Mutant strains of Pseudomonas putida (arvilla) mt-2 which have lost the ability to grow at the expense of mor p-toluate (methylbenzoate) but retain the ability to grow with benzoate arise spontaneously during growth on benzoate; this genetic loss occurs to a lesser extent during growth on nonaromatic carbon sources in the presence of mitomycin C. The mutants have totally lost the activity of the enzymes of the divergent meta pathway with the possible exception of 2-oxopent-4-enoate hydratase and 4-hydroxy-2-oxovalerate aldolase; unlike the wild type they utilize benzoate by the ortho pathway. Evidence is presented that these mutants have lost a plasmid coding for the enzymes of the meta pathway, which may be transmitted back to them or into other P. putida strains. Preliminary results from these mutants and from a mutant defective in the regulation of the plasmid-carried pathway suggest that the wild type contains two benzoate oxidase systems, one on the plasmid which is nonspecific in both its catalysis and its induction and one on the chromosome which is more specific to benzoate as substrate and is specifically induced by benzoate.

1.A reinvestigation of the catabolic pathway(s) used by Pseudomonas putida NCIB 10015 (Dagley's strain) for the degradation of phenol and the cresols has proved the existence of a metabolic divergence after meta cleavage of the eatechols formed by hydroxylation of the primary substrates. The ring-fission products of catechol and 4-methylcatechol are shown to be simultaneously catabolized by two different enzymic activities, an NADf-dependent dehydrogenase and a cofactor-independent hydrolase. The metabolizing activitics of bot,h ring-fission products in extracts of cells grown on phenol and the cresols (0-, m-and p-cresol) were found to be nonspecific ; thermal inactivation of extracts of phenol-grown cells has shown that this nonspecificity is attributable to only one enzyme expressing each activity and that the two activities are locatcd on separate proteins.2. Extracts of cells grown on all four substrates contain high induced levels of the meta cleavage suite of enzymes functional in the dissimilation of catechol, including both the 4-oxalocrotonate branch (NADf-dependent 2-hydroxymuconic semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase and 4-oxalocrotonate decarboxylase) and the hydrolytic branch (2-hydroxymuconic semialdehyde hydrolase).3. The hydroxylase, oxygenase, dehydrogenase and hydrolase activities are shown to be nonspecific and can also act upon the methyl derivatives of their respective substrates. A constant pattern of specificity was found for these enzymes, independent of the monophenolic substrate used for growth.4. From studies with a mutant lacking phenol hydroxylase, the entire suite of meta cleavage enzymes are shown to be coincidently induced from the top by the primary substrate (phenol or the cresols) .5. The evolutionary and physiological implications of the divergent pathway are discussed. [2,3], and that involving the 2-hydroxymuconic semialdehyde dehydrogenase (the 4-oxalocrotonate branch) bears similarities to that

1. Pseudomonas arvilla mt-2 grows a t the expense of benzoate, m-toluate (3-methylbenzoate) and p-toluate (4-methylbenzoate), but not o-toluate (2-methylbenzoate). Under various conditions compounds with spectra characteristic of muconic semialdehydes can be made to accumulate in the media. These spectra indicate that benzoate is metabolised through catechol and 2-hydroxymuconic semialdehyde (2-hydroxy-6-oxohexa-2,4-dienoate), m-toluate through 3-methylcatechol and 2-hydroxy-6-oxohepta-2,4-dienoate and p-toluate through 4-methylcatechol and 2-hydroxy-5-methylmuconic semialdehyde (2-hydroxy-5-methyl-6-oxohexa-2,4-dienoate).2. Freshly harvested cells grown on all three substrates only consume oxygen a t a significant rate when presented with the growth substrates themselves and catechol and the methylcatechols, but not with salicylate, protocatechuate or phenol or the cresols.3. Extracts of cells grown on these substrates contain high induced levels of the suite of meta cleavage enzymes, including both the hydrolytic branch and the 4-oxalocrotonate branch.4. The substrate specificity of the early enzymes of the pathway suggests that only one nonspecific enzyme expresses each activity in cell-free extracts and that it is nonspecifically induced during growth on all three carbon sources. 5.It is suggested that the metabolic role of the hydrolytic branch of the pathway is the assimilation of 3-methylcatechol and its precursor, and that of the 4-oxalocrotonate branch is to assimilate catechol and 4-methylcatechol and their precursors.6. o-Toluate is not metabolised because it is neither a substrate for the benzoate oxidase system nor an inducer of any of the meta pathway enzymes.7 . It appears that benzoate and m-and p-toluates are the substrate inducers of the meta pathway enzymes in this organism. The ortho pathway is induced on incubating cells with catechol and the first inducer appears to be &,cis-muconate or possibly catechol itself.The coexistence of two enzymes able to degrade the meta cleavage product of catechol, 2-hydroxymuconic semialdehyde, was first demonstrated in cell-free extracts of both benzoate-grown Azotobacter species [l] and a naphthalene-grown Pseudomonas strain NCIB 9816 [2]. Sala-Trepat and Evans [3] elucidated the two resulting pathways, which after diverging a t 2-hydroxymuconic semialdehyde converge again a t 2-oxopent-4-enoate ; they concluded that in Azotobacter only the 4-oxalocrotonate branch, involving as the first step the NADf-dependent dehydrogenation of 2-hydroxymuconic semialdehyde, was of any metabolic significance since the 2-hydroxymuconic semialdehyde hydrolase was present Enzymes. Catechol 1,2-oxygenase or catechol: oxygen 1,2-oxidoreductase (EC 1.13.1.1); catechol 2,3-oxygenase or catecho1:oxygen 2,3-oxidoredoctase (EC 1.13.1.2); NAD nucleosidase or NAD glycohydrolase (EC 3.2.2.6). 21 Eur. J. Blochem., Vol.28 in only very low uninducible levels. I n NCIB 9816 all the enzymes of the 4-oxalocrotonate branch are present [4] and again it has been proposed that catechol is metab...

The reaction of 2,4-dinitroanilinomaleimide with sulphite which has been claimed as the basis of a suitable colorimetric assay for the anion was carefully re-examined. The sulphite-imide addition product provides a suitable and specific qualitative test for sulphite after separation by paper chromatography but the method as previously used is probably measuring the hydrolysis of the imide to 2,4-dinitroanilinomaleamic acid and cannot be used for sulphite determination either colorimetrically or in kinetic assays. A new colorimetric method for the determination of sulphite based on its reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) is described and compared for sensitivity with the p-rosaniline-HCHO method. Both methods were used to show the formation of sulphite as the initial product of arylsulphonate metabolism by bacteria. The failure to find sulphite in similar cultures of a third organism was attributed to the very high activities of sulphite oxidase found in extracts. The Ellman reagent was examined as the basis of an indicator medium for the detection of sulphite-excreting colonies.