Chemotaxis and degradation of organophosphate compound by a novel moderately thermo-halo tolerant Pseudomonas sp. strain BUR11: evidence for possible existence of two pathways for degradation

Chemicals

Parathion and its degradation intermediates (such as 4-NP, 4-AP, 4-NC, hydroquinone (HQ), PBQ and 1,2,4 benzenetriol (BT)) as well as TCP were purchased from Sigma. Chlorpyrifos was purchased from Dr. Ehrenstorfer GmbH (Merck, Germany). Solvents used in this study were of HPLC grade. Microbiological media, components, general chemicals were of highest standard and purchased from HiMedia, India.

Isolation of OP degrading bacteria

Soil samples were collected in sterile whirl pack bags (HiMedia, India) from an agricultural field in Burdwan (lat. 23.2500 °N and long. 87.8500 °E), West Bengal, India. For isolation of parathion utilizing bacteria, an enrichment culture was set up in minimal medium (MM) ((g L⁻¹): K₂HPO₄, 0.2; KH₂PO₄, 0.8; MgSO₄7H₂O, 0.2; CaSO₄H₂O, 0.1; NaMoO₄2H₂O, 0.003; FeSO₄7H₂O, 0.005; (NH₄)₂SO₄, 1.0) supplemented with 200 ppm (mg L⁻¹) parathion. One gram of soil sample was added to the 25 mL of sterile MM (in a 100 mL Erlenmeyer flask) supplemented with filter sterilized parathion as the sole source of carbon and energy and was incubated in an orbital shaker (120 rpm) at 37 °C for 1 month. After 1 month, 1 mL of inoculum was transferred to 25 mL of fresh MM with 200 ppm of parathion and incubated for 15 days and the process was repeated three more times. Dilution plating of this enrichment culture on MM agar (1.5% agar) plate supplemented with 200 ppm of parathion and incubation at 37 °C resulted in many isolated colonies which were purified by restreaking on fresh medium. Based on growth response on MM-parathion plate, one isolate designated BUR11 was selected for further studies.

Characterization and identification of isolate BUR11

Phenotypic characterization of strain BUR11 was carried out essentially according to Smibert & Krieg (1994). Growth at various temperatures (20 °C–55 °C) and NaCl concentrations was determined by using basal tryptone soya broth with varying NaCl concentrations (0.5–8.5%), followed by its periodic monitoring at 600 nm using a spectrophotometer. Strain BUR11 was also identified by an automated microbial identification system, VITEK 2 (BioMérieux, Inc., Hazelwood, MO, USA). Genomic DNA was isolated by Marmur’s protocol (Johnson, 1994); amplification of 16S rRNA gene and its sequencing was carried out according to Reddy et al. (2000). Determination of phylogenetic neighbors and pair wise 16S rRNA gene sequence identity was carried out at EzTaxon server (Chun et al., 2007). Phylogenetic tree was constructed according to published methods (Saha & Chakrabarti, 2006) with sequences of close phylogenetic neighbors, retrieved from EzTaxon server.

Growth studies and chemotactic behavior of strain BUR11

The ability of the stain BUR11 to grow on parathion, chlorpyrifos, 4-NP, PBQ, HQ, 4-AP, TCP and BT as sole sources of carbon was determined on MM broth supplemented with 200 ppm of the compounds and incubation at 37 °C with shaking at 120 rpm. Growth was monitored spectrophotometrically (Varion, Cary 50 Bio) at 600 nm, at regular time intervals.

The chemotactic activity of the strain BUR11 towards parathion, chlorpyrifos, TCP, 4-NP and 4-AP were investigated qualitatively (by drop plate and swarm plate assays) and quantitatively by capillary assay following established methods (Pandey et al., 2002; Arora & Bae, 2014). For drop plate assay, culture was grown on MM supplemented with respective chemoattractants, harvested (OD₆₀₀ ∼ 0.7) by centrifugation (6,000g for 10 min) washed twice with phosphate buffered saline (PBS) and resuspended in assay medium (MM with 0.3% agarose), mixed well and poured onto 60 mm Petri plates. The test compounds (either as crystals or as solution) were placed in the centre of the plates which were then incubated at 37 °C. Swarm plate assay was carried out on assay medium (MM with respective toxic compound plus 0.3% agarose) with 60–75 µL of cells suspension in chemotaxis buffer, loaded centrally (in an agar cup that was cut aseptically, using sterile cork borer), in a 60 mm Petri plate. Appearance of bacterial growth in ring pattern (concentric rings in case of drop plate and exocentric in case of swarm plate assay) were recorded as positive chemotactic response. For quantitative capillary assay, the optimum concentration for each of the tested toxic compounds was determined by carrying out chemotaxis assay at various concentrations (from 50 to 500 ppm). For the assay, 10 µL graduated glass capillary tubes were filled with parathion, chlorpyrifos, TCP, 4-NP and 4-AP (in chemotaxis buffer) separately, and the suction end was sealed by sterile agarose gel. Each of these capillary tubes was inserted into a micro centrifuge tube (separately) containing a suspension (10⁸ cells mL⁻¹) of strain BUR11 cells and was incubated at 25 °C for 30 min. The contents of the capillary tubes were then serially diluted and plated onto non-selective tryptone soya agar plate, followed by determination of colony forming units (CFUs count) after overnight incubation at 37 °C. The strength of chemotactic response was expressed in terms of the chemotaxis index (CI), which is the ratio of the number of CFUs produced from the capillary containing the test compound(s) to CFUs produced from a control capillary (i.e., only the chemotaxis buffer without any chemo tactic compound). Citrate was used as the positive control.

Inoculum preparation and extraction of samples for degradation studies

In order to carry out biodegradation studies, strain BUR11 was grown on tryptone soya broth for overnight, cells were harvested by centrifugation, washed thrice with PBS (pH-7.0) to remove traces of medium and resuspended in 1.5 mL of MM. Cell suspension (0.5 OD corresponding to 6.5 × 10⁷ cells mL⁻¹) was inoculated into MM supplemented with parathion (200 ppm). Control flask was prepared exactly the same way without inoculum and all flasks were incubated at 37 °C with shaking at 120 rpm. Entire broth culture was centrifuged and the supernatant was used for extraction and amount of residual parathion in the medium was determined by HPLC after 0, 24, 48, 72 and 96 h of incubation.

For determination of hydrolysis products, after a periodic time interval of growth on MM-parathion, cell free culture supernatant was collected and was subjected to extraction (twice) using equal volume of ethyl acetate, followed by dehydration using anhydrous sodium sulphate (Na₂SO₄) and was dried by a rotary evaporator. The dried sample was dissolved in appropriate volume of acetonitrile, filtered through sterile disposable syringe filter (Millipore, 0.22 µm) and subjected to separation by TLC, LC-MS/MS and GC followed by library search for identification of metabolites.

Analytical methods

Isolation, characterization and identification of parathion degrading bacterium

Several bacterial strains capable of utilizing parathion as sole source of carbon for their growth were isolated from soil of an agricultural field by enrichment culture technique. One of them designated as BUR11 showed comparatively good growth response in MM-parathion broth and MM-parathion-agar plate and was selected for further study.

The isolate BUR11 was Gram negative, rod-shaped, motile and possessed nitrate and nitrite reductase activities. The strain could grow between 20 °C to 50 °C temperatures and could tolerate up to 8% NaCl (Fig. S1). Phenotypic characteristics of the strain are summarized in Table 1. The phylogenetic position of the strain is shown in Fig. 1. Although, it showed high value (99.78%) of 16S rDNA sequence similarity to the type strain of Pseudomonas aeruginosa (JCM 5962^T), in absence of detail polyphasic taxonomic characterization, the strain BUR11 was identified as Pseudomonas sp. The strain has been deposited in Microbial Culture Collection, National Centre for Cell Science, Pune, India (accession number MCC 2328). Nucleotide sequence of the 16S rRNA gene for the strain BUR11 has been deposited in GenBank (accession number KF887018).

Growth studies and chemotactic response of strain BUR11

Study of growth pattern revealed that the Pseudomonas sp. strain BUR11 could utilize parathion, chlorpyrifos (Fig. 2A) and their hydrolytic intermediates (such as 4-NP, 4-AP, HQ, PBQ, BT and TCP; Fig. 2B) as sole source of carbon. The strain could tolerate up to 500 ppm of parathion and chlorpyrifos while its optimum growth was recorded at 200 ppm for both the OP compounds. The ability of this motile strain BUR11, to utilize OP compounds and their hydrolysis intermediates prompted us to look for its chemotactic response towards these compounds.

Results from qualitative (Figs. S2A and S2B; drop plate and swarm plate assay respectively) as well as quantitative (Fig. 3) chemotaxis assays indicated positive chemotaxis for five compounds (parathion, 4-NP, 4-AP, chlorpyrifos and TCP) that were utilized as sole source of carbon for growth by the strain BUR11. The capillary chemotaxis assay indicated concentration dependent chemotaxis. As shown in Fig. 3, the CI values for all five compounds gradually increased with increasing concentrations until the optimal concentrations. Further increase in concentration led to sharp declines for chlorpyrifos, TCP and 4-AP while plateaus for parathion and 4-NP in the strength of the chemotactic response. The optimal chemotactic response concentrations were documented in the range between 200–250 ppm for all the compounds used as sole carbon source. The strongest chemotactic response was observed for chlorpyrifos and TCP, with CI values of 30.1 and 28.5 respectively, at their respective optimal response concentrations whereas CI of 16.25, 18.83 and 24.54 were recorded for parathion, 4-NP and 4-AP. To, the best of our knowledge, this is the first report of chemotaxis towards chlorpyrifos, parathion, 4-AP and TCP, by a moderately thermo-halo tolerant bacterium.

Degradation, hydrolytic intermediates and pathways for parathion degradation by BUR11

Study of the degradation of parathion by Pseudomonas sp. strain BUR11 revealed that the strain could degrade 35% of parathion within 24 h and 62% of the same within 96 h as sole source of carbon (Fig. 4A). As evident from Fig. 4B, within first 24 h, there was increase in concentration of 4-NP, up to 30 ppm and this does not change until 72 h after which its concentration decreased rapidly, indicating its mineralization. Other intermediates (HQ, PBQ and BT) were detected in very low concentration, indicating their rapid conversion into intermediates which were channelized finally into TCA cycle.

Preliminary analysis by TLC (Fig. S3), indicated presence of three metabolites namely, 4-NP, PBQ, BT as major intermediates of hydrolysis. At least three more unidentified spots were detected during TLC analysis. From comparative analyses (by HPLC) of retention time (RT) for peaks of sample extracts and standards, HQ (RT of 2.258 min); BT (RT of 2.195 min), 4-NP (RT of 2.440 min) and PBQ (RT of 2.633 min) were detected as major intermediates of hydrolysis of parathion (Fig. S4). Separation of metabolites by GC followed by library search, detected and identified 4-APar (RT 19.029 min) in the parathion grown culture extract (Fig. S5). In LC-MS/MS analysis, presence of characteristic mass spectra for 4-NP (ion, m/z 137.9 > 108.0 > 92.0), HQ (ion, m/z 110 > 82.0 > 54.0 > 53.0), PBQ (ion, m/z 104.8), 4-AP (ion, m/z 104.9), BT (148.9 > 121.1 > 92.7) and 4-NC (ion, m/z 148.9 > 92.7) were detected (Fig. S6).

LC-MS/MS analysis thus confirmed the presence of six major hydrolysis intermediates, identified as 4-NP, HQ, PBQ, 4-NC, BT and 4-AP; while GC-screening predicted presence of 4-APar. Since both 4-NP (as well as 4-NC, BT, PBQ, HQ) and 4-APar (as well as 4-AP) were detected, we believe that in strain BUR11, possibly, two pathways for degradation of parathion exists. Thus, based on detection of these intermediates we propose possible pathways of degradation of parathion for the strain BUR11 (Fig. 5). The proposed first pathway proceeds via 4-NP that further degrades via two routes; one proceeds via 4-NC and BT whereas the other proceeds via PBQ and HQ. While the second proposed pathway operates through the formation of 4-APar, 4-AP and PBQ (Fig. 5).