Research article

Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis

Yingli Li^{* 1} , Yefeng Qiu^{* 2} , He Gao¹ , Zhaobiao Guo¹ , Yanping Han¹ , Yajun Song¹ , Zongmin Du¹ , Xiaoyi Wang¹ , Dongsheng Zhou¹ and Ruifu Yang¹

¹State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China

²Laboratory Animal Center, Academy of Military Medical Sciences, Beijing 100071, PR China

author email corresponding author email* Contributed equally

BMC Microbiology 2009, 9:128doi:10.1186/1471-2180-9-128

Published:	25 June 2009

Abstract

Background

The zinc uptake regulator Zur is a Zn²⁺-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis.

Results

We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes.

Conclusion

Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.