Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

Abstract

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.

Gaussia princeps Luciferase as a Reporter for Transcriptional Activity, Protein Secretion, and Protein-Protein Interactions in Salmonella enterica Serovar Typhimurium

“…With Gluc-expressing M. smegmatis, there is a linear relationship between luminescence and substrate concentration between 0.1 M and 10 M CTZ (30). Using whole cells or a 1:100 dilution of Salmonella lysates, we tested native CTZ in concentrations between 0.01 M and 100 M (Fig.…”

“…Gluc genes codon optimized for expression in the respective hosts were used in reporter assays with the fungus pathogen Candida albicans (5) and the bacteria Mycobacterium smegmatis (1,30) and Mycobacterium tuberculosis (1) as well as the alga Chlamydomonas reinhardtii (24). Recently, heterologous expression of highly active and soluble Gluc in Escherichia coli was reported, suggesting its usefulness as a reporter gene in Enterobacteriaceae (22).…”

“…A humanized variant of Gluc (hGluc), which was codon optimized for expression in cultured mammalian cells, has been widely used as a reporter gene (26). Gaussia luciferase exhibits an activity up to 1,000-fold higher than to Renilla reniformis luciferase (Rluc), firefly luciferase (Fluc) (26), or bacterial luciferases (LuxAB) (30). The outstanding sensitivity of Gluc-based assays was previously demonstrated detecting as low as 10 Ϫ18 mol purified Gluc (27) or one eukaryotic cell transiently expressing Gluc (25).…”

“…Gluc shares with Rluc its independence of any host-derived cofactors. Additionally, Gluc shows a good robustness regarding changes in pH (12,30), heat shock, and hydrogen peroxide (30). A split form of Gluc was applied as a reporter in protein complementation assays (PCA) to assess protein-protein interactions (15,23).…”

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model

“…smegmatis, using mycobacterial-shuttle plasmid vectors (Blokpoel et al, 2005;Wiles et al, 2005). The expresssion vector used here contains the tetRO region from the Corynebacterium glutamicum TetZ, making expression of genes cloned downstream of tetRO responsive to tetracycline We demonstrate the purification of the recombinant Hybrid-1 proteins from both bacterial hosts and analysis of their biochemical and immunological characteristics, in order to determine whether there are any differences in their immunogenicity.…”