockout method, microarray-based gene expression profiling and conventional biochemical techniques to study the gene regulating targets of Zur and PhoP. The zur and phoP gene in Y. pestis were replaced, respectively, with kanamycin resistance cassette using one-step disruption protocol in this study. The whole-genome DNA microarray of Y. pestis was used to investigate global transcriptional responses. The transcription profile of the wild-type Y. pestis was then compared with that of the mutant for determining upregulated and downregulated genes. These genes composed of Zur or PhoP regulons. Then, the results of transcriptome analysis were proved by real-time reverse-transcription (RT-PCR). Electrophoretic mobility shift assay (EMSA), helped to identify direct target genes of the regulators. DNase I footprinting assay and primer extension analysis were used to study how the transcription and expression of these genes are regulated directly by Zur and PhoP. This will help us to understand the mechanism of how Zur and PhoP can help to control the adaptative ability and pathgenicity of Yersinia pestis in hosts.Through transcriptome analysis, under stimulation of zinc ions, those strains with zur gene deletion had totally 154 genes changed their transcription level comparing with the wild type ones. Among them, 64 genes were upregulated, whereas 90 genes downregulated. We also collected data from published lituratures to predict Zur binding sites.Presenting frequency of four bases at each site of Zur binding sequence was obtained by consensus-matrix and convert-matrix program, then the sequence logo were shown by WebLogo software. Finally, Zur binding box in Yersinia pestis was confirmed to be GAAATGTTATAWTATAACATTTC. Based on the above result and transcriptome analysis of Zur, we screened the conserved sequences of Zur by Matrix scan program and selected four genes(ykgM,znuC,znuA,astA) for further confirming by biochemical experiments. EMSA confirmed three transcriptional units, ykgM,znuC,znuA were regulated by Zur directly. In order to get the precise mechanism of Zur regulation, we used footprinting assay to study the promotors of these transcriptional units. The Zur bingding sequence was obtained, and the predicted conserved Zur box was present in this region. All the three Zur boxes covered -10 elements for blocking RNA polymerase binding. It is possible that this is why Zur inhibits their transcription. By using primer extension analysis, we compared the wild type with ?zur mutant under the stimulation of low zinc ions. Not only determined the transcription start sites of ykgM,znuC and znuA under the regulation of Zur, but also determined the effect of Zur to their transcription. Combined with the result of footprinting assay, -10 and -35 elements for RNA polymerase binding were localized. It was helpful to get more complete information of the promoter region of Zur regulon.In our previous work, the transcriptome analysis of ?phoP mutant and wild type strains under low Mg2+ environments had confirmed PhoP regulon. In the present study, real time RT-PCR were used to test the result of transcriptome analysis. EMSA elucidated that 30 transcriptional units were regulated directly by PhoP. The promotor region of 17 genes were tested by footprinting assay, 19 PhoP conserved binding regions were obtained. Through bioinformatics analysis, PhoP box in Yersinia pestis was confirmed to be TGTTTAWN4TGTTTAW. Primer extension analysis was used to obtain the transcription start sites of these PhoP regulons. Finally, the promoters of the target genes of PhoP were classified into three types: PhoP binding sites lie in upstream of -35 elements, cover -35 elements, and lie in downstream of -35 elements. This study firstly identified the regulons of Zur and PhoP of Yersinia pestis. At the same time, the result confirmed the Zur box and PhoP box of Yersinia pestis and explored the precise mechanism of Zur and PhoP regulation. It is helpful for building up virulence regulators networks and elucidating functional knowledge of these regulons. And further, these resluts will be helpful to investigating pathgenicity of Yersinia pestis.
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