Dominant petroleum hydrocarbon-degrading bacteria in the Archipelago Sea in South-West Finland (Baltic Sea) belong to different taxonomic groups than hydrocarbon degraders in the oceans.

The natural petroleum hydrocarbon degrading capacity of the Archipelago Sea water in S-W Finland was studied in a microcosm experiment. Pristine and previously oil exposed sites were examined. Bacterial community fingerprinting was performed using terminal restriction fragment length polymorphism (T-RFLP) and samples from selected microcosms were sequenced. The abundance of PAH degradation genes was measured by quantitative PCR. Bacterial communities in diesel exposed microcosms diverged from control microcosms during the experiment. Gram positive PAH degradation genes dominated at both sites in situ, whereas gram negative PAH degrading genes became enriched in diesel microcosms. The dominant bacterial groups after a 14 days of diesel exposure were different depending on the sampling site, belonging to the class Actinobacteria (32%) at a pristine site and Betaproteobacteria (52%) at a previously oil exposed site. The hydrocarbon degrading bacteria in the Baltic Sea differ from those in the oceans, where most hydrocarbon degraders belong to Gammaproteobacteria.

Diatom derived polyunsaturated aldehydes do not structure the planktonic microbial community in a mesocosm study.

Several marine and freshwater diatoms produce polyunsaturated aldehydes (PUA) in wound-activated processes. These metabolites are also released by intact diatom cells during algal blooms. Due to their activity in laboratory experiments, PUA are considered as potential mediators of diatom-bacteria interactions. Here, we tested the hypothesis that PUA mediate such processes in a close-to-field mesocosm experiment. Natural plankton communities enriched with Skeletonema marinoi strains that differ in their PUA production, a plankton control, and a plankton control supplemented with PUA at natural and elevated concentrations were observed. We monitored bacterial and viral abundance as well as bacterial community composition and did not observe any influence of PUA on these parameters even at elevated concentrations. We rather detected an alternation of the bacterial diversity over time and differences between the two S. marinoi strains, indicating unique dynamic bacterial communities in these algal blooms. These results suggest that factors other than PUA are of significance for interactions between diatoms and bacteria.

The transcription of l-gulono-gamma-lactone oxidase, a key enzyme for biosynthesis of ascorbate, during development of Persian sturgeon Acipenser persicus.

l-Gulono-gamma-lactone oxidase (GULO) is a key enzyme for the biosynthesis of ascorbate, which is essential for several cellular functions. In the present study, mRNA expression of GULO gene was evaluated during the early development of Persian sturgeon. First, because there are no comparative studies that have established suitable quantitative real-time PCR reference genes in sturgeons for any physiological conditions, we evaluated six candidate reference genes (ACTB, RPL13, UBQ, RPL6, GAPDH and EF1A) during the early development of Persian sturgeon. The most stable mRNA expression was obtained with RPL6 and ACTB, whereas the least stable was RPL13. After normalization using RPL6, ACTB and RPL6/ACTB combination, the mRNA expression of GULO was highest at the embryonic stage (2days before hatching; P<0.05) and started to decline from hatching of larvae to the rest of the developmental time-points. This suggests that the vitamin C requirements are highest during early life stages, and it is likely that the changes in GULO mRNA expression are associated with changes in GULO enzyme activity.

A novel biosensor for the detection of zearalenone family mycotoxins in milk.

In this study, a method for detecting estrogenic mycotoxin residues in milk was developed utilizing bioluminescent whole-cell biosensors. Milk products of various compositions were spiked with the estrogenic mycotoxins zearalenone and its metabolites zearalanone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol and beta-zearalenol. The estrogenic response was detected by a whole-cell biosensor based on a genetically modified Saccharomyces cerevisiae strain that in the presence of an estrogenic compound produces firefly luciferase-enzyme and further light emission within a system provided with D-luciferin substrate. The results show that the yeast sensor reacts to mycotoxins with typical sigmoidal response at nanomolar concentrations. The response differs in different milk products with regard to the fat content of the milk. Due to short assay time of less than 3h and automation the approach can be used as a bioavailability and activity screening method prior to more detailed chemical analysis.

Detecting AhR ligands in sediments using bioluminescent reporter yeast.

Sediments polluted with high concentrations of persistent organic pollutants, many of which are ligands of the aryl hydrocarbon receptor (AhR), are currently of concern around the industrialized world. Bioassays that can detect the presence of AhR ligands in environmental samples offer a relatively rapid and cost-effective means of prioritizing samples before more elaborate, laborious, and costly chemical analyses are applied. This paper presents a new bioluminescent yeast assay based on transcriptional activation of AhR. Its applicability for determining AhR ligands in complex environmental samples was demonstrated by analyzing a set of sediment samples from the River Kymi, Finland. The results from the assay are shown to be consistent with those from both a chemical analysis and an H4IIE-luc bioassay. The yeast assay procedure is simple and can be performed within 1 day. The yeasts grow rapidly, are easy to handle, and do not require continuous cell culturing. Moreover, the robustness of the yeast allows the application of the test to crude extracts or even sediment suspensions. The yeast assay described in this paper can be useful in screening and prioritization of samples prior to chemical analysis. Moreover, the strain can be used in the construction of fibre-optic biosensors.

A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds.

We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control of the androgen response element (ARE). In the presence of androgens, activated hAR binds to the ARE sequences and activates luciferase expression. After addition of D-luciferin, luciferase activity measurements can be performed, and the BL signal is proportional to the androgenic activity of the sample. Cytotoxic effects of the sample are monitored by the use of a control yeast strain that allows BL signal correction according to cell viability. After overnight culture of the recombinant strain, the assay can be accomplished in a 96-well microplate format in 1 working day with a detection limit of 0.05 nM for testosterone and intra- and interassay variability of 14% and 23%, respectively.

Determination of estrogens and estrogenic activity in wastewater effluent by chemical analysis and the bioluminescent yeast assay.

A scheme of bioassay-directed analysis has been developed which combines a yeast assay screening for estrogenic activity with a liquid chromatographic-mass spectrometric (LC-MS/MS) chemical analysis, chromatographic fractionation, solid phase extraction and freeze-drying. The test scheme was applied on effluent samples collected from a municipal sewage treatment plant. The aim was to determine the substances responsible for main portion of the estrogenic activity in the samples and to compare the efficiency of different procedures for isolation and concentration of estogenicity. LC-MS/MS analyses were used for the quantification of 17beta-estradiol, estrone, estriol and 17alpha-ethinylestradiol, and the measured concentrations compared with the activities found in the yeast assay. Following conversion of the concentrations measured by LC-MS/MS to 17beta-estradiol equivalents it was concluded that freeze-drying, solid phase extraction and the chemical analysis gave comparable activities. Since estrone was the major estrogen in the effluent, this estrogen was also the major contributor to the estrogenic activity in the effluent. The estrogenic activity was equivalent to 4-7 ng/L of 17beta-estradiol. The yeast assay results from the tests of the chromatographic fractions showed that the major activity resides in the fraction where estrone, 17beta-estradiol and 17alpha-ethinylestradiol eluted. The activity of this fraction was substantially higher than the activity of the original wastewater sample. The reason for this could in part be explained by an inhibition of activity occurring in the original water sample.

Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices.

In this paper we describe the construction and use of a set of bioluminescent yeast strains for the detection of compounds that can affect androgen or estrogen receptor mediated hormonal signalling. The set includes Saccharomyces cerevisiae strains expressing human androgen receptor (AR), estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta), along with firefly luciferase controlled by a respective hormone responsive promoter. A constitutively luminescent strain was included in the set for determining the cytotoxicity of the sample. Yeast cells were incubated with pure chemicals or complex samples for 2.5 h, after which the signal could be detected from the cell-sample mixture after simply adding the D-luciferin substrate. The assays could be completed in one day and they required no cell lysis or centrifugation steps, which makes them suitable for high-throughput analysis of samples. Due to a short incubation time the assays are directly applicable to different sample matrices, requiring no pretreatment of the samples. The assays were used to assess the hormonal activity in moisturizing lotions as an example of a complex sample matrix known to contain endocrine disrupting chemicals. Six out of eight tested moisturisers showed high estrogenic activity, whereas no androgenic activity was observed in the samples.

A new recombinant cell-based bioluminescent assay for sensitive androgen-like compound detection.

A public concern is continuously arising about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions. These substances, defined as endocrine disrupting compounds (EDC) represent an heterogeneous class of molecules either steroidal or not, sharing the ability of interfering with the endocrine system via nuclear receptor signaling pathways. Therefore there is an urgent need for high throughput screening systems able to detect EDCs and evaluate their biological activity. However, little attention has been dedicated to the development of assays for androgen-like compounds. The present work describes the development and optimization of a new rapid and sensitive bioluminescent yeast-based bioassay for androgen-like compounds in a 96-well microplate format. The bioassay is based on recombinant Saccharomyces cerevisiae cells modified to express human androgen receptor (hAR) and containing the sequence androgen response element (ARE) which drives the expression of Photinus pyralis luciferase, used as reporter gene. A recombinant yeast strain constitutively expressing luciferase was used as external control to correct the light signal accordingly to cell viability and sample matrix aspecific effects. The bioassay responds to testosterone as reference androgen in a concentration-dependent manner from 0.05 to 1000 nM allowing an accurate and precise quantitative evaluation in aqueous environmental samples down to 10(-11)mol/L. Other known androgen-like compounds exhibit similar dose-response behavior, thus permitting the use of the bioassay for an overall detection of androgen-like effect in environmental samples.

Detection of bioavailable heavy metals in EILATox-Oregon samples using whole-cell luminescent bacterial sensors in suspension or immobilized onto fibre-optic tips.

At the EILATox-Oregon Workshop, nine luminescent whole-cell bacterial sensors were used for the determination of bioavailable metals in blind samples (17 synthetic and 3 environmental). A non-inducible luminescent control strain was used to determine sample matrix effects and bacterial toxicity. Whole-cell bacterial sensors capable of determining arsenic, inorganic mercury and its organic derivatives, cadmium, lead or copper were used in suspensions and a bacterial sensor for the detection of inorganic mercury was immobilized onto fibre-optic tips using calcium alginate. Bioavailable amounts of metals were estimated using calibration plots, that were constructed to determine the range of metals giving rise to a linear relationship between luminescence and the amount of metals present in the standard solutions. EILATox-Oregon sample 5, which contained 74 mg l(-1) of Hg, gave a significant response with both formats of the mercury sensor. The bioavailable amounts of mercury according to the measurement of bacterial sensor in suspension and immobilized onto a fibre-optic tip were 76 and 93 mg l(-1), respectively. The bacterial sensor for arsenic and copper showed a response with sample 6 (58 mg l(-1) of As) and sample 8 (400 mg l(-1) of metham sodium), respectively. This study showed that the bacterial sensors in suspension or immobilized onto optical fibres are capable of quantifying bioavailable metals from unknown samples. The measurement protocol of bacterial sensors is simple and possible to perform in the field. Moreover, the samples do not need any pretreatment before analysis. Construction and characterization of the strain for the detection of bioavailable copper are described.