The expression of the Photinus pyralis luciferase gene in Staphylococcus aureus Cowan I allows the development of a live amplifiable tool for immunodetection (original) (raw)
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Journal of Immunological Methods, 1991
The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the fl subunit of luciferase. Only the first fusion protein encoding the entire/3 subunit was able to form an enzymatically active luciferase complex when expressed together with the a subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays.
Alternative luciferase for monitoring bacterial cells under adverse conditions
Applied and …, 2005
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.
International Journal of Medical Microbiology, 2008
Alpha-toxin (Hla, encoded by hla) is a major virulence factor of Staphylococcus aureus. The activity of the hla promoter was analyzed using luxABCDE on an integration vector. The phla-lux construct was introduced in S. aureus Newman and its isogenic sae and sigB regulator mutants. Promoter activity was monitored by bioluminescence in vitro and in the murine tissue-cage model. Hla promoter activity could be followed in real time at repeated time points of infection. The activation of hla in the sigB-deficient strain and the repression to background levels in a sae-deficient strain relative to hla expression in the wild type could be demonstrated in vivo. Subinhibitory concentrations of teicoplanin, imipenem and ciprofloxacin enhanced hla promoter activity in vitro whereas clindamycin and rifampicin did not. Our approach proved to be rapid and adequate to study promoter activity in vitro and in vivo under conditions where high bacterial numbers are reached.
Cell surface display of recombinant proteins on Staphylococcus carnosus
Journal of Bacteriology, 1995
A novel expression system for surface display of heterologous proteins on Staphylococcus carnosus cells has been developed. Taking advantage of the promoter and secretion signals, including a propeptide region, from the lipase gene of Staphylococcus hyicus and the cell wall-spanning and membrane-binding region of protein A from Staphylococcus aureus, efficient surface display of an 80-amino-acid peptide from a malaria blood stage antigen could be achieved. A serum albumin binding protein from streptococcal protein G was used both as a general reporter molecule and to increase the accessibility of the surface-displayed proteins. Immunoblotting, immunogold staining, and immunofluorescence on intact recombinant S. carnosus cells verified the presence of the propeptide, the malaria antigen, and the albumin-binding reporter protein on the bacterial surface. For the first time, fluorescence-activated cell sorting was used to analyze the presence of surface-displayed hybrid receptors on gr...
Fluorescent reporters for Staphylococcus aureus
Journal of Microbiological Methods, 2009
With the emergence of Staphylococcus aureus as a prominent pathogen in community and healthcare settings, there is a growing need for effective reporter tools to facilitate physiology and pathogenesis studies. Fluorescent proteins are ideal as reporters for their convenience in monitoring gene expression, performing host interaction studies, and monitoring biofilm growth. We have developed a suite of fluorescent reporter plasmids for labeling S. aureus cells. These plasmids encode either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling. The reporters were placed under control of characterized promoters to enable constitutive or inducible expression. Additionally, plasmids were assembled with fluorescent reporters under control of the agr quorum-sensing and Sigma factor B promoters, and the fluorescent response with wildtype and relevant mutant strains was characterized. Interestingly, reporter expression displayed a strong dependence on ribosome binding site (RBS) sequence, with the superoxide dismutase RBS displaying the strongest expression kinetics of the sequences examined. To test the robustness of the reporter plasmids, cell imaging was performed with fluorescence microscopy and cell populations were separated using florescence activated cell sorting (FACS), demonstrating the possibilities of simultaneous monitoring of multiple S. aureus properties. Finally, a constitutive YFP reporter displayed stable, robust labeling of biofilm growth in a flow cell apparatus. This toolbox of fluorescent reporter plasmids will facilitate cell labeling for a variety of different experimental applications.
Measurement of Effects of Antibiotics in Bioluminescent Staphylococcus aureus RN4220
Antimicrobial Agents and Chemotherapy, 2001
The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimized, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiotics which is applied to a gram-positive strain, Staphylococcus aureus RN4220. The method utilizes the firefly luciferase reporter gene coupled to the metal-inducible cadA promoter in a plasmid, pTOO24. Correctly timed induction by micromolar concentrations of antimonite rapidly triggers the luciferase gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and translational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain where luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emission from intact living cells is shown to correlate extremely well (r ؍ 0.99) with the conventional overnight growth inhibition measurement. Four of the antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the assay time needed, and it can be performed in 1 to 4 h, depending on the sensitivity needed. Furthermore, the assay can be automatized for high-throughput screening by the pharmaceutical industry.
A destabilized bacterial luciferase for dynamic gene expression studies
Systems and Synthetic Biology, 2006
Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional halflife of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multisubunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression.
2002
The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsred) in whole-cell bacterial sensors. Escherichia coli sensor bacteria were engineered to contain a reporter plasmid that carries the reporter gene under the control of mercury- (mer from Tn21) or arsenite- (ars from R773) responsive regulatory units. Characteristics of the strains were studied by using different arsenite or mercury concentrations and incubation times. The lowest detectable concentration of analytes and the fastest responses were achieved with lucFF or luxCDABE as reporter genes. The fluorescent proteins, GFP and DsRed, gave responses at higher analyte concentrations and after ...