Alternative luciferase for monitoring bacterial cells under adverse conditions (original) (raw)
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Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications
Photochemistry and Photobiology
Copepod luciferases-a family of small secretory proteins of 18.4-24.3 kDa, including a signal peptide-are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine-dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high-throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.
Journal of Biological Chemistry, 2003
Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light ( max ؍ 480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultrahigh throughput screening technologies. . The abbreviations used are: GFP, green fluorescent protein; FL, firefly luciferase; CHO, Chinese hamster ovary; MLuc, Metridia luciferase; IPTG, isopropyl--D-thiogalactopyranoside; NPY2, human neuropeptide Y receptor 2; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
Protein Expression and Purification, 2008
Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (λ max = 480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, E. coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2-3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo posttranslational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis. Although the phenomenon of bioluminescence is widespread most luminous species are marine inhabitants. Many of these marine systems use imidazopyrazinone derivatives as the luciferin and the one most commonly isolated is coelenterazine. Coelenterazine and its derivatives are used by many bioluminescent proteins such as Renilla luciferase [1], the Ca 2+-regulated photoproteins [2,3], luciferase from the scyphozoan medusa Periphylla [4], squid photoproteins [5], etc. The best-characterized examples of such proteins are Ca 2+-regulated photoproteins and Renilla luciferase, the ones responsible for bioluminescence of animals belonging to the classes Hydrozoa and Anthozoa respectively. Crystal structures are now available for a number of coelenterazine-dependent proteins, including several structures of different ligand-dependent conformational states of photoproteins [6-10], Renilla luciferase
2nd International Conference on Biological Research and Applied Science, 2022
Bioluminescence bacteria (BLB) are the most abundant and widely distributed light-emitting organisms that can be found in the marine environment. They are suitable for detecting pollution or integrated into bioluminescent imaging due to their ability to luminesce. However, there are limited studies regarding bioluminescent bacteria in terms of distribution and species present in Malaysia. This study aims to isolate BLB and amplify the luciferase (luxAB) genes. Bioluminescent bacteria were isolated from the guts of marine fish, Selaroidesleptolepis and streak onto luminescence agar (LA). The brightest luminous colony present in the dark was marked and streaked again obtain a pure colony. Then, the pure culture of the colony was subjected to genomic extraction before luxAB genes amplification and phylogenetic analysis. As a result, BLB were successfully isolated and identified to be Photobacteriumleiognathis train SYA2 (MZ491870.1). Genes encoding for luciferase enzyme were also amplified and sequenced with the size of luxA and luxB were 767 bp and 943 bp, respectively. Pairwise distance showed that the isolate has the highest similarity to P. leiognathi (DQ790853) with 99.66% and the lowest similarity to P. kishitanii (AY642227) with 69.84%. The information about the isolate will contribute to the distribution of BLB in Malaysia as well as potential of BLB as a biosensor and bioreporter.
The Sensitized Bioluminescence Mechanism of Bacterial Luciferase
Photochemistry and Photobiology, 2018
After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH2 assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are f...
Biochemical and …, 1988
The nucleotide sequence of a new gene, luxF, located between the luxB and E genes in the bioluminescent system of Photobacterium phosphoreum has been determined. The luxF gene codes for a polypeptide of 231 amino acids which is homologous to the a and 8 subunits of luciferase coded by the luxA and luxB genes, respectively. The degree of homology of the luxF protein is very high with the B subunit of luciferase (~ 30% identity) with greatest similarity to the Vibrio luxB proteins. The !uxF gene appears to have evolved by duplication of the luxB gene followed by deletion of approximately I00 codons just penultimate to the 5'-terminal. The close homology with the luciferase B subunit implicates the luxF protein in a function related to the light-emitting reaction.
Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay1
FEMS Microbiology Ecology, 2000
A 745-bp luxA fragment was amplified from Vibrio harveyi (UM 1503), radiolabeled, and used as a probe to detect and quantify luxA genotypes in culturable bacterial populations from the Chesapeake Bay. DNA samples from 53 reference strains were also examined for this gene. The luxA-positive bacteria comprised from 0^6% of the culturable heterotrophic bacterial community in samples from the Bay. Only those reference strains known to be luminescent contained the luxA gene, as indicated by PCR. Results in all cases were confirmed by PCR of DNA extracts and Southern hybridization analyses, using an internal probe for confirmation of luxA amplification products. Sequence analysis of luxA genes from three nonluminescent bacteria isolated from the Chesapeake Bay indicated little or no differences when compared with luxA sequences from known marine luminescent bacterial species. These three Chesapeake Bay strains and other luxApositive strains were tested with a luminometer and confirmed to be nonluminescent. All of over 7800 bacterial colonies enumerated during this study from Chesapeake Bay samples were non-visibly luminescent. Our results indicate that luxA-positive bacteria isolated from the Chesapeake Bay are not generally luminescent on phenotypic examination, implying that gene probe techniques are required for examining luxA gene distribution in microbial populations present in environmental samples. ß 2000 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
Environmental Toxicology & Water Quality, 1995
The bacterial bioluminescence assay is widely used to estimate chemical cytotoxicity. This assay is performed most often by using luminescent bacteria Vibrio fisheri NRRL-B-11177 (earlier cited as Photobacterium phosphoreum NRRL-B-11177) as a test organism. In this work we have used cloned gram(+) and gram(−) bacterial strains for the evaluation of chemical toxicity. Two types of luciferase genes were used as reporter genes, one of which was ATP-dependent eukaryotic luciferase from Pyrophorus plagiophthalamus and the other was FMN-dependent bacterial luciferase from Vibrio harveyi. These cloned strains were used to evaluate the effects of differences in cell wall structures in the bioluminescence cytotoxicity test using small molecular weight toxic chemicals as model compounds. The strains were found to have remarkably similar behavior and sensitivity toward test toxicants regardless of rather different kinds of membrane structure. However, the eukaryotic luciferase was found in every aspect more useful in toxicity tests than bacterial luciferase mainly because of increased sensitivity and ease of operation. The general behavior of the light emission is described and several aspects affecting the usefulness of the strains are discussed. © by John Wiley & Sons, Inc.
Biochemical and Biophysical Research Communications, 2015
Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SAS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of $3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging.