Cloning of Genes Participating in Aerobic Biodegradation of p-Cumate from Rhodopseudomonas palustris

Abstract

Rhodopseudomonas palustris utilizes p-cumate as a carbon source both under anaerobic light and aerobic dark conditions. A gene cluster was isolated whose sequence showed high homology to genes which have been implicated the degradation of p-cumate in Pseu-domonas putida. Seven structural genes coding for dioxygenase-reductase, dihydroxy-dihydro dehydro-genase, and ring cleavage oxygenases were identified. A putative regulator and its possible recognition site was suggested on the basis of homology data. Mutant cells in which a kanamycin cassette was inserted into the dihydroxy-dihydro dehydrogenase gene could not grow aerobically on p-cumate. The mutation had no effect on growth using the para substituted benzoate derivatives 4-hydroxycinnamate, ferulate, protocate-chuate, and 2,3,4-trihydroxybenzoate as sole carbon source. Moreover, mutant cells showed a growth pattern similar to wild type cells grown on these compounds under photoheterotrophic anaerobic conditions. These data suggest that genes of this operon are involved specifically in aerobic dissimilation of p-cumate. Intermediate products of p-cumate degradation could be detected from extracts of Escherichia coli heterologously expressing the first 5 genes responsible for the first two steps of p-cumate degradation in R. palustris. Primer extension analysis revealed the transcription regulation of the gene cluster which could be induced with para methyl-, ethyl-and isopropyl (cumate) benzoates. This is the first report on genes involved in aerobic degradation of these compounds in photosynthetic bacteria.

Key Words:

• aerobic biodegradation
• molecular cloning
• p-cumate
• Rhodopseudomonas palustris