Pathways for 3-chloro- and 4-chlorobenzoate degradation in Pseudomonas aeruginosa 3mT

A bacterial isolate, Pseudomonas aeruginosa 3mT, exhibited the ability to d egrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l(-1)) and 4-chl orobenzoate (4-CBA 12 g l(-1)) (Ajithkumar 1998). In this study, by delinea ting the initial biochemical steps involved in the degradation of these com pounds, we investigated how this strain can do so well. Resting cells, perm eabilised cells as well as cell-free extracts failed to dechlorinate both 3 -CBA and 4-CBA under anaerobic conditions, whereas the former two readily d egraded both compounds under aerobic conditions. Accumulation of any interm ediary metabolite was not observed during growth as well as reaction with r esting cells under highly aerated conditions. However, on modification of r eaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) acc umulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyr ocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in ex tracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3- dioxygenase) activity against 4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hy droxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating th e presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusivel y, and more than 99% of 4-CBA were degraded through the formation of the re spective chlorocatechol, via a modified ortho-pathway. This defies the trad itional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoate was readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the f ormation of protocatechuate and gentisate, respectively, as intermediary di hydroxy metabolites.