Role of Hsp70 (DnaK-DnaJ-GrpE) and Hsp100 (ClpA and ClpB) chaperones in refolding and increased thermal stability of bacterial luciferases in Escherichia coli cells

The role of chaperones Hsp70 (DnaK–DnaJ–GrpE) and Hsp100 (ClpA–ClpB–ClpX) in refolding of thermoinactivated luciferase from the marine bacterium Photobacterium fischeri and the terrestrial bacterium Photorhabdus luminescens has been studied. These luciferases are homologous, but differ greatly in the rate of thermal inactivation and the rate constant for the luminescence reaction. It was shown that refolding of thermoinactivated luciferases is completely determined by the DnaK–DnaJ–GrpE system. However these luciferases markedly differ in the rate and degree of refolding. The degree of refolding of thermolabile quick Ph. fischeri luciferase reaches 80% of the initial level over several minutes, whereas renaturation of thermostable slow Ph. luminescens luciferase proceeds substantially slower (the degree of renaturation reaches only 7-8% of the initial level over tens of minutes). The measurement of the rate of thermal inactivation of luciferases in vivo in the cells of Escherichia coli wild strain and strains containing mutations in genes clpA, clpB, clpX showed that Ph. luminescens luciferase revealed reduced thermostability in mutant strain E. coli clpA^–. It was shown that this effect was not connected with DnaK-dependent refolding. In the case of thermolabile Ph. fischeri luciferase, mutation in gene clpA has no effect on the shape of the curve of thermal inactivation. These data suggest that denatured Ph. luminescens luciferase has enhanced affinity with respect to chaperone ClpA in comparison with DnaK, whereas thermolabile Ph. fischeri luciferase is characterized by enhanced affinity with respect to chaperone DnaK. Denatured luciferase bound to ClpA does not aggregate and following refolding proceeds probably spontaneously and very quickly (over 1-2 min). It is evident that the process under discussion requires ATP, since the addition of uncoupler of oxidative phosphorylation carbonyl cyanide 3-chlorophenylhydra-zone results in a sharp decrease in thermal stability of luciferase to the level typical of the enzyme in vitro. The enhanced thermosensitivity of luciferases was observed also in E. coli containing mutations in gene clpB. However, this effect, which takes place for Ph. fischeri luciferase as well as for Ph. LuminescensM luciferase, is determined by DnaK-dependent refolding and probably connected with the ability of chaperone ClpB to provide disaggregation of the proteins, resulting in their interaction with chaperones of the Hsp70 family (DnaK–DnaJ–GrpE).     

The Effect of Clp Proteins on DnaK-Dependent Refolding of Bacterial Luciferases

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK–DnaJ–GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.     

Mutation <Emphasis Type="Italic">clp</Emphasis>A::<Emphasis Type="Italic">kan</Emphasis> of the gene for an Hsp100 family chaperone impairs the DnaK-dependent refolding of proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The rate and level of DnaK-dependent refolding of heat-inactivated Vibrio fischeri luciferase in the clp A mutant (clp A:: kan) were considerably lower then in wild-type cells. The decline in refolding level progressed with increasing heat inactivation time. A mutation of clp P had no influence on the kinetics and level of luciferase refolding. Approximately equal amounts of the DnaKJE chaperone were synthesized upon heat shock induction in E. coli clp A + and E. coli clpA::kan cells. It was assumed that, like homologous chaperone ClpB, ClpA is involved in disaggregation of denatured proteins, increasing the refolding efficiency. This in vivo phenomenon occurred only upon a prolonged incubation of cells at a higher temperature, which led to the formation of large protein aggregates that were poorly refoldable by the DnaKJE system.     

Trigger factor assists the refolding of heterodimeric but not monomeric luciferases

The refolding of thermally inactivated protein by ATP-independent trigger factor (TF) and ATP-dependent DnaKJE chaperones was comparatively analyzed. Heterodimeric (αβ) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. In the presence of TF, thermally inactivated heterodimeric bacterial luciferases refold, while monomeric luciferases do not refold. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). Unlike TF, the DnaKJE chaperone system refolds both monomeric and heterodimeric luciferases with equal efficiency.     

Effects of the IbpAB and ClpA chaperones on DnaKJE-dependent refolding of bacterial luciferases in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Rate and level of DnaKJE-dependent refolding of the thermoinactivated Aliivibrio fischeri luciferase are considerably lower in Escherichia coli ibpA and ibpB, clpA mutants than in wild type cells. The rate and level of refolding are lower in E. coli ibpB::kan than in ibpA::kan cells. The decline of refolding level in E. coli clpA::kan makes progress only with the increase of thermoinactivation time of luciferase. Plasmids with genes ibpAB do not compensate clpA mutation. It is supposed that small chaperones IpbAB and chaperone ClpA operate independently in a process of DnaKJE-dependent refolding of proteins at the different stages.     

The effect of Clp proteins on DnaK-dependent refolding of bacterial luciferases

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK-DnaJ-GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.     

Collagen-Like Proteins (ClpA,ClpB, ClpC,and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by Bacillus amyloliquefaciens FZB42

The genes of collagen-like proteins (CLPs) have been identified in a broad range of bacteria, including some human pathogens. They are important for biofilm formation and bacterial adhesion to host cells in some human pathogenic bacteria, including several Bacillus spp. strains. Interestingly, some bacterial CLP-encoding genes (clps) have also been found in non-human pathogenic strains such as B. cereus and B. amyloliquefaciens, which are types of plant-growth promoting rhizobacteria (PGPR). In this study, we investigated a putative cluster of clps in B. amyloliquefaciens strain FZB42 and a collagen-related structural motif containing glycine-X-threonine repeats was found in the genes RBAM_007740, RBAM_007750, RBAM_007760, and RBAM_007770. Interestingly, biofilm formation was disrupted when these genes were inactivated separately. Scanning electron microscopy and hydrophobicity value detection were used to assess the bacterial cell shape morphology and cell surface architecture of clps mutant cells. The results showed that the CLPs appeared to have roles in bacterial autoaggregation, as well as adherence to the surface of abiotic materials and the roots of Arabidopsis thaliana. Thus, we suggest that the CLPs located in the outer layer of the bacterial cell (including the cell wall, outer membrane, flagella, or other associated structures) play important roles in biofilm formation and bacteria-plant interactions. This is the first study to analyze the function of a collagen-like motif-containing protein in a PGPR bacterium. Knocking out each clp gene produced distinctive morphological phenotypes, which demonstrated that each product may play specific roles in biofilm formation. Our in silico analysis suggested that these four tandemly ranked genes might not belong to an operon, but further studies are required at the molecular level to test this hypothesis. These results provide insights into the functions of clps during interactions between bacteria and plants.     

Synergistic coordination of polyethylene glycol with ClpB/DnaKJE bichaperone for refolding of heat‐denatured malate dehydrogenase

The use of polyethylene glycol (PEG) as a refolding additive to a refolding cocktail comprising the molecular bichaperone ClpB and DnaKJE significantly enhances chaperone‐mediated refolding of heat‐denatured malate dehydrogenase (MDH). The critical factor to affect the refolding yield is the time point of introducing PEG to the refolding cocktail. The refolding efficiency reached approximately 90% only when PEG was added at the beginning of refolding reaction. The synergistic coordination of an inexpensive refolding additive PEG with the ClpB/DnaKJE bichaperone system may provide an economical route to further enhance the efficacy of ClpB/DnaKJE refolding cocktail approach, facilitating its implementation in large‐scale refolding processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Photoexcited bacterial bioluminescence. Identity and properties of the photoexcitable luciferase.

Properties of photoexcitable luciferase are compared with those of luciferase, both isolated from the bacterium Beneckea harveyi. The proteins have the same molecular weight, are similarly charged at pH 8, and can be inactivated, with comparable efficiencies, by antibodies against either pure luciferase (a heterodimeric protein) or individual subunits thereof. Compared with luciferase, photoexcitable luciferase has a broader pH range for optimal activity, is more stable under acidic conditions, is less stable under alkaline conditions, and is more resistant at neutral pH to inactivation by heat, urea, and trypsin; A flavine-like chromophore, designated B, can be isolated from photoexcitable luciferase. The binding of B to luciferase restores all the properties characteristic of photoexcitable luciferase. Moreover, photoexcitable luciferases from mutants selected to have heat labile luciferases are also thermally unstable. It is concluded that photoexcitable luciferase actually consists of a luciferase-B complex which is conformationally distinct from luciferase under certain conditions.     

Sensitivity of dark mutants of various strains of luminescent bacteria to reactive oxygen species

Recent studies indicated that bioluminescence of the marine bacterium Vibrio harveyi may both stimulate DNA repair and contribute to detoxification of deleterious oxygen derivatives. Therefore, it was also proposed that these reactions can be considered biological roles of bacterial luminescence and might act as evolutionary drives in development of luminous systems. However, experimental evidence for the physiological role of luciferase in protection of cells against oxidative stress has been demonstrated only in one bacterial species, raising the question whether this is a specific or a more general phenomenon. Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and ferrous ions) growth of dark mutants of different strains of Vibrio fischeri and Photobacterium leiognathi is impaired relative to wild-type bacteria, though to various extents. Deleterious effects of oxidants on the mutants could be reduced (with different efficiency) by addition of antioxidants, A-TEMPO or 4OH-TEMPO. These results support the hypotheses that (1) activities of bacterial luciferases may detoxify deleterious oxygen derivatives, and (2) significantly different efficiencies of this reaction are characteristic for various luciferases.     

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.     

Computational analysis and functional expression of ancestral copepod luciferase

We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species (Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species.     

A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models

Background

Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients.

Methodology/Principal findings

We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT.

Conclusions/Significance

We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin.     

Firefly luciferase genes from the subfamilies Psilocladinae and Ototretinae (Lampyridae, Coleoptera)

Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae (Cyphonocerus ruficollis) and Ototretinae (Drilaster axillaris and Stenocladius azumai), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81-89% identities to each other and 60-81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545-546 nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550-568 nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.     

Expression of luciferase genes from different origins in Bacillus subtilis

Summary A group of vectors for luciferase expression in Bacillus subtilis was constructed. So far, only bacterial luciferases have been expressed in Bacillus, but in this study we wanted also to express genes encoding eukaryotic luciferases to perform direct comparisons of the light levels produced by the two different systems in B. subtilis. The vectors constructed can replicate both in Escherichia coli and B. subtilis, and the luciferase expression is strictly regulated due to the dual plasmid system used. Nearly a 100-fold increase in light production compared to previous results was achieved when genes encoding bacterial luciferase were inserted into the constructs and transformed into B. subtilis. An additional tenfold increase in light production was obtained when luciferase genes from the North American firefly (Photinus pyralis) or a click beetle (Pyrophorus plagiophtalamus) were introduced in a similar fashion into B. subtilis. Measurement of the light emission was performed without disruption of bacterial cells in a real-time manner, which is a common feature when working with all of these constructions. Structures of the shuttle vector constructs and results from light emission measurements are presented.     

Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift

Firefly bioluminescence reaction in the presence of Mg^2 +, ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350–359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7 Å and 2.2 Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351–359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.     

Reporter genes lucFF, luxCDABE, gfp, and dsred have different characteristics in whole-cell bacterial sensors

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 significantly longer incubation times. The results indicate that luciferases are better reporters in whole-cell sensor bacteria.     

In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R² = 0.98) or transcutaneously in the abdominal region of whole animals (R² = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩP_amiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.Among the wide variety of bacteria that colonize the gastrointestinal tracts of mammals, Escherichia coli is the most abundant facultative anaerobe of the human intestinal microflora. Aside from being part of the normal flora, E. coli is also a versatile organism capable of causing a variety of intestinal and extraintestinal diseases (18). The mechanisms that allow commensal E. coli to colonize the intestine and survive successfully in this niche remain poorly characterized. Conventional mice display natural resistance to colonization by commensal E. coli, but oral administration of streptomycin, which alters the intestinal microflora, allows colonization of the mouse large intestine by this species (25). The streptomycin-treated mouse model has been used extensively to study the factors of gram-negative bacteria implicated in the intestinal colonization process. However, this model is limited to the viable plate counts of bacteria in the feces and misses some critical information, such as the kinetics of colonization, the fate of the bacterial cells across the digestive tract, and the site of colonization. A better understanding of colonization would be facilitated by direct in vivo follow-up of this process.Bioluminescence imaging (BLI) technology is emerging as a powerful tool for the study of a wide range of biological processes in live animals, including real-time monitoring of infections (16). Bioluminescence systems emit visible light due to the luciferase-mediated oxidation of a luciferin substrate. A variety of luciferin-luciferase systems with different peak emissions have been identified in nature from numerous species (14). The luciferase of the soil bacterium Photorhabdus luminescens has been expressed successfully in gram-negative and gram-positive bacteria. This system emits blue-green light, with an emission maximum of approximately 490 nm, and does not require the addition of an exogenous substrate since the luciferase operon contains the genes required for synthesis of the substrate. Therefore, this luciferase has been used extensively to monitor bacterial infections in the living mouse. One of the first investigations with Salmonella enterica serovar Typhimurium transformed with the lux operon of P. luminescens evaluated the tissue distribution and the virulence of various S. Typhimurium strains (9). Subsequent modification of the lux operon led to the generation of highly bioluminescent Staphylococcus aureus and allowed the monitoring of infections due to this species in living mice (11). The modified lux operon was engineered into a lux-kan transposon cassette for chromosomal integration in gram-positive bacteria, such as S. aureus, Streptococcus pneumoniae, group A Streptococcus, and Listeria monocytogenes (16). Replication of L. monocytogenes in the lumen of the gall bladder was demonstrated for the first time by BLI (13).Bioluminescent E. coli was used in the neutropenic mouse thigh model of infection to evaluate the in vivo activity of antimicrobial agents (29). Bioluminescence was as indicative of therapeutic efficacy as CFU counts but, in addition, allowed real-time monitoring of the infection and of treatment efficacy in the same animal; however, only short-term monitoring (12 h) could be performed.Because luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. After oral administration of bioluminescent Salmonella to susceptible mice, the bioluminescent signal recorded in the abdominal region was greatly enhanced after air exposure (9). It was therefore assumed that direct bioluminescence imaging of intestine-colonizing microorganisms would not be optimal unless oxygen was provided exogenously or as the result of the close interaction between cells and the bacteria (9). However, the bacterial luciferase was used to trace in real time the colonization dynamics by Citrobacter rodentium of the gastrointestinal tracts of living animals, demonstrating that the gut represents a semianaerobic environment that allows the study of bacterial colonization by BLI (33).Factors essential for colonization are best studied in cocolonization experiments (7, 17). There are several luciferases with distinct emission spectra (34) that could be used in competition experiments to trace simultaneously two bacterial populations in the same living animal. However, in order not to impose additional and different metabolic burdens on the bacteria under study, the exogenous luciferases ideally have to be similar to allow comparison between strains. The thermostable luciferase variants PpyRE-TS and PpyGR-TS, derived from wild-type luciferase from the North American firefly Photinus pyralis, emit red (612 nm) and green (552 nm) light, respectively, at 37°C and are encoded by single genes of 1,650 bp, differing by only 9 bp (4). Bioluminescence color is determined by the Ser₂₈₄Thr (PpyRE-TS) and Val₂₄₁Ile, Gly₂₄₆Ala, and Phe₂₅₀Ser (PpyGR-TS) amino acid changes (5, 34). By use of optical filters, the emission spectra are readily distinguishable (4, 5). Five additional mutations provide enhanced thermostability at 37°C (4), improving the compatibility of the enzymes with bacterial culture conditions and BLI in animal models.While the luciferase mutants and all firefly luciferases use as substrates firefly luciferin and ATP to produce light, in vivo imaging is commonly performed with endogenous ATP and requires only exogenous administration of the luciferase substrate.The aim of this study was to develop a dynamic mouse model using in vivo bioluminescence imaging systems to monitor bacterial colonization in situ and in real time in whole living animals. Various strains of E. coli harboring the genes for the bacterial luciferase from P. luminescens or the firefly luciferase mutants (PpyRE-TS and PpyGR-TS) from P. pyralis have been engineered and used to follow bacterial intestinal colonization in mice. BLI was found to be well adapted to compare the intestine-colonizing capacities of various E. coli strains and to monitor cocolonization in vivo by use of dual bioluminescence emission.     

Interactions between aldehyde derivatives and the aldehyde binding site of bacterial luciferase

The interaction of trizine aldehydes with the aldehyde binding site of bacterial luciferases was investigated using a series of triazine aldehydes with different aldehyde chain length, and substituents on the s-triazine ring. Substrate activity was determined using luciferase from Photobacterium fischeri and Vibrio harveyi in a dithionite-based luciferases assay. The chain length optimum was determined for two triazine aldehyde classes to be C-10 and C-11, respectively. Only the substrate activity of 10-(4-chloro-6-methyithio-s-triazine-2-yl)aminodecanal (5) was as high as n-decanal, the reference aldehyde. All other triazine derivatives reduced light emission, probably by hindered binding of the substrates. The degree of activity reduction correlated with the volume of the triazine ring moiety. The triazine moiety volume of compound 5 was estimated to be 200 × 10^?30 m³. Triazine aldehydes which showed reduced light emission had an estimated volume of 228 × 10^?30 m³ or greater. All triazine aldehydes showed approximately 10-fold lower activities for Vibrio harveyi than for Photobacterium fischeri luciferase. Substrate specificity was the same for both luciferases. A schematic superposition of quinone aldehydes and triazine aldehydes which showed substrate activities equivalent to n-decanal, indicated potential interaction sites of aldehyde substrates with the aldehyde binding site of bacterial luciferases. The in vivo relevance of the results is discussed.     

Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium

The lux genes required for expression of luminescence have been cloned from a terrestrial bacterium, Xenorhabdus luminescens, and the nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits of luciferase determined. The lux gene organization was closely related to that of marine bacteria from the Vibrio genus with the luxD gene being located immediately upstream and the luxE downstream of the luciferase genes, luxAB. A high degree of homology (85% identity) was found between the amino acid sequences of the alpha subunits of X. luminescens luciferase and the luciferase from a marine bacterium, Vibrio harveyi, whereas the beta subunits of the two luciferases had only 60% identity in amino acid sequence. The similarity in the sequences of the alpha subunits of the two luciferases was also reflected in the substrate specificities and turnover rates with different fatty aldehydes supporting the proposal that the alpha subunit almost exclusively controls these properties. The luciferase from X. luminescens was shown to have a remarkably high thermal stability being stable at 45 degrees C (t 1/2 greater than 3 h) whereas V. harveyi luciferase was rapidly inactivated at this temperature (t 1/2 = 5 min). These results indicate that the X. luminescens lux system may be the bacterial bioluminescent system of choice for application in coupled luminescent assays and expression of lux genes in eukaryotic systems at higher temperatures.