title: Repression of btuB gene transcription in Escherichia coli by the GadX protein.
abstract: BACKGROUND
  BtuB (B twelve uptake) 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 of 5' untranslated region of btuB mRNA and the intracellular concentration of adenosylcobalamin (Ado-Cbl) would affect the translational efficiency and RNA stability of btuB gene. 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.
authors.affiliation: Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Guang-Sheng
authors.initials: GS
authors.lastname: Lei
authors.forename: Wan-Jr
authors.initials: WJ
authors.lastname: Syu
authors.forename: Po-Huang
authors.initials: PH
authors.lastname: Liang
authors.forename: Kin-Fu
authors.initials: KF
authors.lastname: Chak
authors.forename: Wensi S
authors.initials: WS
authors.lastname: Hu
authors.forename: Shiau-Ting
authors.initials: ST
authors.lastname: Hu
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000633:toxicity; D051777:AraC Transcription Factor / Q000378:metabolism*; D019677:Artificial Gene Fusion; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis*; D015153:Blotting, Western; D003087:Colicins / Q000378:metabolism / Q000633:toxicity; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D026901:Membrane Transport Proteins / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D051777:AraC Transcription Factor; D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C446316:GadX protein, E coli; D026901:Membrane Transport Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase