Abstract
Pseudomonas spp. MR3 was isolated from the surrounding soil of pesticide manufacturing industries of Ankleshwar, Gujarat. Under laboratory conditions these microbes were able to degrade up to 500 ppm of methyl parathion within 72 h. Genome sequencing of Pseudomonas spp. MR3 was carried out inIon Torrent (PGM), next generation sequencer. The data obtained revealed 1,268 contigs with genome size of 2.99 Mb and G + C content of 60.9 %. The draft genome sequence of strain MR3 will be helpful in studying the genetic pathways involved in the degradation of several pesticides.
Keywords: Methyl parathion, Draft genome, Pseudomonas, Strain MR3
Introduction
Methyl parathion is a broad spectrum insecticide/miticide, formulated as a microencapsulate (20.9 % active ingredient) and as an emulsifiable concentrate (27.59–52.7 % active ingredient). The excessive use of natural resources and large scale synthesis and use of methyl parathion have generated a number of environmental problems such as contamination of air, water and terrestrial ecosystems [1]. Soil microflora is one of the basic agents for detoxification of pesticides. Some investigators found that soil contaminated with pesticides could be decontaminated by inoculation with specifically adapted microorganisms [2]. In the present study, soil samples were collected from the surrounding area of the pesticide manufacturing industries of Ankleshwar, Gujarat, India. Nineteen different bacteria isolated from the soil samples were distinguishable on the basis of their morphology and growth pattern on the selected media. All the isolates were checked for methyl parathion degradation under different concentrations through high performance liquid chromatography (HPLC). Among the nineteen isolates, Pseudomonas spp. MR3 strain showed highest degradation rate with degradation of 500 ppm of methyl parathion within 72 h and hence was selected for whole genome sequencing.
Whole-genome sequencing of Pseudomonas spp. MR3 was done using Ion Torrent (PGM), next generation sequencer (NGS) from Life Technologies at Department of Biotechnology, Junagadh Agricultural University, Junagadh, Gujarat. A 314 chip was loaded with enriched library of 260 bp to generate sequencing data. A total number of 564,810 reads of average length 145 nucleotides (nts), with a coverage of 15× of a 4.5-Mb genome (expected size) were produced by the machine. Assembly was done by using MIRA assembler v 3.4.1.0 [3], which revealed a total of 1,261 contigs. The final genome draft was used for genome annotation by Rapid Annotations using Subsystems Technology (RAST) system [4], RNAmmer 1.2 [5] and ARAGORN software [6]. The size of genome was found to be 2.99 Mb with G + C content of 60.9 %. A total of 79 RNAs genes were found, 3 copies of 5S rRNA were found, 2 copies of 16S rRNA and two for 23S rRNA synthesis. We have also reconfirmed the 16S rRNA gene sequence by Sanger’s sequencing and it was found to be 100 % identical with that obtained from whole genome sequence data analyzed by RNAmmer. The 16S rRNA sequence of Pseudomonas spp. MR3 available in GenBank database (Submission ID: 1626232) showed 99 % identity with the 16S rRNA gene sequence of Pseudomonas putida KT2440 (Accession number NR 074596.1). RAST annotation showed, P. putida KT2440 (score 510), P. putida F1 (score 504), and P. putida GB-1 (score 490) to be the closest neighbors of the strain MR3. This annotation also indicated the presence of genes involved in metabolisms of phosphorus, sulfur, aromatic compounds, nitrogen, protein, potassium and iron acquisition. Genes for Phosphono acetaldehyde hydrolase (EC 3.11.1.1), Salicylate hydroxylase (EC 1.14.13.1), Catechol 1,2-dioxygenase (EC 1.13.11.1), 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase and Monoamine oxidase (1.4.3.4) found to be involved in the catabolism of organophosphate compound were also identified. By using the tblastn, gene sequence of mpd gene at contig 310 with a length of 1,470 bp was identified which lead to the production of hydrolase enzyme responsible for the degradation of methyl parathion.
Nucleotide Sequence Accession Numbers
This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank with the accession number ARYY00000000. The version described in this paper is the first version, NZ_ARYY00000000.1 GI:499136694. Bioproject registered under Accession number: PRJNA203097 ID: 203097.
Acknowledgments
This research was carried out at Department of Biotechnology, Junagadh Agricultural University, Junagadh, Gujarat, India.
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