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. 2020 Apr 2;60(3):325–333. doi: 10.1007/s12088-020-00864-9

Comparative Genomics of a Paddy Field Bacterial Isolate Ochrobactrum sp. CPD-03: Analysis of Chlorpyrifos Degradation Potential

Tanmaya Nayak 1, Ananta N Panda 1, Khushbu Kumari 1, Tapan Kumar Adhya 1, Vishakha Raina 1,✉
PMCID: PMC7329950  PMID: 32655199

Abstract

Ochrobactrum genus is known to catabolize aromatic compounds. This study reports a complete genome sequence of Ochrobactrum sp. CPD-03 (~ 4.6 Mb of chromosomal features) responsible for chlorpyrifos (CP) isolated form a paddy field (20.3588° N, 85.8333° E) in Bhubaneswar, India. A comparative genomics approach was performed between CPD-03 and eight closely related genomes of other Ochrobactrum strains in order to deepen our knowledge, to establish its phylogenetic and functional relationships. The involvement of CP degrading genes indicated a versatile role of CPD-03 in additional field trails. This research would provide the genetic information for its use in natural environment for the depletion of organophosphorus (OP) compounds.

Keywords: Phylogenomics, Chlorpyrifos, Ochrobactrum sp. CPD-03, Arylesterase, Xenobiotic compound degradation

Introduction

The extensive use of organophosphorus pesticides (OPs) have triggered an alarming concern over environmental pollution and food safety apart from contact toxicity. The release of OPs into the ecosystem allows the microbes to have new metabolic pathways and several enzymes to catabolize these complex compounds, either by accessing new sources of carbon and energy or by detoxifying the residues of these contaminants [1]. Nonetheless, given sufficient exposure time, microbial cells are likely to develop pathways to mineralize OPs that have been introduced [2, 3].

Chlorpyrifos (CP; O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) is a mildly toxic among OPs commonly used for protecting agricultural crops from various insects, fungi and other pests. It enters inside the insects through ingestion, by contact or absorbed through cuticles, gut [4] and affects the nervous system by inhibiting the acetylcholinesterase activity ultimately causing death of the target insect [5]. CP hydrolysis results in the formation of a key metabolite TCP (3,5,6-trichloro-2-pyridinol) causing a prevalent pollution in soils and aquatic ecosystem due to greater water solubility [6] with a half-life between 65 and 360 days in soil [7] than CP. Hence, it is essential to remediate CP contaminated sites in order to mitigate the effects of these toxic residues.

Bioremediation as the most appropriate method reported by global researchers and the emphasis has been on microbial degradation, believed to be the primary mechanism for extracting pesticides from soil and water bodies. Ochrobactrum genus, a α-2 subclass of the Proteobacteria [8–10], has shown to degrade various OP compounds such as parathion and methylparathion [11], phenol [12], dimethylformamide [13], petroleum waste [14], chlorothalonil [15], CP & TCP [16]. Earlier studies have reported two complete genome sequences of CP degrading bacterium, Cupriavidus nantongensis X1 [17] and Paracoccus sp. TRP [18]. However, genetic studies with Ochrobactrum genus have not been much explored.

This study reports an efficient CP degrading bacterium, Ochrobactrum sp. CPD-03, which can degrade CP and TCP. Further, a comparative genomics approach has been used to compare CPD-03 and other closely related genomes of Ochrobactrum sp. along with other genomes of CP degraders to deepen our understanding. This analysis would provide valuable source of information to expand its potential as a biodegrader of OPs.

Materials and Methods

Isolation and Screening of CP Degraders

Soil samples were collected from a rice paddy field near KIIT University, Bhubaneswar Odisha, India [20.3551° N, 85.8187° E]. Preliminary screening was performed using Mineral Salt Media (MSM) [contains KH2PO4.H2O, 0.5 g/L; NH4Cl, 0.2 g/L; MgSO4.7H2O, 0.1 g/L; FeSO4.7H2O, 0.01 g/L; CaNO32.4H2O, 0.01 g/L] spiked with CP (100 mg L−1)] pH 7.0 at 30 °C. Analytical grade CP (PESTANAL® 99.99%) and TCP (PESTANAL® 99.99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and commercial grade pesticide mixture for soil enrichment was purchased from local nursery. These soil samples were incubated for 3 weeks at 30 ± 2 °C in shaking condition at 120 rpm. 72–96 h of post incubation, enriched culture was serially diluted and inoculated on MSM agar spiked with CP (100 mg L−1). Out of several isolates, CPD-03 strain was selected for further studies due to its ability to grow and degrade CP up to 100 mg L−1 in a MSM. CPD-03 was found to be gram-negative, small rod-shaped bacterium.

Bacterial Growth and DNA Extraction

Ochrobactrum sp. CPD-03 was grown in MSM supplemented with CP (100 mg L−1)] pH 7.0 at 30 °C. High molecular DNA was obtained by the CTAB extraction method [19] with slight modification and purified by HiYield DNA Ultra Purification Kit (RBC Biosciences). The concentration of DNA was checked by Qubit Fluorometer (Invitrogen) and Nanodrop spectrophotometer (ThermoFisher) and diluted up to 0.2 ng μL−1 concentration.

Genome Sequence, Assembly Statistics and Annotation

In order to comprehend the mechanisms underlying the CP degradation, the complete genome sequence of CPD-03 was determined by means of assembly based on paired-end sequencing in Illumina HiSeq System 2000 (Illumina, Inc.). Sequencing data were checked for full quality control using the NGSQC Toolkit. Velvet (V 1.2.10) [20] was used for primary assembly that resulted a total size of the contig of 4,660,061 bp (~ 4.6 Mb), 31 contigs and 25 Scaffolds (N50: 403.18 kb) (Table 1). De novo genome validation and quality control was performed by Bowtie2 (v 2.2.2) [21]. Quality control of assembled genome based on genomic elements were performed by ARAGORN (v1.2.36) [22]. The whole genome sequence of Ochrobactrum CPD-03 has been submitted in GenBank under the accession RSEU00000000 with a genome coverage of 99.81x.

Table 1.

Genome features of Ochrobactrum sp. CPD-03

Ochrobactrum sp. CPD-03
Genome Size (bp) GC Content (%) CDS rRNA tRNA No. of plasmid
4,660,061 bp (~ 4.6 Mb) 57.67% 4566 3 51 0
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Comparative Genomics

A phylogenomics approach was used to construct a phylogenetic tree using the core conserved loci 16S rRNA sequences of different Ochrobactrum species along with Mycoplana ramose DSM 7292 and Daeguia caeni K107 (as outgroups) in MEGA7.0 [23]. Similarly, an average nucleotide identity (ANI) analysis [24] was performed between the CPD-03 strain and closest organisms retrieved from EzBioCloud (http://www.ezbiocloud.net/eztaxon). Comparative analysis of the two other chlorpyrifos degrading isolates namely Cupriavidus nantongensis strain X1 and Paracoccus sp. Strain TRP were also included in the analysis. Genome comparisons were performed using draft genomes of CPD-03 and other Ochrobactrum strains along with two genomes CP degrading bacteria. Preliminary genome annotations were performed using RAST (Rapid Annotation using Subsystem Technology). Followed by this, KAAS (KEGG Automatic Annotation Server) (https://www.genome.jp/kegg/kaas/) was used to annotate the possible functional pathways of the genomes. Manual inspection of KEGG Orthology (KO), which was expected on the basis of correlations with the KEGG database, deduced metabolic pathways. MinPath was then used to recreate metabolic pathways using protein family predictions that were done in a more restrictive yet more accurate manner and approximate biological pathways according to the query data set. The Genome-to-Genome Distance Calculator (GGDC) 2.1 online software (http://ggdc.dsmz.de) calculator [25] evaluated the genome sequence similarity between Ochrobactrum strains.

Localization of Arylesterases Gene Cluster

Ochrobactrum sp. CPD-03 strain has shown CP degradation in lab scale analysis (Data not shown). Considering this ability, the whole genome of this strain was checked for the presence of the CP degrading core genes/proteins. Moreover, this genome was also compared with the other two previously published genomes two other chlorpyrifos degrading isolates namely Cupriavidus nantongensis X1 and Paracoccus sp. Strain TRP along with 8 more Ochrobactrum strains

Results and Discussion

Isolation and General Features of Ochrobactrum sp. CPD-03 Genome Sequence

The CP degrading bacterium CPD-03 is a Gram-negative, small rod shaped bacterium that was isolated from a paddy field soil sample as mentioned in the materials & methods section. Earlier studies have shown in vitro mineralization of CP by few genera like, Klebsiella sp., Providencia sp., Enterobacter sp., Pseudomonas sp., Lactobacillus sp., Serratia sp. and Synechocystis sp. [26–31]. Moreover, Ochrobactrum sp. has been reported to degrade acetamiprids, pyrethroids and dichlorides [32–34].

Ochrobactrum sp. CPD-03 has shown degradation of CP and TCP in mineral salt media (MSM) spiked with CP (100 mg L−1). Further, CPD-03 strain was able to degrade 100 mg L−1 of CP at a degradation efficiency of 80–82% within 48 h of incubation in the liquid medium with the production of TCP. TCP formation was detected after 40 min of incubation and subsequently it was also degraded post 24–26 h of incubation, TCP was not found in HPLC analysis (Fig. 1). A representative genome map was made using CGView (Circular Genome Viewer) to generate a circular map of Ochrobactrum CPD-03 (Fig. 2).

Fig. 1.

Fig. 1

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Chlorpyrifos degradation by CPD-03 strain. Data analysis were performed using ANOVA with the Prism8. All were tested at the p < 0.0001*** significance level with the mean ± SD of three replicates

Fig. 2.

Fig. 2

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Circular map of Ochrobactrum sp. CPD-03 by CG View

Species Distinction of CPD-03 with Other Ochrobactrum Strains

In order to distinguish Ochrobactrum sp CPD-03 strain, different in silico approaches viz (1) 16S rRNA phylogenetic analysis (2) average nucleotide identity (ANI) analysis by EzBioCloud, (3) GGDC (Genome-to-Genome Distance Calculator) analysis were carried out. Taxonomic classification of CPD-03 confirmed its maximum similarity with Ochrobactrum intermedium LMG 3301T and Ochrobactrum ciceri Ca-34T [35] (Fig. 3). However, previous studies have not provided any information about these strains in relation to OP biodegradation.

Fig. 3.

Fig. 3

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Phylogenetic tree detailing the position of Ochrobactrum sp. CPD-03 genome within the Ochrobactrum genera. Evolutionary analyses were conducted in MEGA 7 [23]

Average Nucleotide Identity (ANI) and Genome-To-Genome Distance Calculator (GGDC) Analysis

The ANI analysis accompanied by GGDC suggested a close relationship between CPD-03 with Ochrobactrum intermedium LMG 3301T with a value of 98.48% and 92.56%, respectively (Table 2), while other strains showed 77–87%. Therefore, this strain can be considered as another subspecies of Ochrobactrum intermedium. According to the previous report, an ANI value between genomes of approximately 95% corresponds to GGDC values of approximately 70% [36] has been accepted for species delineation and provide a valid genome signature for prokaryote microbial taxonomy. Since the whole genome of the few Ochrobactrum strains like Ochrobactrum pecoris 08RB2639, Ochrobactrum haematophilum CCUG 38531, Ochrobactrum tritici SCII24, Ochrobactrum oryzae MTCC 4195, Ochrobactrum pseudintermedium ADV31, Ochrobactrum daejeonense MJ11, Ochrobactrum ciceri Ca-34, Ochrobactrum cytisi ESC1 were not available, hence they were excluded.

Table 2.

Overview of ANI and GGDC compared to Ochrobactrum sp. CPD-03 from the entire genome sequence by using web-based tools

Bacterial strain Accession no. GC % ANI value (%)a GGDC value (%)b
Ochrobactrum thiophenivorans DSM 7216T NZ_NNRJ00000000 51.60 77.32 20.1
Ochrobactrum pseudogrignonense CCUG 30717T NZ_NNRM00000000 54.00 78.02 22.06
Ochrobactrum pituitosum CCUG 50899T NZ_PYSY00000000 53.40 78.59 22.5
Ochrobactrum grignonense OgA9aT NZ_NNRL00000000 54.20 78.70 22.83
Ochrobactrum rhizosphaerae PR17T NZ_NNRK00000000 53.00 77.72 21.16
Ochrobactrum anthropi ATCC 49188T NC_009667 56.15 87.11 47.8
Ochrobactrum lupini LUP21T NZ_NNRN00000000 56.30 87.16 45.76
Ochrobactrum intermedium LMG 3301T NZ_ACQA00000000 57.70 98.48 92.56
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aANI value (%)respect to Ochrobactrum sp. CPD-03; b GGDC value (%)Respect to Ochrobactrum sp. CPD-03

Comparative Genomics for Functional Annotation and Metabolic Pathway Analysis

KAAS (KEGG Automatic Annotation Server) was also used to annotate the orthologous genes that are expected to be involved in the degradation of aromatic and related compounds along with the predicted function in the Ochrobactrum genomes. Upon comparison of the CPD-03 genome with other two CP degrader strain X1 and TRP, it was revealed that all these three strains were able to degrade these aromatic compounds at different rates (Fig. 4a, b). The minimal sets of metabolic pathways were further annotated and reconstructed against the predicted protein families using a web-based approach MinPath [37] and were clustered using ClustVis [38].

Fig. 4.

Fig. 4

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Heatmap and dendrogram of potential abilities of degradation of Aromatic and relative compounds predicted by genomic features of CPD-03 along with other CP two genomes of CP degraders. Information retrieved from MinPath web tool

The overall reconstructed metabolic pathways were also encoded in the genomes of these Ochrobactrum strains including CPD-03. Furthermore, two other CP degraders strains X1 and TRP were analyzed to compare more conservative yet more reliable biological pathway estimates from a sequence dataset, inferring the pathways described in biological sequence samples. It was observed that CPD-03 shared a maximum homology with its closets neighbor LMG 3301T (Fig. 5a, b)

Fig. 5.

Fig. 5

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Functional metabolic pathway comparison between CPD-03 with other Ochrobactrum strains and with other two CP degraders strains X1 and TRP. Information derived from MinPath and mapped in ClustVis for cluster visualization and analysis

Genes Involved in Chlorpyrifos Degradation Present in Ochrobactrum sp. CPD-03

Pesticides are chemicals that are exposed to the environment at an unnaturally high concentration. Microorganisms possess numerous enzymes which have the ability to degrade these compounds and as of now more than ten organophosphorus hydrolase genes were reported to degrade CP [39]. Organophosphorus hydrolase (OPH; EC 3.1.8.1) produced by few microbes, is an ortholog of Arylesterase (EC 3.1.1.2), also known as aryldialkylphosphatase, catalyzes a wide range of OPs by hydrolysis of water-soluble aliphatic esters (short to medium) and distinguished by preferential aromatic ester hydrolysis [40–42].

As of now, many arylesterases were isolated from mammals, known as serum paraoxonases responsible for hydrolyzing paraoxon along with other organophosphorous esters [43]. Such mammalian arylesterases/paraoxonases play a major role in detoxifying organophosphorous compounds. Previous studies have documented arylesterases resistance to oxo-analogs of organophosphorous insecticides such as paraoxon and chlorpyrifos-oxone from insect pests [44]. Bacterial arylesterases can be used for industrial and environmental purposes because they have broad substrates, but their biochemistry is not well understood. It was reported that a unique thermostable arylesterase from an archaeon Sulfolobus solfataricus showed carboxylesterase and paraoxonase activity against OPs. Similar study had reported an esterase chlorpyrifos hydrolase opdB (designated as CPD) from Lactobacillus brevis WCP902 having a sequence similarity of 23.4%, which could play a role in chlorpyrifos hydrolysis [45–47]. Genome analysis between CPD-03 and other Ochrobactrum genomes along with two CP degraders, X1 and TRP, revealed that arylesterase present in CPD-03 shared maximum homology of 100% with LMG 3301T, whereas 36% and 38% sequence similarity with X1 and TRP strains, respectively (Table 3). From this observation, it was evident that CPD-03 could possibly mineralize organophosphate compounds by employing arylesterase enzyme cluster.

Table 3.

Comparative sequence analysis and similarity index (%) of Arylesterase enzyme (EC 3.1.1.2) of CPD-03 and other CP degraders

Microbial strains Similarity index (%)
Ochrobactrum anthropi ATCC 49188 96.23
Ochrobactrum intermedium LMG 3301 100
Ochrobactrum lupini LUP21 96.23
Ochrobactrum pituitosum CCUG 50899 84.52
Ochrobactrum pseudogrignonense CCUG 30717 84.52
Ochrobactrum rhizosphaerae PR17 84.1
Ochrobactrum thiophenivorans DSM 7216 84.52
Ochrobactrum grignonense OgA9a 84.52
Cupriavidus nantongensis X1 37.57
Paracoccus sp. strain TRP 36.56
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Comparative genomic analysis of the orthologous gene clusters was also performed to strengthen our understanding about the genomic structure and function. This was carried out in a web based platform named OrthoVenn [48] which had already been studied for its usefulness in genome wide comparison and visualization of orthologous clusters. As per GGDC analysis, top 6 strains of Ochrobactrum strains were chosen for deciphering the presence of orthologous clusters. An interactive map (Fig. 6) summarized the number of unique clusters present in CPD-03 strain as compared to other Ochrobactrum species. In total 2930 proteins were found in common in all these Ochrobactrum species. It was found that CPD-03 possesses 7 completely unique Orthologous Clusters and this could be a unique feature of CPD-03 strain.

Fig. 6.

Fig. 6

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Venn diagrams provided by OrthoVenn show the number of common and separate clusters of proteins for all six genomes

Conclusion

In the present work, the complete genome sequence of Ochrobactrum sp. CPD-03 was studied. Research into general genome properties and features followed by the ability of CPD-03 to degrade CP revealed that this bacterium has properties similar to other Ochrobactrum strains. Sequence similarities with the available genomes of closely related Ochrobactrum strains followed by ANI (98.48%) and GGDC (92.56%) along with phylogenetic classification validated the closeness of CPD-03 strain with Ochrobactrum intermedium LMG 3301T. Hence, according to the criteria for species distinction, CPD-03 was found to be a subspecies of Ochrobactrum intermedium. Furthermore, the presence of several other genes responsible for xenobiotic degradation were also found in the genome, suggesting a versatile role of CPD-03 in other xenobiotic chemical degradation. Accordingly, genome sequence of CPD-03 supports the ability to hydrolyze OPs.

Acknowledgements

The authors are highly grateful to Bionivid Technology Pvt. Ltd., Bangalore, India for their kind help in the genome sequencing analysis. This research work was funded by the Department of Biotechnology (DBT), Govt of India, New Delhi. Research Grant sanction no. BT/PR7580/BCE/8/1009/2013. All authors remain highly grateful to the Director of the institute for excellent maintenance and infrastructure that enabled us to perform this work.

Footnotes

Publisher's Note

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