Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP

The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from P seudomonas sp. strain ADP was determined. Plasmid pADP-1 was previously sho wn to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of s-triazine ring substituents from the herbicide atrazine to yie ld cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in cat abolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 10 0% amino acid sequence identity to pR751, an IncP beta plasmid previously i solated from Enterobacter aerogenes. pADP-1 was shown to contain a function al mercury resistance operon with 99% identity to Tn5053. Complete copies o f transposases with 99% amino acid sequence identity to TnpA from IS1071 an d TnpA from Pseudomonas pseudoalcaligenes were identified and flank each of the atzA, atzB, and atzC genes, forming structures resembling nested catab olic transposons. Functional analyses identified three new catabolic genes, atzD, atzE, and atzF, which participate in atrazine catabolism. Crude extr acts from Escherichia coli expressing AtzD hydrolyzed cyanuric acid to biur et. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid a midohydrolase, from Pseudomonas sp. strain NRRLB-12227. Two other genes enc oding the further catabolism of cyanuric acid, atzE and atzF, reside in a c ontiguous cluster adjacent to a potential LysR-type transcriptional regulat or. E. coli strains bearing atzE and atzF were shown to encode a biuret hyd rolase and allophanate hydrolase, respectively. atzDEF are cotranscribed. A tzE and AtzF are members of a common amidase protein family. These data rev eal the complete structure of a catabolic plasmid and show that the atrazin e catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, an d thus evolve, to encode the catabolism of compounds recently added to the biosphere.