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

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.

A luminescent reporter evidences active expression of Ralstonia solanacearum type III secretion system genes throughout plant infection

“…We thus decided to develop a short-lived reporter system to precisely measure bacterial gene expression in real time during plant colonization. To this end, we chose the luxCDABE operon, a non-invasive reporter that produces light without addition of any substrate (Craney et al, 2007). The promoters under study were cloned in pRCGent-lux, a gentamicin-selection variant of the pRC vectors that contains a synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria (Craney et al, 2007;Monteiro et al, 2012).…”

“…To this end, we chose the luxCDABE operon, a non-invasive reporter that produces light without addition of any substrate (Craney et al, 2007). The promoters under study were cloned in pRCGent-lux, a gentamicin-selection variant of the pRC vectors that contains a synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria (Craney et al, 2007;Monteiro et al, 2012). This gave rise to pRCGent-Pep-lux and pRCGent-PhB-lux, which were linearized and transformed in R. solanacearum GMI1000 to generate strains with either the Peps : : lux or the PhrpB : : lux fusion inserted in the chromosome (Fig.…”

“…The bacterial operon luxCDABE encodes both the luciferase enzyme and the enzymes required for the production of its substrate, tetradecanal (Craney et al, 2007). As all requirements for the enzyme can be provided by any aerobic living cell, the entire operon is used as a non-disruptive reporter that spontaneously emits light.…”

“…This gave rise to pRCGent-Pep and pRCGent-PhB. GFPuv from pG-GFPuv (Monteiro et al, 2012) and the synthetic luxCDABE gene cluster from pMU1* (Craney et al, 2007) were sequentially cloned using KpnI/BglII and KpnI/EcoRI, respectively. NotI was used instead of EcoRI for cloning in pRCGentPep.…”

Bacterial vectors for imaging and cancer gene therapy: a review

“…A synthetic lux operon lacking TTA codons was constructed and validated for S. coelicolor. 78 Furthermore, codon optimizing the firefly luciferase for M. tuberculosis resulted in a 30-fold increase in signal. 74 However codon optimization may have unforeseen effects; the lux operon codon optimized for M. tuberculosis was found to be nonfunctional 74 reported to be as a result of secondary DNA structures which impede translation.…”