Rhythmic gene expression in a purple photosynthetic bacterium, Rhodobacter sphaeroides

Circadian rhythms are known to exist in all groups of eukaryotic organisms as well as oxygenic photosynthetic bacteria, cyanobacteria. However, little information is available regarding the existence of rhythmic behaviors in prokaryotes other than cyanobacteria. Here we report biological rhythms of gene expression in a purple bacterium Rhodobacter sphaeroides by using a luciferase reporter gene system. Self-bioluminescent strains of Rb. sphaeroides were constructed, which produced a bacterial luciferase and its substrate, a long chain fatty aldehyde, to sustain the luminescence reaction. After being subjected to a temperature or light entrainment regime, the reporter strains with the luciferase genes driven by an upstream endogenous promoter expressed self-sustained rhythmicity in the constant free-running period. The rhythms were controlled by oxygen and exhibited a circadian period of 20.5 h under aerobic conditions and an ultradian period of 10.6-12.7 h under anaerobic conditions. The data suggest a novel endogenous oscillation mechanism in purple photosynthetic bacteria. Elucidation of the clock-like behavior in purple bacteria has implications in understanding the origin and evolution of circadian rhythms.     

Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast

Luciferase reporter genes have been successfully used in a variety of organisms to examine gene expression in living cells, but are yet to be successfully developed for use in chloroplast. Green fluorescent protein (gfp) has been used as a reporter of chloroplast gene expression, but because of high auto-fluorescence, very high levels of GFP accumulation are required for visualization in vivo. We have developed a luciferase reporter for chloroplast by synthesizing the two-subunit bacterial luciferase (lux)AB, as a single fusion protein in Chlamydomonas reinhardtii chloroplast codon bias. We expressed a chloroplast luciferase gene, luxCt, in C. reinhardtii chloroplasts under the control of the ATPase alpha subunit (atpA) or psbA promoter and 5' untranslated regions (UTRs) and the rubisco large subunit (rbcL) 3' UTR. We show that luxCt is a sensitive reporter of chloroplast gene expression, and that luciferase activity can be measured in vivo using a charge coupled device (CCD) camera or in vitro using a luminometer. We further demonstrate that luxCt protein accumulation, as measured by Western blot analysis, is proportional to luminescence, as determined both in vivo and in vitro, and that luxCt is capable of reporting changes in chloroplast gene expression during a dark to light shift. These data demonstrate the utility of the luxCt gene as a versatile and sensitive reporter of chloroplast gene expression in living cells.     

Detection of alkanes, alcohols, and aldehydes using bioluminescence

We report a novel method for the rapid, sensitive, and quantitative detection of alkanes, alcohols, and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light. Primary alcohols with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alcohol dehydrogenase. In addition, alkanes themselves may be detected by conversion to primary alcohols by an alkane hydroxylase, followed by conversion to the aldehyde by alcohol dehydrogenase. We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alcohol dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E. coli host cell line. Alkanes, alcohols, or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader. We demonstrate the application of this method for the detection of alkanes, alcohols, and aldehydes and for the detection of alkane hydroxylase and alcohol dehydrogenase activity in vivo. This method is amenable to the high-throughput screening needs required for the identification of novel catalysts.     

Action of phenobarbital on bacterial luciferase of Photobacterium fischeri

The effect of phenobarbital, a typical substrate of monooxygenases from higher organisms, on bioluminescence of the marine bacterium Photobacterium fischeri and bacterial luciferase was studied. Phenobarbital was shown to be an effective quenching agent owing to the interaction with cytochrome P-450, a terminal luciferase component. A competitive interrelation was found between phenobarbital and an aliphatic aldehyde, the substrate of the luminescent reaction.     

Molecular biology of bacterial bioluminescence.

The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.     

A thermostable Clostridium thermocellum lichenase-based reporter system for studying the gene expression regulation in prokaryotic and eukaryotic cells

A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells. The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter. The gene product was active and did not undergo modification in heterologous hosts. Simple and sensitive methods were used to detect and to quantitate the lichenase activity. Inducible licBM2 expression was demonstrated with E. coli and yeast cells, allowing the system to be employed in dynamic studies.     

Bacterial DNA methylation and gene transfer efficiency

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam(+), dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in eukaryotic cells or organisms.     

Toxin–antitoxin-stabilized reporter plasmids for biophotonic imaging of Group A streptococcus

Bioluminescence is a rapid and cost-efficient optical imaging technology that allows the detection of bacteria in real-time during disease development. Here, we report a novel strategy to generate a wide range of bioluminescent group A streptococcus (GAS) strains by using a toxin–antitoxin-stabilized plasmid. The bacterial luciferin–luciferase operon (lux) or the firefly luciferase gene (ffluc) was introduced into GAS via a stabilized plasmid. The FFluc reporter gave significantly stronger bioluminescent signals than the Lux reporter, and was generally more stable. Plasmid-based luciferase reporters could easily be introduced into a variety of GAS strains and the signals correlated linearly with viable cell counts. Co-expression of the streptococcal ω–ε–ζ toxin–antitoxin operon provided segregational stability in the absence of antibiotics for at least 17 passages in vitro and up to 7 days in a mouse infection model. In addition, genome-integrated reporter constructs were also generated by site-specific recombination, but were found to be technically more challenging. The quick and efficient generation of various M-type GAS strains expressing plasmid-based luciferase reporters with comparable and quantifiable bioluminescence signals allows for comparative analysis of different GAS strains in vitro and in vivo.     

Bacterial toxin-antitoxin gene system as containment control in yeast cells

The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae. Expression of the relE gene was highly toxic to yeast cells. However, expression of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae. Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial fermentation processes in which the escape of genetically modified cells would be considered highly risky.     

Bacterial and firefly luciferase genes in transgenic plants: Advantages and disadvantages of a reporter gene

Genes encoding light-emitting luciferase were recently isolated from luminous marine bacteria and fireflies. Expression of luciferase genes in diverse organisms is a unique way for studying gene expression by simple and sensitive measurement of light. Recent advances in application of luciferase reporter genes are reviewed and documented by examples of in vivo visualization of their expression in transgenic plants.     

Rapid screening method for the detection of antimicrobial substances

Bioluminescence is phenomenon where living organisms produce light and this production is directly dependent on metabolic activity of the organism. Genes encoding enzymes, luciferases, responsible for light production can be cloned into indicator strains, thus allowing sensitive detection of antimicrobial activity. This study utilized bacterial luciferase genes cloned into Staphylococcus aureus, Escherichia coli and Salmonella enterica serovar Typhimurium indicator strains and showed that the detection of antimicrobial activity can be obtained already in 2 h without laborious plate counting and overnight incubation. Indicator strains used in the study harboured luxAB genes responsible of producing light as well as luxCDE genes for synthesis of long-chain fatty aldehyde as substrate for light production. As a consequence, no exogenous aldehyde addition was needed allowing stable light production. Furthermore, the method was used for the detection of antimicrobial activity from lactic acid bacteria after the effect of organic acids was eliminated.     

Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging

Optical imaging is a modality that is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. For this study, we used a green fluorescent protein (GFP)- and luciferase-expressing mouse tumor model to compare and contrast the quantitative and qualitative capabilities of a fluorescent reporter gene (GFP) and a bioluminescent reporter gene (luciferase). We describe the relationship between tumor volume, tumor mass, and bioluminescent/fluorescent intensity for both GFP and luciferase. Bioluminescent luciferase imaging was shown to be more sensitive than fluorescent GFP imaging. Luciferase-expressing tumors were detected as early as 1 day after tumor cell inoculation, whereas GFP-expressing tumors were not detected until 7 days later. Both bioluminescent and fluorescent intensity correlated significantly and linearly with tumor volume and tumor weight, as measured by caliper. Compared to bioluminescent imaging, fluorescent imaging does not require the injection of a substrate and may be appropriate for applications where sensitivity is not as critical. Knowing the relative strengths of each imaging modality will be important in guiding the decision to use fluorescence or bioluminescence.     

The spirochetal chpK-chromosomal toxin-antitoxin locus induces growth inhibition of yeast and mycobacteria

Toxin-antitoxin systems encoded by bacterial plasmids and chromosomes typically consist of a toxin that inhibits growth of the host cell and a specific antitoxin. In this report, the chpK gene from the chromosomal toxin-antitoxin locus of the spirochete Leptospira interrogans was studied in both prokaryotic and eukaryotic systems. Cloning of the the spirochetal chpK gene into a mycobacterial expressing vector led to dramatic reductions of transformation efficiency in both Mycobacterium smegmatis and Mycobacterium bovis BCG. However, few mycobacterial transformants were obtained. This result could be due to plasmid structural modifications leading to disruption of chpK expression, suggesting that L. interrogans ChpK is highly toxic for mycobacteria. Presence of the L. interrogans chpK gene was also found to inhibit cell growth of the yeast Saccharomyces cerevisiae. These results show that ChpK possesses a broad activity against both prokaryotes and eukaryotes, suggesting that the cellular target of the toxin is conserved in these organisms.     

Red-emitting luciferases for bioluminescence reporter and imaging applications

North American firefly Photinus pyralis luciferase, which emits yellow-green light (557 nm), has been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. Luciferase variants with red-shifted bioluminescence and high specific activity can be paired with green-emitting counterparts for use in dual-color reporter assays or can be used alone for in vivo imaging. Beginning with a previously reported red-emitting thermostable mutant and using mutagenesis techniques, we engineered two luciferases with redder emission maxima while maintaining satisfactory specific activities and thermostability. The novel enzymes were expressed in HEK293 cells, where they performed similarly to Promega’s codon-optimized click beetle red luciferase in model reporter assays. When the firefly luciferase variants were codon-optimized and retested using optimized substrate concentrations, they provided 50- to 100-fold greater integrated light intensities than the click beetle enzyme. These results suggest that the novel enzymes should provide superior performance in dual-color reporter and in vivo imaging applications, and they illustrate the importance of codon optimization for assays in mammalian cells.     

Candida albicans, a distinctive fungal model for cellular aging study

The unicellular eukaryotic organisms represent the popular model systems to understand aging in eukaryotes. Candida albicans, a polymorphic fungus, appears to be another distinctive unicellular aging model in addition to the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe. The two types of Candida cells, yeast (blastospore) form and hyphal (filamentous) form, have similar replicative lifespan. Taking the advantage of morphologic changes, we are able to obtain cells of different ages. Old Candida cells tend to accumulate glycogen and oxidatively damaged proteins. Deletion of the SIR2 gene causes a decrease of lifespan, while insertion of an extra copy of SIR2 extends lifespan, indicating that like in S. cerevisiae, Sir2 regulates cellular aging in C. albicans. Interestingly, Sir2 deletion does not result in the accumulation of extra-chromosomal rDNA molecules, but influences the retention of oxidized proteins in mother cells, suggesting that the extra-chromosomal rDNA molecules may not be associated with cellular aging in C. albicans. This novel aging model, which allows efficient large-scale isolation of old cells, may facilitate biochemical characterizations and genomics/proteomics studies of cellular aging, and help to verify the aging pathways observed in other organisms including S. cerevisiae.     

The carboxyl-terminal tripeptide serine-lysine-leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast.

Firefly luciferase is imported into peroxisomes in insects, mammals, plants, and yeast, which implies that the mechanism of protein translocation into peroxisomes has been conserved during eukaryotic evolution. The carboxyl-terminal tripeptide serine-lysine-leucine in luciferase acts as a peroxisomal import signal in mammalian cells. We have investigated whether this tripeptide is also involved in translocation of firefly luciferase into peroxisomes in yeast (Saccharomyces cerevisiae). We show by gene fusion experiments that the carboxyl-terminal 104 amino acids of luciferase can direct a heterologous protein to yeast peroxisomes. Luciferase mutant proteins were tested for their ability to be imported into yeast peroxisomes in vivo. We demonstrate that mutations in the carboxyl-terminal serine-lysine-leucine tripeptide abolish translocation of the protein into yeast peroxisomes. However, when a passenger protein was tagged at its carboxyl terminus with this tripeptide the fusion protein did not go to peroxisomes. These results indicate that, in yeast, the tripeptide is necessary but not sufficient for peroxisomal import.