Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

Abstract

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.

Autoren

Susanne Gebhard
Jason N Busby
Georg Fritz
Nicole J Moreland
Gregory M Cook
J Shaun Lott
Edward N Baker
Victoria A Money

DOI

10.1128/jb.01965-14

eISSN

1098-5530

ISSN

0021-9193

Ausgabe der Veröffentlichung

19

Zeitschrift

Journal of Bacteriology

Sprache

en

Paginierung

3472 - 3481

Datum der Veröffentlichung

2014

Status

Published

Herausgeber

American Society for Microbiology

Herausgeber URL

http://dx.doi.org/10.1128/jb.01965-14

Datum der Datenerfassung

2021

Titel

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

Ausgabe der Zeitschrift

196

Data source: Crossref

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.

Addresses

Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Department Biology I, Mikrobiologie, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany susanne.gebhard@bio.lmu.de v.a.money@durham.ac.uk.

Autoren

Susanne Gebhard
Jason N Busby
Georg Fritz
Nicole J Moreland
Gregory M Cook
J Shaun Lott
Edward N Baker
Victoria A Money

DOI

10.1128/jb.01965-14

eISSN

1098-5530

Externe Identifier

PubMed Identifier: 25049090
PubMed Central ID: PMC4187667

Open access

false

ISSN

0021-9193

Ausgabe der Veröffentlichung

19

Zeitschrift

Journal of bacteriology

Schlüsselwörter

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

Sprache

eng

Medium

Print-Electronic

Online publication date

2014

Paginierung

3472 - 3481

Datum der Veröffentlichung

2014

Status

Published

Datum der Datenerfassung

2014

Titel

Crystal structure of PhnF, a GntR-family transcriptional regulator of phosphate transport in Mycobacterium smegmatis.

Sub types

Research Support, Non-U.S. Gov't
research-article
Journal Article

Ausgabe der Zeitschrift

196

Files

https://jb.asm.org/content/jb/196/19/3472.full.pdf https://europepmc.org/articles/PMC4187667?pdf=render

Data source: Europe PubMed Central

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.

Autoren

Susanne Gebhard
Jason N Busby
Georg Fritz
Nicole J Moreland
Gregory M Cook
J Shaun Lott
Edward N Baker
Victoria A Money

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/25049090

DOI

10.1128/JB.01965-14

eISSN

1098-5530

Externe Identifier

PubMed Central ID: PMC4187667

Ausgabe der Veröffentlichung

19

Zeitschrift

J Bacteriol

Schlüsselwörter

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

Sprache

eng

Country

United States

Paginierung

3472 - 3481

PII

JB.01965-14

Datum der Veröffentlichung

2014

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2014

Titel

Crystal structure of PhnF, a GntR-family transcriptional regulator of phosphate transport in Mycobacterium smegmatis.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

196

Data source: PubMed

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

Files

Tools