Improved bioluminescence-based endotoxin measurement method using a salt-resistant luciferase mutant.

Endotoxin is a component of the cell wall of gram-negative bacteria and causes fever and shock symptoms upon entering the bloodstream. We previously demonstrated that the bioluminescence-based Limulus amebocyte lysate test is highly sensitive and rapid for measuring endotoxin. However, as the firefly luciferase reaction is inhibited in the presence of sodium chloride, the endotoxin detection method did not meet the validation guidelines under medical dialysis conditions (range of 75-125% of the measured values tested in water). Here, we used a salt-resistant luciferase mutant, which met the criteria for validation of endotoxin measurement.

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride.

OBJECTIVES: Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition. RESULTS: We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. CONCLUSIONS: The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.

Endotoxin assay by bioluminescence using mutant firefly luciferase.

The Limulus reaction is an application of the defense mechanism of horseshoe crab for endotoxin detection. Endotoxin is a component of the cell wall in the outer membrane of gram-negative bacteria, and causes fever or shock when it enters the human blood stream. For endotoxin detection, gel formation or turbidity of the coagulation factor chromogen or fluorescence-modified peptide is used. However, these conventional methods have problems with regard to their measurement time or sensitivity. We recently obtained a mutant firefly luciferase that has a luminescence intensity over 10-fold higher than that of the wild type. Therefore, we developed a new endotoxin detection method that combines the Limulus reaction and bioluminescence using mutant luciferase. The new method detects 0.0005EU/ml of endotoxin within 15min.

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles.

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76(N). The backpressure on the columns was 0.03 MPa in 1-10 ATP amplification cycles, and no increases in backpressure were observed.

Detection of living Salmonella cells using bioluminescence.

ATP-based bioluminescence using mutant firefly luciferase was combined with an immunochromatographic lateral flow test strip assay for Salmonella enteritidis detection. In this combination method, the Salmonella-antibody-gold complex captured at the test line on the test strip was lysed by heat-treatment, and the ATP released from the cells was measured using mutant luciferase. This method resulted in approximately 1,000 times higher sensitivity in the detection of Salmonella (i.e. 10(3) c.f.u./ml) compared to immunochromatographic lateral flow assay.

Single bacterial cell detection using a mutant luciferase.

Previously, we constructed a genetically modified luciferase of Photinus pyralis that generates more than 10-fold higher luminescence intensity than the wild-type enzyme. In this study, we demonstrate that this modified luciferase enables us to detect ATP at 10(-18) mol, which is almost equal to the quantity contained in a single bacterial cell. Consequently,we have been able to detect bacterial contamination in samples as low as one colony-forming unit (c.f.u.) for both Escherichia coli and Bacillus subtilis.

Enhanced dibenzothiophene biodesulfurization in a microchannel reactor.

A microchannel reactor system was used in a biodesulfurization process in which the rate of biodesulfurization in the oil/water phase of the microchannel reaction was more than nine-fold that in a batch (control) reaction. In addition, the microchannel reaction system using a bacterial cell suspension degraded alkylated dibenzothiophene that was not degraded by the batch reaction system. This work provides a foundation for the application of a microchannel reactor system consisting of biological catalysts using an oil/water phase reaction.

Evaluation of cell wall binding domain of Staphylococcus aureus autolysin as affinity reagent for bacteria and its application to bacterial detection.

We evaluated the cell wall binding (CWB) domain of Staphylococcus aureus autolysin as an affinity reagent for bacteria. A fusion of CWB domain and green fluorescent protein (CWB-GFP) bound to S. aureus with a dissociation constant of 15 nM. CWB-GFP bound to a wide range of gram-positive bacteria, but not to most gram-negative bacteria. We suspected that the outer membrane of gram-negative bacteria inhibits the access of CWB-GFP to peptidoglycan layer. Indeed, CWB-GFP bound to gram-negative bacteria when they were treated with benzalkonium chloride. Because CWB-GFP bound to the bacterial peptidoglycan layer, it appeared to be an effective affinity reagent for bacteria and CWB fusion with reporter proteins could be applied to detect bacteria. We also constructed a fusion of CWB and luciferase, which can be used for the rapid detection of bacteria.

Increase in bioluminescence intensity of firefly luciferase using genetic modification.

Firefly luciferase is widely used for enzymatic measurement of ATP, and its gene is used as a reporter for gene expression experiments. From our mutant library, we selected novel mutations in Photinus pyralis luciferase with higher luminescence intensity. These included mutations at Ile423, Asp436, and Leu530. Luciferase is structurally composed of a large N-terminal active site domain (residues 1-436), a flexible linker (residues 436-440) peptide, and a small C-terminal domain (residues 440-550) facing the N domain. Thus, the mutations are located at the junction of the N-terminal domain and the flexible linker, in the flexible linker peptide, and in the tip of the C-terminal domain, respectively. Substitution of Asp436 with a nonbulky amino acid such as Gly remarkably increased the substrate affinity for ATP and d-luciferin. Substitution of Ile423 with a hydrophobic amino acid such as Leu and that of Leu530 with a positively charged amino acid such as Arg increased the substrate affinity and the turnover rate. Combining these mutations, we obtained luciferases that generate more than 10-fold higher luminescence intensity than the wild-type enzyme.

Recombinant Pseudomonas putida carrying both the dsz and hcu genes can desulfurize dibenzothiophene in n-tetradecane.

Pseudomonas putida IFO13696, a recombinant strain with dsz desulfurization genes, desulfurized dibenzothiophene (DBT) in water but not in n-tetradecane. By introducing into this recombinant strain the hcuABC genes that take part in the uptake of DBT in the oil phase into the cell, 82% of 1 mM DBT in n-tetradecane was degraded in 24 h by resting cells. The products of hcuABC genes thus acted in the uptake of DBT in n-tetradecane into the cells and were effective in desulfurization of DBT in the hydrocarbon phase.

Desulfurization of 2,4,6,8-tetraethyl dibenzothiophene by recombinant Mycobacterium sp. strain MR65.

Recombinant Mycobacterium sp. strain MR65 harboring dszABCD genes was used to desulfurize alkyl dibenzothiophenes (Cx-DBTs) in n-hexadecane. The specific desulfurization activity for 2,4,6,8-tetraethyl DBT (C8-DBT) by DszC enzyme was about twice that for 4,6-dipropyl DBT (C6-DBT). However, the degradation rate of 2,4,6,8-tetraethyl DBT in n-hexadecane by resting cells of strain MR65 was only about 40% of that of 4,6-dipropyl DBT. These results indicated that the desulfurization ability for Cx-DBTs by resting cells depends on carbon number substituted at positions 4 and 6 and that the rate-limiting step in the desulfurization reaction of highly alkylated Cx-DBTs is the transfer process from the oil phase into the cell.

Enhanced desulfurization in a transposon-mutant strain of Rhodococcus erythropolis.

The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l(-1) to 70 mg l(-1) in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%-41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of delta9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.

Cloning of a rhodococcal promoter using a transposon for dibenzothiophene biodesulfurization.

The expression of biodesulfurization genes (dsz) in Rhodococcus erythropolis strain KA2-5-1 is repressed by sulfate which is the product of biodesulfurization. The application of a sulfate non-repressible promoter could be effective in enhancing biodesulfurization. A promoter-probe transposon was constructed using the promoterless, red-shifted green fluorescence protein gene (rsgfp). A 340 bp putative promoter element, designated kap1, was isolated from a strain KA2-5-1 recombinant that had shown high fluorescence intensity. The activity of kap1 was not affected by 1 mM sulfate. It gave about a 2-fold greater activity than the 16S ribosomal RNA promoter in R. erythropolis strain KA2-5-1 and is therefore useful for expressing desulfurization genes in rhodococcal strains.

Selective cleavage of 10-methyl benzo[b]naphtho[2,1-d]thiophene by recombinant Mycobacterium sp. strain.

Recombinant Mycobacterium sp. strain MR65 carrying dszABCD genes was used for desulfurization of 10-methylbenzo[b]naphtho[2,1-d]thiophene (10-methyl BNT) in the hexadecane phase. The specific activity was 25% of that of dibenzothiophene (DBT). One of two major metabolites of 10-methyl BNT produced by strain MR65 was identified as 1-methoxy-2-(3-methylphenyl)naphthalene by 1H and 13C NMR. The other major metabolite and two minor metabolites were determined as 1-hydroxy-2-(3-methylphenyl)naphthalene, 2-(2-methoxy-3-methylphenyl)naphthalene and 2-(2-hydroxy-3-methylphenyl)naphthalene, respectively, by HPLC and GC-MS. The production ratio of the two desulfurization metabolite isomers was 0.99:0.01, calculated on the basis of peak GC areas. These results indicated that the C-S bond adjacent to the naphthalene skeleton was selectively cleaved to form the two major compounds.

Thermophilic biodesulfurization of hydrodesulfurized light gas oils by Mycobacterium phlei WU-F1.

Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by conventional hydrodesulfurization (HDS) treatment using metallic catalysts. The thermophilic DBT-desulfurizing bacterium Mycobacterium phlei WU-F1 grew in a medium with hydrodesulfurized LGO as the sole source of sulfur, and exhibited high desulfurizing ability toward LGO between 30 and 50 degrees C. When WU-F1 was cultivated at 45 degrees C with B-LGO (390 ppm S), F-LGO (120 ppm S) or X-LGO (34 ppm S) as the sole source of sulfur, biodesulfurization resulted in around 60-70% reduction of sulfur content for all three types of hydrodesulfurized LGOs. In addition, when resting cells were incubated at 45 degrees C with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v) oils, biodesulfurization reduced the sulfur content from 390 to 100 ppm S (B-LGO), from 120 to 42 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO). Gas chromatography analysis with an atomic emission detector revealed that the peaks of alkylated DBTs including 4-methyl-DBT, 4,6-dimethyl-DBT and 3,4,6-trimethyl-DBT significantly decreased after biodesulfurization. Therefore, thermophilic M. phlei WU-F1, which could effectively desulfurize HDS-treated LGOs over a wide temperature range up to 50 degrees C, may be a promising biocatalyst for practical biodesulfurization of diesel oil.

Isolation of a recombinant desulfurizing 4,6-diproply dibenzothiophene in n-tetradecane.

Rhodococcus erythropolis strain KA2-5-1 is unable to desulfurize 4,6-dipropyl dibenzothiophene (DBT) in the oil phase. The dsz desulfurization gene cluster from R. erythropolis strain KA2-5-1 was transferred into 22 rhodococcal and mycobacterial strains using a transposon-transposase complex. The recombinant strain MR65, from Mycobacterium sp. NCIMB10403, was able to grow on a minimal medium supplemented with 1.0 mM 4,6-dipropyl DBT in n-tetradecane (50%, v v ) as the sole sulfur source. Resting cells of recombinant strain MR65 could desulfurize 68 mg l- of sulfur in light gas oil (LGO) containing 126 mg sulfur l-. Strain MR65 had about 1.5-times the LGO desulfurization activity of R. erythropolis strain KA2-5-1. The application of a recombinant, which is able to utilize 4,6-dipropyl DBT in the oil phase, was effective in enhancing LGO biodesulfurization.

Isolation of the Pseudomonas aeruginosa gene affecting uptake of dibenzothiophene in n-tetradecane.

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa strain NCIMB9571 using a transposon vector. All of the recombinant strains completely desulfurized 1 mM dibenzothiophene (DBT) in n-tetradecane (n-TD) except one, named strain PARMI. Strain PARMI was unable to desulfurize DBT in n-TD, but was able to desulfurize it in water. The n-alkane utilization ability, the biosurfactant production and the fatty acid composition of cells in strain PARMI were the same level as those of the other recombinants. The transposon insertion area of strain PARMI was analyzed by transposon tagging. We cloned three possible open reading frames (ORFs), designated hcuA, hcuB and hcuC, from the genomic DNA of P. aeruginosa NCIMB9571 using the transposon insertion area of strain PARMI as a DNA probe. Examination of the sequence revealed the transposon was inserted into hcuA. The full length of the hcuABC genes transformed into strain PARMI achieved 87% recovery of the desulfurization activity of DBT in n-TD, but partial hcuABC genes achieved only 0-12%. These results indicate that DBT desulfurization in the oil phase by recombinant P. aeruginosa strain NCIMB9571 requires the full length of the hcuABC gene cluster. The hcuABC gene cluster relates to DBT uptake from the oil phase to inside of the cell, and the uptake ability is independent of the n-alkane utilization ability, the biosurfactant production and the fatty acid composition of cells.

Recombinant Rhodococcus sp. strain T09 can desulfurize DBT in the presence of inorganic sulfate.

The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus-E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source.