Involvement of the global Crp regulator in cyclic AMP-dependent utilization of aromatic amino acids by Pseudomonas putida

J Bacteriol. 2012 Jan;194(2):406-12. doi: 10.1128/JB.06353-11. Epub 2011 Nov 11.

Abstract

The phhAB operon encodes a phenylalanine hydroxylase involved in the conversion of L-phenylalanine into L-tyrosine in Pseudomonas putida. The phhAB promoter is transcribed by RNA polymerase sigma-70 and is unusual in that the specific regulator PhhR acts as an enhancer protein that binds to two distant upstream sites (-75 to -92 and -132 to -149). There is an integration host factor (IHF) binding site that overlaps the proximal PhhR box, and, consequently, IHF acts as an inhibitor of transcription. Use of L-phenylalanine is compromised in a crp-deficient background due to reduced expression from the phhAB promoter. Electrophoretic mobility shift assays and DNase I footprinting assays reveal that Crp binds at a site centered at -109 only in the presence of cyclic AMP (cAMP). We show, using circular permutation analysis, that the simultaneous binding of Crp/cAMP and PhhR bends DNA to bring positive regulators and RNA polymerase into close proximity. This nucleoprotein complex promotes transcription from phhA only in response to L-phenylalanine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids, Aromatic / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Conditioning, Operant
  • Cyclic AMP / genetics
  • Cyclic AMP / metabolism*
  • Cyclic AMP Receptor Protein / genetics
  • Cyclic AMP Receptor Protein / metabolism*
  • Gene Expression Regulation, Bacterial / physiology
  • Models, Molecular
  • Phenylalanine / metabolism
  • Pseudomonas putida / genetics*
  • Pseudomonas putida / metabolism*
  • Tyrosine / metabolism

Substances

  • Amino Acids, Aromatic
  • Bacterial Proteins
  • Cyclic AMP Receptor Protein
  • Tyrosine
  • Phenylalanine
  • Cyclic AMP