Browsing by Author "Pieper, Dietmar H."

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Open Access
The 2‐methylpropene degradation pathway in Mycobacteriaceae family strains
(2023) Helbich, Steffen; Barrantes, Israel; dos Anjos Borges, Luiz Gustavo; Pieper, Dietmar H.; Vainshtein, Yevhen; Sohn, Kai; Engesser, Karl‐Heinrich
Mycolicibacterium gadium IBE100 and Mycobacterium paragordonae IBE200 are aerobic, chemoorganoheterotrophic bacteria isolated from activated sludge from a wastewater treatment plant. They use 2‐methylpropene (isobutene, 2‐MP) as the sole source of carbon and energy. Here, we postulate a degradation pathway of 2‐methylpropene derived from whole genome sequencing, differential expression analysis and peptide‐mass fingerprinting. Key genes identified are coding for a 4‐component soluble diiron monooxygenase with epoxidase activity, an epoxide hydrolase, and a 2‐hydroxyisobutyryl‐CoA mutase. In both strains, involved genes are arranged in clusters of 61.0 and 58.5 kbp, respectively, which also contain the genes coding for parts of the aerobic pathway of adenosylcobalamin synthesis. This vitamin is essential for the carbon rearrangement reaction catalysed by the mutase. These findings provide data for the identification of potential 2‐methylpropene degraders.
Open Access
(+)-4-Carboxymethyl-2,4-dimethylbut-2-en-4-olide as dead-end metabolite of 2,4-dimethylphenoxyacetic acid or 2,4-dimethylphenol by alcaligenes eutrophus JMP 134
(1990) Pieper, Dietmar H.; Engesser, Karl-Heinrich; Knackmuss, Hans-Joachim
2,4-Dimethylphenoxyacetic acid and 2,4-dimethylphenol are not growth substrates for Alcaligenes eutrophus JMP 134 although being cooxidized by 2,4-dichlorophenoxyacetate grown cells. None of the relevant catabolic pathways were induced by the dimethylphenoxyacetate, 3,5-Dimethylcatechol is not subject to metacleavage. The alternative ortho-eleavage is also unproductive and gives rise to (+)-4-carboxymethyl-2,4-dimethylbut-2-en-4-olide as a dead-end metabolite. High yields of this metabolite were obtained with the mutant Alcaligenes eutrophys JMP 134-1 which constitutively expresses the genes of 2,4-dichlorophenoxyacetic acid metabolism.
Open Access
Assemblage of ortho cleavage route for simultaneous degradation of chloro- and methylaromatics
(1987) Rojo, Fernando; Pieper, Dietmar H.; Engesser, Karl-Heinrich; Knackmuss, Hans-Joachim; Timmis, Kenneth N.
Genetic engineering is a powerful means of accelerating the evolution of new biological activities and has considerable potential for constructing microorganisms that can degrade environmental pollutants. Critical enzymes from five different catabolic pathways of three distinct soil bacteria have been combined in patchwork fashion into a functional ortho cleavage route for the degradation of methylphenols and methylbenzoates. The new bacterium thereby evolved was able to degrade and grow on mixtures of chloro- and methylaromatics that were toxic even for the bacteria that could degrade the individual components of the mixtures. Except for one enzymatic step, the pathway was fully regulated and its component enzymes were only synthesized in response to the presence of pathway substrates.
Open Access
Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134
(1988) Pieper, Dietmar H.; Reineke, Walter; Engesser, Karl-Heinrich; Knackmuss, Hans-Joachim
Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.
Open Access
Modified ortho-cleavage pathway in Alcaligenes eutrophus JMP134 for the degradation of 4-methylcatechol
(1985) Pieper, Dietmar H.; Engesser, Karl-Heinrich; Don, Robert H.; Timmis, Kenneth N.; Knackmuss, Hans-Joachim
Methylsalicylate-grown cells of Pseudomonas sp. WR 401 cometabolized 3-, 4- and 5-substituted halosalicylates to the corresponding halocatechols. Further degradation was unproductive due to the presence of high levels of catechol 2,3-dioxygenase. This strain acquired the ability to utilize 3-chlorobenzoate following acquisition of genes from Pseudomonas sp. B 13 which are necessary for the assimilation of chlorocatechols. This derivative (WR 4011) was unable to use 4- or 5-chlorosalicylates. Derivatives able to use these compounds were obtained by plating WR 4011 on 5-chlorosalicylate minimal medium; one such derivative was designated WR 4016. The acquisition of this property was accompanied by concomitant loss of the methylsalicylate phenotype. During growth on 4- or 5-chlorosalicylate the typical enzymes of chlorocatechol assimilation were detected in cell free extracts, whereas catechol 2,3-dioxygenase activity was not induced. Repeated subcultivation of WR 4016 in the presence of 3-chlorosalicylate produced variants (WR 4016-1) which grew on all three isomers.
Open Access
Purification and characterization of 4-methylmuconolactone methylisomerase, a novel enzyme of the modified 3-oxoadipate pathway in the gram-negative bacterium Alcaligenes eutrophus JMP 134
(1990) Pieper, Dietmar H.; Stadler-Fritzsche, Karin; Knackmuss, Hans-Joachim; Engesser, Karl-Heinrich; Bruce, Neil C.; Cain, Ronald B.
4-Carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone) isomerase, transforming 4-methyl-2-enelactone to 3-methyl-2-enelactone, was purified from a derivative strain of Pseudomonas sp. B13, named B13 FR1, carrying the plasmid pFRC2OP. This plasmid contained the isomerase gene cloned from Alcaligenes eutrophus JMP 134, which uses 4-methyl-2-enelactone as a carbon source. The enzyme consists of a single peptide chain of Mr 40,000 as judged by SDS/PAGE. In addition to 4-methyl-2-enelactone, the putative reaction intermediate, 1-methyl-3,7-dioxo-2,6-dioxy-bicyclo[3.3.0]octane (1-methylbislactone), was a substrate for the enzyme, but kinetic data presented did not favour its role as a reaction intermediate. Isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides were neither substrates nor inhibitors. Possible reaction mechanisms are discussed.
Open Access
Regulation of catabolic pathways of phenoxyacetic acids and phenols in Alcaligenes eutrophus JMP 134
(1989) Pieper, Dietmar H.; Engesser, Karl-Heinrich; Knackmuss, Hans-Joachim
Alicaligenes eutrophus JMP 134 is able to grow on 2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxy acetic acid. The unsubstituted phenoxyacetic acid, however, is no growth substrate due to very poor induction of the 2,4-D monooxygenase. Spontaneous mutants of Alcaligenes eutrophus JMP 134 capable of growth with phenoxyacetic acid were selected on agar plates. One of these mutants, designated Alcaligenes eutrophus JMP 134-1, shows constitutive production of six enzymes of the 2,4-D pathway, which were known to be localized in at least three different transcriptional units. A common regulatory gene is postulated to be mutated. 2,4-Dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid were the inducers of the enzymes of the ldquochloroaromatic pathwayrdquo in Alcaligenes eutrophus JMP 134. Phenol and 2-methylphenol, metabolites of the degradation of phenoxyacetic acid and 2-methylphenoxyacetic acid, were shown to be inducers of the meta-cleavage pathway, whereas 2,4-dichlorophenol and 4-chloro-2-methylphenol were not. Thus efficient regulation prevents chloroaromatics from being misrouted into the unproductive meta-cleavage pathway. Because 2,4-dichloro-and 4-chloro-2-methylphenol did not show any induction potential, they were growth substrates only for the mutant strain JMP 134-1.
Open Access
Simultaneous degradation of chloro- and methylaromatics via ortho pathway by genetically engineered bacteria and natural soil isolates
(1989) Engesser, Karl-Heinrich; Pieper, Dietmar H.; Rojo, Fernando; Timmis, Kenneth N.; Knackmuss, Hans-Joachim
The simultaneous bacterial metabolism of chloro- and methylaromatics via ortho- or metapathway, normally results in incomplete degradation and death of the organisms. This is caused by misrouting of central intermediates. i.e. substituted catechols into unproductive pathways and suicide inactivation of the key enzyme of meta pathway, (catechol 2,3-dioxygenase). The meta pathway proved to be definitely unsuited for productive metabolism of chloroaromatics. Therefore two strategies were used for simultaneous degradation of mixtures of chloro- and methylaromatics via ortho pathways: Methyllactons or certain mixtures of chloro- and methylaromatics were used as enrichment substrates, yielding strains which metabolized these compounds almost exclusively via the desired pathway. Alternatively relevant enzymes from five different catabolic pathways of three distinct soil bacteria were combined in a patchwork fashion generating a functional ortho cleavage route for methylaromatics coexisting with the ortho cleavage pathway of chloroaromatics.