Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene

Abstract

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...

Degradation of chloroaromatics by Pseudomonas putida GJ31: assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome

“…The supernatant collected after centrifugation at 100 000 g was used as the enzyme extract. P. putida GJ31 was grown as previously described (Mars et al, 1997).…”

“…These are usually further degraded through the modified ortho-cleavage pathway (Dorn & Knackmuss, 1978a, b;Kaschabek & Reineke, 1992;. In contrast, Pseudomonas putida strain GJ31 has been shown to be able to degrade toluene and chlorobenzene simultaneously (Oldenhuis et al, 1989), and rapidly degrades chlorobenzene via 3-chlorocatechol using a novel type of metacleavage pathway instead of the modified ortho-cleavage pathway (Mars et al, 1997). The chlorocatechol 2,3-dioxygenase CbzE of strain GJ31 productively converts 3-chlorocatechol, and stoichiometric displacement of chloride leads to the production of 2-hydroxymuconate (Kaschabek et al, 1998), which is further converted through the meta-cleavage pathway.…”

“…2,3-Dihydroxyphenylpropionate 1,2-dioxygenase activity was measured by determining product formation from 2,3-dihydroxyphenylpropionate at 394 nm (e 2-hydroxy-6-oxo-nona-2,4-diene 1,9-dicarboxylate 519 150 l mol 21 cm 21 ) (Bugg, 1993). 2-Hydroxymuconic semialdehyde hydrolase activity was measured by determining the NAD + -independent decrease in concentration of 2-hydroxymuconic semialdehyde (0.06 mM) at 375 nm (Mars et al, 1997). 2-Hydroxymuconic semialdehyde dehydrogenase activity was measured by determining the NAD + -dependent decrease in concentration of 2-hydroxymuconic semialdehyde (0.06 mM) at 375 nm.…”

Biodegradation of 2,4-dichlorophenol using Mycoplana dimorpha extracts and evaluation of kinetic parameters

“…J. Biotechnol. indicates that immobilized crude extract degradation is much faster than microbial degradation (Axcell and Geary 1973;Grund et al, 1975;Hartmann et al, 1979;Chang et al, 1992;Spiess and Gorisch 1996;Beil et al, 1997;Mars et al, 1997;Beil et al, 1998;Pollmann et al, 2001;Alfreider et al, 2003;Rapp and Gabriel-Jurgens 2003;Manikandan et al, 2007a;Manikandan et al, 2007b). The similar trend of decomposition was observed with the initial concentrations of 60 and 80 ppm with the percentage degradation at the end of 30 min is 72% for 60 ppm and 75% for 80 ppm (Manikandan et al, 2007d).…”

“…Mars et al (1997) have studied the microbial degradation of chloroaromatics and the use of the metacleavage pathway for mineralization of chlorinated compounds. They demonstrated the degradation studies with Pseudomonas putida GJ31 on toluene and chlorobenzene.…”