Evaluation and optimization of PCR primers for selective and quantitative detection of marine ANME subclusters involved in sulfate-dependent anaerobic methane oxidation.

Since the discovery that anaerobic methanotrophic archaea (ANME) are involved in the anaerobic oxidation of methane coupled to sulfate reduction in marine sediments, different primers and probes specifically targeting the 16S rRNA gene of these archaea have been developed. Microbial investigation of the different ANME subtypes (ANME-1; ANME-2a, b, and c; and ANME-3) was mainly done in sediments where specific subtypes of ANME were highly enriched and methanogenic cell numbers were low. In different sediments with higher archaeal diversity and abundance, it is important that primers and probes targeting different ANME subtypes are very specific and do not detect other ANME subtypes or methanogens that are also present. In this study, primers and probes that were regularly used in AOM studies were tested in silico on coverage and specificity. Most of the previously developed primers and probes were not specific for the ANME subtypes, thereby not reflecting the actual ANME population in complex samples. Selected primers that showed good coverage and high specificity for the subclades ANME-1, ANME-2a/b, and ANME-2c were thoroughly validated using quantitative polymerase chain reaction (qPCR). From these qPCR tests, only certain combinations seemed suitable for selective amplification. After optimization of these primer sets, we obtained valid primer combinations for the selective detection and quantification of ANME-1, ANME-2a/b, and ANME-2c in samples where different ANME subtypes and possibly methanogens could be present. As a result of this work, we propose a standard workflow to facilitate selection of suitable primers for qPCR experiments on novel environmental samples.

Growth and activity of ANME clades with different sulfate and sulfide concentrations in the presence of methane.

Extensive geochemical data showed that significant methane oxidation activity exists in marine sediments. The organisms responsible for this activity are anaerobic methane-oxidizing archaea (ANME) that occur in consortia with sulfate-reducing bacteria. A distinct zonation of different clades of ANME (ANME-1, ANME-2a/b, and ANME-2c) exists in marine sediments, which could be related to the localized concentrations of methane, sulfate, and sulfide. In order to test this hypothesis we performed long-term incubation of marine sediments under defined conditions with methane as a headspace gas: low or high sulfate (±4 and ±21 mM, respectively) in combination with low or high sulfide (±0.1 and ±4 mM, respectively) concentrations. Control incubations were also performed, with only methane, high sulfate, or high sulfide. Methane oxidation was monitored and growth of subtypes ANME-1, ANME-2a/b, and ANME-2c assessed using qPCR analysis. A preliminary archaeal community analysis was performed to gain insight into the ecological and taxonomic diversity. Almost all of the incubations with methane had methane oxidation activity, with the exception of the incubations with combined low sulfate and high sulfide concentrations. Sulfide inhibition occurred only with low sulfate concentrations, which could be due to the lower Gibbs free energy available as well as sulfide toxicity. ANME-2a/b appears to mainly grow in incubations which had high sulfate levels and methane oxidation activity, whereas ANME-1 did not show this distinction. ANME-2c only grew in incubations with only sulfate addition. These findings are consistent with previously published in situ profiling analysis of ANME subclusters in different marine sediments. Interestingly, since all ANME subtypes also grew in incubations with only methane or sulfate addition, ANME may also be able to perform anaerobic methane oxidation under substrate limited conditions or alternatively perform additional metabolic processes.

Growth of anaerobic methane-oxidizing archaea and sulfate-reducing bacteria in a high-pressure membrane capsule bioreactor.

Communities of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) grow slowly, which limits the ability to perform physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated SRB are affected by it. Here, we report on the growth of ANME-SRB in a membrane capsule bioreactor inoculated with Eckernförde Bay sediment that combines high-pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2-m-pore-size membrane. The results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. The rates of oxidation of labeled methane were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was enriched only at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2 and Eel-1 groups and the Desulfuromonadales and not to the typically found SEEP-SRB1 group. The increase of ANME-2a/b occurred in parallel with the increase of SEEP-SRB2, which was previously found to be associated only with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence the growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at a high methane partial pressure.

Inhibition of binding of the AB5-type enterotoxins LT-I and cholera toxin to ganglioside GM1 by galactose-rich dietary components.

Cholera, travelers' diarrhea, or colibacillosis in pigs can possibly be prevented or attenuated by dietary provision of competitive inhibitors that react with the GM1-binding sites of the enterotoxins cholera toxin (CT), human Escherichia coli heat-labile enterotoxin of serogroup I (LTh-I), and porcine LT-I (LTp-I). The interfering efficiency of natural substances with binding of the toxins to the gangliosid receptor GM1 was tested using a specially adapted GM1-coated-microtiter-well enzyme-linked immunosorbent assay. The substances tested for their GM1 displacing capacity were galactose-containing or -related saccharides from bovine milk, skim milk powder, galactan from gum arabic, food stabilizers as well as ground fenugreek seed and soy bean constituents that contain galactomannans, the galactopolysaccharides agar and agarose, and larch wood and other plant materials that contain arabinogalactans. Skim milk powder, compared with the pure milk saccharides tested, interfered to a higher extent with LTh-I (65-66% inhibition at 5 mg test substance/mL) and CT binding (63-67% inhibition at 5 mg test substance/mL) when supplied before or simultaneously with the toxins in the GM1-enzyme-linked immunosorbent assay. Ground fenugreek seed counteracted GM1 binding of 5 ng LTh-I/mL as well as 5 ng and 1 microg LTp-I/mL (43-65% inhibition at 5 mg test substance/mL), and 4 ng CT/mL (61-92% inhibition at 5 mg test substance/mL) very efficiently when supplied before the toxin-GM1 complex had formed. With 50 mg/mL fenugreek seed, inhibition percentages of even 92-99% were reached for LTh-I and CT binding. Efforts to resolve already bound toxin from GM1 with the test substances were less effective than preincubations and concurrent incubations.

Effects of atipamezole, detomidine and medetomidine on release of steroid hormones by porcine adrenocortical cells in vitro.

The 4-substituted imidazole type alpha2-adrenoceptor ligands atipamezole, detomidine, and medetomidine were screened for actions on the release of aldosterone by a suspension of porcine adrenocortical cells with deoxycorticosterone (1 microM) as substrate. Progesterone, pregnenolone or corticosterone (all at 1 microM) were also used as substrates. With pregnenolone as substrate, drug-induced effects on the output of nine steroids (aldosterone, corticosterone, cortisol, deoxycortisol, testosterone, progesterone, 17alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone) were monitored simultaneously. The alpha2-adrenoceptor antagonist atipamezole was a potent inhibitor of aldosterone release (range 10-1000 nM). The sedative alpha2-adrenoceptor agonists medetomidine and detomidine also inhibited aldosterone release (range 10-1000 nM). With pregnenolone as substrate, the inhibition induced by 4-substituted imidazoles of the release of corticosterone and cortisol was more pronounced than that of aldosterone. Androstenedione and deoxycortisol release was enhanced. The 4-substituted imidazoles atipamezole, detomidine, and medetomidine inhibited mitochondrial cytochrome P450(11beta/18) in vitro. This inhibition was unrelated to their alpha2-adrenoceptor actions. The 4-substituted imidazole type alpha2-adrenoceptor ligands used to control sedation/anaesthesia can alter the steroid-based defence mechanisms of the body.

Effects of alpha1-antagonists on production and release of aldosterone and other steroid hormones by porcine adrenocortical cells in vitro.

Quinazoline type alpha1-adrenoceptor antagonists (range 10-100 microM) inhibited aldosterone release of a cell suspension of porcine adrenocortical cells, potency order: doxazosin > prazosin > trimazosin. Phenoxybenzamine also inhibited the aldosterone release at a concentration of 100 microM. Alpha1-adrenoceptor antagonists from other chemical classes had no measurable effect on the aldosterone output from adrenocortical cells in vitro. Agonists selective for either alpha1- or beta-adrenoceptors did not affect the aldosterone release. The inhibition of the aldosterone release induced by quinazolines was similar with different substrates. The small differences between the drug-induced inhibitions could be ranked as corticosterone = progesterone > pregnenolone = deoxycorticosterone. The doxazosin (10 microM)-induced changes in the release of nine steroids indicated that quinazoline-type alpha1-antagonists interfere with enzymes of the aldosterone biogenesis pathway involved in C18-oxidation and C21beta-hydroxylation, reducing the release of both aldosterone and corticosterone. At higher concentrations (100 microM), the C21beta-hydroxylation in the cortisol biogenesis pathway is also affected, decreasing the output of cortisol and deoxycortisol, but increasing the output of progesterone and OH-progesterone. Simultaneously, the C17-oxidation and side-chain cleavage is also inhibited, decreasing the output of androstenedione. The rank order of phenoxybenzamine (100 microM)-induced inhibition of the aldosterone release with different substrates is pregnenolone > corticosterone = progesterone > deoxycorticosterone. With pregnenolone as substrate, the output of aldosterone, corticosterone, and cortisol was reduced to the same extent. The dehydroepiandrosterone, androstenedione, and progesterone release was enhanced. It seems that phenoxybenzamine is a rather selective inhibitor of the mitochondrial P450(11beta/18) enzymes.

Differential effects of nitrofurans on the production/release of steroid hormones by porcine adrenocortical cells in vitro.

Changes in the biogenesis of corticosteroids caused by nitrofurans were studied. The three nitrofurans used: furazolidone, furaltadone and nitrofurantoin, altered the steroid production/release by porcine adrenocortical cells in vitro during 1 h incubations. With pregnenolone as a substrate the nitrofurans inhibited aldosterone production/release. Although the nitrofurans differed in potency (nitrofurantoin > furazolidone > furaltadone) maximum inhibition occurred at 100 microM. In this concentration the nitrofurans changed also the release/production of other corticosteroids. The output of corticosterone and cortisol decreased by 50%. The production/release of deoxycortisol stayed the same. In contrast the output of progesterone and 17alpha-hydroxyprogesterone increased to more than 200% of control. The nitrofurans slightly reduced the output of androstenedione. No significant increases of the production/release of other steroids (testosterone, dehydroepiandrosterone, estradiol-17beta and estrone) by the cell suspension could be observed. The profile of the nitrofuran-induced changes lead to the conclusion that nitrofurans interfere with mitochondrial enzymes. These enzymes, presumably cytochrome P450(11,18) mediate the hydroxylation and the oxidation at C11 and C18, the final steps in the biogenesis of aldosterone, corticosterone and cortisol. The rapid and reversible fall in the output of these steroids occurs in vitro at concentrations which are below therapeutic blood concentrations seen in vivo. At higher concentrations the nitrofurans hinder the biogenesis of androgens. Thus nitrofurans can also affect steps in the steroid biogenesis located in the endoplasmatic reticulum.

Screening for drug-induced alterations in the production and release of steroid hormones by porcine adrenocortical cells in vitro.

To screen drugs rapidly and at minimal expense for their potential to alter steroidogenesis, an in vitro model using porcine adrenocortical cells was developed. Pregnenolone, progesterone, deoxycorticosterone or corticosterone (all at 1 muM) were used as substrates. Drug-induced changes in the production/release of aldosterone were measured after 1-hr incubation. With pregnenolone, drug-induced effects on the release of nine steroids (aldosterone, corticosterone, cortisol, deoxycortisol, testosterone, progesterone, HO-progesterone, androstenedione, dehydroepiandrosterone) were monitored simultaneously. For assessment of cell viability and the amount of steroids produced/released, a cheap, simple modified Krebs solution was at least comparable to an elaborate cell culture medium. Within the conditions adopted, the cell suspension reacted to varying potassium concentrations as expected. ACTH stimulated steroid production/release only without added substrate. 11 agents known to interfere with steroid biogenesis were tested at 0.1-100 muM. Although all known points of action of the test compounds were located, several showed additional activity. Spironolactone shifted steroid biogenesis from aldosterone and cortisol towards androgenic steroids. Aminoglutethimide inhibited the release of aldosterone with corticosterone as substrate, but not with deoxycorticosterone or progesterone as substrate, revealing an alternative pathway in the biogenesis of aldosterone by-passing corticosterone. Trilostane (0.1-1 muM) completely blocked conversion of pregnenolone to progesterone and OH-progesterone; the release of androstenedione was at most only halved, whereas the release of dehydroepiandrosterone and testosterone was greatly enhanced. This implies isoenzymes of 3beta-hydroxysteroid dehydrogenase/isomerase with different sensitivities towards trilostane. Mitotane, metyrapone, ketoconazole and etomidate all inhibited the mitochondrial P450 (11beta 18 ) enzymes. In addition, mitotane and ketoconazole also inhibited (albeit to a lesser extent) endoplasmic enzymes involved in transformations at C21 and at C17, respectively. Cyproheptadine blocks all transformations with progesterone or HO-progesterone as starting point. Finasteride reduced the release of most steroids, except the androgens, presumably by inhibition of transformations at C3 and at C11. Carbadox and related quinoxalines inhibited not only C18 oxidation but also C21 hydroxylation. Steroidogenesis in these porcine adrenocortical cells in vitro could be described as similar to that in other mammals. A notable feature was that inhibition of the release/production of a steroid hormone was usually accompanied by an increased release of other steroid hormones. This screening model also yields information about the point of action of drugs interfering with steroidogenesis.

Effects of feed additives and veterinary drugs on aldosterone production and release by porcine adrenal cells in vitro.

The preparation of suspensions of porcine adrenocortical cells is described. Within the conditions adopted, the cell suspension responded to various agents as expected. It was possible to screen drugs (standard range 0.3-100 microM, incubation period 1 h) for actions on the production/release of aldosterone by the cortical cells using 1 microM deoxycorticosterone as substrate. Progesterone, pregnenolone or corticosterone were also used as substrates. Feed additives of the quinoxaline type induced a slowly developing inhibition of aldosterone production/release by the cell suspension, which was virtually irreversible. During the standard 1 h incubation period inhibitions of up to 22 +/- 2% of control were observed, which increased upon prolongation of the incubation by 2 h. With 100 microM cyadox the inhibition increased from 19 +/- 2% to 35 +/- 2% with prolonged incubation. Ten nitrofuran compounds exerted a more rapidly developing inhibition (by up to 79 +/- 1% of control) of aldosterone production/release, which was reversible. A submaximal inhibition with 10 microM furazolidone of 21 +/- 5% increased to 40 +/- 1% with longer incubation. The concentrations at which these compounds exerted this effect in vitro were well below the peak blood plasma concentrations encountered after administration of the drugs in therapeutic doses. Polyether-antibacterials/ionophores rapidly inhibited aldosterone production/release (to 26 +/- 1% of control) and this effect was completely reversible. The nitroimidazole compounds tested did not affect aldosterone production/release when deoxycorticosterone or progesterone were used as substrates. With use of corticosterone and to a lesser extent with pregnenolone as substrates a clear inhibition (to 73 +/- 3% of control) of aldosterone production was obtained. Amprolium in concentrations up to 100 microM, with deoxycorticosterone as substrate, did not induce a significant change in aldosterone production/release by the suspension of adrenocortical cells. In the same dose range tylosin and roxarsone induced a small but significant inhibition (by up to 10 +/- 3% of control) of aldosterone production/release, which was not dose-dependent. It is concluded that a wide range of growth-promoting drugs may be able to change aldosterone production/release in the animal.