H.G. Rast

Degradation of 1,4-dichlorobenzene by enriched and constructed bacteria

SummaryThree strains, RHO1, R3 and B1, tentatively identified as a Pseudomonas sp., an Alcaligenes sp. and a Pseudomonas sp. which were able to use 1,4-dichlorobenzene as the sole carbon and energy source were isolated from water of the Rhine river and from the sewage plant at Leverkusen-Bürrig. A hybrid strain, WR1313, which uses chlorobenzene as the growth substrate, was obtained by mating the benzene-growing Pseudomonas putida strain F1 with strain B13, a Pseudomonas sp. degrading chlorocatechols. Further selection of this strain for growth on 1,4-dichlorobenzene allowed the isolation of strain WR1323. During growth on 1,4-dichlorobenzene the strains released stoichiometric amounts of chloride. The affinity of the organisms to 1,4-dichlorobenzene was measured with strain R3 showing a Ks value of 1.2 mg/l. Respiration data and enzyme activities in cell extracts as well as the isolation of 3,6-dichlorocatechol from the culture fluid are consistent with the degradation of 1,4-dichlorobenzene via 3,6-dichlorocatechol, 2,5-dichloro-cis,cis-muconate, 2-chloro-4-carboxymethylenebut-2-en-4-olide.

Metabolism of o-phthalic acid by different gram-negative and gram-positive soil bacteria

Different bacteria, isolated from soil by the enrichment method, were able to grow on phthalic acid as carbon source. Protocatechuate was identified as intermediate in phthalate metabolism. All phthalategrown bacteria oxidized phthalate and protocatechuate rapidly without having a lag-period. Benzoic acid, terephthalic acid, protocatechuic acid, salicylic acid, di- and mono-butyl phthalate were also metabolized by some of the organisms, benzoic acid being degraded via catechol and terephthalic acid via protocatechuate as intermediate. All organisms tested cleaved protocatechuate or catechol, respectively, by the “ortho” fission, when grown on phthalate, terephthalate, or benzoate as carbon source. A characterization and tentative identification of the organisms is given.

Bacterial metabolism of substituted phenols

Abstract4-(Methylmercapto)-phenol (MMP) and 4-(methylsulfinyl)-phenol (MSP) are oxidized by the soil isolate Nocardia spec. DSM 43251, which is closely related to Nocardia calcarea. The rate of degradation depends on the capability of a substrate to support growth and is strongly enhanced in the presence of a second carbon source under the conditions of cooxidation. MMP and MSP are cometabolized by hydroxylation of the benzene ring with the formation of the substituted catechol following by ring cleavage between carbon atoms 2 and 3 (“meta”fission) to give 2-hydroxy-5-methylmercapto-or 2-hydroxy-5-methylsulfinylmuconic semialdehyde. Oxidation of MMP to MSP represents a bypath of MMP-oxidation. The intermediates were identified on the basis of their physical properties. The enzymes responsible for the metabolism of MMP and MSP are induced by growth with MMP or MSP, but not with glucose. MMP- and MSP-induced cells catalyze the oxidation of a variety of substituted phenols. This indicates a rather low substrate specificity of the enzymes induced by MMP and MSP.

Analytik bakterientoxischer Effekte mit Hilfe genetisch konstruierter Leuchtbakterien

SummaryLuminescent bacteria bioassays represent an elegant method for fast and reproducible determination of microbial toxicity. A general application of this method is however prevented by the fact that the capability for bioluminescence is restricted to a relatively small group of bacteria with extreme environmental demands. Therefore it seems questionable whether results obtained with those organisms can be used for all interesting applications. It is shown in this paper, that the field of applications for bacterial luminescent assays can be widened using gentechnologically constructed luminescent bacteria.ZusammenfassungLeuchtbakterienteste stellen eine elegante Methode zur schnellen, reproduzierbaren Bestimmung bakterientoxischer Effekte dar. Einer allgemeinen Anwendung steht allerdings entgegen, daß die Luminescenzeigenschaften auf relativ wenige Bakterienfamilien mit zum Teil extremen Standortansprüchen beschränkt sind. Die Übertragbarkeit der Meßergebnisse auf interessierende Anwendungen ist deshalb in vielen Fällen nicht gegeben. In der vorliegenden Arbeit wird am Beispiel eines gentechnologisch konstruierten luminescenten Abwasserstammes gezeigt, daß durch Verwendung von anwendungsspezifischen Testorganismen das Anwendungsspektrum von Leuchtbakterientesten erweitert werden kann.

2,3-Cleavage of Substituted Catechols in Nocardia sp. DSM 43251 (Rhodococcus rubrus)

Summary A catechol 2.3-dioxygenase, isolated from Nocardia sp. DSM 43251 (Rhodococcus rubrus) after induction with unpolar substituted phenols e.g. 4-(methylthio)-phenol or 4-methoxyphenol, was purified 79-fold by ammonium sulfate precipitation, acetone precipitation, and sephadex G 200 gel filtration. The molecular weight, as determined by gel filtration, was 125,000. SDS-polyacrylamide gel electrophoresis revealed three non-identical subunits with molecular weights from 40,000 to 50,000. Highest activity was obtained at pH 7.5 to 8.5. The enzyme was stable in the presence of oxygen, reducing and oxidizing agents, and at high temperatures with an optimum temperature of 60 °C. Atomic absorption spectrometry proved one atom of iron per molecule of enzyme which could not be removed by dialysis. The ring cleavage reaction was inhibited by metal ions like Ag+, Hg++ and Cu++ (10−6–10−5 M), and by 2,2′-bipyridyl (10−3 M). Catechol 2,3-dioxygenase from Nocardia sp. DSM 43251 showed a strict specificity for catechols with a substituent at C-4. Maximum reaction velocity was strongly influenced by the electronegativity of these substituents. Additional methyl groups at C-3 or C-5 decreased the affinity of the enzyme for the substrate as well as the reaction velocity. The structure of the reaction product and linear correlation of the reaction velocity with the tfp-values of the different catechols tested clearly indicate a 2,3-cleavage-mechanism.