SELF-MOBILIZATION AND ORGANIZATION OF THE GENES ENCODING THE TOLUENE METABOLIC PATHWAY OF PSEUDOMONAS-MENDOCINA KR1

The toluene metabolic pathway of Pseudomonas mendocina KR1 is chromosomally encoded, but the pathway could be transferred by conjugation from strain KR1 to the chromosome of P. aeruginosa or P. putida. Such transconjugants utilized toluene, p-cresol, and p-hydroxybenzaldehyde. However, transconjugants were unable to further transfer toluene genes to other recipients unless Pseudomonas sex factor R68.45 was present in trans. Although the genes encoding the upper pathway for toluene metabolism in P. mendocina KR1 are sufficiently linked to permit their coordinate mobilization, they were found to be encoded in three independently regulated units: one encoding toluene-4-monooxygenase, a second encoding p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase, and a third encoding p-hydroxybenzoate hydroxylase. The last two regulatory units were cloned from the chromosome of a P. aeruginosa transconjugant onto a plasmid designated pRO1999. Analysis of pRO1999 showed that genes encoding p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase are organized as an operon; the gene encoding p-hydroxybenzaldehyde dehydrogenase is transcribed first, and this is followed by transcription of the gene encoding p-cresol methylhydroxylase. This operon is regulated by a positively acting regulator. The P. mendocina KR1 gene encoding p-hydroxybenzoate hydroxylase was linked to, but independently regulated from, the genes encoding toluene-4-monooxygenase, p-cresol methylhydroxylase, and p-hydroxybenzaldehyde dehydrogenase.