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

Abstract

The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of s-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPbeta plasmid previously isolated from Enterobacter aerogenes. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn5053. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS1071 and TnpA from Pseudomonas pseudoalcaligenes were identified and flank each of the atzA, atzB, and atzC genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes, atzD, atzE, and atzF, which participate in atrazine catabolism. Crude extracts from Escherichia coli expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from Pseudomonas sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid, atzE and atzF, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator. E. coli strains bearing atzE and atzF were shown to encode a biuret hydrolase and allophanate hydrolase, respectively. atzDEF are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, and thus evolve, to encode the catabolism of compounds recently added to the biosphere.

FIG. 1

Proposed pathway for the degradation of cyanuric acid by atrazine- and melamine-degrading bacteria. Cyanuric acid is hydrolyzed to biuret and is hypothesized to be subsequently hydrolyzed to urea, carbon dioxide, and ammonia.

FIG. 2

Physical circular map of the catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP. The map positions of selected restriction sites, genes, and operons on pADP-1 are indicated. Regions with 99% identity to the tra and trb operons of plasmid pR751 are boxed. Genes involved in atrazine catabolism are indicated. Copies of similar putative transposons have the same shading.

FIG. 3

Linear map showing graphical representation of ORFs present on the pADP-1 sequence. Boxes above the lines refer to ORFs on the top strand, while those below the line are from the complementary strand. Genes of the same type (operons, insertion sequence [IS] elements, and catabolic genes) have similar shading, and ORFs with putative functions are listed by their numbers. ORFs and genes have been numbered relative to the origin of replication (oriV). Distance between tick marks is 1.3 kb.

FIG. 4

(A) Degradation of cyanuric acid by crude cell extracts from E. coli DH5α(pBMZ1). Plasmid pBMZ1 contains atzD from pADP-1 cloned into the BamHI site of pKT230. Symbols: ●, E. coli DH5α(pBMZ1); ○, E. coli DH5α. (B) Degradation of biuret by crude cell extracts from E. coli DH5α (atzE). Symbols: ▪, E. coli DH5α (atzE); □, E. coli DH5α(pUC18). (C) Degradation of allophanate by crude cell extracts of E. coli DH5α (atzF). Symbols: ●, E. coli DH5α (atzF); ○, E. coli DH5α(pUC18). Values are the means of three replicates. Bars indicate standard deviations of the mean.

FIG. 5

Sequence alignment of ORF101 and ORF102 with members of the amidase protein family. Identical amino acids are boxed. The arrows indicate residues that have been shown to be important for amidase activity in a fatty acid amide hydrolase (FAAH). Residues common to the amidases and aspartic proteases are denoted by the asterisks. The amidase sequences used for the alignment were as follows: ORF102 from the pADP-1 plasmid sequence; Dur (1, 2), urea amidolyase from Saccharomyces cerevisiae (accession number CAA85172); Nicam, nicotinamidase from Mycobacterium smegmatis (accession number AAC77368.1); Rho, amidase from Rhodococcus (accession number M74531); GluAT, Glu-tRNA amidotransferase from Bacillus subtilis (accession number gi:25899195); Nylam, 6-aminohexanoate cyclic dimer hydrolase, Flavobacterium sp. (accession number gi:148711), EI, 6-aminohexanoate-cyclic-dimer hydrolase from Flavobacterium sp. (accession number M26953); ORF101 from the pADP-1 plasmid sequence; N-774, amidase from Rhodoccocus sp. strain N-774 (accession number X54074); J1-L, amidase from Rhodococcus rhodochrous J1 (accession number D16207); B23, amidase from Pseudomonas chlororaphis (accession number D90216); VDHAP, vitamin D₃ hydroxylase-associated protein, Gallus domesticus (accession number gi:1079452); FAAH1, fatty acid amide hydrolase, Rattus norvegicus (accession number gi:1680722); Celeg, predicted amidase from Caenorhabditis elegans (accession number gi:6425411); IAAH, indoleacetamide hydrolase, Pseudomonas syringae (accession number gi:77820); AMD, acetamidase, Emericella nidulans (accession number gi:101782).

FIG. 6

Ammonia released after incubation of biuret with cell extracts from E. coli DH5α (atzE) and E. coli DH5α (atzF). Symbols: □, 3 mM biuret plus crude cell extracts from E. coli DH5α (atzE) and E. coli DH5α (atzF); ▿, 3 mM biuret plus cell extract from E. coli DH5α (atzE); ▾, 3 mM urea plus cell extract from E. coli DH5α (atzF); ○, 3 mM biuret plus cell extract from E. coli DH5α (atzF); ●, 3 mM biuret plus cell extract from E. coli DH5α(pUC18). Values are the means of results from three replicates. Error bars indicate standard deviations of the means.

FIG. 7

Complete catabolic pathway for atrazine degradation by Pseudomonas sp. strain ADP. Genes and potential ORFs involved at each catabolic step are indicated.