Mycobacterium gilvum PYR-GCK에서 Omics 기술을 이용한 피렌 분해에 대한 분자적 고찰

Abstract

다환 방향족 탄화수소 (Polycyclic aromatic hydrocarbons; PAHs)는 원유, 크레오소트와 콜타르의 주 성분이다. 이는 여러 환경에 존재하는 유독성 오염물질로 인류 건강에 큰 위협이 되고 있다. 다환 방향족 탄화수소를 분해하는 방법 중의 하나로 미생물에 의한 분해가 있다. 몇몇 미생물 균주은 이들 화합물을 대사 작용하여 성장에 필요한 에너지로 활용하는데, 이를 통해 다환 방향족 탄화수소의 독성을 줄이거나 완전히 제거할 수 있다. Mycobacterium gilvum PYR-GCK는 인디아나 북서쪽에 위치한 그랜드 캘류멧 강에서 분리되었다. 이 미생물은 환경에 존재하는 다환 방향족 탄화수소인 피렌에서 증식하며 이를 탄소와 에너지원으로 활용한다. 전세계적으로 일어나는 기름 유출 사건으로 인해 미생물 기술을 기반으로 한 다환 방향족 탄화수소의 생물학적 교정 연구가 시작되었다. 이 균주를 연구하면 이 미생물의 피렌 분해 활동의 전반적인 내용을 알 수 있게 되며, 생물학적 교정 목적으로 활용하는데 필요한 절차를 형성하는데 큰 기여를 할 것이다. 이 균주의 피렌 분해 과정 연구를 위해 최신 기술인 가스 크로마토그래피, 질량분석법, 불꽃이온화 감지법을 도입한 대사체 연구를 진행하였다. 이를 통해 4원구조의 기질을 3원, 2원 그리고 단원 형태로 분해하는 능력이 탁월하며 결국에는 트리카르복시산 회로의 중간대사물질로 들어가 이화작용 및 동화작용에 사용됨을 확인하였다. 단백질체 분석을 통해서는 피렌 분해를 유도하는 중요한 촉매제들을 발견하였다. 피렌 분해 경로 내 각 단계에서 작용하는 단백질 또는 효소를 찾기 위해 1차 겔 전기영동 및 액체 크로마토그래피/질량분석법을 도입하였다. 주 경로에 작용하는 효소와 대사물질이 사용되는 대체 경로에 작용하는 단백질을 발견하였다. 발현에 변화가 있는 유전자를 찾기 위해 엎에서 분석한 결과를 주요 대사작용에 존재하는 단백질과 비교하였다. 최신기술인 RNA 서열분석으로 피렌 분해 시 조절되는 전사체를 확인하였다. 피렌에 의해 발현되는 기능 및 복합 조절 요소 등의 전사체 목록을 확인할 수 있었다. 단백질체와 전사체 결과에 대한 비교를 통해 전사에 대한 정성 분석이 이루어졌으며 이를 통해 Mycobacterium gilvum PYR-GCK 내 피렌 분해의 분자적 정교함에 대한 정보를 밝혀냈다. 초기 분석에서 별다른 정보를 얻지 못한 관계로, 다양한 산성도와 염도 조건하의 균주 내 유전자 발현 분석을 실행하였다. 이 연구를 통해 다른 조건 내 방향족 원형 구조 절단에 관여하는 유전자의 활성도를 확인하였다. 연구한 유전자는 phdF, phdI, pcaG and pcaH로 각각 extradiol dioxygenase, 1-hydroxy-2-naphthoate dioxygenase/gentisate-1, 2-dioxygenase, protocatechuate-3,4-dioxygenase의 알파 및 베타 서브유닛을 암호화하고 있다. 이 연구로 피렌 분해 활성은 중성에서 약염기 조건에서 가장 높고, 균주 자체의 내염성 및 피렌 분해가 무염 조건 및 해수보다 높은 염 농도 58%에서도 가능함을 밝혀냈다. 박테리아 내 전사체와 유전자 발현에 대한 보고는 매우 제한적이며 이는 다환 방향족 탄화수소 분해 관련의 연구 분야에서는 보기 어렵기에, 이번 연구는 미생물 분야에서 앞서가며 Mycobacterium group 내 PAH 분해생물 분야에서 처음으로 유전자 발현 및 전사체를 다룬 연구이다. |Polycyclic aromatic hydrocarbons (PAHs) are major constituents of crude oil, creosote and coal tar. They are toxic pollutants that are present in a variety of environments and pose serious risk to human health. One of the primary processes by which PAHs can be eliminated from the environment is through microbial activities. Some bacterial species have the ability to metabolize these compounds and use them as energy sources for growth, thereby rendering them less toxic or totally safe. Mycobacterium gilvum PYR-GCK was isolated from the sediment of the Grand Calumet River in Northwestern Indiana based on its ability to grow on pyrene. This isolate can grow on pyrene (an environmentally persistent PAH) utilizing it as a sole source of carbon and energy. Microbiological studies centered on bioremediation of PAH-contaminated soils have been initiated due to the concerns of people over the major oil spills occurring worldwide. In depth study of this bacterial strain gives an overall view of its activity during pyrene degradation and this knowledge will enable proper steps to be taken during its use for PAH bioremediation purposes. A metabolomics study, using the latest technologies of gas chromatography / mass spectrometry / flame ionization detection in the field, was applied to the pyrene degradation process in the strain. This revealed the excellent degrading ability of the strain in breaking down the four-ringed substrate into subsequent metabolites of three-, two- and one-ringed nature and eventually into the simplest intermediates that are absorbed totally into the tricarboxylic acid cycle to be used in catabolism and anabolism processes. A global proteomics analyses revealed the important catalysts driving the degradation process of the pyrene substrates. The technologies of 1-dimensional gel electrophoresis and liquid chromatography / mass spectrometry were employed to elucidate the proteins/enzymes responsible for each step in the pyrene degradation pathway. The basic enzymes responsible for the main pathway were identified along with proteins involved in alternative pathways by which the metabolites were used up. This analysis was compared with the proteins expressed in the central metabolism pathway in order to identify differentially expressed genes. The new technique of RNA-seq was employed to give a global view of the transcripts regulated during the process of pyrene degradation. A total cataloguing of transcripts expressed by pyrene induction was revealed, showing functional elements and complex regulatory elements. A global qualitative classification of the transcriptional activity was made by comparing the proteomics data with the transcriptomics and this revealed a ton of information on the molecular intricacies of pyrene degradation in Mycobacterium gilvum PYR-GCK. With a little of the information gotten from the earlier analyses, a gene expression study of the strain’s molecular activity in various conditions of pH and NaCl concentrations, was undertaken. This study revealed the activities of the critical process of aromatic ring cleavage genes at the different experimental conditions. The genes studied included phdF, phdI, pcaG and pcaH, coding for an extradiol dioxygenase, 1-hydroxy-2-naphthoate dioxygenase/gentisate-1, 2-dioxygenase, alpha and beta subunits of protocatechuate-3,4-dioxygenase, respectively. The study revealed high pyrene degrading activities at neutral to slightly alkaline conditions, the halotolerant nature of the strain and its pyrene degrading ability in null salinity as well as NaCl concentrations of up to 58%, above seawater salinity concentration. There are limited reports on transcriptomics and gene expression studies in bacteria talk less of a similar study on any PAH substrate degradation, therefore this study will be the first of this kind in this field and in the Mycobacterium group of PAH degraders.; Polycyclic aromatic hydrocarbons (PAHs) are major constituents of crude oil, creosote and coal tar. They are toxic pollutants that are present in a variety of environments and pose serious risk to human health. One of the primary processes by which PAHs can be eliminated from the environment is through microbial activities. Some bacterial species have the ability to metabolize these compounds and use them as energy sources for growth, thereby rendering them less toxic or totally safe. Mycobacterium gilvum PYR-GCK was isolated from the sediment of the Grand Calumet River in Northwestern Indiana based on its ability to grow on pyrene. This isolate can grow on pyrene (an environmentally persistent PAH) utilizing it as a sole source of carbon and energy. Microbiological studies centered on bioremediation of PAH-contaminated soils have been initiated due to the concerns of people over the major oil spills occurring worldwide. In depth study of this bacterial strain gives an overall view of its activity during pyrene degradation and this knowledge will enable proper steps to be taken during its use for PAH bioremediation purposes. A metabolomics study, using the latest technologies of gas chromatography / mass spectrometry / flame ionization detection in the field, was applied to the pyrene degradation process in the strain. This revealed the excellent degrading ability of the strain in breaking down the four-ringed substrate into subsequent metabolites of three-, two- and one-ringed nature and eventually into the simplest intermediates that are absorbed totally into the tricarboxylic acid cycle to be used in catabolism and anabolism processes. A global proteomics analyses revealed the important catalysts driving the degradation process of the pyrene substrates. The technologies of 1-dimensional gel electrophoresis and liquid chromatography / mass spectrometry were employed to elucidate the proteins/enzymes responsible for each step in the pyrene degradation pathway. The basic enzymes responsible for the main pathway were identified along with proteins involved in alternative pathways by which the metabolites were used up. This analysis was compared with the proteins expressed in the central metabolism pathway in order to identify differentially expressed genes. The new technique of RNA-seq was employed to give a global view of the transcripts regulated during the process of pyrene degradation. A total cataloguing of transcripts expressed by pyrene induction was revealed, showing functional elements and complex regulatory elements. A global qualitative classification of the transcriptional activity was made by comparing the proteomics data with the transcriptomics and this revealed a ton of information on the molecular intricacies of pyrene degradation in Mycobacterium gilvum PYR-GCK. With a little of the information gotten from the earlier analyses, a gene expression study of the strain’s molecular activity in various conditions of pH and NaCl concentrations, was undertaken. This study revealed the activities of the critical process of aromatic ring cleavage genes at the different experimental conditions. The genes studied included phdF, phdI, pcaG and pcaH, coding for an extradiol dioxygenase, 1-hydroxy-2-naphthoate dioxygenase/gentisate-1, 2-dioxygenase, alpha and beta subunits of protocatechuate-3,4-dioxygenase, respectively. The study revealed high pyrene degrading activities at neutral to slightly alkaline conditions, the halotolerant nature of the strain and its pyrene degrading ability in null salinity as well as NaCl concentrations of up to 58%, above seawater salinity concentration. There are limited reports on transcriptomics and gene expression studies in bacteria talk less of a similar study on any PAH substrate degradation, therefore this study will be the first of this kind in this field and in the Mycobacterium group of PAH degraders.