DarR, a TetR-like transcriptional factor, is a cyclic di-AMP-responsive repressor in Mycobacterium smegmatis

J Biol Chem. 2013 Feb 1;288(5):3085-96. doi: 10.1074/jbc.M112.428110. Epub 2012 Dec 17.

Abstract

Cyclic dinucleotides, including cyclic di-AMP (c-di-AMP), are known to be ubiquitous second messengers involved in bacterial signal transduction. However, no transcriptional regulator has been characterized as a c-di-AMP receptor/effector to date. In the present study, using a c-di-AMP/transcription factor binding screen, we identified Ms5346, a TetR family regulator in Mycobacterium smegmatis, as a c-di-AMP receptor in bacteria. Ms5346 could specifically bind c-di-AMP with K(d) of 2.3 ± 0.5 μM. Using EMSA and DNase I footprinting assays, c-di-AMP was found to stimulate the DNA binding activity of Ms5346 and to enhance its ability to protect its target DNA sequence. A conserved 14-bp palindromic motif was identified as the DNA-binding site for Ms5346. Further, Ms5346 was found to negatively regulate expression of three target genes including Ms5347 (encoding a major facilitator family transporter), Ms5348 (encoding a medium chain fatty acyl-CoA ligase), and Ms5696 (encoding a cold shock protein, CspA). Ms5346 is the first cyclic di-AMP receptor regulator to be identified in bacteria, and we have designated it as DarR. Our findings enhance our understanding of the function and regulatory mechanism of the second messenger c-di-AMP in bacteria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • DNA, Bacterial / metabolism
  • Dinucleoside Phosphates / pharmacology*
  • Fatty Acids / biosynthesis
  • Gene Deletion
  • Gene Expression Regulation, Bacterial / drug effects
  • Genes, Bacterial / genetics
  • Inverted Repeat Sequences / genetics
  • Molecular Sequence Data
  • Mycobacterium smegmatis / genetics
  • Mycobacterium smegmatis / growth & development
  • Mycobacterium smegmatis / metabolism*
  • Mycobacterium smegmatis / ultrastructure
  • Nucleotide Motifs / genetics
  • Promoter Regions, Genetic / genetics
  • Protein Binding / drug effects
  • Protein Binding / genetics
  • Protein Structure, Tertiary
  • Repressor Proteins / chemistry
  • Repressor Proteins / metabolism*

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Dinucleoside Phosphates
  • Fatty Acids
  • Repressor Proteins
  • cyclic diadenosine phosphate