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I R McDonald

Publications and source records attributed to I R McDonald.

At least 19 recordsLinked to original sources
PubMed1998

A novel pink-pigmented facultative methylotroph, Methylobacterium thiocyanatum sp. nov., capable of growth on thiocyanate or cyanate as sole nitrogen sources.

The isolation and properties of a novel species of pink-pigmented methylotroph, Methylobacterium thiocyanatum, are described. This organism satisfied all the morphological, biochemical, and growth-substrate criteria to be placed in the genus Methylobacterium. Sequencing of the gene encoding its 16S rRNA confirmed its position in this genus, with its closest phylogenetic relatives being M. rhodesianum, M. zatmanii and M. extorquens, from which it differed in its ability to grow on several diagnostic substrates. Methanol-grown organisms contained high activities of hydroxypyruvate reductase -3 micromol NADH oxidized min-1 (mg crude extract protein)-1], showing that the serine pathway was used for methylotrophic growth. M. thiocyanatum was able to use thiocyanate or cyanate as the sole source of nitrogen for growth, and thiocyanate as the sole source of sulfur in the absence of other sulfur compounds. It tolerated high concentrations (at least 50 mM) of thiocyanate or cyanate when these were supplied as nitrogen sources. Growing cultures degraded thiocyanate to produce thiosulfate as a major sulfur end product, apparently with the intermediate formation of volatile sulfur compounds (probably hydrogen sulfide and carbonyl sulfide). Enzymatic hydrolysis of thiocyanate by cell-free extracts was not demonstrated. Cyanate was metabolized by means of a cyanase enzyme that was expressed at approximately sevenfold greater activity during growth on thiocyanate [Vmax 634 +/- 24 nmol NH3 formed min-1 (mg protein)-1] than on cyanate [89 +/- 9 nmol NH3 min-1 (mg protein)-1]. Kinetic study of the cyanase in cell-free extracts showed the enzyme (1) to exhibit high affinity for cyanate (Km 0.07 mM), (2) to require bicarbonate for activity, (3) to be subject to substrate inhibition by cyanate and competitive inhibition by thiocyanate (Ki 0.65 mM), (4) to be unaffected by 1 mM ammonium chloride, (5) to be strongly inhibited by selenocyanate, and (6) to be slightly inhibited by 5 mM thiosulfate, but unaffected by 0.25 mM sulfide or 1 mM thiosulfate. Polypeptides that might be a cyanase subunit (mol.wt. 17.9 kDa), a cyanate (and/or thiocyanate) permease (mol.wt. 25.1 and 27.2 kDa), and a putative thiocyanate hydrolase (mol.wt. 39.3 kDa) were identified by SDS-PAGE. Correlation of the growth rate of cultures with thiocyanate concentration (both stimulatory and inhibitory) and the kinetics of cyanase activity might indicate that growth on thiocyanate involved the intermediate formation of cyanate, hence requiring cyanase activity. The very high activity of cyanase observed during growth on thiocyanate could be in compensation for the inhibitory effect of thiocyanate on cyanase. Alternatively, thiocyanate may be a nonsubstrate inducer of cyanase, while thiocyanate degradation itself proceeds by a carbonyl sulfide pathway not involving cyanate. A formal description of the new species (DSM 11490) is given.

Cyanates↗
PubMed1997

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.

DNA Probes↗
PubMed1997

Autotrophic growth on carbon disulfide is a property of novel strains of Paracoccus denitrificans.

Three distinct strains (KL1, KS1, and KS2) of facultatively chemolitho-autotrophic bacteria able to use carbon disulfide or carbonyl sulfide as sole energy substrates were identified as novel strains of Paracoccus denitrificans. Evidence for their identity as biovars of P. denitrificans and as close relatives of Paracoccus versutus is based on their DNA composition, total sequencing of the genes for their 16S rRNA, muropeptide profiles, amino acid composition of peptidoglycan, kinetics of murein degradation by lysozyme, possession of large plasmids (91-98 kb) and megaplasmids (> 450 kb), and plasmid transfer between the strains and with P. denitrificans and P. versutus. No functions have been identified for the 91- to 98-kb plasmids of strains KL1 and KS2, but curing strain KL1 of its plasmid did not affect growth on carbon disulfide, thiosulfate or succinate. Emendation of the formal description of Paracoccus denitrificans is presented. Autotrophic growth on carbon disulfide and thiosulfate was confirmed by 14CO2 fixation. Evidence is presented for initiation of carbon disulfide oxidation by an NADH-dependent oxygenase. Cell-free extracts catalyzed (1) NADH-stimulated uptake of oxygen in the presence of carbon disulfide, and (2) carbon-disulfide-stimulated oxidation of NADH. The activity was not sedimented at 50,000 x g. Intermediates in aerobic carbon disulfide metabolism were shown by GC and GC/MS to include carbonyl sulfide and hydrogen sulfide, but anaerobic production of COS and H2S from carbon disulfide did not occur. SDS-PAGE of cell-free extracts showed polypeptides that were unique to growth on carbon disulfide, common to carbon disulfide and carbonyl sulfide, or found after growth on carbon disulfide, carbonyl sulfide or thiosulfate. The possible identity of these as proteins involved in sulfur compound metabolism is discussed.

Bacterial Proteins↗
PubMed1997

The soluble methane monooxygenase gene cluster of the trichloroethylene-degrading methanotroph Methylocystis sp. strain M.

In methanotrophic bacteria, methane is oxidized to methanol by the enzyme methane monooxygenase (MMO). The soluble MMO enzyme complex from Methylocystis sp. strain M also oxidizes a wide range of aliphatic and aromatic compounds, including trichloroethylene. In this study, heterologous DNA probes from the type II methanotroph Methylosinus trichosporium OB3b were used to isolate souble MMO (sMMO) genes from the type II methanotroph Methylocystis sp. strain M. sMMO genes from strain M are clustered on the chromosome and show a high degree of identity with the corresponding genes from Methylosinus trichosporium OB3b. Sequencing and phylogenetic analysis of the 16S rRNA gene from Methylocystis sp. strain M have confirmed that it is most closely related to the type II methanotroph Methylocystis parvus OBBP, which, unlike Methylocystis sp. strain M, does not possess an sMMO. A similar phylogenetic analysis using the pmoA gene, which encodes the 27-kDa polypeptide of the particulate MMO, also places Methylocystis sp. strain M firmly in the genus Methylocystis. This is the first report of isolation and characterization of methane oxidation genes from methanotrophs of the genus Methylocystis.

Amino Acid Sequence↗
PubMed1997

The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for methanotrophs and methylotrophs.

The methanol dehydrogenase gene mxaF, encoding the large subunit of the enzyme, was amplified from the DNA of a number of representative methanotrophs, methyletrophs, and environmental samples by PCR using primers designed from regions of conserved amino acid sequence identified by comparison of three known sequences of the large subunit of methanol dehydrogenase. The resulting 550-bp PCR products were cloned and sequenced. Analysis of the predicted amino acid sequences corresponding to these mxaF genes revealed strong sequence conservation. Of the 172 amino acid residues, 47% were conserved among all 22 sequences obtained in this study. Phylogenetic analysis of these MxaF sequences showed that those from type I and type II methanotrophs form two distinct clusters and are separate from MxaF sequences of other gram-negative methylotrophs. MxaF sequences retrieved by PCR from DNA isolated from a blanket bog peat core sample formed a distinct phylogenetic cluster within the MxaF sequences of type II methanotrophs and may originate from a novel group of acidophilic methanotrophs which have yet to be cultured from this environment.

Alcohol Oxidoreductases↗
PubMed1996

Taxonomic relationships of Thiobacillus halophilus, T. aquaesulis, and other species of Thiobacillus, as determined using 16S rDNA sequencing.

Total base sequences of the 16S rRNA genes of Thiobacillus halophilus and Thiobacillus aquaesulis show that these bacteria fall into the gamma- and beta-subdivisions, respectively of the Proteobacteria. The closest relative of T. halophilus is Thiobacillus hydrothermalis (with 98.7% similarity), and the closest relative of T. aquaesulis is Thiobacillus thioparus (93.2% similarity). Physiological properties and mol% G+C content of their DNA serve to confirm that these four organisms are all distinct species. It is reiterated that the species currently assigned to the genus Thiobacillus are clearly so diverse that they need reclassification into several genera. The type species, T. thioparus, is unequivocally placed in the beta-subdivision of the Proteobacteria, thus requiring that the use of the genus name Thiobacillus be restricted to the chemolithoautotrophic species falling into that group. T. aquaesulis and T. thioparus may thus be regarded as true species of Thiobacillus. The relatively large number of obligately chemolithoautotrophic Thiobacillus species falling in the gamma-subdivision of the Proteobacteria need further study in order to assess the case for reclassification into one or more new or different genera.

Bacteriological Techniques↗
PubMed1995

Factors affecting the electroporation of Bacillus subtilis.

Bacillus subtilis 168 trp- was found to be transformable with the tetracycline resistance plasmid pAB124 by electroporation of whole cells, inconsistently and at very low frequencies. Supplementation of the growth medium with glycine, or particularly DL-threonine, produced cells that could be electrotransformed much more efficiently at frequencies up to 2.5 x 10(3) transformants per microgram plasmid DNA. Transformation was optimal with cells grown in medium containing a racemic mixture of the D- and L-isomers of threonine, and no transformants were obtained when pure forms of the D- and L-threonine isomers were used. The cell walls of B. subtilis grown in the presence or absence of D-, L- and DL-threonine had a similar amino acid composition which did not include threonine. A more complex biochemical explanation of the enhancement of electroporation by growth in DL-threonine is likely, and this is discussed. Lysozyme treatments to weaken the cell wall and possibly mimic the effect of DL-threonine did not yield any transformants. The effects of buffer composition and culture incubation time were also determined and the electroporation protocol optimized accordingly. The response of a range of other B. subtilis strains to electroporation by the method produced was found to be variable. In all cases, transformation was verified by recovery of the plasmid DNA from putative transformants.

Amino Acids↗
PubMed1995

Detection of methanotrophic bacteria in environmental samples with the PCR.

We designed PCR primers by using the DNA sequences of the soluble methane monooxygenase gene clusters of Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and these primers were found to be specific for four of the five structural genes in the soluble methane monooxygenase gene clusters of several methanotrophs. We also designed primers for the gram-negative methylotroph-specific methanol dehydrogenase gene moxF. The specificity of these primers was confirmed by hybridizing and sequencing the PCR products obtained. The primers were then used to amplify methanotroph DNAs in samples obtained from various aquatic and terrestrial environments. Our sequencing data suggest that a large number of different methanotrophs are present in peat samples and also that there is a high level of variability in the mmoC gene, which codes for the reductase component of the soluble methane monooxygenase, while the mmoX gene, which codes for the alpha subunit of the hydroxylase component of this enzyme complex, appears to be highly conserved in methanotrophs.

Alcohol Oxidoreductases↗
PubMed1993

Adrenalectomy and steroid replacement in a small macropodid marsupial, the quokka (Setonix brachyurus): metabolic and renal effects.

Contrary to previously accepted observations, the quokka (Setonix brachyurus), a small macropodid marsupial, survival bilateral adrenalectomy indefinitely if injected with appropriate doses of cortisol and aldosterone, either singly or combined. Normal behavior and plasma solute concentrations could be maintained indefinitely by daily i.m. injection of 0.2 mg cortisol acetate and 0.01 mg aldosterone/kg. Plasma glucose concentration was maintained at the control value when the supplement contained cortisol but was significantly reduced with only a mineralocorticoid. Cortisol also caused a dose-related enhancement of food intake. There was no significant effect of supplementation on plasma urea concentration. Deoxycorticosterone, with or without cortisol, caused Na+ retention but aldosterone had this effect only when combined with cortisol. Urinary K+ excretion was not significantly affected by any of these corticosteroids. Withdrawal of hormone supplements caused muscular weakness and cessation of food intake within 3-6 days, accompanied by a moderate fall in Na+ and rise in K+ concentration of blood plasma as well as instability of plasma glucose concentration with hypoglycemic episodes. Renal excretion of both Na+ and K+ exceeded intake only when food intake had declined to negligible levels. Glomerular filtration rate (GFR) and renal plasma flow (RPF) fell when corticoids were withheld, without change in inulin space. However, p-aminohippurate (PAH) space fell, as did the intercompartmental exchange parameters of both inulin and PAH. Cortisol and aldosterone or aldosterone alone restored GFR, but not RPF, to the normal range. The fractional reabsorption of K+, but not Na+, was reduced in the absence of corticoids. It is suggested that, in this species, the circulatory effects of corticoids are essential for normal tissue perfusion and their influence on renal circulation is important in the control of salt and water balance. Metabolic effects are probably mainly permissive, as in other macropodid marsupials.

Adrenal Cortex Hormones↗
PubMed1988

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)

Animals↗
PubMed1988

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.

Animals↗
PubMed1986

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)

Adrenal Cortex Hormones↗
PubMed1986

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)

Adrenal Cortex↗
PubMed1981

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.

Adrenocorticotropic Hormone↗