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Nucleic Acids Research Advance Access published online on March 1, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm086
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Arryn Craney, Tobias Hohenauer, Ye Xu, Naveen Kumar Navani, Yingfu Li and Justin Nodwell*

Biochemistry & Biomedical Sciences, McMaster University, Health Sciences Centre,1200 Main Street W Hamilton, Ontario L8N 3Z5, Canada

*To whom correspondence should be addressed. Tel: +1-905 525 9140; Fax: +1-905 522 9033; Email: nodwellj{at}mcmaster.ca

Received December 11, 2006. Revised January 29, 2007. Accepted January 30, 2007.

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.

The authors wish it to be known that in their opinion the first two authors should be regarded as joint First Authors.


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