Alasdair M. Cook

Initial steps in the degradation of benzene sulfonic acid, 4-toluene sulfonic acids, and orthanilic acid in Alcaligenes sp. strain O-1

Alcaligenes sp. strain O-1 grew with benzene sulfonate (BS) as sole carbon source for growth with either NH4+ or NH4+ plus orthanilate (2-aminobenzene sulfonate, OS) as the source(s) of nitrogen. The intracellular desulfonative enzyme did not degrade 3- or 4-aminobenzene sulfonates in the medium, although the enzyme in cell extracts degraded these compounds. We deduce the presence of a selective permeability barrier to sulfonates and conclude that the first step in sulfonate metabolism is transport into the cell. Cell-free desulfonation of BS in standard reaction mixtures required 2 mol of O2 per mol. One mol of O2 was required for a catechol 2,3-dioxygenase. When meta ring cleavage was inhibited with 3-chlorocatechol in desalted extracts, about 1 mol each of O2 and of NAD(P)H per mol of BS were required for the reaction, and SO32- and catechol were recovered in high yield. Catechol was shown to be formed by dioxygenation in an experiment involving 18O2. 4-Toluene sulfonate was subject to NAD(P)H-dependent dioxygenation to yield SO32- and 4-methylcatechol, which was subject to meta cleavage. OS also required 2 mol of O2 per mol and NAD(P)H for degradation, and SO32- and NH4+ were recovered quantitatively. Inhibition of ring cleavage with 3-chrorocatechol reduced the oxygen requirement to 1 mol per mol of OS SO32- (1 mol) and an unidentified organic intermediate, but no NH4+, were observed.

Degradation of p-toluenesulphonic acid via sidechain oxidation, desulphonation and meta ring cleavage in Pseudomonas (Comamonas) testosteroni T-2.

Pseudomonas (Comamonas) testosteroni T-2 completely converted p-toluenesulphonic acid (TS) or p-sulphobenzoic acid (PSB) to cell material, CO2 and sulphate, with growth yields of about 5 g protein (mol C)-1. PSB and sulphite were excreted as transient intermediates during growth in TS-salts medium. All reactions of a catabolic pathway involving sidechain oxidation and cleavage of the sulphonate moiety as sulphite were measurable in the soluble portion of cell extracts. Degradation of TS and PSB was inducible and apparently involved at least two regulons. TS was converted to p-sulphobenzyl alcohol in a reaction requiring NAD(P)H and 1 mol O2 (mol TS)-1. This alcohol was in an equilibrium (in the presence of NAD+) with p-sulphobenzaldehyde, which was converted to PSB in an NAD(P)+-dependent reaction. PSB was desulphonated to protocatechuic acid in a reaction requiring NAD(P)H and 1 mol O2 (mol PSB)-1. Experiments with 18 O2 confirmed involvement of a dioxygenase, because both atoms of this molecular oxygen were recovered in protocatechuate. Protocatechuate was converted to 2-hydroxy-4-carboxymuconate semialdehyde by a 4.5-dioxygenase.

Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran in Sphingomonas sp. strain RW1

Sphingomonas sp. strain RW1, when grown in salicylate-salts medium, synthesized the enzymes for the degradation of dibenzofuran. The reaction subsequent tometa cleavage of the first benzene ring was found to be catalyzed by two isofunctional hydrolases, H1 and H2, which were purified by chromatography on anion exchange, hydrophobic interaction and gel filtration media. Each enzyme was able to hydrolze 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate to produce salicylate and benzoate, respectively. SDS/PAGE of each purified enzyme showed a single band ofMr31 000 (H1) or 29 000 (H2). The N-terminal amino acid sequences of the two proteins showed 50% homology.

Dichloromethane utilized by an anaerobic mixed culture: acetogenesis and methanogenesis.

Dichloromethane (8.9 mg/l) was eliminated from industrially polluted, anaerobic groundwater in a fixed-bed reactor (43 m3) which was packed with activated charcoal and operated continuously for over three years. The elimination of dichloromethane over this period was some ten-fold in excess of the sorptive capacity of the charcoal, and the elimination (3.7 mg/h·[kg of charcoal]: residence time, 49 h) was tentatively attributed to dehalogenative microorganisms immobilized on the charcoal. Anaerobic enrichment cultures, with dichloromethane as the sole added source of carbon and energy, were inoculated with material from the reactor. Reproducibly complete substrate disappearance in subcultures was observed when traces of groundwater (1%) or yeast extract (0.01%) were supplied. Fed-batch experiments under an atmosphere of CO2 plus N2 led to the conversion in 11 days of 11 mM dichloromethane to 3 mM acetate and 2 mM methane, with a growth yield of 0.4 g of protein/mol of dichloromethane; insignificant amounts (<1 μM) of chloromethane accumulated. Methanogenesis could be inhibited by 50 mM 2-bromoethane sulfonate without any effect on the dehalogenation rate. The maximum dehalogenation rate was 0.13 mmol dichloromethane/h·l (2.6 mkat/kg of protein).

Co-culture of defined bacteria to degrade seven sulfonated aromatic compounds : efficiency, rates and phenotypic variations

SummaryA co-culture, consisting of five defined bacteria [e.g., T. Thurnheer, T. Köhler, A. M. Cook, and T. Leisinger: J Gen Microbiol 132:1215–1220], was able to degrade at least seven substituted benzenesulfonic acids in continuous culture. HPLC, total organic carbon analyses and colourimetric analyses showed that the sulfonated compounds could be completely degraded to biomass, SO42-, NH4+and CO2. The maximum observed degradation rate was 138 mg of C/h·1. The five organisms were Alcaligenes sp. strain 0–1 (substrates benzenesulfonic acid, 4-methylbenzenesulfonic acid and 2-aminobenzenesulfonic acid), two Pseudomonas spp., strains T-2 (substrates 4-methylbenzenesulfonic acid and 4-sulfobenzoic acid) and PSB-4 (substrate 4-sulfobenzoic acid) and two unidentified rods, strains M-1 (substrates benzenesulfonic acid, 4-methylbenzenesulfonic acid and 3-aminobenzenesulfonic acid) and S-1 (substrates 4-aminobenzenesulfonic acid and 4-hydroxybenzenesulfonic acid). The system was operated for over 18 months with five sulfonates, and no competition was detected amongst the four organisms present, because all organisms were still present (100% of the population after 7 months, 55% after 18 months). Many bacteria isolated from the continuous culture after 18 months showed substrate ranges different from those of the original strains. The most common occurrence (33% of the population) was the appearance of organisms which could degrade 2-aminobenzenesulfonic acid and 4-sulfobenzoic acid. Several cases of the loss of a character were seen but only rarely (1%) was a net gain of characters observed. After 30 months, only two (of five) parents were present (35% of the population) and some isolates could utilize all seven substrates on solid media.

Monochloro- and dichloroacetic acids as carbon and energy sources for a stable, methanogenic mixed culture

A stable methanogenic mixed culture was enriched from an industrial environment to utilize chloroacetate as sole carbon and energy source for growth. It immobilized spontaneously on activated charcoal and grew reproducibly on this carrier in a fluidized bed reactor when supplied with an anaerobic mineral salts medium. Substrate disappearance was complete. Methane, CO2 and chloride ions were conclusively identified as the metabolic products and quantified. The growth yield from chloroacetate was about 1 g of protein/mol of carbon. The calculated degradation rate in the fluidized bed reactor was 0.2 to 0.8 mmol/l·h. The first metabolic intermediate from [2−13C]monochloroacetate in portions of biofilm-coated carrier was shown by 13C-NMR to be glycolate, from which 13CO2 and 13CH4 were formed. Glycolate was formed in an oxygen-insensitive hydrolysis, but its conversion to CO2 and CH4 was strictly anaerobic and sensitive to inhibition by bromoethanesulfonate. Degradation of [1-14C]-and [2-14C]-chloroacetate each yielded the same amount of [14C]-methane. We thus presume glycolate to be cleaved to CO2 and H2, which were the substrates for methanogenesis. Dehalogenation was limited to chlorobromo-, iodo- and dichloroacetate. These four compounds and glycolate were utilized as the sole carbon and energy sources by the methanogenic mixed culture.

Degradation of p-toluic acid (p-toluenecarboxylic acid) and p-toluenesulphonic acid via oxygenation of the methyl sidechain is initiated by the same set of enzymes in Comamonas testosteroni T-2

Summary: Comamonas testosteroni T-2 utilizes p-toluate (TC, 4-toluenecarboxylate) as sole source of carbon and energy for growth. Cells grown in TC-salts medium oxygenated terephthalate (PcB, 4-carboxybenzoate) and contained protocatechuate 4,5-dioxygenase but no detectable (methyl)catechol dioxygenase. The intermediates 4-carboxybenzyl alcohol (COL), 4-carboxybenzaldehyde (CYD) and PcB were detected during the metabolism of TC. A TC methyl-monooxygenase system, a COL dehydrogenase and a CYD dehydrogenase were detected, analogous to the known degradative pathway and enzymes for 4-toluenesulphonate (TS) to 4-sulphobenzoate (PSB) (Locher et al., Journal of General Microbiology 135, 1969-1978, 1989). Genetic evidence indicated that the steps from TS to PSB and from TC to PcB were catalysed by the same enzymes. This hypothesis was substantiated by purifying or separating the appropriate enzymes from cells grown in TS-salts and TC-salts media. The behaviour of pairs of enzymes was effectively identical in all chromatographic and catalytic properties that were compared. The data support the existence of a novel pathway for the degradation of TC, with the same initial pathway enzymes being used to metabolize TS.