Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Integrative analysis of cellular responses of Pseudomonas sp. HK-6 to explosive RDX using its xenA knockout mutant

Pseudomonas sp. HK-6의 xenA 돌연변이체를 이용하여 RDX 폭약에 노출된 세포반응들의 통합적 분석

  • Lee, Bheong-Uk (Division of Biological Sciences and Chemistry, Kosin University) ;
  • Choi, Moon-Seop (Department of Life Science and Biotechnology, Soonchunhyang University) ;
  • Seok, Ji-Won (Department of Life Science and Biotechnology, Soonchunhyang University) ;
  • Oh, Kye-Heon (Department of Life Science and Biotechnology, Soonchunhyang University)
  • 이병욱 (고신대학교 의생명과학과) ;
  • 최문섭 (순천향대학교 생명시스템학과) ;
  • 석지원 (순천향대학교 생명시스템학과) ;
  • 오계헌 (순천향대학교 생명시스템학과)
  • Received : 2018.07.25
  • Accepted : 2018.09.10
  • Published : 2018.12.31
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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.

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 첨가 배지에서 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 dnaA와 groEL의 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.

Keywords

MSMHBQ_2018_v54n4_343_f0001.png 이미지

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.

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Fig. 2. Survival rates of wild-type (A) and xenA mutant (B) Pseudomonas sp. HK-6 following exposure to RDX.

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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.

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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).

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Fig. 5. Analysis of dnaK (A) and groEL (B) gene expression under RDX stress conditions.

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Fig. 6. 2-DE patterns of total proteins in wild-type (A) and xenA mutant (B) of Pseudomonas sp. HK-6.

Table 1. PCR primers used in this study

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Table 2. Comparative analysis of RDX-induced proteins in Pseudomonas sp. HK-6 and its xenA mutant strain by MALDI-TOF fingerprinting

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