Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

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.     

Engineering an Enhanced,Thermostable, Monomeric Bacterial Luciferase Gene As a Reporter in Plant Protoplasts

The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.     

PHOSPHORUS BIOAVAILABILITY MONITORING BY A BIOLUMINESCENT CYANOBACTERIAL SENSOR STRAIN 1

Phosphorus (P) is widely considered to be the main nutrient limiting the productivity of freshwater phytoplankton, but an assessment of its bioavailability in natural samples is highly complex. In an attempt to provide a novel tool for this purpose, the promoter of the alkaline phosphatase gene, phoA, from Synechococcus sp. PCC 7942 was fused to the luxAB luciferase genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus sp. PCC 7942 genome to yield strain APL, which emitted light when inorganic P concentrations fell below 2.3 μM. Light emission of P‐deprived cells decreased rapidly upon inorganic P readdition. The reporter was demonstrated to be a sensitive tool for monitoring the bioavailability of both inorganic and organic P sources. In water samples taken from a natural freshwater environment (Lake Kinneret, Israel), the luminescence measured correlated with total dissolved phosphate concentrations.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Peculiarities of luminescent response of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strain bearing cloned <Emphasis Type="Italic">Vibro harveyi lux AB</Emphasis> genes to the action of thermostable blood serum compounds

A strain of Bacillus subtilis previously used as the test-organism in bacteriological and nephelometry methods for detection of thrombocytes cation protein (TCP) has been transformed by a plasmid pLFlux containing cloned luxAB genes of a sea luminescent bacterium Vibro harveyi. The designed luminescent biosensor B. subtilis of The All-Russia Collection of Industrial Microorganisms (Moscow) B-10191 demonstrated specific response to the thermostable fraction of guinea pig blood serum. Sensitivity to chromatographically purified TCP as part of the system has been analyzed and the correlation between luminescence inhibition and direct bacterial effect on the target cells has been demonstrated. The obtained results are considered as the first stage of the design of the bioluminescent technology for TCP detection in biological liquids with complicate composition of the components.     

Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish,Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes

Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a luxAB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase subunit gene luxA of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G+C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.The nucleotide sequence reported in this article has been deposited in Genbank under accession number M36597     

Spectral sensitivity of the compound eyes in two day-active fireflies (Coleoptera: Lampyridae: Lucidota)

Summary The electroretinographic visual spectral sensitivity functions in day-active fireflies Lucidota luteicollis and Lucidota atra show a broad green sensitivity and a shoulder in the near-ultraviolet region of the spectrum (Figs. 1, 2) as is commonly found among day-active insects. The nomogram for P530 visual pigment matches the spectral sensitivity curves in the green. The adult L. luteicollis retains its larval bioluminescent light organ which has a peak emission at 562 nm. The _max of the ERG spectral sensitivity does not match the bioluminescent peak (Fig. 1B) as it does in twilight- and dark-active fireflies. Some relevant behavioural observations with respect to mating are presented.     

A bioluminescence assay to detect nitrification inhibitors released from plant roots: a case study with Brachiaria humidicola

A bioluminescence assay using recombinant Nitrosomonas europaea was adopted to detect and quantify natural nitrification inhibitors in plant–soil systems. The recombinant strain of N. europaea produces a distinct two-peak luminescence due to the expression of luxAB genes, introduced from Vibrio harveyi, during nitrification. The bioluminescence produced in this assay is highly correlated with NO₂⁻ production (r ² = 0.94). Using the assay, we were able to detect significant amounts of a nitrification inhibitor produced by the roots of Brachiaria humidicola (Rendle) Schweick. We propose that the inhibitory activity produced/released from plants be termed ‘biological nitrification inhibition’ (BNI) to distinguish it from industrially produced inhibitors. The amount of BNI activity produced by roots was expressed in units defined in terms of the action of a standard inhibitor allylthiourea (AT). The inhibitory effect from 0.22 μM AT in an assay containing 18.9 mM of NH₄⁺ is defined as one AT unit of activity. A substantial amount of BNI activity was released from the roots of B. humidicola (15–25 AT unit g⁻¹ root dry wt day⁻¹). The BNI activity released was a function of the growth stage and N content of the plant. Shoot N levels were positively correlated with the release of BNI activity from roots (r ² = 0.76). The inhibitor/s released from B. humidicola roots suppressed soil nitrification. Additions of 20 units of BNI per gram of soil completely inhibited NO₃⁻ formation in a 55-day study and remained functionally stable in the soil for 50 days. Both the ammonia monooxygenase and the hydroxylaminooxidoreductase enzymatic pathways in Nitrosomonas were effectively blocked by the BNI activity released from B. humidicola roots. The proposed bioluminescence assay can be used to characterize and determine the BNI activity of plant roots, thus it could become a powerful tool in genetically exploiting the BNI trait in crops and pastures.     

Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation

A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m⁻²) and solar irradiation (≈620 W m⁻²). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py⁺-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.     

Bioluminescent bioreporter integrated-circuit sensing of microbial volatile organic compounds

A bioluminescent bioreporter for the detection of the microbial volatile organic compound p-cymene was constructed as a model sensor for the detection of metabolic by-products indicative of microbial growth. The bioreporter, designated Pseudomonas putida UT93, contains a Vibrio fischeri luxCDABE gene fused to a p-cymene/p-cumate-inducible promoter derived from the P. putida F1 cym operon. Exposure of strain UT93 to 0.02–850 ppm p-cymene produced self-generated bioluminescence in less than 1.5 h. Signals in response to specific volatile organic compounds (VOCs) such as m- and p-xylene and styrene, also occurred, but at two-fold lower bioluminescent levels. The bioreporter was interfaced with an integrated-circuit microluminometer to create a miniaturized hybrid sensor for remote monitoring of p-cymene signatures. This bioluminescent bioreporter integrated-circuit device was capable of detecting fungal presence within approximately 3.5 h of initial exposure to a culture of p-cymene-producing Penicillium roqueforti.     

Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe

We demonstrate a novel rapid direct detection method for immunohistochemistry, using a bioluminescent probe. An anti‐CEA antibody‐fused far‐red bioluminescent protein can monitor the accumulation of this type of probe in tumour tissues. The bimodal spectrum (λ_max = 460 and 675 nm) of this bioluminescent probe is extremely stable under different conditions of pH and ion concentration. The sensitivity of our bioluminescent labelling was at the same level of enzymatic labelling, e.g. peroxidase, as an indirect system. Our novel technique is simple and can shorten the pretreatment time of paraffin sections to around 30 min. The utility of our bioluminescent labelling covers all imaging in vitro, in vivo and ex vivo, suggesting that our antibody‐fused bioluminescent probe has the potential to detect tumour antigens with a high sensitivity in routine immune histological examinations. Copyright © 2012 John Wiley & Sons, Ltd.     

A rice (Oryza sativa L.) MAP kinase gene, OsMAPK44, is involved in response to abiotic stresses

We have isolated and characterized a putative rice MAPK gene (designated OsMAPK44) encoding for a protein of 593 amino acids that has the MAPK family signature and phosphorylation activation motif, TDY. Alignment of the predicted amino acid sequences of OsMAPK44 showed high homology with other rice MAPKs. Under normal conditions, the OsMAPK44 gene is highly expressed in root tissues, but relatively less in leaf and stem tissues of the japonica type rice plant (O. sativa L. Donggin). mRNA expression of the gene is highly inducible by salt and drought treatment, but not by cold treatment. Moreover, the mRNA level of the OsMAPK44 is up-regulated by exogenously applied Abscisic acid (ABA) and H₂O₂. When we compared the OsMAPK44 gene expression level between a salt sensitive indica cultivar (IR64) and a salt resistant indica cultivar (Pokkali), they showed some difference in expression kinetics with the salt treatment. OsMAPK44 gene expression in Pokkali was slightly up-regulated within 30 min and then disappeared rapidly, while IR64 maintained its expression for 1 h following down-regulation. Under the salinity stress, OsMAPK44 overexpression transgenic rice plants showed less damage and greater ratio of potassium and sodium than OsMAPK44 suppressed transgenic lines did, suggesting that OsMAPK44 may have a role to prevent damages due to working for favorable ion balance in the presence of salinity.     

Cloning and Characterization of the Xyn11A Gene from <Emphasis Type="Italic">Lentinula edodes</Emphasis>

Hemicellulose represents a rich source of biomass that can be converted into useful chemical feedstocks. One of the main components of hemicellulose is xylan, a polymer of xylose residues. Xylanase enzymes that hydrolyze xylan are therefore of great commercial interest. We have cloned a gene (xyn11A) that encodes a 283-amino acid xylanase enzyme from the fungus Lentinula edodes. The enzyme has a pI of 4.6 and belongs to the highly conserved glycosyl hydrolase family 11. The xylanase gene was cloned into a Pichia pastoris expression vector that secretes active enzyme into both solid and liquid media. The optimal reaction conditions were at pH 4.5 and 50°C. The enzyme had a K_m of 1.5 mg/ml and a V_max of 2.1 mmol/min/mg. Xyn11A produced primarily xylobiose, xylotriose, and xylotetraose from a birchwood xylan substrate. This is the first report on the cloning of a hemicellulase gene from L. edodes.     

A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi

In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X₂-CX₁₇-C-X₂-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X₂-C-X₁₈-C-X₂-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light.     

Preliminary characterization of wine lactobacilli able to degrade arginine

Lactobacillus strains able to degrade arginine were isolated and characterized from a typical red wine. All the strains were gram-positive, catalase-negative and produced both D- and L-lactate from glucose. Strains L2, L3, L4, and L6 were able to produce CO₂ from glucose; however, production of CO₂ from glucose was not observed in strains L1 and L5, suggesting that they belong to the homofermentative wine lactic acid bacteria (LAB) group. All of the lactobacilli were tested for their ability to ferment 49 carbohydrates. The sugar fermentation profile of strain L1 was unique, suggesting that this strain belonged to Lactococcus lactis ssp. cremoris, a non-typical wine LAB. Furthermore, a preliminary typing was performed by using a random amplified polymorphic DNA analysis (RAPD-PCR analysis).     

Heterologous protein production in Escherichia coli using the propionate-inducible pPro system by conventional and auto-induction methods

We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella enterica prpBCDE promoter (P_prpB) and compared it to that from the strongest IPTG-inducible promoter, P_T7. In contrast to our previous study showing slightly higher production of green fluorescent protein (GFP) from the pPro system compared to that from the T7 system, production of two plant proteins CMT and NCS from P_prpB was 2- to 4-fold higher than that from P_T7. Unlike P_T7, expression from P_prpB did not reduce cell growth even when highly induced, indicating that this propionate-inducible system is more efficient for overproduction of proteins that result in growth inhibition. In an auto-induction experiment, which does not require monitoring the culture or adding inducer during cell growth, the pPro system exhibited much higher protein production than the T7 system. These results strongly indicate that the pPro system is well-suited for overproduction of recombinant proteins.