DEGRADATIVE PATHWAYS FOR P-TOLUENECARBOXYLATE AND P-TOLUENESULFONATE AND THEIR MULTICOMPONENT OXYGENASES IN COMAMONAS-TESTOSTERONI STRAINS PSB-4 AND T-2

Three multicomponent oxygenases involved in the degradation of p-tolue nesulfonate and p-toluenecarboxylate and the regulation of their synth esis have been examined in three strains (T-2, PSB-4 and TER-1) of Com amonas testosteroni. Strain T-2 utilizes p-toluenesulfonate as a sourc e of carbon and energy for growth via p-sulfobenzoate and protocatechu ate, and p-toluenecarboxylate via terephthalate and protocatechuate, a nd has the unusual property of requiring the reductase (TsaB) of the t oluenesulfonate methyl monooxygenase system (TsaMB) in an incompletely expressed sulfobenzoate dioxygenase system (PsbAC) [Schlafli Oppenber g, H. R., Chen, G., Leisinger, T. & Cook, A. M. (1995). Microbiology 1 41, 1891-1899]. The independently isolated C. testosteroni PSB-4 utili zed only sulfobenzoate and terephthalate via protocatechuate. Mutant T ER-1, derived from strain T-2, utilized only terephthalate via protoca techuate. We detected no enzymes of the pathway from toluenesulfonate to sulfobenzoate in strains PSB-4 and TER-1, and confirmed by PCR and Southern blot analysis that the genes (tsaMB) encoding toluenesulfonat e monooxygenase were absent. We concluded that, in strain PSB-4, the r egulatory unit encoding the genes for the conversion of toluenesulfona te to sulfobenzoate was missing, and that generation of mutant TER-1 i nvolved deletion of this regulatory unit and of the regulatory unit en coding desulfonation of sulfobenzoate. The degradation of sulfobenzoat e in strain PSB-4 was catalysed by a fully inducible sulfobenzoate dio xygenase system (PsbAC(PSB-4)), which, after purification of the oxyge nase component (PsbA(PSB-4)), turned out to be indistinguishable from the corresponding component from strain T-2 (PsbA(T-2)). Reductase Psb C(PSB-4), which we could separate but not purify, was active with oxyg enase PsbA(PSB-4) and PsbA(t-2). Oxygenase PsbA(PSB-4) was shown by el ectron paramagnetic resonance spectroscopy to contain a Rieske [2Fe-2S ] centre. The enzyme system oxygenating terephthalate was examined and the oxygenase component purified and characterized. The oxygenase com ponent in strains T-2 (and mutant TER-1) and PSB-4 were indistinguisha ble. The reductase component, which we separated but failed to purify, was active with the oxygenase from all strains. Gains and losses of b locks of genes in evolution is discussed.