title: Characterization of a gene cluster involved in 4-chlorocatechol degradation by Pseudomonas reinekei MT1.
abstract: Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12O(ccaA), a novel (chloro)muconate cycloisomerase, MCI(ccaB), which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes, ccaA (C12O(ccaA)) and ccaB (MCI(ccaB)), were located in a 5.1-kb genomic region clustered with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12O(ccaA) and MCI(ccaB) are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCI(ccaB) and the previously identified C12O(salD), rather than C12O(ccaA), are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization.
authors.affiliation: Department of Microbial Pathogenesis, HZI Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Cámara
authors.forename: Patricia
authors.initials: P
authors.lastname: Nikodem
authors.forename: Piotr
authors.initials: P
authors.lastname: Bielecki
authors.forename: Roberto
authors.initials: R
authors.lastname: Bobadilla
authors.forename: Howard
authors.initials: H
authors.lastname: Junca
authors.forename: Dietmar H
authors.initials: DH
authors.lastname: Pieper
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D002396:Catechols / Q000378:metabolism*; D019753:Intramolecular Lyases / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics* / Q000502:physiology*; D010802:Phylogeny; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002396:Catechols; D019753:Intramolecular Lyases; C021811:muconate cycloisomerase; C055773:chloromuconate cycloisomerase; C038861:4-chlorocatechol