Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

Fast Monitoring of Indoor Bioaerosol Concentrations with ATP Bioluminescence Assay Using an Electrostatic Rod-Type Sampler

A culture-based colony counting method is the most widely used analytical technique for monitoring bioaerosols in both indoor and outdoor environments. However, this method requires several days for colony formation. In this study, our goal was fast monitoring (Sampling: 3 min, Detection: < 1 min) of indoor bioaerosol concentrations with ATP bioluminescence assay using a bioaerosol sampler. For this purpose, a novel hand-held electrostatic rod-type sampler (110 mm wide, 115 mm long, and 200 mm tall) was developed and used with a commercial luminometer, which employs the Adenosine triphosphate (ATP) bioluminescence method. The sampler consisted of a wire-rod type charger and a cylindrical collector, and was operated with an applied voltage of 4.5 kV and a sampling flow rate of 150.7 lpm. Its performance was tested using Staphylococcus epidermidis which was aerosolized with an atomizer. Bioaerosol concentrations were measured using ATP bioluminescence method with our sampler and compared with the culture-based method using Andersen cascade impactor under controlled laboratory conditions. Indoor bioaerosol concentrations were also measured using both methods in various indoor environments. A linear correlation was obtained between both methods in lab-tests and field-tests. Our proposed sampler with ATP bioluminescence method may be effective for fast monitoring of indoor bioaerosol concentrations.     

An ATP Bioluminescence Assay Applicable to Rapid Fluconazole Susceptibility Testing of Dermatophytes

An ATP bioluminescence assay as a rapid reference method for fluconazole (FLCZ) susceptibility testing of dermatophytes, as well as yeasts, was developed and evaluated by comparing it with viability, turbidity and fungal protein content-based conventional methods. FLCZ susceptibility results obtained with strains of Candida albicans and dermatophytes by the bioluminescence method in high-resolution medium were well correlated with those obtained by conventional methods currently used in clinical microbiology laboratories or reported previously, including a broth dilution method by the National Committee for Clinical Laboratory Standards (NCCLS). Thus, ATP bioluminescence assay can be used to monitor fungal growth in liquid culture media. The procedure has considerable potential for the rapid testing of FLCZ susceptibility of dermatophytes and other fungi.     

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.     

Solid-Phase Contact Assay That Uses a lux-Marked Nitrosomonas europaea Reporter Strain To Estimate Toxicity of Bioavailable Linear Alkylbenzene Sulfonate in Soil

Information about in situ toxicity of the bioavailable pools of adsorptive soil pollutants is a prerequisite for proper ecological risk assessment in contaminated soils. Such toxicity data may be obtained by assays allowing for direct exposure of introduced test microorganisms to the toxicants, as they appear in solid solution equilibria in the natural soil. We describe a novel sensitive solid-phase contact assay for in situ toxicity testing of soil pollutants based on a recombinant bioluminescent reporter strain of Nitrosomonas europaea. A slurry of the reporter strain and soil sample was shaken for 1 h, after which bioluminescence was measured either directly (soil slurry protocol) or in the supernatant obtained after centrifugation (soil extract protocol). The assay was validated for both protocols by using linear alkylbenzene sulfonate (LAS) as a toxic and adsorptive model compound in the soil samples. Interestingly, LAS showed the same toxicity to the reporter strain with either soil incubation (both protocols) or pure culture, suggesting that adsorbed LAS pools contributed to the observed toxicity. The solid-phase contact assay that used the reporter strain of lux-marked N. europaea was slightly more sensitive for the detection of LAS toxicity in soil than activity-based assays targeting indigenous nitrifiers and much more sensitive than assays targeting indigenous heterotrophic microbes. We conclude that the new solid-phase contact assay, which is based on direct interaction of the test microorganisms with bioavailable pools of the toxicants in soil, provides a most sensitive and relevant method for evaluating the in situ toxicity and assessing the risks of soil contaminants.     

A new,fast, and very sensitive bioluminescence assay for phospholipases A and C

A new, simple, and very sensitive assay for phospholipase A and C is described. The assay is based on the bioluminescence developed by the mutant of the bacterium Beneckea harveyi as a response to myristic acid released from dimyristoyl phosphatidylcholine by either phospholipase A or by a phospholipase C-lipase coupled system. It is possible to assay these enzymes at a rate corresponding to a release of as little as 1 to 2 pmol of myristic acid per minute.     

Immobilization of bioluminescent Escherichia coli cells using natural and artificial fibers treated with polyethyleneimine

Biosensors based on whole-cell bioluminescence have the potential to become a cost-effective alternative to conventional detection methods upon validation of target selectivity and sensitivity. However, quantitative analysis of bioluminescence is greatly hindered due to lack of control over the total number of cells in a suspending culture. In this study, the effect of surface properties of genetically engineered luminous E. coli cells and fibrous matrices on the immobilization capacity and effectiveness under various environmental conditions were characterized. Four different fibers, including cotton, polyester, viscose rayon, and silk, were investigated. Although cell adhesion was observed on untreated viscose and cotton fibers, viscose fiber pretreated with 0.667% polyethyleneimine (PEI) was found capable of immobilizing the most viable E. coli DPD2234 cells, followed by viscose treated with 0.33% and 1% PEI. The cells immobilized on PEI-treated viscose remained viable and yielded 20% or more bioluminescence signals immediately upon contact with the inducer up to 72 h without feeding nutrients to the cells, suggesting that viscose treated with 0.667% PEI could provide a stable immobilization mechanism for bioluminescent E. coli cells with long sensing period, quick response time, and good signal reproducibility.     

Dead-end ultrafiltration concentration and IMS/ATP-bioluminescence detection of Escherichia coli O157:H7 in recreational water and produce wash

The purpose of this study was to develop a detection method for viable E. coli O157:H7 in fresh produce and recreational water. The method was evaluated using eight samples of produce wash and recreational water with or without spiked E. coli O157:H7 at ≤ 10² CFU·ml^− 1 and concentrated using dead-end ultrafiltration (DEUF) to produce primary and secondary retentates. Fifty-four matrix replicates of undiluted secondary retentates or dilutions (1:2 or 1:10 in buffer) were evaluated using an IMS/ATP bioluminescence assay (IMS/ATP). Combining primary and secondary DEUF yielded a 2-4 log₁₀ increase in E. coli O157:H7 concentrations in spiked samples and resulted in signal-to-noise ratios 2-219 fold higher than controls, depending on the sample type. DEUF increased the concentration of E. coli O157:H7 to within the detectable limits of IMS/ATP. The combined assay provided detection of viable E. coli O157:H7 in produce and recreational water. Accurate detection of microbial pathogens using DEUF and IMS/ATP could reduce disease outbreaks from contaminated water sources and food products.     

A Nisin Bioassay Based on Bioluminescence

A Lactococcus lactis subsp. lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods.     

Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

利用ATP扩增反应与生物发光法结合检测微量微生物

摘要:【目的】腺苷酸激酶（adenylate kinase, ADK）和多聚磷酸盐激酶（polyphosphate kinase, PPK）偶联催化的ATP扩增反应结合生物发光检测法能够对微量微生物进行检测。但是PPK当中结合的内源性的ADP会产生背景干扰,影响测定。本文旨在融合表达ADK和PPK,并建立一种方便有效的内源性ADP的去除方法,降低背景,使之与传统生物发光法结合,实现高灵敏生物发光法检测微量ATP及微生物。【方法】PCR扩增得到PPK、ADK基因,插入表达载体pET28a (+)中构建重组表达质粒pET28a (+)-PPKADK,表达PPK-ADK融合蛋白。利用表面包裹聚胺醇（Polyurethane）的磁珠（magnetic beads）,通过化学反应将腺苷酸双磷酸酶（apyrase）固定于磁珠表面,制备固相腺苷酸双磷酸酶（Beads-apyrase）,用于除去与融合蛋白结合的内源性ADP,降低ATP扩增反应的背景,从而使之与生物发光反应相结合,测定微量外源ATP及细菌菌落数。【结果】表达的融合蛋白具有PPK和ADK的活性,利用Beads-apyrase可以方便而有效的去除内源性ADP,显著地降低反应背景,从而实现了利用ATP扩增反应与传统生物发光反应结合,测定了小于1 fmol的外源微量ATP,使生物发光法检测ATP及微生物的灵敏度提高至少100倍。【结论】利用Beads-apyrase能够方便、有效地降低PPK-ADK中的ADP背景,从而使PPK-ADK催化的ATP扩增反应能够与传统生物发光法相结合,极大地提高了生物发光法的灵敏度。     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Rapid detection of Pseudomonas aeruginosa in mouse feces by colorimetric loop-mediated isothermal amplification

A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1 g feces (3.25 CFU/reaction). The assay was completed within 2 h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection.     

Novel NanoLuc-type substrates with various C-6 substitutions

NanoLuc (NLuc)-furimazine bioluminescence system offers several advantages over established systems, including improved stability, smaller size, and >150-fold enhancement in bioluminescence. Herein, we designed and synthesized a series of bioluminescent substrates with varying at the C-6 position of furimazine for NLuc-furimazine bioluminescence system. Among all derivatives, compounds A6 and A11 provided excellent bioluminescence characteristics compared with furimazine in vitro and in vivo. We believe that these new NLuc substrates can broaden the application of NLuc bioluminescence techniques, especially in vivo bioluminescent imaging.     

An acid precipitation technique: A strip assay for the large-scale DNA polymerase activity screening

A new format of a very rapid, low-cost and high-productive analysis based on the acid precipitation of radiolabeled DNA was developed. By contrast to the conventional processing of a large number of GF/C discs, the method employs one GF/C strip containing samples on individual teeth. The strip assay was validated by comparison with the glass fiber disk technique; the efficiency was demonstrated by screening E. coli superproducers and fractions obtained at the steps of Bst DNA polymerase, Large Fragment purification by the protocol we developed. The principle proposed allows simultaneous assaying many samples for the activity of different polymerases.     

Enumeration of phagocytosed Staphylococcus aureus inside cells of the mouse macrophage cell line J774 by bioluminescence,fluorescence and viable counting techniques

In order to quantify intracellular Staphylococcus aureus within a macrophage-like cell line by a bioluminescence technique, the mouse cell line J774 and opsonized Staphylococcus aureus were incubated together to allow phagocytosis to occur. Experiments using UV microscopy and fluorescent stained S. aureus were performed to determine an estimate of the mean intracellular bacterial numbers. For enumeration of intracellular bacteria by a bioluminescence technique, extracellular bacteria were removed by washing, the macrophages lysed mechanically and osmotically and treated with apyrase to remove somatic ATP. Bacterial cells were washed and the intracellular ATP measured by firefly luciferase bioluminescence in a luminometer. This new method of enumerating intracellular bacteria was compared to the conventional method of viable counts and found to correlate (r = 0.78). The bioluminescence assay developed was found to be a relatively rapid alternative method to the techniques currently used to enumerate intracellular bacteria and could prove advantageous in studies of intracellular killing and effects of antimicrobial agents on intracellular pathogens.     

Applications of luciferin biosynthesis: Bioluminescence assays for l-cysteine and luciferase

Based on the biosynthetic pathway of firefly bioluminescence substrate d-luciferin, the concentration of l-cysteine can be quantified using a simple protocol and a conventional luminescence detector. The lower limit of quantification (signal/noise ratio [S/N] = 10) was 0.26 μM. Using our method, the total amount of free/reduced and disulfide/oxidized l-cysteine could be measured successfully in human serum. In addition, biosynthetic precursors such as 2-cyano-6-hydroxybenzothiazole and l-luciferin could replace d-luciferin in the cell-based luciferase assay. Our results suggest that the bioluminescence reaction associated with the biosynthesis of bioluminescence substrates can provide a fast and cost-effective assay method.     

Grb14 inhibits FGF receptor signaling through the regulation of PLCγ recruitment and activation

To decipher the mechanism involved in Grb14 binding to the activated fibroblast growth factor receptor (FGFR), we used the bioluminescence resonance energy transfer (BRET) technique and the Xenopus oocyte model. We showed that Grb14 was recruited to FGFR1 into a trimeric complex containing also phospholipase C gamma (PLCγ). The presence of Grb14 altered FGF-induced PLCγ phosphorylation and activation. Grb14-FGFR interaction involved the Grb14-SH2 domain and the FGFR pY766 residue, which is the PLCγ binding site. Our data led to a molecular model whereby Grb14 binding to the phosphorylated FGFR induces a conformational change that unmasks a PLCγ binding motif on Grb14, allowing trapping and inactivation of PLCγ.

Structured summary

MINT-8019680: Grb14 (uniprotkb:O88900) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by anti tag coimmunoprecipitation (MI:0007)MINT-8019693, MINT-8019727: Grb14 (uniprotkb:O88900) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by bioluminescence resonance energy transfer (MI:0012)MINT-8019714, MINT-8019746: PLC gamma1 (uniprotkb:P19174) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by bioluminescence resonance energy transfer (MI:0012)     

EVALUATION OF ADENOSINE TRIPHOSPHATE (ATP) BIOLUMINESCENCE FOR ESTIMATING BACTERIA ON SURFACES OF BEEF CARCASSES

A rapid (<15 min), inexpensive and simple method has been developed to estimate the concentration of bacteria on surfaces of beef carcasses using adenosine triphosphate (ATP) bioluminescence. Surfaces (5x5 cm²) of beef carcasses (n= 159) were collected by excision. An ATP assay and aerobic plate count were performed on each sample. A significant (p < 0.001) positive linear relationship (r = 0.83) between plate count and ATP assay was obtained for 159 beef carcass samples. When thresholds levels were set at 1 × 10⁴, 1 × 10⁵ and 1 × 10⁶ CFU/cm², there was moderate to good agreement between the ATP bioluminescence assay and the aerobic plate count as determined by the k-statistic. The application of this ATP bioluminescence test to HACCP systems for beef slaughter processes is discussed.