GENETIC-CHARACTERIZATION AND EXPRESSION IN HETEROLOGOUS HOSTS OF THE 3-(3-HYDROXYPHENYL)PROPIONATE CATABOLIC PATHWAY OF ESCHERICHIA-COLI K-12

We report the complete nucleotide sequence of the gene cluster encodin g the 3-(3-hydroxyphenyl)propionate (3-HPP) catabolic pathway of Esche richia coli K-12. Sequence analysis revealed the existence of eight ge nes that map at min 8 of the chromosome, between the lac and hemB regi ons, Six enzyme-encoding genes account for a flavin-type monooxygenase (mhpA), the extradiol dioxygenase (mhpB), and the meta-cleavage pathw ay (mhpCDFE). The order of these catabolic genes, with the sole except ion of mhpF, parallels that of the enzymatic steps of the pathway, The mhpF gene may encode the terminal acetaldehyde dehydrogenase (acylati ng) not reported previously in the proposed pathway, Enzymes that cata lyze the early reactions of the pathway, MhpA and MhpB, showed the low est level of sequence similarity to analogous enzymes of other aromati c catabolic pathways, However, the genes mhpCDFE present the same orga nization and appear to be homologous to the Pseudomonas xy1, dmp, and nah meta-pathway genes, supporting the hypothesis of the modular evolu tion of catabolic pathways and becoming the first example of this type of catabolic module outside the genus Pseudomonas. Two bacterial inte rspersed mosaic elements were found downstream of the mhpABCDFE locus and flank a gene, orfT, which encodes a protein related to the superfa mily of transmembrane facilitators that might be associated with trans port, All of the genes of the 3-HPP cluster are transcribed in the sam e direction, with the sole exception of mhpR. Inducible expression of the mhp catabolic genes depends upon the presence, in the cis or trans position, of a functional mhpR gene, which suggests that the mhpR gen e product is the activator of the 3-HPP biodegradative pathway, The pr imary structure of MhpR revealed significant similarities to that of m embers of the IclR subfamily of transcriptional regulators, A 3-HPP ca tabolic DNA cassette was engineered and shown to be functional not onl y in enteric bacteria (E. coli and Salmonella typhimurium) but also in Pseudomonas putida and Rhizobium meliloti, thus facilitating its pote ntial application to improve the catabolic abilities of bacterial stra ins for degradation of aromatic compounds.