Enzyme regulation in in vivo‐constructed Pseudomonas putida strains with two alternative routes for oxidative degradation of phenol

Abstract

Based on plate matings using P. putida strain BLlO as the recipient and either P.purida strain PG405 or PG125 as the donor, the transconjugant strains TKI and TK3 were obtained. These were shown to harbour a single plasmid of a molecular size comparable to that of PGH 1 (in the donor strain PG405) and pPGH I I (in the donor strain PG125), respectively. Clear-cut evidence was presented that a t least the transconjugant strain TK3 has the genetic potential of degrading phenol via both the or-tho-pathway (encoded by chromosomal genes) and the meta-pathway (encoded by plasmid-localized genes) due to two complete alternative enzyme sequences. However, in both transconjugant strains T K l and TK3 only the meta-pathway enzymes were found to operate during cell growth on phenol as the sole carbon source. Several lines of investigation indicated the observed predominance of the meta-pathway in T K l and TK3 cells growing on phenol lo be a specific phenomenon resulting from coordinate induction of the respective plasmid-encoded enzymes by the primary substrate.Catechol is known to be the central metabolite in oxid'ative biodegradation of numerous monocyclic aromatic compounds. In the case of phenol degradation, it is directly produced from the primary substrate due to ortko-hydroxylation by NAD(P)H-depending enzymes of the phenol 2-monooxygenase type (NEUJAHR and GAAL 1973, KRUG and STRAUBE 1986, STRAUBE 1987, ADAMS and RIBBONS 1988). Further metabolism of catechol may proceed via two alternate routes : (i) the ortho-pathway initiated by intradiolic ring-fission due to catechol 1,2-dioxygenase (C 120), (ii) the meta-pathway initiated by extradiolic ring-fission due to catechol2,3-dioxygenase (C230). These routes have been found to differ with respect to regulation of de novo synthesis of the individual degradative enzymes involved: While the rneta-pathway enzymes are subject to coordinative induction "from the top", i.e. by the primary substrate or certain non-metabolizable analogous ( FEIST and HECEMAN 1969 1987), sequential enzyme induction (with the C120 being induced by the ortho-cleavage product cis, cis-muconic acid) takes place in the Fase of the ortho-pathway (STANIER and ORNSTON 1973). Typically, bacteria degrading phenol via the nwta-pathway can employ the same enzyme sequence for total degradation of the cresol isomers (RIBBONS 1970, SALA-TREPAT et (11. 1972, BUSWELL 1975, JANKE et al. 1981. In contrast, the ortho-pathway enzymes are usually inefficient at processing total degradation of methylarenes through methylcatechols (KNACKMUSS et al. 1976).The genetic basis of oxidative phenol degradation has been examined so far only for a few Pseudornonas strains. WONG and DUNN (1976) reported Pseudomonas purida strain PP 1-2 (derived from strain ATCC 17453) to degrade phenol via the orfho-pathway by products of chromosomal genes. Recent analysis of P . putidu strain H indicated a non-conjugative 200-220 kb plasmid (pPGH1) to carry all the genes required for degradation