A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria

Nucleic Acids Res. 2007;35(6):e46. doi: 10.1093/nar/gkm086. Epub 2007 Mar 1.

Abstract

The luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens has proven to be a superb transcriptional reporter. It encodes a luciferase (LuxA and LuxB) and the enzymes that produce its substrate (LuxC, LuxD and LuxE) so cells that express the cluster emit the 490-nm light spontaneously. The sequence of these genes is AT-rich (>69%) and for this and other reasons, they are not expressed efficiently in high-GC bacteria like Streptomyces coelicolor. We therefore constructed a synthetic luxCDABE operon encoding the P. luminescens Lux proteins optimized for expression in high-GC bacteria. We tested the genes using transcriptional fusions to S. coelicolor promoters having well-established expression profiles during this organism's life cycle. The hrdB gene encodes a housekeeping sigma factor; while ramC is important for the formation of the spore-forming cells called aerial hyphae and whiE is required for the production of a grey, spore-associated pigment that is deposited in the walls of developing spores. Using these fusions we demonstrated that our synthetic lux genes are functional in S. coelicolor and that they accurately report complex developmental gene expression patterns. We suggest that this lux operon and our procedure for generating synthetic high-GC genes will be widely useful for research on high-GC bacteria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Cloning, Molecular
  • DNA, Bacterial / chemistry
  • DNA-Binding Proteins / genetics
  • GC Rich Sequence
  • Genes, Reporter*
  • Genes, Synthetic*
  • Luciferases, Bacterial / genetics*
  • Luminescent Agents*
  • Operon*
  • Photorhabdus / genetics*
  • Polymerase Chain Reaction / methods
  • Promoter Regions, Genetic
  • Recombinant Fusion Proteins / analysis
  • Sigma Factor / genetics
  • Streptomyces coelicolor / genetics*
  • Streptomyces coelicolor / metabolism

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • DNA-Binding Proteins
  • Luminescent Agents
  • Recombinant Fusion Proteins
  • Sigma Factor
  • HrdB protein, Streptomyces
  • Luciferases, Bacterial