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Open Access Highly Accessed Research article

Repression of btuB gene transcription in Escherichia coli by the GadX protein

Guang-Sheng Lei1, Wan-Jr Syu1, Po-Huang Liang2, Kin-Fu Chak3, Wensi S Hu4 and Shiau-Ting Hu15*

Author Affiliations

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

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BMC Microbiology 2011, 11:33  doi:10.1186/1471-2180-11-33

Published: 11 February 2011

Abstract

Background

BtuB (

    B
 
    t
welve
    u
ptake) is an outer membrane protein of Escherichia coli, it serves as a receptor for cobalamines uptake or bactericidal toxin entry. A decrease in the production of the BtuB protein would cause E. coli to become resistant to colicins. The production of BtuB has been shown to be regulated at the post-transcriptional level. The secondary structure switch of 5' untranslated region of butB and the intracellular concentration of adenosylcobalamin (Ado-Cbl) would affect the translation efficiency and RNA stability of btuB. The transcriptional regulation of btuB expression is still unclear.

Results

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%.

Conclusions

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.