Bioluminescent Bacterium Photobacterium Phosphoreum Research Articles

Application of luminescent Photobacterium Phosphoreum T3 for the detection of zearalenone and estimating the efficiency of their enzymatic degradation

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes enormous economic losses in the food and feed industries. Simple, rapid, low-cost, and quantitative analysis of ZEN is particularly urgent in the fields of food safety and animal husbandry. Using the bioluminescent bacterium Photobacterium phosphoreum T3, we propose a bioluminescence inhibition assay to evaluate ZEN levels quickly. The limit of detection (LOD), limit of quantification (LOQ), and quantitative working range of this bioluminescence inhibition assay were 0.1 µg/mL, 5 µg/mL, and 5-100 µg/mL, respectively. The concentration-response curve of the bioluminescence inhibition rate and ZEN concentration was plotted within the range 5 to 100 μg/mL, as follows: y = 0.0069x2 – 0.0190x + 7.9907 (R2 = 0.9943, y is luminescence inhibition rate, x is ZEN concentration). First, we used the bioluminescence inhibition assay to detect the remaining ZEN in samples treated with purified lactonohydrolase ZHD101. The bioluminescence inhibition assay results showed a strong correlation with the HPLC analysis. Furthermore, we successfully evaluated the overall toxicity of samples treated with purified peroxidase Prx and H2O2 using the P. phosphoreum T3 bioluminescence inhibition assay. The results indicate that the degradation products of ZEN created by purified peroxidase Prx and H2O2 showed little toxicity to P. phosphoreum T3. In this study, a simple, rapid, and low-cost assay method of zearalenone by bioluminescent P. phosphoreum T3 was developed. The bioluminescence inhibition assay could be used to estimate the efficiency of enzymatic degradation of ZEN.

Degradation of trichloroacetic acid by an efficient Fenton/UV/TiO2 hybrid process and investigation of synergetic effect

An efficient hybrid degradation process combined Fenton with TiO2 photocatalytic oxidation for trichloroacetic acid (TCAA) was first investigated, and then the synergetic degradation effect between Fenton reagent and TiO2 photocatalytic oxidation was discussed in this study. The dominant factors, such as dosages of TiO2 and Fe2+, TCAA concentration and pH were researched and optimized. The cooperation effect between Fenton and TiO2 photocatalytic oxidation probably occurred because (1) more OH radicals were produced in the Fe(OH)2+ and H2O2 decomposition reactions irradiated by UV light; and (2) photoinduced electrons voluntarily participated in the reduction from Fe3+ to Fe2+, improving the efficient separation of the photogenerated electrons and holes. Adsorption and degradation were both involved in the removal of TCAA in this hybrid process, and the removal of TCAA followed the Langmuir model and the degradation kinetics accorded with first order degradation kinetics. Toxicity test using a bioluminescent bacterium Photobacterium phosphoreum as the indicator proved that the hybrid process was a safe treatment because the toxicity decreased greatly with the degradation time.

Improved detection of toxic chemicals by Photobacterium phosphoreum using modified Boss medium

A bioluminescent bacterium Photobacterium phosphoreum has been used widely as an indicator of pollutants where the presence of toxic chemicals decreases light output. The optimum conditions for the growth and bioluminescence of P. phosphoreum KCTC 2852 were investigated using modified Boss medium and two environmental conditions (pH and temperature). Optimized conditions that supported growth of P. phosphoreum with high bioluminescence intensities were: NaCl (20 g/L), glycerol (2.5 g/L), peptone (1.0 g/L), and yeast extract (1.0 g/L). Growth and bioluminescence gradually increased as pH increased reaching a maximum at pH 7.0. The maximum temperature for growth and bioluminescence was 20 °C. Based on these optimum conditions for bioluminescence, a continuous culture reactor of P. phosphoreum was operated. During the continuous operation, the optimized medium maintained bioluminescence with a high relative light unit (RLU) around 13,600 for more than 120 h. When zinc (0.5 mg/L) was added to the reactor, an EC 50 was observed in 15 min. The detection limit was improved by using the modified Boss medium since the modified Boss medium contained less organic matter which minimizes the complexation and precipitation of heavy metals compared to other enrichment media with high levels of organic matter.

Improved Agar Diffusion Method for Detecting Residual Antimicrobial Agents

The improved agar diffusion method for determination of residual antimicrobial agents was investigated, and the sensitivities of various combinations of test organisms and assay media were determined using 7 organisms, 5 media, and 31 antimicrobial agents. Bacillus stearothermophilus and synthetic assay medium (SAM) showed the greatest sensitivity for screening penicillins (penicillin G and ampicillin). The combination of Bacillus subtilis and minimum medium (MM) was the most sensitive for tetracyclines (oxytetracycline and chlortetracycline), B. stearothermophilus and SAM or Micrococcus luteus and Mueller-Hinton agar (MHA) for detecting tylosin and erythromycin, B. subtilis and MHA for aminoglycosides (streptomycin, kanamycin, gentamicin, and dihydrostreptomycin), B. stearothermophilus and SAM for polyethers (salinomycin and lasalocid), and B. subtilis and MM or Clostridium perfringens and GAM for polypeptides (thiopeptin, enramycin, virginiamycin, and bacitracin). However, gram-negative bacterium Escherichia coli ATCC 27166 and MM were better for screening for colistin and polymixin-B. For detecting the synthetic drugs tested, the best combination was B. subtilis and MM for sulfonamides, E. coli 27166 and MM for quinolones (oxolinic acid and nalidixic acid), B. subtilis and MM for furans (furazolidone), and the bioluminescent bacterium Photobacterium phosphoreum and luminescence assay medium for chloramphenicol and oxolinic acid. The results showed that the use of four assay plates, B. stearothermophilus and SAM, B. subtilis and MM, M. luteus and MHA, and E. coli 27166 and MM, was superior to the currently available techniques for screening for residual antimicrobial agents in edible animal tissues.

Detection of toxicity of cyanobacterial strains using Artemia salina and MicrotoxR assays compared with mouse bioassay results

Abstract Cyanobacterial samples were collected from ponds, water supplies and rivers in Brittany 1991–1993. Strains belonging to Microcystis, Anabaena, Aphanizomenon and Oscillatoria were isolated and maintained in culture. Toxicity of the strains was assessed using larvae of the brine shrimp Artemia salina each and by using the bioluminescent bacterium Photobacterium phosphoreum (Cohn) Ford (Microtox® assay). It was compared with the mouse bioassay, which indicated that the toxic strains only produced hepatotoxins. Artell1ia salina larvae were sensitive to hepatotoxins, and the results showed good correlation with the mouse bioassay. In contrast, Microtox® responses were inconsistent with the mouse assay. In most cases, data obtained with Microtox® showed discrepancies: similar EC50 values were found for both toxic and non-toxic strains. Compounds other than hepatotoxins seemed to be responsible for toxic response in the Microtox® assay.

Methylenetetrahydrofolate dehydrogenase-cyclohydrolase from Photobacterium phosphoreum shares properties with a mammalian mitochondrial homologue

The marine bioluminescent bacterium Photobacterium phosphoreum expresses a bifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase with dual cofactor specificity. An investigation of the kinetic parameters of the P. phosphoreum enzyme indicate that its utilization of dinucleotide cofactors shares similarities with the human mitochondrial dehydrogenase-cyclohydrolase. Both enzymes exhibit dual cofactor specificity and the NAD +-dependent dehydrogenase activities from both enzymes can be activated by inorganic phosphate. Furthermore, an analysis of multiply aligned dehydrogenase-cyclohydrolase sequences from 11 species revealed that bacterial and mitochondrial enzymes are more closely related to each other than to the dehydrogenase-cyclohydrolase domains from eukaryotic bifunctional enzymes, and that the bacterial and mitochondrial enzymes share a common point of divergence. Since the NADP + cofactor is kinetically favoured by a factor of 18 over NAD +, and is therefore likely to be the preferred in vivo cofactor, we propose that the P. phosphoreum enzyme and the human mitochondrial enzyme evolved from a common ancestral dehydrogenase-cyclohydrolase with dual cofactor specificity, but that cofactor preference in these two enzymes diverged in response to different metabolic requirements.

Use of the bioluminescent bacterium Photobacterium phosphoreum to detect potentially biohazardous materials in water.

Use of the bioluminescent bacterium Photobacterium phosphoreum to detect potentially biohazardous materials in water.

Fatty acid reductase in bioluminescent bacteria. Resolution from aldehyde reductases and characterization of the aldehyde product.

Fatty acid reductase from the bioluminescent bacterium Photobacterium phosphoreum, has been partially purified free of aldehyde reductase activity and with a low endogenous fatty acid content permitting the characterization of the aldehyde product of the reaction. Two aldehyde reductases, both dependent on NADH, were separated by anion-exchange chromatography from the fatty acid reductase activity. The partially purified fatty acid reductase catalyzed the synthesis exclusively of long chain aldehydes from fatty acids in the presence of ATP and NADPH as demonstrated by the conversion of [3H]tetradecanoic acid to [3H]aldehyde. Comparison of the amount of [3H]aldehyde produced with the bioluminescence responses of luciferase to the aldehyde product and standard aldehydes, both with respect to maximum light intensity and luminescent decay, established that tetradecanoic acid has been converted to tetradecanal, the aldehyde of the same chain length. These results are consistent with a mechanism involving activation of the fatty acid with ATP followed by reduction of a fatty acyl intermediate to the corresponding aldehyde.

Protein-ligand interactions in lumazine protein and in Desulfovibrio flavodoxins from resonance coherent anti-Stokes Raman spectra.

The resonance coherent anti-Stokes Raman technique was used to obtain vibrational spectra of flavin in flavodoxins from Desulfovibrio gigas and Desulfovibrio vulgaris and of the simpler 6,7-dimethyl-8-ribityllumazine chromophore in the blue fluorescence lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. In the region examined, 1100-1700 cm-1, the Raman spectrum of the lumazine is less crowded than that of the flavin and this facilitates assignment of observed frequencies to particular vibrational modes. Certain modes are not affected by chromophore binding to the protein, but others are changed in frequency or intensity in a way that can be rationalized by expected interactions of the chromophore with the amino acid residues of the binding site. For example, a tentative assignment of change in hydrogen bonding at N(5) is suggested as the cause of the frequency shift for the chromophore in both flavodoxins (free-bound, 1582-1572 cm-1) and for C(4)=O in glucose oxidase (1359-1364 cm-1) and lumazine protein (1359-1362 cm-1). Shifts of the C(2)-N(3) mode in D2O may arise from hydrogen-bonding changes at C(2)=O in lumazine protein (1284-1291 cm-1), flavodoxin (1300-1280 cm-1), and glucose oxidase (1297-1287 cm-1). Bonding at N(3)-H may be the origin of changes in the frequency or intensity of the amide III mode in riboflavin binding protein and glucose oxidase. A stacking interaction is suggested for the change in a pyrimidine ring mode in FAD (1508 cm-1) since this mode is found at 1504 cm-1 in 30% Me2SO/H2O, where the adenine and pyrimidine are unstacked. The results clearly indicate different interactions in the binding sites of those proteins studied to date.

Lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. Purification and characterization.

Lumazine protein, a novel protein containing 6,7-dimethyl-8-ribityllumazine as a bound prosthetic group, is one of the several major proteins produced by the bioluminescent bacteria, Photobacterium phosphoreum. purification to complete homogeneity from cell extracts is achieved in six steps. Lumazine protein is a near spherical, monomeric protein of average molecular weight 20,000; in amino acid composition it is acidic with two isoelectric isomers, pI 4.9 and 5.0, and is hydrophilic (974 cal/residue) with single methionine and tryptophan residues and two accessible cysteines. It contains no carbohydrate. Reaction of the cysteines with dithionitrobenzoic acid results in quenching of the bound lumazine fluorescence but is otherwise reversible. Estimates of protein by dry weight results in a mole ratio of one bound lumazine group per protein.

Lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. A fluorescence study of the protein-ligand equilibrium.

The changes of fluorescence spectral distribution, polarization, and lifetime of the lumazine protein from Photobacterium phosphoreum can be interpreted in terms of an equilibrium between the protein and its dissociated prosthetic group 6,7-dimethyl-8-(1'-D-ribityl)lumazine. The equilibrium is rapidly attained, 1:1, and Kd is 5 x 10(-8) M (4 degrees C, pH 7, 67 mM phosphate). A change in solution conditions like an increase in temperature or dilution or a decrease in pH or ionic strength favors dissociation of the ligand from the protein. The dissociation was confirmed by separating the free ligand by ultrafiltration, and the apoprotein was reconstituted with the authentic lumazine derivative. A van't Hoff analysis of the dissociation constant allows evaluation of the thermodynamic parameters: delta H degrees = 28 kcal/mol and delta S degrees = 67 eu. By analogy to published results on the binding of FMN to apoflavodoxin, the contribution of hydrophobic interaction and of hydrogen bonding to the binding enthalpy can be estimated. The decay of the emission anisotropy of lumazine protein following a 0.5-ns laser pulse excitation can be fitted with a single correlation time characteristic of a 30 000-dalton spherical protein.