Repression of btuB gene transcription in Escherichia coli by the GadX protein
1 Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei 11221, Taiwan
2 Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
3 Institute of Biochemistry, National Yang Ming University, Taipei, 11221, Taiwan
4 Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei 11221, Taiwan
5 Department of Education and Research, Taipei City Hospital, Taipei, Taiwan
BMC Microbiology 2011, 11:33 doi:10.1186/1471-2180-11-33Published: 11 February 2011
To determine whether the btuB gene is also transcriptionally controlled by trans-acting factors, a genomic library was screened for clones that enable E. coli to grow in the presence of colicin E7, and a plasmid carrying gadX and gadY genes was isolated. The lacZ reporter gene assay revealed that these two genes decreased the btuB promoter activity by approximately 50%, and the production of the BtuB protein was reduced by approximately 90% in the presence of a plasmid carrying both gadX and gadY genes in E. coli as determined by Western blotting. Results of electrophoretic mobility assay and DNase I footprinting indicated that the GadX protein binds to the 5' untranslated region of the btuB gene. Since gadX and gadY genes are more highly expressed under acidic conditions, the transcriptional level of btuB in cells cultured in pH 7.4 or pH 5.5 medium was examined by quantitative real-time PCR to investigate the effect of GadX. The results showed the transcription of gadX with 1.4-fold increase but the level of btuB was reduced to 57%.
Through biological and biochemical analysis, we have demonstrated the GadX can directly interact with btuB promoter and affect the expression of btuB. In conclusion, this study provides the first evidence that the expression of btuB gene is transcriptionally repressed by the acid responsive genes gadX and gadY.