Identification and Mining of Hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX) Degradation Bacteria through Molecular Techniques

The explosive hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX) is commonly applied to both military and industrial applications. Nowadays, RDX is considering a powerful environmental pollutant as a results of accumulation mainly from weapons manufactures and trainings [1,2]. Also, RDX was listed as possible human carcinogen by U.S. Environmental Protection Agency. Thus, applied some ranges of RDX have been restricted as a result of soil and groundwater pollution (U.S. Environmental Protection Agency, 2017). Bacterial degradation for RDX which consider a xenobiotic compound has been detected. RDX biodegradation mechanism may be understated in the light of comprising and fusing unusual cytochrome P450 system of flavodoxin domain to the N terminus of a cytochrome P450 (CYP177A1, XplA). XplB system played a key role as RDX biodegradation through flavodoxin reduction which dedicated to RDX degradation in Rhodococcus rhodochrous strain 11Y and catalyzes the reductive denotation of RDX, in final, under aerobic and anaerobic condition, RDX ring was cleavage [3]. 16S rRNA gene-based specific primers consider Received: November 30, 2017; Accepted: December 12, 2017; Published: December 18, 2017 Abstract


Introduction
The explosive hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX) is commonly applied to both military and industrial applications. Nowadays, RDX is considering a powerful environmental pollutant as a results of accumulation mainly from weapons manufactures and trainings [1,2]. Also, RDX was listed as possible human carcinogen by U.S. Environmental Protection Agency. Thus, applied some ranges of RDX have been restricted as a result of soil and groundwater pollution (U.S. Environmental Protection Agency, 2017). Bacterial degradation for RDX which consider a xenobiotic compound has been detected. RDX biodegradation mechanism may be understated in the light of comprising and fusing unusual cytochrome P450 system of flavodoxin domain to the N terminus of a cytochrome P450 (CYP177A1, XplA). XplB system played a key role as RDX biodegradation through flavodoxin reduction which dedicated to RDX degradation in Rhodococcus rhodochrous strain 11Y and catalyzes the reductive denotation of RDX, in final, under aerobic and anaerobic condition, RDX ring was cleavage [3]. 16S rRNA gene-based specific primers consider Identification and Mining of Hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX) Degradation Bacteria through Molecular Techniques powerful genetic biomarkers tools that used as indicators for potential contaminant biodegradation. Regarding to previous findings, applied biomarkers targeting conserved regions of 16S rRNA gene to identify microbial organisms on taxonomic and phylogenetic levels are effective when biodegradation is dependent on a specific microbial strain and has been widely accepted as a sensitive and reliable technique to identify intestinal micro biota [4][5][6]. This investigation was carried out to achieve two main goals. Firstly, identify bacterial genera with capability of RDX components biodegradation. Secondly, evaluate biodegradation activity for RDX components. EPA-method 8510 (Nitroaromatics, Nitramines, and nitrate esters by High Performance Liquid Chromatography, HPLC) was applied for extraction of explosives from soil and Agilent 1100 system (Agilent, Santa Clara, CA) with Chromosphere C18 reverse phase column (5 μm, 250 × 4.6 mm) was applied. The method detection limit was approximately 2 µg/L for RDX. The detection limit was 5 µg/L for the RDX breakdown products hexahydro-3,5-dinitro-1-nitroso-1,3,5-triazine (MNX), hexahydro-5-nitro-1, 3-dinitroso-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5triazine (TNX), TNT or related nitroaromatics were undetected in the non-contaminated soil (<0.01 ppm detection limit). Upper 5-10 cm of tested soil was applied via taken 50 grams. Pooling of soil samples was compounded from thee replicating to make one sample and kept at 4°C during transportation. Sieves (2 mm) were employed to homogenize the samples. Obtaining rhizosphere soil was achievement via shaking for 10 min at max. Speed in sterile P-buffer (per l: 2.36 g Na 2 HPO 4 ; 1.80 g NaH 2 PO 4 , 85.0 g NaCl and 200 μl Tween 80; pH 6.8). The tubes were subjected to centrifugation (2,500 g, 20 min), resulting pellet was kept. All soil samples were stored at −80°C till DNA extrication.

Amplification, cloning and sequence of 16S rDNA
16S rRNA gene diversity analysis was employed to identify five bacterial which isolate from five explosive contaminated sites at Riyadh province, Saudi Arabia. Total genomic DNA was extracted and purified using E.Z.N.A.®Soil DNA Kit (D5625-01, OMEGA bio-tek, USA) according to manufacturer protocol. 16S rDNA fragments were amplified by PCR technique using 27F and 1492R primers (Table 1). Biobasics, Canada) according to DeLong [7]. PCR amplification was carried out in a themal cycler (peqSTAR 96X Universal Gradient, Germany) under conditions as follows: 5-min initial denaturation at 95°C, 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by 10 min at 72°C and incubation at 4°C [8]. Gel documentation system (Geldocit, UVP, England), was applied for data analysis using Total lab analysis software. The PCR amplicons were blunt-ended with Klenow polymerase and ligated to an Eco RV-digested pBluescript II plasmid (Stratagene) and transformed into Escherichia coli JM 109 electro competent cells according to the manufacturer's instruction. Transformed ligated cells were cultured on LB agar plates containing 100 mg/ml Ampicillin, 100 mg/ml IPTG, and 100 mg/ml X-Gal. Genomics DNA of transformed white colonies were sequenced by Applied Biosystems® 3730 DNA Analyzer. GenBank database was compared with 16S rRNA nucleotide sequences [9] through the BLASTn program [10] and identified with Ribosomal Database Project (RDP) [11] and Alignments sequences were analysed on NCBI website using BLAST to confirm their identity. The Genetic distances and Multi Alignments were computed by Pairwise Distance method using ClusteralW software analysis.

Mining xpl RDX degrading genes
XplA and XplB (RDX degrading genes) encoding a novel flavodoxin fused cytochrome P450 were mining, amplifying and sequence through xplA-HindIIIF and xplB-KpnF1 specific primers (Table 1) from transformed bacterial cells, for xplA and xplB respectively. Thermo Scientific™ DreamTaq™ Hot Start Master Mix (Thermo Scientific, USA, K9011) was applied according to manufacturer protocol. PCR started with an initial denaturation step of 95°C for 5 minutes, followed by 30 cycles of denaturation step of 95°C (30 s), annealing step (varied temperature determined from primers melting temperature) and extension step of 72°C (1-3 minutes). A final extension cycle at 72°C for 10 minutes was performed [12]. Germany) and filter paper (Whatman, 3 mm chromatography paper) were applied for performing western blotting technique. After protein transfer, membrane was incubated with anti-rabbit polyclonal antibodies were prepared by using purified XplA and XplB proteins, respectively, in rabbit [13] and XplB [14] for one hour and reaction was stopped by washing the membrane with TBS, 0.1% Tween 20, TBS and water according to Chong [12]. Data analysis was performed via ChemiDoc-It® Imager (UVP, England).

Results and Discussion
This investigation aimed to identify and evaluate biodegradation capability of RDX components for different bacterial isolates. To achieve these goals, 16 rRNA specific genes were applied for identified thirty nine isolates from different four explosive contaminated sites at Riyadh province, Saudi Arabia. As shown in Figure 1 all crude bacterial which were extracted and purified form four locations reflected clear 16 rRNA specific fragments of distinguishing ~ 950-base-pair. Then, the amplified 16S rDNA fragments were cloned and sequence. Thirty eight clones were successfully sequence. Sequence results indicated that, Proteobacterium, Burkholderia and Rhodococcus were the major dominant identified genera sequences among partial-sequence analyses of 38 clones from each four location libraries from four explosive contaminated sites at Riyadh province, Saudi Arabia.  and Enterobacter were dominant for third location isolates. Fourth isolates location were identified generally as Rhodococcus, Burkholderia, β proteobacterium. Genetic similarity between thirty nine isolates and within location isolates were estimated and phyllogenetic tree was constructed for all studied locations as shown in Figure 2. More light was added to our findings through using 16S rRNA gene sequence similarity in identified bacterial genera was added [15]. They identified genus Comamonas based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phyllogenetic tree analysis. Also, our finding was in agreements on Chong et al. [11]. They constructed Phyllogenetic analysis of the 16S rRNA gene sequences to compare the four RDX degrading Rhodococcus isolates. Also, for identified monooxygenase-associated genes in Pseudonocardia dioxanivorans CB1190 used as biomarkers to biodegrade 1,4-dioxane in pure cultures and environmental samples [6].
Likewise of our results of applied bacteria-specific primers to amplify the 16S rRNA genes to construct phylogenetic analysis for bacterial biodegradation of explosives in groundwater [16] Clostridium and Pseudomonas known explosive-degrading multiple sequences strains were identified and grouped. With agreement on dominance of α-and β-proteobacteria which isolated and identified from RDX-contaminated saturated soils samples [17] identified the same bacterial phyla using terminal restriction fragment length polymorphism (tRFLP) analysis of 16S rRNA genes.
XplA and XplB (RDX degrading genes) were applied as molecular marker for evaluating RDX components biodegradation capability of previous identified isolates. As shown in Figure 3, specific differentiation for XplA and XplB proteins level were detected for thirty eight identified bacterial genera which isolated from four different locations.  03-17-38 (JF_763852.1) and Burkholderia alpina strain PO-04-17-38 (NR_146371.1). All bacterial isolates from fourth location showed the lowest proteins level for XplA and XplB genes. By contrary of our findings, xplA, hydA, onr, xenA, and xenB genes which associated with RDX degradation were disappeared in all of the groundwater RDX contaminant samples [16]. Our previous findings for quantifying XplA protein level through western blotting were supported by presented results of Chong [12]. Who detected only a single band with 47 kDa with XplA antibody cross-reactivity? One of important explanation for XplA and XplB expression level variation which detecting in our findings were cleared in the light of Chong [3] through applying Western blot analysis of crude extracts probed with XplA and XplB antibodies and indicated that that XplA and XplB protein levels were distinguishably decrease in. Also, open reading frame of xplB was not disrupted which cleared possibility for affection of mutations transcript stability or translational efficiency.

Conclusion
Our findings for identification and mining for biodegradation of hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX) summarized that different bacterial genera with certain biodegradable activity were identified via 16S rRNA molecular marker technique. Furthermore, specific biodegradation genes, XplA and XplB showed different expression levels consistent with locations of bacterial isolation. Further studies were requirement to understand biodegradation molecular metabolic interaction for isolating from different locations.