Crystal Structure of PhnF, a GntR-family Transcriptional Regulator of Phosphate Transport in Mycobacterium Smegmatis

J Bacteriol. 2014 Oct;196(19):3472-81. doi: 10.1128/JB.01965-14. Epub 2014 Jul 21.

Abstract

Bacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Biological Transport
  • Crystallography, X-Ray
  • Gene Expression Regulation, Bacterial
  • Membrane Transport Proteins / genetics*
  • Membrane Transport Proteins / metabolism
  • Multigene Family
  • Mycobacterium smegmatis / chemistry
  • Mycobacterium smegmatis / genetics
  • Mycobacterium smegmatis / metabolism*
  • Operon
  • Phosphates / metabolism*
  • Protein Binding
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Bacterial Proteins
  • Membrane Transport Proteins
  • Phosphates
  • Transcription Factors

Associated data

  • PDB/1G1B
  • PDB/2FA1
  • PDB/2IKK
  • PDB/2OGG
  • PDB/2OOI
  • PDB/2P19
  • PDB/2WV0
  • PDB/2WVO
  • PDB/3BWG
  • PDB/3F8L
  • PDB/3F8M