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Abstract. 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 eutroplws 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 hydroxylascs in this strain. 3,5-Dichlorocatechol and 5-ch loro-3-methylca techol, metabolites of the degradation of 2,4-diehlorophenoxyacetic acid and 4-cWoro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxy. acetic acid-grown cells showed sim ultaneous induction of meta-and ortho-cleavage enzymes. Two catechol 1,2-dioxygcnases responsible for o rlho-cleavage of the intcr~ mediate 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 ofmuconates was found , which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatcchol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.

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Stimulation of transcription from positively regulated promoters involves regulatory proteins that have been activated, generally as a consequence of binding low molecular weight effector molecules. To define essential structural features of effectors for one positively acting gene regulator, the xylS-encoded protein, which activates the TOL plasmid meta-cleavage pathway operon promoters, effector activities of a wide range of benzoate derivatives have been systematically analyzed, and mutant xylS-encoded proteins exhibiting altered effector specificities have been generated and characterized. Cloned mutant xylS genes were trans dominant in partial diploids containing the wild-type xylS allele and could therefore be used to effect expansion of the range of aromatic compounds completely or partially degraded by Pseudomonas bacteria. The method developed to isolate mutant xylS-encoded proteins has general applicability and could in principle be used to isolate gene regulator specificity mutants of any inducible regulatory system.

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The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation ( K 2 ) were 1.62 × 10 −3 sec −1 for 3-chlorocatechol and 2.38 × 10 −3 sec −1 for 3-fluorocatechol. The inhibitor constants ( K i ) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta -cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.

Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho-and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxycis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.Various bacterial cultures which can use chloroaromatics as the single source of carbon and energy for growth, resulting in the formation of carbon dioxide, chloride, and biomass, have been described. These organisms can be differentiated on the basis of the catabolic pathways dealing with the substituents. Chlorosubstituents can be removed by initial oxygenolytic, reductive, or hydrolytic reactions. Further mineralization can then occur via classical pathways such as the 3-oxoadipate and the meta-cleavage pathways. However, the majority of the organisms able to mineralize chlorinated aromatics do not possess enzyme systems capable of initial dechlorination. They transform chlorinated aromatics to chlorocatechols, which are further metabolized by the enzymes of the modified orthocleavage pathway, and dechlorination occurs after ring cleavage (32).It is generally accepted that degradation of chloroaromatics does not proceed via the meta-cleavage pathway (20,26,29). An explanation for this has been found in the production of an acylchloride from 3-chlorocatechol by the catechol 2,3-dioxygenase of the meta-cleavage pathway, which leads to rapid suicide inactivation of the enzyme (3). Therefore, meta cleavage is considered to be unsuitable for the mineralization of haloaromatics that are degraded via halocatechols.Whereas chlorocatechols are mineralized via ortho-cleavage pathways, methylaromatics are commonly mineralized via meta-cleavage routes. Simultaneous metabolism of chloro-and methylcatechols often creates biochemical anarchy. meta cleavage leads to substrate misrouting in the case of 4-chlorocatechol or formation of a suicide product in the case of 3-chlorocatechol. Formation of dead-end methyllactones ca...