Isolation of mutants of the obligate methanotroph Methylomonas albus defective in growth on methane

A strain of Methylomonas albus BG8WM, a type 1 obligate methanotroph, which had been maintained for 2 ycars by serial passage on solid medium containing methanol as a carbon source was found to mutate at a frequency of 10^-5-10^-6 to resistance to dichloromethane (DCM^R), the parental strain BG8 did not give rise to DCM^R colonies. DCM^R strains were no longer capable of growth on methane as a carbon cource and exhibited greatly reduced or undetectable methane mono-oxygenase activity. The mutants fell into three groups on the basis of SDS-PAGE analysis of the polypeptide profiles of the particulate fraction of cell extracts. One or more of four polypeptides of Mr 70,000, 50,000, 25,000 and 23,000 were implicated as being components of the methane mono-oxygenase. Spontaneous reversion to growth on methane and sensitivity to dichloromethane occurred at a frequency of about 10^-8. The loss of methane mono-oxygenase activity was not associated with loss of the resident 55 kb plasmid.Abbreviations DCM^R dichloromethane-resistant - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - NMS nitrate minimal salts medium     

The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.     

Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

The anaerobic decomposition of benzoic acid during methane fermentation

Anaerobic rupture of the benzoic acid ring was investigated. Carbon 4 was converted primarily to carbon dioxide. Following ring rupture during methane fermentation, propanoic acid is an intermediate, and carbon 4 of benzoate becomes its carboxyl.Contribution No. 1285-j, Division of Biology, Kansas State University, Manhattan, KS 66506. This work was supported in part by funds from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506. Paper II of this series is Fina and Fiskin (1960)     

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

Controlled generation of reactive oxygen species orchestrates numerous physiological signaling events (Finkel, T. (2011) Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15). A major cellular target of reactive oxygen species is the thiol side chain (RSH) of Cys, which may assume a wide range of oxidation states (i.e. −2 to +4). Within this context, Cys sulfenic (Cys-SOH) and sulfinic (Cys-SO₂H) acids have emerged as important mechanisms for regulation of protein function. Although this area has been under investigation for over a decade, the scope and biological role of sulfenic/sulfinic acid modifications have been recently expanded with the introduction of new tools for monitoring cysteine oxidation in vitro and directly in cells. This minireview discusses selected recent examples of protein sulfenylation and sulfinylation from the literature, highlighting the role of these post-translational modifications in cell signaling.     

Nitrosating activity in Escherichia coli

Nitrosation activity was measured in Escherichia coli isolates and a range of nitrite reductase (nir) mutants. Activity was only detected in intact cells and could be inhibited by a number of treatments such as sonication and osmotic shock. Aerobically-grown cells had highest nitrosation activity compared to oxygen-limited ones. Inclusion of nitrite in growth media induced high activities of nitrite reductase and for some isolates, nitrosation. Analysis of nir mutants identified two which were unable to nitrosate. This result suggested that NADH-dependent nitrite reductase was implicated either directly or indirectly in nitrosation.     

Waterlogging responses in dune,swale and marsh populations of Spartina patens under field conditions

Summary Soil waterlogging responses were examined in three Spartina patens populations along a steep flooding gradient in coastal Louisiana. Root anatomy and physiological indicators of anaerobic metabolism were examined to identify and compare flooding responses in dune, swale and marsh populations, while soil physicochemical factors were measured to characterize the three habitats. Soil waterlogging increased along the gradient from dune to marsh habitats and was accompanied by increases in root porosity (aerenchyma). Aerenchyma in marsh roots was apparently insufficient to provide enough oxygen for aerobic respiratory demand, as indicated by high root alcohol dehydrogenase activities and low energy charge ratios. Patterns of root metabolic indicators suggest that dune and swale roots generally respired aerobically, while anaerobic metabolism was important in marsh roots. However, in each population, relatively greater soil waterloging was accompanied by differences in enzyme activities leading to malate accumulation. In dune and swale roots under these circumstances, depressed adenylate energy charge ratios may have been the result of an absence of increased ethanol fermentation. These trends suggest that: 1) Aerenchyma formation was an important, albeit incomplete, long-term adaptation to the prevalent degree of soil waterlogging. 2) All populations adjusted root metabolism in response to a relative (short-term) increase in soil waterlogging.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Anaerobic respiration in the Rhodospirillaceae: characterisation of pathways and evaluation of roles in redox balancing during photosynthesis

Abstract Recent discoveries relating to pathways of anaerobic electron transport in the Rhodospirillaceae are reviewed. The main emphasis is on the organism Rhodobacter capsulatus ** but comparisons are made with Rhodobacter sphaeroides ** f. sp. denitrificans and Rhodopseudomonas palustris . The known electron acceptors for anaerobic respiration in Rhodobacter capsulatus are trimethylamine- N -oxide (TMAO), dimethyl sulphoxide (DMSO), nitrate and nitrous oxide. In each case respiration generates a proton electrochemical gradient and in some cases can support growth on non-fermentable carbon sources. However, the principal objective of this review is to discuss the possibility that, apart from a role in energy conservation, anaerobic respiration in the photosynthetic bacteria may have a special function in maintaining redox balance during photosynthetic metabolism. Thus the electron acceptors mentioned above may serve as auxiliary oxidants: (a) to maintain an optimal redox poise of the photosynthetic electron transport chain; (b) to provide a sink for electrons during phototrophic growth on highly reduced carbon substrates.
Molecular properties of the nitrate reductase, nitrous oxide reductase and a single enzyme responsible for reduction of TMAO and DMSO are discussed. These enzymes are all located in the periplasm. Electrons destined for all three enzymes can originate from the rotenone-sensitive NADH dehydrogenase but do not proceed through the antimycin- and myxothiazol-sensitive cytochrome b/c₁ complex. It is likely, therefor, that the pathways of anaerobic respiration overlap with the cyclic photosynthetic electron transport chain only at the level of the ubiquinone pool. Redox components which might be involved in the terminal branches of anaerobic respiration are discussed.