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

Lei GS, Syu WJ, Liang PH, Chak KF, Hu WS, Hu ST - BMC Microbiol. (2011)

Bottom Line: 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.The results showed the transcription of gadX with 1.4-fold increase but the level of btuB was reduced to 57%.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan.

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.

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Structures of pGAD10, pGadXY, pGadX, and pGadY. pGAD10 was the vector used to clone gadXY, gadX, and gadY. pGadXY has a 1,470-bp fragment containing gadX, gadY, and a portion of gadW of E. coli K-12 genomic DNA inserted into the EcoRI site of pGAD10. pGadX contains a DNA fragment carrying the 825-bp gadX also inserted into the EcoRI site of pGAD10. pGadY is derived from pGadXY by deleting the 601-bp NcoI-DraIII fragment and thus contains a truncated gadX, the entire gadY, and a portion of gadW. Nucleotide sequences of the promoter regions of gadX and gadY are shown. The orientation of gadX is opposite to that of gadY. The sigma factor S (RpoS) recognition site and the Shine-Dalgarno (SD) sequence are shown in the 5' end region of gadX. PADH is the promoter of GAL4-AD and is not functional in E. coli.
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Figure 1: Structures of pGAD10, pGadXY, pGadX, and pGadY. pGAD10 was the vector used to clone gadXY, gadX, and gadY. pGadXY has a 1,470-bp fragment containing gadX, gadY, and a portion of gadW of E. coli K-12 genomic DNA inserted into the EcoRI site of pGAD10. pGadX contains a DNA fragment carrying the 825-bp gadX also inserted into the EcoRI site of pGAD10. pGadY is derived from pGadXY by deleting the 601-bp NcoI-DraIII fragment and thus contains a truncated gadX, the entire gadY, and a portion of gadW. Nucleotide sequences of the promoter regions of gadX and gadY are shown. The orientation of gadX is opposite to that of gadY. The sigma factor S (RpoS) recognition site and the Shine-Dalgarno (SD) sequence are shown in the 5' end region of gadX. PADH is the promoter of GAL4-AD and is not functional in E. coli.

Mentions: To search for genes that can confer E. coli resistance to ColE7, plasmids in the genomic library were transformed into the ColE7-sensitive E. coli strain DH5α, and the transformants were plated on LB agar plates containing 50 μg/ml of ampicillin and 5.0 ng/ml of His6-tagged ColE7/ImE7. Two colonies were seen after incubation at 37°C overnight. The plasmids of each colony were isolated after culturing in 3 ml LB medium containing 50 μg/ml of ampicillin then retransformed into DH5α. The new transformants were plated on agar plates containing 0, 1.3, 2.6, 3.9, or 5.2 ng/ml of His6-tagged ColE7/ImE7 to confirm their resistance to ColE7. The insert in the plasmid that conferred DH5α resistance to 5.2 ng/ml His6-tagged ColE7/ImE7 was sequenced. A 1,470-bp DNA region on the chromosome at position 3662617 to 3664086 was analyzed that contains both complete gadX and gadY genes. The plasmid was thus named pGadXY (Figure 1).


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

Lei GS, Syu WJ, Liang PH, Chak KF, Hu WS, Hu ST - BMC Microbiol. (2011)

Structures of pGAD10, pGadXY, pGadX, and pGadY. pGAD10 was the vector used to clone gadXY, gadX, and gadY. pGadXY has a 1,470-bp fragment containing gadX, gadY, and a portion of gadW of E. coli K-12 genomic DNA inserted into the EcoRI site of pGAD10. pGadX contains a DNA fragment carrying the 825-bp gadX also inserted into the EcoRI site of pGAD10. pGadY is derived from pGadXY by deleting the 601-bp NcoI-DraIII fragment and thus contains a truncated gadX, the entire gadY, and a portion of gadW. Nucleotide sequences of the promoter regions of gadX and gadY are shown. The orientation of gadX is opposite to that of gadY. The sigma factor S (RpoS) recognition site and the Shine-Dalgarno (SD) sequence are shown in the 5' end region of gadX. PADH is the promoter of GAL4-AD and is not functional in E. coli.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050690&req=5

Figure 1: Structures of pGAD10, pGadXY, pGadX, and pGadY. pGAD10 was the vector used to clone gadXY, gadX, and gadY. pGadXY has a 1,470-bp fragment containing gadX, gadY, and a portion of gadW of E. coli K-12 genomic DNA inserted into the EcoRI site of pGAD10. pGadX contains a DNA fragment carrying the 825-bp gadX also inserted into the EcoRI site of pGAD10. pGadY is derived from pGadXY by deleting the 601-bp NcoI-DraIII fragment and thus contains a truncated gadX, the entire gadY, and a portion of gadW. Nucleotide sequences of the promoter regions of gadX and gadY are shown. The orientation of gadX is opposite to that of gadY. The sigma factor S (RpoS) recognition site and the Shine-Dalgarno (SD) sequence are shown in the 5' end region of gadX. PADH is the promoter of GAL4-AD and is not functional in E. coli.
Mentions: To search for genes that can confer E. coli resistance to ColE7, plasmids in the genomic library were transformed into the ColE7-sensitive E. coli strain DH5α, and the transformants were plated on LB agar plates containing 50 μg/ml of ampicillin and 5.0 ng/ml of His6-tagged ColE7/ImE7. Two colonies were seen after incubation at 37°C overnight. The plasmids of each colony were isolated after culturing in 3 ml LB medium containing 50 μg/ml of ampicillin then retransformed into DH5α. The new transformants were plated on agar plates containing 0, 1.3, 2.6, 3.9, or 5.2 ng/ml of His6-tagged ColE7/ImE7 to confirm their resistance to ColE7. The insert in the plasmid that conferred DH5α resistance to 5.2 ng/ml His6-tagged ColE7/ImE7 was sequenced. A 1,470-bp DNA region on the chromosome at position 3662617 to 3664086 was analyzed that contains both complete gadX and gadY genes. The plasmid was thus named pGadXY (Figure 1).

Bottom Line: 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.The results showed the transcription of gadX with 1.4-fold increase but the level of btuB was reduced to 57%.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan.

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.

Show MeSH
Related in: MedlinePlus