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Integrative analysis of cellular responses of Pseudomonas sp. HK-6 to explosive RDX using its xenA knockout mutant
Korean J. Microbiol 2018;54(4):343-353
Published online December 31, 2018
© 2018 The Microbiological Society of Korea.

Bheong-Uk Lee1, Moon-Seop Choi2, Ji-Won Seok2, and Kye-Heon Oh2,*

1Division of Biological Sciences and Chemistry, Kosin University, Busan 49104, Republic of Korea,
2Department of Life Science and Biotechnology, Soonchunhyang University, Asan 31091, Republic of Korea
Correspondence to: E-mail: kyeheon@sch.ac.kr; Tel.: +82-41-530-1353; Fax: +82-41-530-1493
Received July 25, 2018; Revised September 10, 2018; Accepted September 10, 2018.
Abstract

Our previous research demonstrated the essential role of the xenB gene in stress response to RDX by using Pseudomonas sp. HK-6 xenB knockout. We have extended this work to examine the cellular responses and altered proteomic profiles of the HK-6 xenA knockout mutant under RDX stress. The xenA mutant degraded RDX about 2-fold more slowly and its growth and survival rates were several-fold lower than the wild-type HK-6 strain. SEM revealed more severe morphological damages on the surface of the xenA mutant cells under RDX stress. The wild-type cells expressed proportionally-increased two stress shock proteins, DnaK and GroEL from the initial incubation time point or the relatively low RDX concentrations, but slightly less expressed at prolonged incubation period or higher RDX. However the xenA mutant did not produced DnaK and GroEL as RDX concentrations were gradually increased. The wild-type cells well maintained transcription levels of dnaA and groEL under increased RDX stress while those in the xenA mutant were decreased and eventually disappeared. The altered proteome profiles of xenA mutant cells under RDX stress also observed so that the 27 down-regulated plus the 3 up-regulated expression proteins were detected in 2-DE PAGE. These all results indicated that the intact xenA gene is necessary for maintaining cell integrity under the xenobiotic stress as well as performing an efficient RDX degradation process.

Keywords : Pseudomonas sp. HK-6, xenA gene, proteomic analysis, RDX stress
Body
exahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is one of the most powerful and commonly used military explosives and is released into ecosystems principally as the result of military activities (Juhasz and Naidu, 2007). RDX is s toxic compound that threatens public health as a consequence of this contamination, and has been proposed as a possible human carcinogen (McLellan et al., 1992). The widespread contamination by RDX necessitates the remediation processes of contaminated soil and groundwater. Studies have reported on the biodegradation of RDX in water and soil (Kitts et al., 1994; Ronen et al., 2008). Many bacterial and fungal isolates, including Pseudomonas sp., Stenotrophomonas sp., and Rhodococcus sp., are known to be capable of metabolizing RDX, and many different types of intermediates and end products have been identified (Binks et al., 1995; Fuller et al., 2010; Lee et al., 2013).

Extensive researches on the biodegradation of RDX have been performed over the last decades. Most of the researches were carried out anaerobically on environmental samples such as soil, sewage sludge, horse manure compost, and so on (Osman and Klausmeiser, 1973; McCormick et al., 1981; Kitts et al., 1994). Relatively small numbers of aerobic RDX degradation have been reported (Binks et al., 1995). Although most researches have focused on the ecological and metabolic aspects of the RDX-degrading isolates, several enzymes (especially xenobiotic nitroreductase) that are involved in RDX biodegradation have been the target of considerable interest for the development of cost-effective biological alternatives, such as the construction of transgenic microorganisms or plants (Jackson et al., 2007; Fuller et al., 2009; Lorenz et al., 2012).

The degradation of RDX (Fuller et al., 2009) and TNT (Pak et al., 2000) by xenobiotic reductase (XenA or XenB) from the Ps. putida II-B and Ps. fluorescens I-C has been explored in detail. Both XenA and XenB belong to the Old Yellow Enzyme (OYE) family of flavoprotein oxidoreductases, which have been found in bacteria, yeast, and plants (Williams and Bruce, 2002). These enzymes were known to more quickly transform RDX or other energetic compounds under anaerobic conditions compared to aerobic conditions (Fuller et al., 2009). However, very little data is available on the characteristics of the involvement of xenobiotic reductase A (XenA) in the biodegradation of RDX. We previously reported the characterization of the xenB mutant under RDX stress (Lee et al., 2015). In many aspects, the results obtained from the xenA mutant were quite similar to those of the xenB mutant, yet it would be worthy of comparing of them.

In order to investigate the role of xenA gene in RDX degradation process and under RDX stress condition, the Pseudomonas sp. HK-6 xenA mutant was constructed by a homologous recombination with a partial xenA gene fragment lacking start and stop codons. Several aspects of the xenA mutant were compared with the wild-type strain in order to elucidate the role of this gene in RDX biodegradation and the stress response. The RDX degradability by the xenA mutant was measured and compared to the wild-type bacteria under aerobic conditions. The survival rates and the expression levels of the stress shock proteins (SSPs) DnaK and GroEL under RDX stress were examined. In addition, mRNA expression levels of xenA mutant and wild-type cells were examined under RDX stress. Furthermore, scanning electron microscopy (SEM) analysis was carried out to see the morphological changes occurred in the cell envelope of both strains after RDX exposure. Finally, the different proteome profiles of the wild-type HK-6 strain and the xenA mutant under RDX stress were compared.

Materials and Methods

Bacterial strains, culture conditions and molecular manipulation of the xenA gene

For RDX degradation, HK-6 cells were grown in liquid medium composed of 25–75 µM RDX, 10 mM K2HPO4, 5 mM NaH2PO4, 1 mM MgSO4・7H2O, 0.07 mM CaCl2・2H2O, 0.04 mM FeCl3・6H2O, 0.0005 mM MnCl2・4H2O, and 0.00035 mM ZnSO4・7H2O and 2 mM fructose as a supplemental carbon source. The aerobic cultivation and maintenance of Pseudomonas strains have been previously described in detail (Chang et al., 2004).

The xenA knockout mutant was constructed by integration of a 931-bp internal DNA fragment into the HK-6 xenA original locus. An internal DNA fragment of xenA was amplified with the primers 5’-AATTCAAGCTTGCTGCCGTGCCGACGAAG-3’ and 5’-AATTGAATTCTCGTTCAGTGGCGCATCC-3’. The partial xenA DNA product was inserted in pBGS18 and transformed into HK-6 cells, and its integration in the original xenA locus was confirmed by another PCR with chromosomal DNA as a template. Other molecular manipulation techniques were performed as described by Miller (1972).

Survival test

The wild-type and xenA mutant cells grown on Luria-Bertani (LB) media were harvested by centrifugation at 2,000 × g for 10 min. These cells were washed three times with 10 mM phosphate buffer (pH 7.0), and then inoculated to approximately 107 cells/ml of liquid basal medium in 100 ml Erlenmeyer flasks containing 25, 50, or 75 µM RDX. The cell survival was examined by removing aliquots of cells at selected intervals during 60 days of incubation; then, the colonies were counted by plating them on LB agar incubated at 30°C.

SDS-PAGE, western blot, and real-time qPCR analyses

RDX-treated cells were analyzed to analyze for the expression of the SSPs, DnaK, and GroEL by Western blot technique with anti-DnaK and anti-GroEL monoclonal antibodies (StressGen Biotechnologies Corp.), which were induced by heat shocking Escherichia coli (70 kDa for DnaK and 60 kDa for GroEL). Isolation of the SSPs from the HK-6 wild-type and xenA mutant strains was performed using 12% acrylamide for the separating gel and 5% acrylamide for the stacking gel according to the methods described by Bollag et al. (1996). Western blot analysis was performed by methods with some modification, described by Lee et al. (2000).

Total RNA was extracted from the wild-type and xenA mutant cells with an RNA extraction kit (Macherey-Nagel Inc.) according to the manufacturer’s instructions. Then, total RNA was treated with RNase-free DNase and quantified at 260 nm with a Tecan Multi-Reader spectrometer (Männodorf). For RT-qPCR analysis, we used dnaK-F/dnaK-R, groEL-F/groEL-R and internal control primers 16S rRNA-F/16S rRNA-R (Table 1). RT-qPCR analysis was performed using an iScript™ One-Step RT-qPCR kit with SYBR Green (Bio-Rad) in a Bio-Rad CFX96 RT-PCR System. RT-qPCR samples were run in triplicate, and the data were analyzed using the Bio-Rad CFX Manager software.

PCR primers used in this study

PrimerSequenceSource or reference
dnaK-F5’-TTCGGTCATCGAAATCGCCGAAGT-3’This work
dnaK-R5’-TGCCCGACTCTTTCTTGAACTCGT-3’This work
groEL-F5’-ATCCGTGCCCAGATCG1AAGAAACT-3’This work
groEL-R5’-CAACGCGGGCTTTCTTCTCTTTCA-3’This work
16S rRNA-F5’-AAGGAACACCAGTGGCGAAGG-3’This work
16S rRNA-R5’-CCAGGCGGTCAACTTAATGCG-3’This work

SEM analysis

Colonies of wild-type HK-6 and the xenA mutant grown on LB agar plates for 24 h were excised as small agar blocks. The agar blocks that containing a colony were then exposed to 50 µM RDX in minimal salt medium for 8 h. Both wild-type and xenA mutant cells were fixed, dehydrated, air dried, and coated with gold and examined under a Hitachi S-2500C scanning electron microscope (SEM) (Hitachi) as previously described (Ng et al., 1985).

Proteomic analysis

Two-dimensional electrophoresis (2-DE) was performed with the proteomes from the wild-type and xenA mutant cells treated with 50 μM RDX. A 2-DE analysis was conducted according to previously described methods (Heukeshoven and Dernick, 1985; Kim et al., 2002). The protein spots were excised from the silver-stained 2-D gels and digested with trypsin in accordance with the previously described technique (Ho et al., 2004). The MALDI fingerprint data were analyzed using MS-Fit (http://prospector.ucsf.edu/prospector/4.0.8/html/msfit.htm) against the NCBI database (Perkins et al., 1999).

Results and Discussion

Construction of the xenA knockout mutant

The complete 1,092 bp xenA gene was amplified from the HK-6 strain using PCR primers 5’-ATGGATCCACCACGCTTTTCGATCCGATC-3’ and 5’-ATCCCTGCAGTCACAACCGCGGATAATCGATG-3’ which were designed based on the previously reported xenA gene sequences in GenBank. The deduced amino acid sequences of XenA from HK-6 showed 99% and 94% identity to those from Ps. putida KT2440 and Ps. fluorescens, respectively (data not shown). The preliminary crystal structure of XenA from Ps. fluorescens I-C was determined to a 2.3 Å resolution (Orville et al., 2004) and the substrate-binding amino acid residues of XenA have been determined (Spiegelhauer et al., 2010). A multiple sequence alignment of 40 previously reported OYEs revealed two conserved active sites (HG and YGGS) (Nizam et al., 2014), which were also present in the HK-6 XenA protein (data not shown). However, the amino acid residues that are essential for the catalytic reactions of XenA require further elucidation.

To construct the xenA null mutant, a 931 bp-long internal xenA gene fragment lacking both initiation and termination codons was amplified and inserted into pBGS18 and transformed into the HK-6 strain. The transformed internal xenA fragment was integrated into its original locus via single crossover homologous recombination. This event resulted in the generation of two nonfunctional partial xenA genes: one that did not have the start codon and another that did not have the stop codon. The integration of the partial xenA fragment also conferred kanamycin-resistance, which was attributed by the presence of the simultaneously integrated pBGS18 plasmid. To verify the proper integration of the plasmid harboring the partial xenA DNA fragment, the entire chromosome of the putative xenA mutant was purified and subjected to PCR with primers that targeted a portion of the plasmid DNA sequence. In the xenA mutant, the amplified DNA fragment from the inserted plasmid was detected; however, it was not detected in the wild-type strain (data not shown).

Comparative degradation of RDX between HK-6 wild-type and its xenA mutant

A previous study reported that XenA of Ps. Fluorescens I-C was able to transform RDX under aerobic and anaerobic conditions, although the degradation rate was always faster under anaerobic conditions (Fuller et al., 2009). To examine the role of xenA in a RDX degradation process, the residual RDX in the supernatants of aerobic cultures of the HK-6 and the xenA mutant cells was measured (Fig. 1). HK-6 was shown to completely degrade 25 and 50 µM RDX in 40 and 50 days, respectively (Fig. 1A and B). However, the xenA mutant degraded less than 30% of the RDX over the same time period. Even after 60 days of aerobic incubation, the xenA mutant achieved only approximately 27% and 21% degradation of 25 and 50 µM RDX, respectively. About 52% of the RDX was still remained in the supernatant of HK-6 aerobic culture which contained 75 µM RDX (Fig. 1C). Taken altogether, XenA appeared to be an important enzyme in the RDX degradation pathways, yet it was not the only enzyme involved in the process since the xenA knockout mutant also more or less showed an ability to reduce The concentration of RDX. This means that there are at least more than two degradation pathways of RDX exist in the HK-6 strain. No metabolic intermediates during RDX degradation were detected by HPLC in this work.

Fig. 1.

Bacterial growth (open symbol) and RDX degradation (closed symbol) of wild-type (circle) and the xenA knockout mutant (square) of Pseudomonas sp. HK-6. The growth rate was measured as the optical density at 600 nm, and the rate of RDX degradation was determined by HPLC every 10 days throughout the 60-day incubation period. In this experiment, RDX was initially supplied at concentrations of 25 (A), 50 (B), and 75 μM (C), respectively. The error bars indicate the standard deviations of the means.


Survival rates and scanning electron microscopy

The survival rates of the xenA mutant were 102–104-fold lower than the rates of the HK-6 strain in the presence of 25, 50, and 75 µM RDX (Fig. 2A and B). It may need a further investigation to find precise reasons of this higher lethality of xenA mutant. However it may be partly caused by a higher concentration of RDX in the media due to the slower degradation rate of the xenA mutant.

Several studies reported that aromatic hydrocarbons at high concentrations induced toxic effects on the cells due to the disruption of membrane components (Ramos et al., 1995; Sikkema et al., 1995), ultimately leading to cell death. We also previously showed that toxic chemicals have substantial cytotoxic impacts on target cells, causing perforations and changes in shape; these changes can be observed under scanning electron microscopy (Chang et al., 2004; Lee et al., 2008a). To observe morphological cellular changes after exposure to RDX, the wild-type and xenA mutant cells were cultured in LB broth supplemented with 50 µM RDX for 8 h. Both wild-type cells and xenA mutant cells grown in LB medium without RDX exhibited a typical rod shape with a smooth surface (data not shown). Obvious morphological change was little observed in wild-type cells treated with 50 µM RDX for 8 h (Fig. 3A). However significant morphological changes were shown in the xenA mutant cells under the same conditions (Fig. 3B). The xenA mutant had several destructive openings and a higher frequency of irregular rod forms with wrinkled cell surfaces. These results indicated that the removal of toxic RDX by XenA might not be a sole reason to keep cell integrity; nonetheless, it probably did help it. In addition to this, two stress shock proteins (SSPs), DanK and GroEL appeared not to be sufficiently expressed in xenA mutant background (Table 2). This probably contributed a lower survival rate as well as more damages on cell surface of xenA mutant.

Comparative analysis of RDX-induced proteins inPseudomonassp. HK-6 and itsxenAmutant strain by MALDI-TOF fingerprinting

Spot No.Identification proteinGenBank IDSequence coverage (%)Fold change WT/MT
Chaperones
1DnaKYP_00470398454↑/-
2GroELWP_01671358521↑/-
3Trigger factorYP_00876256936↑/-

Metabolism
4Xenobiotic reductase A, XenANP_74341457↑/-
5NAD(P)H quinone oxidoreductaseWP_00457581633↑/-
6Membrane dipeptidaseYP_00166658028↑/-
7Ribose-phosphate pyrophosphokinase, PRPS1YP_00126610440↑/-
8Bifunctional aconitate hydratase 2/2-methyl isocitrate dehydratase, AcnBYP_00126874134↑/-
92,3,4,5-Tetrahydropyridine-2,6-carboxylate N-succinyltransferaseNP_74368733↑/-
10NAD synthetaseWP_01909891336↑/-
11Gamma-carboxygeranoyl-CoA hydratase, LiuCWP_01909633838↑/-
12Aldo-keto reductaseWP_01909804225↑/-
132-Oxoisovalerate dehydrogenase subunit beta, BCKDHBYP_00809242260-/↑

Transcription, Translation, and Biosynthesis
14Elongation factor G, EF-GWP_01909659338↑/-
15Polynucleotide phosphorylase/polyadenylase, PnpWP_02304899424↑/-
16Adenylosuccinate synthetaseWP_01909735142↑/-
17Cysteine desulfuraseNP_74300329↑/-
18DNA-directed RNA polymerase subunit alpha, rpoAYP_00811154942↑/-
19Elongation factor P, EF-PWP_00940658253↑/-
20Putative dihydroorotateYP_00811610336↑/-

Transport, Binding protein, and Protein export
21Ribosome-binding factor A, RbfAYP_00470396867↑/-
22Glycine/betaine ABC transporter substrate-binding proteinWP_01909685522↑/-
23Branched-chain amino acid ABC transporter substrate-binding proteinYP_00809760830↑/-
24Amino acid ABC transporter, periplasmic binding protein, AapJYP_00593204553↑/-
25Extracellular solute-binding proteinYP_00174777860↑/-
26Iron ABC transporter substrate-binding proteinWP_01909810052↑/-

Secondary metabolites biosynthesis
27Phenylacetate-CoA oxygenase subunit, PaaINP_74541932↑/-

Cell envelope
28Outer membrane protein, OprQAAN6589928↑/-
29Outer membrane protein H1, OprHYP_00470345156-/↑

Signal transduction
30Alginate biosynthesis sensor histidine kinase/GAF domain proteinAAN7080735-/↑

↑, Arrows indicate up-regulation of protein expression level based on comparison of protein sizes

-, not significantly changed


Fig. 2.

Survival rates of wild-type (A) and xenA mutant (B) Pseudomonas sp. HK-6 following exposure to RDX. The cells were exposed to concentrations of 25 (circle), 50 (square), and 75 μM (triangle) RDX. At intervals, the number of colonies (CFU/ml) was measured. The error bars indicate the standard deviations of the means.


Fig. 3.

Scanning electron micrographs of the wild-type (A) and its xenA mutant (B) of Pseudomonas sp. HK-6 treated with 50 μM RDX for 8 h. The upper left in each micrograph includes the control strains.


Western blot and RE-qPCR analyses

Environmental stresses including explosives such as RDX, induce SSPs. The expression levels of the 70-kDa DnaK and 60-kDa GroEL proteins under RDX stress conditions in the HK-6 wild-type and the xenA mutant were compared using SDS-PAGE and western blotting with anti-DnaK and anti-GroEL monoclonal antibodies (Fig. 4). In the wild-type, the two SSPs were induced by RDX and were found to increase in proportion to the length and concentration of the RDX treatment, which is consistent with previous observations (Fig. 4A, Fig. 4B, and C) (Chang et al., 2004). However, the fates of the two SSPs in the xenA mutant appeared to be quite different. Both DnaK and GroEL were more highly expressed in the xenA mutant in the absence of RDX (Fig. 4D, Fig. 4E, and F). However, the levels of the two SSPs began to decrease in proportion to the elapsed treatment time in the presence of 50 µM RDX. Interestingly, DnaK and GroEL exhibited a small amount of expression in the presence of 50 and 75 µM RDX, while their expression appeared to be enhanced in the presence of 25 µM RDX. The levels of relative gene expression measured by RT-qPCR were consistent with the western blotting results (Fig. 5). The expression of the dnaK and groEL genes was reduced in the XenA genetic background. These results indicated that increased lethality and other phenotypic changes might occur in the presence of RDX stress due to the limited expression of these SSPs. Many SSPs, such as DnaK and GroEL, are chaperone proteins that assist in the proper folding of essential enzymes under environmental stress conditions.

Fig. 4.

Induction of stress shock proteins (SSPs) in wild-type and xenA mutant Pseudomonas sp. HK-6 treated with 50 μM RDX for different exposure times (A, B, and C), and for different RDX concentrations (D, E, and F). The SSPs were analyzed by SDS-PAGE (A, D) and western blot with anti-DnaK (B, E) and anti-GroEL (C, F) monoclonal antibodies. The initial colony-forming units per ml (CFU/ml) were approximately 1.46 × 10 8, and samples with equal amounts of protein (40 μg) were subjected to western blotting.


Fig. 5.

Analysis of dnaK (A) and groEL (B) gene expression under RDX stress conditions. Induction of the dnaK and groEL genes in cells treated with 0, 25, 50, and 75 μM RDX for 8 h. The DnaK and GroEL concentrations were analyzed using western blotting with the anti-DnaK and anti-GroEL monoclonal antibodies, and dnaK and groEL gene expression was measured using RT-qPCR of the wild-type (square) and xenA (shaded square) mutant cells. The numbers on the x-axis of the RT-qPCR graph and above the western blot photos indicate the time points for the samples collected. The numbers on the y-axis represent relative gene expression.


Analysis of the proteome profiles

The proteomic profiles of the wild-type and xenA mutant cells cultured in LB medium in the presence of RDX were compared (Fig. 6). A total of 30 unambiguous spots were extracted, digested with trypsin, and analyzed by MALDI-TOF MS. The MALDI-TOF MS analysis was independently conducted at least three times. The total proteins were classified into 7 groups based on function: chaperone, metabolism, transcription and translation, transport and binding protein, secondary metabolite biosynthesis, cell envelope, and signal transduction. The MS analysis showed 27 proteins with decreased expression or no expression in the xenA mutant (Table 2). Interestingly, there was little or no expression of the SSPs (e.g., DnaK, GroEL, and the trigger factor) in the xenA mutant under RDX stress despite the fact that SSPs are usually induced by stress. In fact, TNT stress was reported to cause the accumulation of the chaperones GroEL and RpoH; also these proteins were more highly accumulated in the algA mutant, which did not produce alginate, a matrix to potentially protect cells exposed to stresses (Lee et al., 2008b). Furthermore, the gene AAN70807, which is related to alginate biosynthesis, was highly induced under RDX stress conditions (Table 2). It remains unclear whether the increased expression of this gene was directly connected to the production of alginate. However, the xenA mutant cells would produce more alginate if they needed stronger protection against the higher concentrations of RDX.

Fig. 6.

2-DE patterns of total proteins in wild-type (A) and xenA mutant (B) of Pseudomonas sp. HK-6. Numbers associated with the spots identified by MALDI-TOF are listed in Table 2.


Outer membrane (OM) proteins are involved in many aspects of growth and development of the bacterial cell (Hancock and Brinkman, 2002). In particular, OM proteins of Ps. aeruginosa play a significant role in membrane permeability, nutrient uptake, antibiotic resistance, and virulence at the infection site (Arhin and Boucher, 2010). Our MS analysis identified 2 OM proteins: up-regulated OprH and down-regulated OprQ. In our proteome analysis, OprH, which might be necessary for cell survival in harsh environments such as RDX exposure, was highly expressed in the xenA mutant. OprH has been reported to stabilize the OM by increasing its protection from membrane perturbation (Edrington et al., 2011). In contrast, OprQ was down-regulated and not detected in the xenA mutant. OprQ has been reported to participate in nutrient uptake from the environment and is involved (directly or indirectly) in the regulation of other proteins/porins that are important for Ps. aeruginosa growth. OprQ is also required for survival under conditions where Ps. aeruginosa is exposed to adverse growth conditions (Arhin and Boucher, 2010). Based on previous reports, these results indicate that the xenA mutant cells exposed to RDX demonstrated reduced survival due to a lack of OprQ production.

Previous comparison of proteome profiles between the wild-type and the xenB cells revealed 22 differently-expressed proteins (Lee et al., 2015). Among them, 13 proteins were consistent with those of 2-DE gels from the xenA mutant cells while 9 proteins were not identical in the profile of the xenA mutant proteome. 17 proteins from 2-DE gel in this xenA mutant study were newly identified. The exact roles of these differently-expressed proteins under RDX stress in xenA or xenB genetic background would not be totally understandable. However, accumulation of proteome profiles from the independently-performed analyses will generate more valuable data regarding proteins involved in xenobiotics stress responses.

The XenA protein had not been extensively studied in Pseudomonas species. However, there have been an increasing number of reports regarding the biodegradation or bioremediation of the RDX and TNT explosives using the XenA and XenB enzymes. A broader and more intensive understanding of the involvement of XenA in RDX biodegradation will create more opportunities for a variety of biotechnological applications. The enzyme can be improved by mutagenesis or genome shuffling techniques for a more efficient bioremediation process (Ronen et al., 2008). Additionally, studies of XenA, XenB, or related enzymes involved in the microbiological degradation of xenobiotics may provide information that can be used to improve the degradation function of these enzymes. Furthermore, a more powerful strain with a greater capability for RDX degradation can be developed by the introduction of the genes derived from these enzymes into a single strain, enabling the construction of transgenic microorganisms or plants that can be used for RDX bioremediation.

적 요

이전 연구에서 우리는 RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) 분해세균 Pseudomonas sp. HK-6에서 xenobiotic reductase B를 암호화하는 xenB 유전자의 돌연변이 균주를 이용하여 RDX 스트레스에 대한 xenB 유전자의 역할에 관하여 연구를 보고하였다[Lee et al. (2015)Curr. Microbiol. 70(1): 119–127]. 본 연구에서는 Pseudomonas sp. HK-6 xenA 돌연변이 균주로 연구 범위를 확대하여 RDX 스트레스 조건에서 세포반응과 프로테옴 프로필의 변화를 분석하였다. RDX 첨가배지에서 xenA 돌연변이 균주는 야생균주와 비교하여 RDX를 약 2배 정도 느리게 분해하였으며, RDX 스트레스 하에서 xenA 돌연변이 균주의 생장률과 생존율은 야생균주와 비교하여 낮았다. RDX 스트레스에 의한 심한 형태적 손상이 xenA 돌연변이 균주의 세포 표면에 발생하는 것이 주사전자현미경을 통해서 확인되었다. RDX 스트레스 하에서 야생균주에서 발현된 충격단백질인 DnaK 및 GroEL의 양은 배양 초기 혹은 상대적으로 낮은 RDX 농도에서는 증가하였으나, 배양시간이 길어지거나 높은 RDX 농도에서는 다소 감소하였다. 그러나 xenA 돌연변이 균주에서는 DnaK와 GroEL의 발현양은 RDX 농도가 증가함에 따라 점차 감소되었다. RT-qPCR에 의해 측정된 야생균주에서 dnaAgroEL의 전사 수준은 RDX 스트레스가 증가된 상태에서 잘 유지되었으나, xenA 돌연변이 균주에서는 점차 감소되어 결국에는 소멸되었다. RDX 스트레스에서 xenA의 돌연변이에 의한 프로테옴 프로필의 변화를 2-DE PAGE를 통해서 관찰한 결과에 따르면 27개 단백질이 감소하고 3개가 증가한 것으로 나타났다. 이들 결과로 보아, 정상적인 xenA 유전자는 RDX 스트레스 하에서 세포의 온전한 형태 유지와 효율적인 RDX 분해 과정을 수행하기 위해서 필요하다는 것을 의미하였다.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (2011-0026690), and by the Soonchunhyang University Research Fund. We thank the ROK Agency for Defense Development for providing the RDX used in this study.

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