1. A cell-free system from Pseudononaw fluorescens catalysed the oxidative demethylation and subsequent ring-cleavage of vanillate, with uptake of 2-5 moles of oxygen/mole of substrate. 2. Demethylation involved absorption of 0-5mole of oxygen/mole, and required reduced glutathione (GSH) and nucleotide (probably NADPH) as cofactors, with further possible requirements, the natures of which are discussed. 3. Incomplete evidence suggested that the aromatic ring was opened via protocatechuate and the appropriate oxygenase, with absorption of 1mole of oxygen/mole of substrate, eventually yielding ,-oxoadipate. 4. The methyl group was removed sequentially as formaldehyde, formate and carbon dioxide, the steps catalysed respectively by formaldehyde dehydrogenase, which required GSH and NAD+, and formate dehydrogenase. Each enzyme was cytochrome-linked and accounted for absorption of 0-5 mole of oxygen/mole of substrate. 5. All enzymes except formate dehydrogenase, which was a cell-wall enzyme, resided in the soluble fraction of the extract. The demethylase could not be resolved because of unknown cofactor requirements.The interpretation of the results of bacterial attack on lignin is difficult because of the chemical complexity of this material. To obtain essential background information for this difficult problem we have examined the bacterial degradation of substances that could be products of lignin catabolism or that, by dehydrogenation and condensation reactions, could be converted into lignin in plants.We now present, for the first time, observations on demethylation processes by partly resolved bacterial enzymes. Whereas Axelrod (1956) and Brodie et al. (1955) demonstrated salient features of the enzymic demethylation of methoxyphenyl derivatives in mammalian tissues, no detailed information has been reported on the process in micro-organisms; although, for example, Henderson (1957Henderson ( , 1961 showed that demethylated products arise from the attack by fungal cells on methoxybenzoic acids.

Recent work on the dissimilation of nitrate by microorganisms and plants suggests that, during reduction to the amino level, nitrite is 'fixed' as an organic nitro derivative (de la Haba, 1950). The existence of enzyme systems capable of metabolizing nitro compounds in liver (Egami & Itahishi, 1951), Neurospora crassa (Little, 1951) and pea plants (Little, 1957) lends further evidence for the possibility that nitro compounds are widespread functional intermediates in nitrogen metabolism. In none of the three systems mentioned was there prior contact with environmental nitro compounds, so that adaptation to these materials could not explain the presence ofnitro-metabolizing enzymes. Although nitro compounds, e.g. chloromycetin

has been observed. The fundamental importance of this reaction is emphasized by the work of de la Haba (1950) and McElroy & Spencer (1956), from which the suggestion arose that nitro compounds are probably intermediates in the reduction of nitrates by green plants and microorganisms , and the finding that moulds and higher plants, in the absence of added nitro compounds or their presumed precursors, elaborate enzymes capable of metabolizing nitro compounds (Bush, Touster & Brockman, 1951; Shimoda, 1951; Raistrick & St6ssl, 1958; Little, 1957). The appearance of arylamines in cultures of Nocardia species growing on nitrobenzoic acids was noted by Cain (1958a), but Cartwright & Cain (1959) found that these compounds appeared to have little significance in the oxidative breakdown of p-and m-nitrobenzoates, although this is less certain with the ortho-isomer. The study of the reduction of nitrobenzoates by Nocardia species and a strain of Pseudomona8 fluorescen8 described in this paper includes investigations with cell-free extracts and the stimulation of a nitroreductase by flavin adenine dinucleotide, an effect which had previously been observed only in E8cherichia coli among the bacteria (Saz & Martinez, 1956). EXPERIMENTAL Organi8m The organisms used were strains of Nocardia erythropolis, N. opaca, Nocardia M 1 and P8eudomona8fluore8cens isolated by enrichment methods and grown in bulk on a chemically defined medium by the techniques described by Cain (1958a). The procedures for obtaining adapted cells, washed suspensions and cell-free extracts were those described by Cartwright & Cain (1959). The particular strain of organisms

It has previously been suggested that the lignans are derived by oxidative coupling of n-propenylphenol derivatives a t the 8-carbon atom of the side chain. This type of oxidative coupling has now been experimentally realised by the conversion of ferulic acid into " dehydrodiferulic acid " (20% yields) by the action of either ferric chloride or ammonium persulphate. Constitution (111; R = H) assigned to the oxidation product has been established by hydrolysis of the dimethyl ether to veratraldehyde and veratrylidenesuccinic acid, and also by cyclisation and dehydrogenation to the anhydride of 6 : 7-dimethoxy-l-(3' : 4'-dimethoxyphenyl)naphthalene-2 : 3-dicarboxylic acid.

Hydrogenation of citrinin is accompanied by aromatisation of the nucleus, giving rise to dihydrocitrinin which on esterification and methylation yields methyl 0-dimethyldihydrocitrinin. On oxidation the latter compound furnished a lactone, methyl 0-dimethylcitrinone, and the acid obtained by the hydrolysis of the carbmethoxy-group of this lactone underwent simultaneous decarboxylation and demethylation when heated with glycerol, yielding a phenolic lactone, identical with the product formed by the action of hot hydriodic acid on methyl 0-dimethyldihydrocitrinone. On being boiled with aqueous sodium hydroxide this lactone gave rise to phenol (A) by simultaneous hydration and loss of carbon dioxide. These reactions in conjunction with the conversion of dihydrocitrinin into (VIII) by means of hydriodic acid have made it possible to deduce rational structural formulae for the phenolic lactone (VI), methyl 0-dimethyldihydrocitrinone (V; R = Me), 0-dimethyldihydrocitrinin (IV; R = H), and for dihydrocitrinin (111; R = H). The expression (11) appears best to represent the properties of citrinin.Methylation of methyl citrinin gave a complex mixture from which a crystalline compound (M), C,,H,,O,(OMe), has been isolated in comparatively small yield. On alkaline hydrolysis this substance yielded acetaldehyde, diethyl ketone, and formic acid, together with a phenolic * As in the analogous case of the methyl ester of the carboxylic acid citromycetin (Part 111, this vol., p. 848) we refer to the methyl ester of citrinin as methyl citrinin. The composition of trivial names for derivatives of these acids in so far as they are necessary and convenient would have been much easier and more systematic had the originators of the names followed the normal custom in naming acids, e.g., citrinic and citromycetinic acid. This also applies to the more recently isolated unsaturated ketone, trichothecin (Freeman and Morrison, Nature, 1948, 162, 30), for which a more appropriate name would have been, e.g., trichothecenone."