Abstract

Because the phosphoenolpyruvate:sugar phosphotransferase system plays multiple regulatory roles in addition to the phosphorylation-coupled transport of many sugars in bacteria, synthesis of its protein components is regulated in a highly sophisticated way. Thus far, the cAMP receptor protein (CRP) complex and Mlc are known to be the major regulators of ptsHIcrr and ptsG expression in response to the availability of carbon sources. In this report, we performed ligand fishing experiments by using the promoters of ptsHIcrr and ptsG as bait to find out new factors involved in the transcriptional regulation of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, and we found that the anaerobic regulator ArcA specifically binds to the promoters. Deletion of the arcA gene caused about a 2-fold increase in the ptsG expression, and overexpression of ArcA significantly decreased glucose consumption. In vitro transcription assays showed that phospho-ArcA (ArcA-P) represses ptsG P1 transcription. DNase I footprinting experiments revealed that ArcA-P binds to three sites upstream of the ptsG P1 promoter, two of which overlap the CRP-binding sites, and the ArcA-P binding decreases the CRP binding that is essential for the ptsG P1 transcription. These results suggest that the response regulator ArcA regulates expression of enzyme IICB(Glc) mediating the first step of glucose metabolism in response to the redox conditions of growth in E. coli.

Highlights

  • Because the phosphoenolpyruvate:sugar phosphotransferase system plays multiple regulatory roles in addition to the phosphorylation-coupled transport of many sugars in bacteria, synthesis of its protein components is regulated in a highly sophisticated way

  • We performed ligand fishing experiments by using the promoters of ptsHIcrr and ptsG as bait to find out new factors involved in the transcriptional regulation of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, and we found that the anaerobic regulator ArcA binds to the promoters

  • It was reported that expression of the pts operon and the ptsG gene increases a few fold during growth on glucose or other phosphotransferase system (PTS) substrates [1], and the functional cAMP1⁄7CRP complex is essential for the expression of the ptsG gene [1, 11]

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Summary

Genotype or phenotype

W3110 ⌬lacU169 gal490 ␭CI857 ⌬(cro-bioA) FϪ ␭Ϫ lacIq lacPL8 ampC::Ptrp cI FϪ araD139 ⌬(argF-lac)U169 flbB5301 thiA deoC1 relA1 rbsR rpsL150 ptsF25 Wild type E. coli MC4100 suhX1 ptsG::cat DY330 arcA::cat MC4100 arcA::cat MC4100 ␭ ͓⌽(ptsGЈ-ЈlacZ)͔ YJ2002 arcA::cat DY330 mlc::TetR YJ2002 mlc::TetR YJ2003 mlc::TetR.

This study
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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