Phylogenetic delineation of the novel phylum Armatimonadetes (former candidate division OP10) and definition of two novel candidate divisions.

Small-subunit (SSU) rRNA gene sequences associated with the phylum Armatimonadetes were analyzed using multiple phylogenetic methods, clarifying both the phylum boundary and the affiliation of previously ambiguous groupings. Here we define the Armatimonadetes as 10 class-level groups and reclassify two previously associated groups as candidate divisions WS1 and FBP.

Evaluation of lactic acid bacterium from chilli waste as a potential antifungal agent for wood products.

AIMS: The aim of this study was to isolate lactic acid bacteria from chilli waste and evaluate metabolites produced for the ability to arrest wood decay. METHODS AND RESULTS: Using an optical density screening method, one bacterium (isolate C11) was identified as having pronounced antifungal properties against Oligoporus placenta. This isolate was identified as Lactobacillus brevis by 16S rRNA gene sequencing. To determine antifungal activity in wood, Pinus radiata blocks were impregnated with Lact. brevis [C11] cell-free supernatant and exposed to brown rot fungi O. placenta, Antrodia xantha and Coniophora puteana. The treated timber demonstrated resistance to degradation from all fungi. The antifungal metabolites were heat stable and not affected by proteinase K, but were affected by neutralization with NaOH suggesting the metabolites were of an acidic nature. The presence of lactic and acetic acid was confirmed by HPLC analysis. CONCLUSIONS: Lactobacillus brevis [C11] produced acidic metabolites that were able to inhibit the growth of wood decay fungi and subsequent wood decay. SIGNIFICANCE AND IMPACT OF THE STUDY: Traditional wood treatments are becoming an environmental issue as the public demands more benign options. The use of lactic acid bacteria which are considered safe for general use is a potential alternative to the conventional heavy metal chemicals currently in use.

Regulation of expression of methane monooxygenases by copper ions.

Many methanotrophs contain both a soluble and a particulate methane monooxygenase. A unique metabolic switch, mediated by copper ions, regulates the expression of these enzymes. When the copper-to-biomass ratio of the cell is low, the soluble enzyme is expressed, and when the copper-to-biomass ratio is high, the particulate enzyme is expressed. A model for the mechanism of this switch is proposed.

Corticosteroid binding by plasma proteins and the effects of oestrogens in the marsupial brush-tailed opossum, Trichosurus vulpecula (Kerr).

Using the techniques of equilibrium dialysis at 36 degrees C and gel filtration at 4 degrees C, a high-affinity, transcortin-like, corticosteroid binding system has been demonstrated in the blood plasma of the marsupial Trichosurus vulpecula. The affinity constant for non-albumin binding was 4-018 +/- 1-032 X 10(7) (S.C.) 1/mol for males and 4-046 +/- 0.981 X 10(7) for females. The concentration of non-albumin binding sites was 1-82 +/- 0.76 X 10(-7) mol/1 in males and 1-86 +/- 0-57 X 10(-7) mol/1 for females. Oestrogen administration, sufficient to cause marked hypertrophy of the genital tract in the femlaes, had no effect on the affinity constant or the concentration of the non-albumin binding sites in either males or females. The general condition of the animals deteriorated during oestrogen administration and there were significant falls in the concentrations of albumin and cortisol in the blood plasma. In one animal which died during oestrogen treatment, the adrenal glands were significantly hypertrophied.

Secretion of aldosterone in the monotreme mammal, Tachyglossus aculeatus.

The secretion of aldosterone and its regulation by ACTH and angiotensin II were investigated in conscious, unrestrained echidnas with chronically implanted jugular catheters. Aldosterone was measured by radioimmunoassay after extraction and isolation by silica gel thin-layer chromatography. The mean concentration of aldosterone in blood plasma of five male and three female echidnas was only 5.4 +/- 1.3 (S.E.M.) pg/ml. During stress (surgery and anaesthesia) the mean concentration increased to 17.6 +/- 3.8 pg/ml. Infusion of beta 1-24 ACTH at a rate of 5 units/kg per h increased the plasma concentration of aldosterone to 53.8 +/- 9.8 pg/ml. Infusion of angiotensin II at rates of 100 and 500 ng/kg per h also increased aldosterone concentration, to 24.1 +/- 8.6 and 35.1 +/- 10.9 pg/ml respectively. Production and metabolic clearance rates were measured by the constant-rate infusion of [3H]aldosterone and found to be 5.0 +/- 2.2 ng/kg per h and 14.3 +/- 1.3 ml/kg per min respectively in the unstimulated state. Production rate was increased approximately sevenfold by the infusion of ACTH at 5 units/kg per h and fourfold and sixfold by infusion of angiotensin II at 100 and 500 ng/kg per h respectively. Metabolic clearance decreased following the infusion of ACTH or angiotensin II. Direct measurement of secretion rate by the collection of adrenal venous blood from three anaesthetized, laparotomized echidnas gave values of 9.4, 15.6 and 8.8 mg/kg per h. It is concluded that the adrenal secretion of aldosterone in the echidna is extremely low compared with that in other mammals but the response to stress, ACTH and angiotensin II indicates the presence of typical mammalian control mechanisms for its secretion.

Adrenocortical functions in a macropodid marsupial Thylogale billardierii.

In a study of adrenocortical functions in macropodid marsupials, measurements were made of the effects of ACTH infusion, ether stress and adrenaline infusion on plasma corticosteroid and glucose concentrations in wallabies (Thylogale billardierii) provided with indwelling venous catheters. The mean plasma total glucocorticoid concentration in undisturbed males and females was 80 +/- 5 (S.E.M.) micrograms/l, of which more than 90% was cortisol. This fraction declined to 68% of the total at the highest ACTH-stimulated concentration of 225 micrograms/l, due to an increase in the contribution by 11-deoxycortisol. Although maximal ACTH stimulation (4.5 i.u./kg per h) caused a five- to sixfold increase in cortisol secretion rate, as measured by isotope dilution during constant-rate tracer infusion, plasma cortisol concentration rose only two- to threefold, due to a marked increase in metabolic clearance. Plasma glucose concentration did not change significantly during either short-term (1 h) i.v. infusion or long-term (8 days) i.m. injection of ACTH, even though plasma cortisol concentration was significantly increased. Ether anaesthesia caused a marked hyperglycaemia that preceded an increase in plasma cortisol concentration and was not sustained while plasma cortisol concentration continued to increase. Infusion of adrenaline i.v. at rates sufficient to cause a similar hyperglycaemia had no significant effect on plasma cortisol concentration. A marked hyperglycaemia during xylazine anaesthesia was not associated with an increase in plasma cortisol concentration and was attributable to suppression of insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic effects of cortisol, corticosterone and adrenocorticotrophin in a prototherian mammal Tachyglossus aculeatus (Shaw).

The effects of injections of cortisol, corticosterone and ACTH on indices of carbohydrate, fat and protein metabolism were investigated in the conscious echidna, Tachyglossus aculeatus. Intravenous infusion of cortisol and corticosterone for 2 h at rates of 3 and 30 microgram/kg/h respectively did not cause significant changes in the plasma concentrations of glucose, urea or amino acids during a 12.5 h observation period. In contrast, a dose-related increase in plasma free fatty acid (FFA) concentration was observed. Infusion of synthetic ACTH at 2 i.u./kg/h for 2 h caused a minor, short-lived increase in FFA concentration. Daily i.m. injections of 0.2 mg cortisol or corticosterone acetates/kg, which raised plasma total corticosteroid concentrations to levels characteristic of maximal ACTH stimulation, did not cause glycosuria nor was there any change in body weight, nitrogen intake or urinary nitrogen excretion. However, there was a minor, but significant, increase in plasma glucose concentration. The liver glycogen content of 24 h fasted, corticosteroid-treated animals was similar to that of fasted control animals. It is concluded that cortisol, corticosterone and ACTH have only minor effects on carbohydrate and protein metabolism and that the main action of these hormones may be to mobilize fat reserves.

Effect of cortisol on utilization and hepatic release of glucose in the marsupial brush-tailed opossum, Trichosurus vulpecula.

Brush-tailed opossums were prepared surgically with indwelling heatic and jugular venous catheters for blood sampling without disturbance in the conscious state. Hepatic extraction of Rose Bengal was 21 +3 (s.d.) % and hepatic clearance, used as a measure of hepatic blood flow, was 42.5 +7 ml/kg/min. Hepatic relaease of new glucose, calculated from the thorias vena caval-hepatic venous difference in glucose specific activity at equilibrium during i.v. infusion of [14C]glucose and hepatic blood flow, was 3.5 + o.0 mg/kg/mim. This was not changed by i.v. infusions of 10% ethanolic aline or cortisol in ethanolic saline, at 1mg/kg/h for 90 min, although the cortisol infusion caused the peripheral blood glucose concentration to rise form 56.5 + 7.3 to 83.2 + 10.3 mg/100ml. The rate of metabolic clearance of glucose fell from 6.1 +1.1 to 4.2 +0.9 ml/kg/min during i.v. cortisol infusion. Daily i.m. injection of 1 mg cortisol accetate/kg for 5 days caused an increase in hepatic new glucose release to 8.0 + 1.6 mg/kg/min. The findings support the propostion that, in the marsupial, the short-term effect of cortisol on plasma glucose concentration is due to inhibition of peripheral glucose utilization, whereas the long-term effect is due to increased hepatic glucose production.

Adrenocortical function in a prototherian mammal, Tachyglossus aculeatus (Shaw).

The peripheral plasma concentrations and production rates of corticosterone and cortisol were measured in the conscious, unrestrained echidna (Tachyglossus aculeatus) under basal conditions and during maximal ACTH stimulation. Using Sephadex LH-20 column chromatography and radioligand assay, only cortisol and corticosterone could be detected in the peripheral blood plasma at very low concentrations of 0-07 +/- 0-03 (S.E.M.) mug/100 ml and 0-14 +/- 0-07 mug/100 ml respectively. Two-hourly sampling over periods of 36-48 h disclosed a diurnal periodicity in the combined plasma concentration of these corticosteroids, the high concentrations corresponding to periods of behavioural activity. Marked, short-term fluctuations in plasma corticosteroid concentration were also observed during periods of more frequent (20 min) sampling. Constant rate i.v. infusion of synthetic ACTH increased the plasma concentrations of both steroids to maximal values of 0-42 +/- 0-23 mug cortisol/100 ml and 1-06 +/- 0-56 mug corticosterone/100 ml at infusion rates of 1 i.u. ACTH/kg/h. This is approximately 1/160 of the potency of this ACTH in man. The production rates of corticosterone and cortisol, measured by isotope dilution during constant rate i.v. infusion of 3H-labelled tracers, were only 0-35 +/- 0-21 and 0-56 +/- 0-26 mug/kg/h respectively during saline infusion, and increased to 2-86 +/- 3-47 and 2-74 +/- 2-07 mug/kg/h during the infusion of 1 i.u. ACTH/kg/h. The metabolic clearance rate of cortisol was greater than that of corticosterone and both were depressed by ACTH. Plasma corticosteroid concentrations were increased after surgery during ether anaesthesia and in sick animals with heavy worm infestation. It is concluded that the adrenal cortex of echidnas responds to ACTH stimulation and stress in a similar way to eutherians, but the level of activity is much lower.

Metabolic actions of cortisol in a macropodid marsupial Thylogale billardierii.

In undisturbed pademelon wallabies (Thylogale billardierii) with indwelling jugular venous catheters, an increase in the plasma cortisol concentration from 0.25 +/- 0.05 to 1.35 +/- 0.15 (S.E.M.) mumol/l in 2 h, during i.v. infusion of cortisol at 1.0 mg/kg per h, caused no significant change in the plasma glucose concentration from the control value of 4.26 +/- 0.25 mmol/l. The rates of appearance (Ra) and metabolic clearance (MCR) of glucose, measured by steady-state isotope dilution, also did not change significantly from the control values of 14.9 +/- 0.7 mumol/kg per min and 3.52 +/- 0.19 ml/kg per min respectively. Twice-daily i.m. injections of 7 mg cortisol/kg for 7 days caused increases in plasma concentrations of cortisol, from 0.26 +/- 0.02 to 0.66 +/- 0.04 mumol/l on day 7, and glucose, from 5.1 +/- 0.1 to 7.2 +/- 0.6 mmol/l by day 5. The concentration of glycogen in the liver of wallabies fasted for 24 h increased from the control level of 1.17 +/- 0.56 to 5.92 +/- 1.14 g/100 g on day 7 (P less than 0.01), but mean glucose Ra and MCR did not change significantly. Plasma concentrations of alpha-amino nitrogen rose from 2.73 +/- 0.13 to 3.22 +/- 0.12 mmol/l on day 1 and remained at this level. Plasma concentrations of urea rose from 8.59 +/- 0.62 to 9.70 +/- 0.32 mmol/l on day 1, but then declined below the control level. Food intake and urinary excretion of nitrogen did not change in undisturbed animals. However, fasting followed by liver biopsy was accompanied by urinary excretion of nitrogen in excess of food intake, persisting until day 2 of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids in the blood plasma of the platypus Ornithorynchus anatinus.

Blood samples were obtained from two male and two female platypuses at various times after capture and anaesthesia for other experimental purposes. In samples obtained during ketamine-xylazine or pregnanediol anaesthesia 15-24 h after capture, the concentration of total glucocorticoids, measured as 'cortisol equivalent' in a radioligand assay, was 207-620 nmol/l. In samples taken 14-35 h after injection of dexamethasone (0.2 mg/kg) total glucocorticoid concentration was 79-88 nmol/l. Individual glucocorticoids were isolated on columns of Sephadex LH-20 and measured separately against appropriate standards. In all except two haemolysed samples obtained from a male that died 25 h after capture, the major glucocorticoid behaved as cortisol, contributing 77-94% of the total. The remainder was made up of varying proportions of substances behaving as corticosterone, 11-deoxycortisol and cortisone. In the haemolysed samples from the moribund animal the major reactive substance, contributing 52-54% of the total, behaved as cortisone. The total adrenal gland weight of this animal was 747 mg, compared with 200-286 mg in two others, suggesting preceding exposure to stress. Equilibrium dialysis and polyacrylamide gel electrophoresis (PAGE) revealed no evidence for a transcortin-like glucocorticoid- and progesterone-binding protein in platypus plasma. However, as in the echidna, there was a heat-labile, high-capacity binding system migrating with albumin on PAGE. Glucose was undetectable in the plasma of the moribund animal and only 1.7-2.8 mmol/l in the initial plasma samples from the others. In two animals, injection of glucose i.p. and dexamethasone i.m. was followed by an increase in the plasma concentration of glucose to the range 3.8-9.9 mmol/l and commencement of normal swimming and feeding activity for the next 36-48 h.

Failure of glucocorticoid feedback in males of a population of small marsupials (Antechinus swainsonii) during the period of mating.

In an investigation of the factors leading to the increase in the concentration of plasma free glucocorticoid, which results in immunosuppression and death after mating of all males in natural populations of a small shrew-like marsupial, the dusky antechinus (Antechinus swainsonii), the integrity of the glucocorticoid feedback control of the concentration of plasma cortisol was examined by use of dexamethasone-suppression tests. Injection of 0.2 mg dexamethasone/kg i.m. caused a marked fall in the concentration of plasma cortisol 17 h later, approximately 2 months and 2 weeks before the annual mating period in mid-July. However, the same dose had no significant effect on the increased concentration of plasma cortisol characteristic of the mid- to late July mating period. Injection of 100 i.u. ACTH/kg i.m. caused a significant increase in the concentration of plasma cortisol 6-7 h later on all occasions, indicating that the responsiveness of the adrenal cortex to ACTH did not change. Pretreatment with dexamethasone had no effect on the ACTH-stimulated cortisol concentration, ruling out a possible direct effect of dexamethasone on adrenocortical secretion in this species. Dexamethasone also reduced the concentration of plasma testosterone when the level was low, before the mating period, but not when the level was high, at the beginning of the mating period. It is concluded that, in association with a rapid increase in the concentration of plasma testosterone, an increase in aggression and intense mating activity, glucocorticoid feedback control of ACTH secretion is impaired.(ABSTRACT TRUNCATED AT 250 WORDS)

The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs.

The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.

Methyl chloride utilising bacteria are ubiquitous in the natural environment.

Enrichment and isolation of methyl chloride utilising bacteria from a variety of pristine terrestrial, freshwater, estuarine and marine environments resulted in the detection of six new methyl chloride utilising Hyphomicrobium strains, strain CMC related to Aminobacter spp. and to two previously isolated methyl halide utilising bacteria CC495 and IMB-1, and a Gram-positive isolate SAC-4 phylogenetically related to Nocardioides spp. All the pristine environments sampled for enrichment resulted in the successful isolation of methyl chloride utilising organisms.

Stable isotope probing: technical considerations when resolving (15)N-labeled RNA in gradients.

RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. (15)N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using (15)N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used (15)N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with (15)N-ammonium chloride or (15)N(2) we assessed the fractionation resolution of (15)N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between (15)N- and (14)N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments.