pestis CO92, these Zur-dependent genes were distributed in 15 functional categories (Additional file 3). Their products included regulators, membrane-related proteins, transport/binding proteins,
biosynthesis selleck and metabolism related proteins and lots of unknown proteins. Additional file 4 showed the complete list of differentially regulated genes, giving an overall picture of the alteration of the global gene transcription pattern of Y. pestis affected by Zur with sufficient zinc. The microarray data (GSE15183) had been deposited in Gene Expression Omnibus (GEO). Validation of microarray data by Real-time RT-PCR Microarray results are influenced by various factors, and thereby should be validated by at least one traditional method. Accordingly, the real-time quantitative RT-PCR, using RNA preparations as described in the microarray analysis, was performed to validate the microarray data. Based on
gene classification, genomic location and transcriptional changes, 17 genes were chosen for RT-PCR (Additional file 5). The log-transformed change in relative quantity of mRNA level between WT and Δzur was calculated for each gene. The resulting real-time RT-PCR data were then plotted against the average log ratio values PF-02341066 mouse obtained by microarray analysis. There was a strong positive correlation (R2 = 0.796) between the two techniques (Additional file 5). It should be noted that these 17 genes gave a 100% consistency for differential regulation between microarray and RT-PCR data, confirming the reliability of our microarray data. Characterization of DNA-binding ability of Zur by EMSA We prepared a recombinant Y. pestis Zur protein by overproducing it in E. coli and examined its DNA-binding
activity by EMSA (Fig. 1). Increasing amounts (from 0 to 160 pmol) of the purified Zur protein were incubated with 10 fmol of32P-labeled znuA promoter region (it contained a strongly predicted Zur binding site; see Fig. 1a) in the presence of 100 μM ZnCl2 (Fig. 1b). From 1.25 pmol of Zur, the Zur-DNA complex (i.e. gel retardation) emerged; with the Zur amount increased, gel retardation appeared more and more heavily and reached to the peak at 80 pmol of Zur. Figure 1 DNA binding ability of Zur. The upstream region of znuA almost (panel a) or rovA (f), with or without a predicted Zur binding site, respectively, was amplified by PCR and used as target DNA probe in EMSA. For EMSA, the [γ-32P]-labeled target DNA probes (1000 to 2000 c.p.m/μl) were incubated with the Zur protein in the presence or absence of 100 μM ZnCl2. Increasing amounts of Zur (b and g), ZnCl2(c), or EDTA (d and e) were employed. The mixtures were directly subjected to 4% polyacrylamide gel electrophoresis. The rovA gene was used as negative control. It should be noted that the target DNA was progressively and continuously retarded (i.e.