Modulation of CRP‐dependent transcription at the Escherichia coli acsP2 promoter by nucleoprotein complexes: anti‐activation by the nucleoid proteins FIS and IHF

• Published in: Molecular microbiology 2004-01, Vol.51 (1), p.241-254
• Main Authors: Browning, Douglas F., Beatty, Christine M., Sanstad, Erik A., Gunn, Kathryn E., Busby, Stephen J. W., Wolfe, Alan J.
• Format: Article
• Language: English
• Subjects: Acetate-CoA Ligase - genetics; Bacteriology; Base Sequence; Binding Sites; Biological and medical sciences; Cyclic AMP Receptor Protein; DNA Primers; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA-Directed RNA Polymerases - metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial - genetics; Microbiology; Miscellaneous; Molecular Sequence Data; Nuclear Proteins - genetics; Nucleoproteins - genetics; Promoter Regions, Genetic; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Regulatory Sequences, Nucleic Acid; Sequence Alignment; Sequence Homology, Nucleic Acid; Transcription Factors; Transcription, Genetic
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.2003.03824.x

Summary acs encodes acetyl‐coenzyme A synthetase, a high‐affinity enzyme that allows cells to scavenge for acetate during carbon starvation. CRP activates acs transcription by binding tandem DNA sites located upstream of the major promoter, acsP2. Here, we used electrophoretic mobility shift assays and DNase I footprint analyses to demonstrate that the nucleoid proteins FIS and IHF each bind multiple sites within the acs regulatory region, that FIS competes successfully with CRP for binding to their overlapping and neighbouring sites and that IHF binds independently of either FIS or CRP. Using in vitro transcription assays, we demonstrated that FIS and IHF independently reduce CRP‐dependent acs transcription. Using in vivo reporter assays, we showed that disruption of DNA sites for FIS or deletion of DNA sites for IHF increases acs transcription. We propose that FIS and IHF each function directly as anti‐activators of CRP, each working independently at different times during growth to set the levels of CRP‐dependent acs transcription.