Substrate specificity of chlorophenoxyalkanoic acid-degrading bacteria is not dependent upon phylogenetically related tfdA gene types

The phenoxyalkanoic acid herbicides constitute a group of chemically relate d molecules that have been widely used for over 50 years. A range of bacter ia have been selected from various locations for their ability to degrade t hese compounds. Previously reported strains able to utilise 2,4-dichlorophe noxyacetic acid (2,4-D) include, Ralstonia eutropha JMP134, Burkholderin sp . RASC and Variovorax paradoxus TV1 and Sphingomonas sp. AW5 able to utilis e 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In addition a novel set of m ecoprop-degrading strains including Alcaligenes dentrificans, Alcaligenes s p. CS1 and Ralstonia sp. CS2 are here described. It has been reported recen tly that TfdA enzymes, initially reported to have a role in 2,4-D catabolis m are also involved in the first-step cleavage of related phenoxyalkanoate herbicides. However, a diversity of tfdA gene sequences have been reported. We relate the tfdA gene type to the metabolic ability of these strains. Th e tfdA-like genes were investigated by polymerase chain reaction amplificat ion using a set of specific tfdA primers. Degradation ability was observed via phenol production from a range of unsubstituted and substituted phenoxy alkanoics including, 2,4-D, 2-methyl 4-chlorophenoxyacetic acid (MCPA), rac emic mecoprop, (R)mecoprop, 2-(2,4-dichlorophenoxy) propionic acid (racemic 2,4-DP), 2,4,5-T, 2,4-dichlorophenoxybutyric acid (2,4-DB), 4-chloro-2-met hylphenoxybutyric acid (MCPB) and phenoxyacetate. Mecoprop-degrading strain s showed partial tfdA sequences identical to the one described for V. parad oxus TV1 (a strain isolated on 2,4-D). However, substrate specificity was n ot identical as V. paradoxus exhibited greatest activity towards 2,4-D and MCPA only, whereas the mecoprop-degrading strains showed intense activity t owards 2,4-D, MCPA, racemic mecoprop and (R)-mecoprop as substrates. Howeve r, Sphingomonas sp. AW5 which has been shown to carry a very different tfdA -like gene was the only strain to utilise the phenoxybutyric acid MCPB as a sole carbon source. In this study, we thus demonstrate that sequence diver sity is not related to substrate specificity within the tfdA-like gene fami ly. However, phylogenetically unrelated sequences may govern substrate spec ific activity.