Comparative Analysis Of Fis-dependent Transcriptomes In Yersinia Pestis And Characterization Of DNA-binding Activity Of Fis

Yersinia pestis is the causative agent of plague. Fis is an important bacterial regulator that controls a large collection of genes at the transcriptional level. The entire coding region of fis was replaced by the kanamycin resistance cassette using the one-step inactivation method based on the lambda Red phage recombination system, to generate the fis mutant of Y. pestis. By using cDNA microarray, the mRNA profiles were compared for the wild-type and fis mutant strains grown at exponential and stationary phases. The microarray analysis disclosed 323 and 352 genes whose transcription is dependent of Fis at exponential and stationary phases, respectively. The microarray data were further validated by real-time RT-PCR. The microarray and RT-PCR data showed a significant positive correlation with a correlation coefficient (r) of 0.93. The Fis regulator is able to regulate some virulence genes, especially including psaAB, pla, rovA, and those encoding effectors of type III secretion system, at both exponential and stationary phases. This indicates that Fis participates in the coordinated regulation of metabolic and virulence genes in Y. pestis.The entire coding region of the fis gene of Y. pestis,was amplified by PCR, and his cloned into the BamHI and HindIII sites of the pET28a vector. The recombinant plasmid pET28a-fis was inducted into BL21λDE3. Over-expression of His-Fis in the LB medium was induced by addition of 1 mM IPTG (isopropyl-b-D-thiogalactoside). The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). A bioinformatics analysis indicated the Fis consensus-like sequences were of presence within the upstream regions of the Fis-dependent genes maeB, yscN, ompX, metA, and YPO3655. The electrophoretic mobility shift assay (EMSA) was carried out to analyze the DNA-binding activities of the Fis proteins in vitro. It was shown that Fis was able to bind to the upstream regions of the above five genes, which confirmed that Fis recognized the AT-rich target regions.