Isolation and ecophysiological characterization of a polycyclic aromatic hydrocarbon-degrading bacterium, Alteromonas sp. SN2, from a crude oil-contaminated tidal flat

국문 초록 (Abstract)

원유는 연안 환경에서 주요한 오염물질로서, 지속적으로 지층으로부터 유출되는 유류 또는 유류유출사고에 의해 연안의 심각한 유류오염이 발생하게 된다. 갯벌환경은 갯벌에 서식하는 미생물의 풍부도와 다양성에 의존하여 높은 1차 생산능력과 빠른 영양소의 순환속도를 나타내며, 갯벌에서 석유화합물인 원유는 분해되거나 산화되기 어려움에도 불구하고 미생물의 대사과정에 의해 제거될 수 있다. 이에 따라 본 연구에서는 유류로 오염된 태안 갯벌이 방향족 탄화수소 분해 미생물의 군집의 저장소가 될 것이라 가정하고, i)유류로 오염된 갯벌환경으부터 다환성 방향족 탄화수소 (Polycyclic Aromatic Hydrocarbon; PAH)를 분해하는 미생물을 농화배양한 후 미생물 군집의 다양성 및 구성을 조사하고, ii)농화배양된 미생물 군집으로부터 다환성 방향족 탄화수소 분해하는 능력을 갖는 단일 미생물을 분리하여 그들의 방향족 탄화수소 분해 능력을 검정하고, iii)대사체분석 및 비교유전체분석을 이용해 방향족 탄화수소의 분해경로와 생태적 생존전략 및 유전적 특성을 파악하고, iv) 유전체의 전반적인 유전자 발현양상 관찰을 통해 생태생리학적 성질을 규명하기 위해 해당 연구를 진행하였다.
첫번째 연구에서는 나프탈렌을 단일 탄소원으로 공급하고 바닷물을 배지로 사용하는 환경모방형 농화배양을 구축하였고, 그 결과 농화배양기간동안 다른 미생물군집에 비해 활성이 있는것으로 보이는 Alteromonas sp. SN2를 분리할 수 있었다. 실험실에서 실시된 분해테스트를 통해, Alteromonas sp. SN2는 나프탈렌 이외에 페난스렌, 안트라센 및 파이렌을 분해할 수 있는 것으로 조사되었으며, 유류로 오염되었던 갯벌토양으로 추출된 DNA로부터 방향족 탄화수소 분해에 요구되는 naphthalene 1,2- dioxygenase(NDO) 유전자를 PCR 기법을 통해 검출할 수 있었다. 이런 결과를 토대로 오염된 갯벌시료에서 추출한 DNA와 RNA를 사용하여NDO 유전자를 검출한 결과, Cycloclasticus계열의 유전자는 DNA로부터 증폭된 반면에 Alteromona계열의 NDO유전자는 DNA뿐 아니라 RNA에서 조차 검출할 수 있었다. 또한 2년간 현장에서 채취한 갯벌 시료로부터 16S rRNA 유전자 및 NDO유전자의 양적 변화를 관찰한 결과, 여름보다 겨울에 Alteromonas sp. SN2의 개체수가 20배가 넘게 증가한 것을 알 수 있었으며, NDO유전자의 활성도 유지되는 것을 관찰할 수 있었다. 이런 결과를 통해 Alteromonas sp. SN2가 유류로 오염된 갯벌 현장에서 겨울과 방향족 탄화수소의 분해의 핵심미생물로서 존재함을 견론지을 수 있었다.
두번째 연구에서는 유류로 오염된 태안으로부터 분리된 Alteromonas sp. SN2가 분류동정학적 관찰에 의해 기존의 Alteromonas 속의 다른 종과는 다른 새로운 종의 미생물로 제시하였다. 그람음성 미생물로서 일반적인 호염미생물의 특성을 갖고 있으나, 다른 종의 미생물들과 다르게 유류로 오염된 갯벌에서 분리되었으며 다환성 방향족 탄화수소를 분해할 수 있는 능력을 보인다. 균주 SN2는 절대 호기성 미생물, 카탈라아제 및 산화제의 활성, 편모 운동성 등의 특성을 갖고 있다. 뿐만 아니라, 다양한 온도, pH 및 염도에서 성장실험을 수행하여 균주 SN2가 최적 성장온도 (25?30°C), 최적 pH (pH 7.0?7.5) 그리고 최적 성장염도 (2.0%)를 갖는 것을 확인하였다. 유일한 퀴논성분으로는 유비퀴논 8 (Q-8)이 아이소프레노이드 퀴논으로 분석되었으며, 주요 지방산은 C16:1 ω7c and/or iso-C15:0 2-OH, C16:0, C18:1 ω7c 그리고 C12:0 등으로 분석되었다. 또한, 전체 유전자의 G+C 함량이 43.5 mol%으로 분석되었고, 계통학적 분석에 의해 16S rRNA gene의 염기서열 유사도를 분석한 결과 같은 속의 Alteromonas 계열의 미생물들과 그룹을 형성하는 것을 알 수 있었다. 균주 SN2와 16S rRNA유전자와 염기서열이 가장 유사한 세가지 균주 Alteromonas stellipolaris LMG 21861, Alteromonas addita R10SW13 and Alteromonas macleodii ATCC 27126 와 염기서열의 유사도가 97%을 넘지만 DNA-DNA 간의 유사도가 모두 70% 이하(48.7 ± 6.6 %, 24.9 ± 7.5 % and 27.9 ± 8.4 %)로 분석되었다. 이러한 형태학적, 화학적동정실험결과를 통해 기존의 Alteromonas 속의 다른 종과는 다른 새로운 종의 미생물로 제시하였다
마지막 세번째 연구에서는 유전체 전반의 유전자발현을 종합적으로 분석하여 갯벌과 바닷물에서 strain SN2의 환경에 대한 반응과 생태생리학적 특성을 관찰하였다. 네가지 환경적 모방 조건으로 갯벌-나프탈렌, 갯벌-피루빈산, 바닷물-나프탈렌 그리고 바닷물-피루빈산을 구성하였으며, illumine mRNA-seq기법을 사용하여 종합적이고 정량적으로 유전체 전반적인 유전자 발현양상을 평가하였다. 전사체 발현양상은 서식환경에 의한 영향으로 인해 좀더 강력한 클러스터를 형성하였으며, 유전체 전반의 전사체 발현은 탄소원의 영향보다 서식환경에 의한 영향과 더운 밀접한 관계가 있는 것으로 보여진다. KEGG pathway에 cDNA read의 metabolic mapping분석은 strain SN2가 주어진 환경조건과 관계없이 에너지 대사, 전사체의 번역 및 운동성과 관련이 있는 대사조절 유전자들이 네조건 모두에서 높게 나타난 것을 관찰할 수 있었으며 이는 strain SN2가 기회주의적 해양 r-strategy 미생물로서 copiotrophic 성질을 보이는 것을 암시한다. 또한 네가지 조건에서 다르게 발현된 유전자 발현패턴의 비교를 통해 네가지 환경적 변이요소(갯벌, 바닷물, 나프타렌, 피부브산)에 의해 strain SN2가 각 환경조건에 특이적으로 조절한 유전자를 검출할 수 있었는데, 방향족 탄화수소 대사를 포함한 스트레스 반응 및 chaperone 단백질과 긴축 운동성 관련 유전자들이 갯벌조건에서 특이적으로 높게 발현된 것을 보아 이들 유전자에 의해 strain SN2가 계절적으로 저온이되는 갯벌 환경에 생태학적으로 적응한 것으로 보여진다.

다국어 초록 (Multilingual Abstract)

Crude oil is one of the most important organic pollutants in coastal environments and the oil contamination is occured regularly by releases of petroleum hydrocarbons and oil spill. Although petroleum hydrocarbon compounds are difficult to be degraded or oxidized, some hydrocarbons are degraded by microbial metabolic process. Because of high primary production and nutrient cycling rates, sea-tidal flats are may rely upon high microbial abundance and diversity. We hypothesized that the oil contaminated Taean tidal flat may also harbor diverse aromatic hydrocarbon-degrading microbial communities. The present study therefore aimed to investigate (i) the diversity and composition of the aromatic hydrocarcon (especially PAH and BTEX)-degrading bacterial populations enriched from the crude oil-contaminated sea-tidal flat on the Taean coast; (ii) the isolation of PAH or BTEX biodegrading bacteria from the enriched bacterial consortia and their aromatic hydrocarbons degradation abilities; (iii) the characteristics of ecological survival strategy of isolates by metabolites analysis and comparative genomic analysis; (iv) the ecophysiological properties by genome-wide transcriptional analysis; (v) and the optimal conditions of variable factors (nitrogen and phosphate) for efficient degradation of pollutants in sea-tidal flats.
In the first study, enrichment cultures were established using seawater and modified minimal media containing naphthalene as sole carbon source. The enriched microbial community was characterized by 16S rRNA-based DGGE profiling; sequencing selected bands indicated Alteromonas (among others) were active. Alteromonas sp. SN2 was isolated and was able to degrade PAH (naphthalene, phenanthrene, anthracene, and pyrene) in laboratory-incubated microcosm assays. PCR-based analysis of DNA extracted from the sediments revealed naphthalene dioxygenase (NDO) genes of only two bacterial groups: Alteromonas and Cycloclasticus, having gentisate and catechol metabolic pathways, respectively. However, reverse transcriptase PCR (RT-PCR)-based analysis of field-fixed mRNA revealed in situ expression of only the Alteromonas-associated NDO genes. A qPCR-based two-year data set monitoring Alteromonas-specific 16S rRNA genes and NDO transcripts in sea-tidal flat field samples showed that the abundance of bacteria related to strain SN2 during the winter season was 20-fold higher than in the summer season. Based on the above data, we conclude that strain SN2 and its relatives are site natives? key players in PAH degradation and adapted to winter conditions in these contaminated sea-tidal-flat sediments. Moreover, Alteromonas sp. SN2 was suggested as a new species of genus Alateromonas by invesitigation of taxonomic properties.
In second study, a novel PAH-degrading bacterium, strain SN2, belonging to the genus Alteromonas was isolated from sea-tidal flat sediment contaminated with crude oil and its taxonomic properties were characterized using a polyphasic approach. A Gram-staining-negative and moderately halophilic bacterium, designated SN2, capable of polycyclic aromatic hydrocarbon biodegradation, was isolated from sea-tidal flat sediment contaminated with crude oil in South Korea. Cells were strictly aerobic, catalase- and oxidase-positive motile rods with a single polar flagellum. Growth of strain SN2 was observed at 4?37°C (optimum, 25?30°C), at pH 6.0?9.0 (optimum, pH 7.0?7.5) and in the presence of 0.5?9.0 % (w/v) NaCl (optimum, 2.0 %). Only ubiquinone-8 (Q-8) was detected as the isoprenoid quinone and summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH), C16:0, C18:1 ω7c and C12:0 were observed as the major cellular fatty acids. The G+C content of the genomic DNA was 43.5 mol %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SN2 formed a distinct phylogenetic lineage within the genus Alteromonas. Strain SN2 was most closely related to Alteromonas stellipolaris LMG 21861T, Alteromonas addita R10SW13T and Alteromonas macleodii ATCC 27126T with the 16S rRNA gene sequence similarities of 99.5 %, 99.3 % and 98.4 %, respectively and their DNA-DNA relatedness values were 48.7 ± 6.6 %, 24.9 ± 7.5 % and 27.9 ± 8.4 %, respectively. On the basis of phenotypic, chemotaxonomic and molecular features, strain SN2 clearly represents a novel species of the genus Alteromonas, for which the name Alteromonas naphthalenivorans sp. nov. is proposed. The type strain is SN2 (=KCTC 11700BPT =JCM 17741T).
In third study, a comprehensive analysis of genome-wide gene expression was performed to investigate the ecophysiological properties and the environmental behaviors of strain SN2 in sea-tidal flat and seawater. Four environmental mimic conditions consisting of tidal flat-naphthalene (TF-N), tidal flat-pyruvate (TF-P), seawater-naphthalene (SW-N), and seawater-pyruvate (SW-P) were prepared and the genome-wide transcriptional expressions of strain SN2 were comprehensively and quantitatively assessed using an Illumina mRNA-seq approach. The transcriptional profiles were clustered by the habitats (TF-N/TF-P and SW-N/SW-P), not by carbon sources, suggesting that environmental habitat may have more influences on the genome-wide expression of strain SN2. The mapping analysis of the cDNA reads on the KEGG pathway of strain SN2 showed that metabolic and regulatory genes associated with energy metabolism, translation, and cell motility were highly expressed in all four test conditions, implicating copiotrophic properties of strain SN2 as an opportunistic marine r-strategist. The comparison of differential gene expressions showed that strain SN2 has specific cellular responses of strain SN2 to respective environmental variables (tidal flat, seawater, naphthalene, and pyruvate). In addition, the comparison of differential gene expressions displayed that strain SN2 may have ecological fitness traits including aromatic hydrocarbon metabolism to seasonally cold tidal flat habitats by high expressions of stress response and chaperone proteins and twitching motility proteins.

목차 (Table of Contents)

• LIST OF FIGURES v
• LIST OF TABLES vii
• I. GENERAL INTRODUCTION 1
• 1.1. Petroleum hydrocarbon: an overview 2
• 1.2. Description of sea-tidal flat and Hebei spirit oil spill 3
• 1.3. Microbial biodegradation of aromatic hydrocarbons 4
• 1.4. Mechanism of aromatic hydrocarbons degradation 6
• 1.5. The genus Alteromonas 8
• 1.6. Objectives of this study 9
• II. CHAPTER 1. Alteromonas as a key agent of polycyclic aromatic hydrocarbon biodegradation in crude oil-contaminated coastal sediment 11
• 2.1. INTRODUCTION 14
• 2.2. MATERIALS AND METHODS 14
• 2.2.1. Sampling of crude oil-contaminated sea-tidal flat sediment 14
• 2.2.2. Monitoring of PAH compounds in the crude oil-contaminated sea-tidal flat 14
• 2.2.3. Enrichment cultures and denaturing gradient gel electrophoresis (DGGE) analysis 15
• 2.2.4. Isolation of naphthalene degrading bacteria from enrichment cultures and PAH transformations tests 18
• 2.2.5. Naphthalene degradation tests of bacterial isolates 18
• 2.2.6. DNA sequencing and phylogenetic analysis 19
• 2.2.7. PAH transformations of strain SN2 in liquid media and slurry systems 19
• 2.2.8. Analysis of the naphthalene dioxygenase gene of strain SN2 and PCR primer designs 20
• 2.2.9. PCR amplification of naphthalene dioxygenase genes from genomic DNA and mRNA extracted from the contaminated sea-tidal flat 21
• 2.2.10. Quantitative real time-PCR (qRT-PCR) to determine 16S rRNA copy number of total bacteria and Alateromonas species in the crude oil-contaminated sea-tidal flat 22
• 2.2.11. Real time-reverse transcriptase PCR (RT-reverse transcriptase PCR) for the expressional analysis of the naphthalene catabolic gene of Alteromonas sp. and its relatives 23
• 2.2.12. Sediment microcosms: GC-MS analysis of naphthalene metabolites (catechol and gentisate) and nucleic acid extraction 23
• 2.3. RESULTS 24
• 2.3.1. Monitoring of PAH compounds in the crude oil-contaminated tidal flat sediments 24
• 2.3.2. Enrichment cultures and DGGE analysis 26
• 2.3.3. Isolation of bacteria from enrichment cultures and their naphthalene degradation abilities 29
• 2.3.4. Biodegradation of PAHs by Alateromonas sp. strain SN2 in liquid media and slurry systems 32
• 2.3.5. PCR primer design for NDO genes and their application to strain SN2 35
• 2.3.6. Analysis of the NDO gene sequences from genomic DNA and mRNA extracted from the contaminated sea-tidal flat 37
• 2.3.7. Using microcosm slurry incubations to refine metabolite and nucleic acid (enumeration and gene expression) biomarker assays for Alteromonas-catalyzed naphthalene degradation 37
• 2.3.8. Applying the nucleic-acid and metabolite biomarker assays to the sea-tidal flat field site: quantitative seasonal monitoring of total bacteria and Alteromonas species and Alateromonas-related NDO gene expression 41
• 2.4. DISCUSSION 44
• III. CHAPTER 2. Alteromonas naphthalenivorans sp. nov., a polycyclic aromatic hydrocarbon degrading bacterium, isolated from sea-tidal flat sediment 48
• 3.1. INTRODUCTION 49
• 3.2. MATERIALS AND METHODS 50
• 3.2.1. Isolation and cultivation 50
• 3.2.2. Phenotypic and physiological tests 50
• 3.2.3. Chemotaxonomy 51
• 3.2.4. Determination of 16S rRNA gene sequence and phylogenetic analyses 51
• 3.2.5. DNADNA hybridization (DDH) 52
• 3.3 RESULTS 53
• 3.3.1. Phenotypic characteristics 53
• 3.3.2. Phylogenetic analysis 56
• 3.3.3. DNA-DNA hybridization 56
• 3.3.4. Chemotaxonomic characteristics and G + C content 56
• 3.4 CONCLUSION 60
• IV. CHAPTER 3. Genome-wide transcriptional analysis of Alteromonas naphthalenivorans SN2 in contaminated seawater and coastal marine sediment 62
• 4.1. INTRODUCTION 63
• 4.2. MATERIALS AND METHODS 65
• 4.2.1. Collection of sea-tidal flat sediment and seawater 65
• 4.2.2. Preparation of four environmental conditions and growth of strain SN2 65
• 4.2.3. RNA extraction and mRNA purification 67
• 4.2.4. Illumina sequencing and bioinformatic data analysis 68
• 4.3. RESULTS AND DISCUSSION 69
• 4.3.1. Preparation of TF-N, TF-P, SW-N, and SW-P condition 69
• 4.3.2. Growth of strain SN2 and mRNA purification 70
• 4.3.3. Illumina sequencing and bioinformatic data process 76
• 4.3.4. Genome-wide transcriptional profiles of strain SN2 78
• 4.3.5. Genome-wide functional gene expressions of strain SN2 83
• 4.3.6. Genome-wide differential gene expressions of strain SN2 under four different environmental conditions 93
• 4.3.7. Genes of strain SN2 with differential expressions under different environmental conditions 121
• 4.3.8. Expressional analysis of the PAH degradation genes of strain SN2 under different environmental conditions 125
• 4.4. CONCLUSIONS 128
• V. CONCLUDING REMARKS 129
• VI. REFERENCES 131
• VII. 국문초록 152
• VIII. ABSTRACT 155
• IX. ACKNOWLEDGEMENT 158