Abstract
Carbaryl (1-naphthyl N-methylcarbamate) is a most widely used carbamate pesticide in the agriculture field. Soil isolate, Pseudomonas sp. strain C5pp mineralizes carbaryl via 1-naphthol, salicylate and gentisate, however the genetic organization and evolutionary events of acquisition and assembly of pathway have not yet been studied. The draft genome analysis of strain C5pp reveals that the carbaryl catabolic genes are organized into three putative operons, ‘upper’, ‘middle’ and ‘lower’. The sequence and functional analysis led to identification of new genes encoding: i) hitherto unidentified 1-naphthol 2-hydroxylase, sharing a common ancestry with 2,4-dichlorophenol monooxygenase; ii) carbaryl hydrolase, a member of a new family of esterase; and iii) 1,2-dihydroxy naphthalene dioxygenase, uncharacterized type-II extradiol dioxygenase. The ‘upper’ pathway genes were present as a part of a integron while the ‘middle’ and ‘lower’ pathway genes were present as two distinct class-I composite transposons. These findings suggest the role of horizontal gene transfer event(s) in the acquisition and evolution of the carbaryl degradation pathway in strain C5pp. The study presents an example of assembly of degradation pathway for carbaryl.
Highlights
Carbaryl (1-naphthyl N-methylcarbamate) is a most widely used carbamate pesticide in the agriculture field
We report the genetic organization of degradation genes and functional identification of carbaryl hydrolase (CH), 1-naphthol 2-hydroxylase (1NH) and 1,2-dihydroxynaphthalene dioxygenase (12DHNDO) involved in carbaryl metabolism in Pseudomonas sp. strain C5pp
The G1-DNA showed amplification of genes viz. salicylaldehyde dehydrogenase (SalDH, 0.5 kb), salicylate 5-hydroxylase (S5H) β-subunit (0.5 kb) and gentisate dioxygenase (GDO, 0.25 kb) by PCR using primers reported earlier[17], G1 cells failed to grow on carbaryl or salicylate
Summary
Construction and functional screening of genomic DNA library. The genomic DNA library of Pseudomonas sp. strain C5pp was constructed in CopyControl fosmid pCC2FOS with a phage titer of 3 × 106 CFU. ml−1. N-terminal (MLXNIFLKDE) and partial peptide (FTLLTGIGGEGWIR) sequences of 1-NH purified from strain C5pp showed identity (100%) with amino acid sequence derived from mcbC (1773 bp, 590 a.a.) suggesting the gene probably encodes 1NH (Fig. 2). Gene mcbH (903 bp, 300 a.a.), a part of the ‘middle’ pathway segment (salicylate degradation) encodes a McbH and showed 67–69% identity to a group of NagR/DntR/NahR type LysR transcriptional regulators from Pseudomonas sp. The observed gene organization with respective regulator into three distinct ‘upper’, ‘middle’ and ‘lower’ pathways for the carbaryl degradation in strain C5pp corroborates well with biochemical studies reported earlier[17]. The information becomes important giving avenue to engineer a strain for effective degradation of array of aromatic compounds
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