The CRP-cAMP complex and downregulation of the glnAp2 promoter provides a novel regulatory linkage between carbon metabolism and nitrogen assimilation in Escherichia coli.
In Escherichia coli, glnA (encoding glutamine synthetase) is transcribed from two promoters (glnAp1 and glnAp2). The glnAp1 is a sigma(70)-dependent promoter that is activated by the cAMP receptor protein (CRP). Under nitrogen-deficient growth conditions, glnAp1 is repressed by NtrC-phosphate. The downstream glnAp2 promoter is sigma(54)-dependent and is activated by NtrC-phosphate. Here, we show that glnAp2 expression is affected by different carbon sources and that the CRP-cAMP complex inhibits the glnAp2 promoter activity. Primer extension and KMnO4 footprinting analysis indicate that the inhibitory effect is at the transcriptional level in vivo. When glnAp2 is activated by NifA, a similar inhibitory effect by CRP-cAMP is observed. Site-directed mutagenesis and deletion analysis indicate that the characterized and putative CRP-binding sites located in the upstream region of the glnAp2 promoter are not essential for the inhibitory effect. CRP-cAMP inhibits sigma(54)-dependent glnAp2 strongly, by 21-fold. By activating glnAp1 and downregulating glnAp2, the overall effect of CRP-cAMP on glnA expression is an approximately fourfold reduction, which correlates with the reduction of gamma-glutamyl transferase activities in the cells. We propose therefore that a physiological role of CRP-cAMP activation of glnAp1 is to partially compensate for CRP-cAMP downregulation of glnAp2, allowing a low but non-negligible level of expression of the important genes transcribed from it. A novel regulatory linkage between carbon and nitrogen regulons is proposed.
Tian
Z X
ZX
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China.
Li
Q S
QS
Buck
M
M
Kolb
A
A
Wang
Y P
YP
D002244:Carbon / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015536:Down-Regulation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D016296:Mutagenesis; D009584:Nitrogen / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism; D014158:Transcription, Genetic
D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D012808:Sigma Factor; C497751:rpoN protein, E coli; D002244:Carbon; D000242:Cyclic AMP; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; C063842:dnaK protein, E coli; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen