John T. G. Hamilton

Methane emissions from terrestrial plants under aerobic conditions

Methane is an important greenhouse gas and its atmospheric concentration has almost tripled since pre-industrial times. It plays a central role in atmospheric oxidation chemistry and affects stratospheric ozone and water vapour levels. Most of the methane from natural sources in Earths atmosphere is thought to originate from biological processes in anoxic environments. Here we demonstrate using stable carbon isotopes that methane is readily formed in situ in terrestrial plants under oxic conditions by a hitherto unrecognized process. Significant methane emissions from both intact plants and detached leaves were observed during incubation experiments in the laboratory and in the field. If our measurements are typical for short-lived biomass and scaled on a global basis, we estimate a methane source strength of 62–236 Tg yr-1 for living plants and 1–7 Tg yr-1 for plant litter (1 Tg = 1012 g). We suggest that this newly identified source may have important implications for the global methane budget and may call for a reconsideration of the role of natural methane sources in past climate change.

Biochemistry: Biosynthesis of an organofluorine molecule

Although fluorine in the form of fluoride minerals is the most abundant halogen in the Earths crust, only 12 naturally occurring organofluorine compounds have so far been found, and how these are biosynthesized remains a mystery. Here we describe an enzymatic reaction that occurs in the bacterium Streptomyces cattleya and which catalyses the conversion of fluoride ion and S-adenosylmethionine (SAM) to 5′-fluoro-5′-deoxyfluoroadenosine (5′-FDA). To our knowledge, this is the first fluorinase enzyme to be identified, a discovery that opens up a new biotechnological opportunity for the preparation of organofluorine compounds.

Methoxyl groups of plant pectin as a precursor of atmospheric methane: evidence from deuterium labelling studies

* The observation that plants produce methane (CH4) under aerobic conditions has caused considerable controversy among the scientific community and the general public. It led to much discussion and debate not only about its contribution to the global CH4 budget but also about the authenticity of the observation itself. Previous results suggested that methoxyl groups of the abundant plant structural component pectin might play a key role in the in situ formation process of CH4. Here, this effect is investigated using an isotope labelling study. * Polysaccharides, pectin and polygalacturonic acid, with varying degrees of trideuterium-labelled methyl groups in the methoxyl moieties, were investigated for CH4 formation under UV irradiation and heating. * A strong deuterium signal in the emitted CH4 was observed from these labelled polysaccharides. * Results clearly demonstrate that ester methyl groups of pectin can serve as a precursor of CH4, supporting the idea of a novel chemical route of CH4 formation in plants under oxic environmental conditions.

Haloalkane degradation and assimilation by Rhodococcus rhodochrous NCIMB -13064

The bacterium Rhodococcus rhodochrous NCIMB 13064, isolated from an industrial site, could use a wide range of 1-haloalkanes as sole carbon source but apparently utilized several different mechanisms simultaneously for assimilation of substrate. Catabolism of 1-chlorobutane occurred mainly by attack at the C-1 atom by a hydrolytic dehalogenase with the formation of butanol which was metabolized via butyric acid. The detection of small amounts of gamma-butyrolactone in the medium suggested that some oxygenase attack at C-4 also occurred, leading to the formation of 4-chlorobutyric acid which subsequently lactonized chemically to gamma-butyrolactone. Although 1-chlorobutane-grown cells exhibited little dehalogenase activity on 1-chloroalkanes with chain lengths above C10, the organism utilized such compounds as growth substrates with the release of chloride. Concomitantly, gamma-butyrolactone accumulated to 1 mM in the culture medium with 1-chlorohexadecane as substrate. Traces of 4-hydroxybutyric acid were also detected. It is suggested that attack on the long-chain chloroalkane is initiated by an oxygenase at the non-halogenated end of the molecule leading to the formation of an omega-chlorofatty acid. This is degraded by beta-oxidation to 4-chlorobutyric acid which is chemically lactonized to gamma-butyrolactone which is only slowly further catabolized via 4-hydroxybutyric acid and succinic acid. However, release of chloride into the medium during growth on long-chain chloroalkanes was insufficient to account for all the halogen present in the substrate. Analysis of the fatty acid composition of 1-chlorohexadecane-grown cells indicated that chlorofatty acids comprised 75% of the total fatty acid content with C14:0, C16:0, C16:1 and C18:1 acids predominating. Thus the incorporation of 16-chlorohexadecanoic acid, the product of oxygenase attack directly into cellular lipid represents a third route of chloroalkane assimilation. This pathway accounts at least in part for the incomplete mineralization of long-chain chloroalkane substrates. This is the first report of the coexistence of a dehalogenase and the ability to incorporate long-chain haloalkanes into the lipid fraction within a single organism and raises important questions regarding the biological treatment of haloalkane containing effluents.

Large carbon isotope fractionation associated with oxidation of methyl halides by methylotrophic bacteria

The largest biological fractionations of stable carbon isotopes observed in nature occur during production of methane by methanogenic archaea. These fractionations result in substantial (as much as ≈70‰) shifts in δ13C relative to the initial substrate. We now report that a stable carbon isotopic fractionation of comparable magnitude (up to 70‰) occurs during oxidation of methyl halides by methylotrophic bacteria. We have demonstrated biological fractionation with whole cells of three methylotrophs (strain IMB-1, strain CC495, and strain MB2) and, to a lesser extent, with the purified cobalamin-dependent methyltransferase enzyme obtained from strain CC495. Thus, the genetic similarities recently reported between methylotrophs, and methanogens with respect to their pathways for C1-unit metabolism are also reflected in the carbon isotopic fractionations achieved by these organisms. We found that only part of the observed fractionation of carbon isotopes could be accounted for by the activity of the corrinoid methyltransferase enzyme, suggesting fractionation by enzymes further along the degradation pathway. These observations are of potential biogeochemical significance in the application of stable carbon isotope ratios to constrain the tropospheric budgets for the ozone-depleting halocarbons, methyl bromide and methyl chloride.

Biosynthesis of fluorinated secondary metabolites by streptomyces cattleya

The biosynthesis of organofluorine compounds by Streptomyces cattleya NRRL 8057 was examined using 19F NMR spectroscopy. The organism produced 1.2 mM fluoroacetate and 0.5 mM 4-fluorothreonine as secondary metabolites when cultured for 28 d on a chemically defined medium containing 2 mM fluoride. Cell suspensions from batch cultures harvested at the growth maximum of 4 d were not capable of fluoride uptake or fluorometabolite biosynthesis, but by 6 d had developed an efficient fluoride-uptake system and biosynthesized the two fluorometabolites in almost equal proportions. As the harvest age increased, the proportion of fluoroacetate to 4-fluorothreonine formed by cell suspensions rose progressively so that 16-d-old cells showed a ratio of 76:26 for the two compounds. Fluoride uptake and fluorometabolite production by cell suspensions were highly dependent on pH, with both processes showing a maximum rate at pH 6.0 but declining rapidly at higher pH values. This decrease was particularly marked in the case of fluoroacetate biosynthesis which was barely detectable at pH 7.5. Fluoroacetate and 4-fluorothreonine showed only low levels of interconversion by cell suspensions, suggesting that the carbon skeleton of neither was derived by metabolism of the other. The limited interconversion observed is explicable in terms of a small degree of biological defluorination occurring with each compound, followed by reincorporation of the resulting fluoride ion into the organic form by the active fluorinating system, a phenomenon also noted on incubation of cell suspensions with a number of other fluorinated biochemical intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloromethane biosynthesis in poroid fungi

Abstract A survey of the distribution of chloromethane (MeCl), biosynthesis amongst polypores indicates that the trait is expressed widely in genera of the Hymenochaetaceae. Of 63 species examined in this family, including species from the genera Phellinus , Inonotus, Hymenochaete, Onnia, Phaeolus and Fomitoporia , 34 (54%) were capable of MeCl biosynthesis. The magnitude of MeCl production was dependent on the nature of the medium and the fungal species; however 21 species were capable of converting more than 10% of chloride ion in the medium to MeCl on at least one of the media employed. Of 27 species screened from the Ganodermataceae and the Polyporaceae, only one Fomitopsis cytisina was capable of MeCl biosynthesis. Of the 23 MeCl biosynthesizing species in the genus Phellinus , 12 were also capable of producing methyl benzoate and, in most instances, methyl esters of salicylic and 2-furoic acid. Ester production was not observed in Phellinus species not possessing the MeCl biosynthesis trait. It is postulated that the biosynthesis of esters and MeCl is biochemically linked; ester biosynthesis being a more advanced trait in evolutionary terms. The results suggest that the potential for MeCl production in nature by wood-rotting fungi is considerable. The environmental implications of a significant fungal contribution to the atmospheric MeCl burden are considered.

The distinctive isotopic signature of plant-derived chloromethane: possible application in constraining the atmospheric chloromethane budget.

Chloromethane (CH(3)Cl) is the most abundant halocarbon in the atmosphere. Although largely of natural origin it is responsible for around 17% of chlorine-catalysed ozone destruction. Sources identified to date include biomass burning, oceanic emissions, wood-rotting fungi, higher plants and most recently tropical ferns. Current estimates reveal a shortfall of around 2 million ty(-1) in sources versus sinks for the halocarbon. It is possible that emissions from green plants have been substantially underestimated. A potentially valuable tool for validating emission flux estimates is comparison of the delta13C value of atmospheric CH(3)Cl with those of CH(3)Cl from the various sources. Here we report delta13C values for CH(3)Cl released by two species of tropical ferns and show that the isotopic signature of CH(3)Cl from pteridophytes like that of CH(3)Cl from higher plants is quite different from that of CH(3)Cl produced by biomass burning, fungi and industry. delta13C values for CH(3)Cl produced by Cyathea smithii and Angiopteris evecta were respectively -72.7 per thousand and -69.3 per thousand representing depletions relative to plant biomass of 42.3 per thousand and 43.4 per thousand. The characteristic isotopic signature of CH(3)Cl released by green plants should help constrain their contribution to the atmospheric burden when reliable delta13C values for all other major sources of CH(3)Cl are obtained and a globally averaged delta13C value for atmospheric CH(3)Cl is available.

High levels of monofluoroacetate in Dichapetalum braunii

Abstract Monofluoroacetate occurs in young leaves and seeds of Dichapetalum braunii Engl. & K. Krause from southeast Tanzania at concentrations of 7200 and 8000 ppm, respectively, on a dry weight basis. This is the highest level so far reported from a plant source. The fluoride and monofluoroacetate (MFA) concentrations in this species are compared with those of six other Dichapetalum spp. collected from the same locality in Tanzania. The seeds of D. braunii , unlike D. toxicarium , do not contain any ω-fluorinated fatty acids.

Dioxygenase-catalyzed cis-dihydroxylation of pyridine-ring systems

Toluene dioxygenase-catalyzed dihydroxylation, in the carbocyclic rings of quinoline, 2-chloroquinoline, 2-methoxyquinoline, and 3-bromoquinoline, was found to yield the corresponding enantiopure c...