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Researchers transferred a gene cluster for the chlorocatechol ortho-cleavage pathway into Pseudomonas putida KT2442. This research is based on efforts to genetically modify bacteria so they can biodegrade haloaromatic compounds.

Several possibilities were investigated to clarify the initial steps of the degradative pathway in bacterial strain LW1 of the family Comamonadaceae, which uses 1-chloro-4-nitrobenzene as a a sole source of carbon, nitrogen and energy. Results indicated that the initial attack on the nitro group was reductive, given the accumulation of ammonia but not of nitrite in conversion experiments performed with resting cells grown with 1C4NB and the observed 1C4NB reductase activity in LW1 cell extracts. Partial reduction of the nitro group was suggested by the complete conversion of 1C4NB to 2-amino-5-chlorophenol in the absence of oxygen.

The aerobic mineralization of 1,2,3,4-tetrachlorobenzene by a pure culture of Pseudomonas chlororaphis RW71 was demonstrated. The reaction produced stoichiometric amounts of chloride. The transient excretion of tetrachlorocatechol in the early growth phase suggested an initial attack by a dioxygenase to form the corresponding dihydrodiol which rearomatizes to the catechol. Extraordinarily high activity was found in chlorocatechol 1,2-dioxygenase in crude cell extracts.

Microbiological research shows the mineralization of the polyhalogenated dibenzofuran 4-chlorodibenzofuran using a coculture of dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 and the chlorosalicy-late-degrading bacterium Burkholderia sp. strain JWS (13,15). The complete degradation of 4-chlorodibenzofuran with concomitant release od Cl- and formation of biomass is observed.

The metabolism of 2-chloro and 3-chlorotoluene by Burkholderia sp. strain PS12 and Pseudomonas putida F1 was analyzed to determine the biochemical characteristics of the doixygenolytic pathway. The toluene dioxygenase of Pseudomonas putida F1 and the chlorobenzene dioxygenase of Burkholderia sp. PS12 strain catalyzed the conversion of 2-, 3- and 4-chlorotoluene to dihydrodiols. The synthetic process was characterized by the formation of minor products such as methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes from 2- and 3-chlorotoluene.

The metabolism of 4-chlorocatechol via the 3-oxoadipate pathway produces the antibiotic protoanemonin that is toxic to indigenous and supplemented 4-chlorobiphenyl-cometabolizing bacteria in soil. The survival of soil bacteria and fungi is adversely affected by the direct addition of protoanemonin, but is unaffected by its degradation. The addition of organisms that contain the meta-cleavage pathway for 4-chlorocatechol metabolism increases the survival of the soil bacteria. This suggests that protoanemonin is formed via the ortho-cleavage pathway.

The bacterium Sphingomonas sp. strain RW1 degrades many mono- and dichlorinated dibenzofurans, and dibenzo-p-dioxins, to the mono- and dichlorinated salicylates and catechols. The degradation is apparently a dioxygenolytic attack on the substituted and nonsubstituted aromatic nucleus of the target compounds. The strain RW1 uses monochlorinated dibenzo-p-dioxins and dibenzofurans as the carbon source.

A study of 11 substituted dibenzofurans by the Sphingomonas sp. strain which is dibenzofuran-degrading indicates high regioselectivity of these monosubstituted dibenzofurans for the ether bond cleavage with the preference for the nonsubstituted aromatic nucleus. The HH69 strain developed on 2-methoxydibenzofurans after getting adapted to the utilization of 5-methoxysalicyclic acid. Hydrolysis of ester bonds stimulates the degradation of acetoxydibenzofurans.

Sphingomonas sp. strain SS3, with the ability to metabolize diphenyl ether (DE) and its 4-fluoro, 4-chloro and 4-bromo derivatives, was isolated from soil samples from an industrial waste deposit. The bacterium was enriched and characterized. The results showed that SS3 could cometabolize the monohalogenated DEs, resulting in the production of phenol and catechol as intermediates. These were then channeled through the 3-oxoadipate pathway. In general, the degradation steps showed characteristics of the 1,2-dioxygenation system, which in this bacterium, apparently acts nonspecifically.

A bacterium, identified asSphigomonas sp. strain RW1, with the ability to mineralize dibenzo-para-dioxins(DD) and dibenzofuran (DF), was isolated from enrichment cultures inoculated with water samples from the river Elbe. The proposed pathway for degradation ofthe biaryl ethers begins with ring cleavage of the substrate at the angular position adjacent to the ether bridge. The resulting 2,2',3-trihyroxy compoundsare meta cleaved, with the resulting catechol undergoing both ortho and meta cleavage.

Sphingomonas sp. strain SS33 was successfully modified to be employed in a few dihalogenated diphenyl ethers. Sphingomonas sp. strain SS3 is an organism that can mineralize diphenyl ether and many other 4-halodiphenyl ethers. The bacterium utilized 4,4-dichlorodiphenyl ether to aid growth. Mineralization of intermediary halocatechols also occur through the ortho pathway.

The homogeneous preparation of type II hydride transferases of the old yellow enzyme family of flavoproteins, XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli is reported. The flavoproteins responsible for the reduction of the polynitroaromatic compound 2,4,6-trinitro-toluene (TNT) have important environmental implications for TNT remediation.

A set of old yellow enzyme (OYE) homologs of Pseudomonas putida is utilized to study the potential complementary activities of multiple enzymes belonging to the same family within a single microorganism. The in vitro analysis of six OYE homologs identified in P. putida KT2440 offer insight into the catalytic activity of enzymes against 2,4,6-trinitrotoluene (TNT), which serves as a nitrogen source for microorganisms in vivo.