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Draft Genome Sequence of Pseudomonas sp. Strain ADP, a Bacterial Model for Studying the Degradation of the Herbicide Atrazine.

ABSTRACT: We report here the 7,259,392-bp draft genome of Pseudomonas sp. strain ADP. This is a bacterial strain that was first isolated in the 1990s from soil for its ability to mineralize the herbicide atrazine. It has extensively been studied as a model to understand the atrazine biodegradation pathway. This genome will be used as a reference and compared to evolved populations obtained by experimental evolution conducted on this strain under atrazine selection pressure.

SUBMITTER: Devers-Lamrani M

PROVIDER: S-EPMC4751332 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Draft Genome Sequence of Pseudomonas sp. Strain ADP, a Bacterial Model for Studying the Degradation of the Herbicide Atrazine.

Devers-Lamrani Marion M Spor Aymé A Mounier Arnaud A Martin-Laurent Fabrice F

Genome announcements 20160211 1

We report here the 7,259,392-bp draft genome of Pseudomonas sp. strain ADP. This is a bacterial strain that was first isolated in the 1990s from soil for its ability to mineralize the herbicide atrazine. It has extensively been studied as a model to understand the atrazine biodegradation pathway. This genome will be used as a reference and compared to evolved populations obtained by experimental evolution conducted on this strain under atrazine selection pressure. ...[more]

PMID: 26868408

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Project description: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.

Project description:AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.

Dataset Information

Draft Genome Sequence of Pseudomonas sp. Strain ADP, a Bacterial Model for Studying the Degradation of the Herbicide Atrazine.

Publications

Draft Genome Sequence of Pseudomonas sp. Strain ADP, a Bacterial Model for Studying the Degradation of the Herbicide Atrazine.

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