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Molecular action mechanisms of cyanocidal natural compounds on Microcystis aeruginosa

Microcystis aeruginosa에 대한 천연 살조 화합물의 분자적 작용 메커니즘 연구

  • Wonjae Kim (김원재, 대학원 환경생태공학과 생물재료공학전공)

초록/요약

Harmful cyanobacterial blooms, an environmental issue in freshwater ecosystems worldwide, are primarily caused by the dominant species Microcystis aeruginosa, which accounts for 60% of such occurrences. These blooms result in water pollution and adverse impacts on aquatic ecosystems due to toxins, including microcystins, produced by M. aeruginosa and the rapid depletion of dissolved oxygen by Microcystis-bacterial aggregates. Controlling the growth of M. aeruginosa is essential for managing these blooms. However, current non-specific control methods, such as red clay dispersal and hydrogen peroxide treatment, have the downside of causing secondary environmental pollution. Utilizing plant-derived extracts containing substances like amentoflavone has been proven effective in preventing such collateral environmental and ecological damage, aside from halting harmful cyanobacterial blooms. Understanding the molecular-level cell death mechanisms is necessary for controlling the growth of M. aeruginosa specifically. However, research excluding general cell death mechanisms like photosynthesis inhibition and reactive oxygen species stress is scarce due to the challenges in isolating single Microcystis strains and limitations of genetic manipulation in M. aeruginosa cells. Metagenomic analysis of xenic M. aeruginosa communities, cultured from environmental isolates, specifically revealed the absence of amino acid degradation genes like cadA encoding a lysine decarboxylase in the genome of M. aeruginosa, while these genes were found in the genomes of associated bacteria. Consequently, M. aeruginosa cells were unable to grow in nutrient-rich media, such as Luria Bertani (LB) and Tryptone/yeast extract/glucose (TYG), and exhibited significant growth inhibition, particularly under the lysine and threonine treatment conditions. Excessive lysine-mediated peptidoglycan layer impairment in M. aeruginosa cells was elucidated using 14C-L-lysine and fluorescent D-amino acids. Additionally, treatment with threonine in M. aeruginosa cells led to keto acid generation through a moonlighting function of threonine synthase and induced amino acid imbalances, negatively impacting the integrity of the cell membrane and cell wall, thus leading to rapid cell death. Therefore, novel findings of this study demonstrate a sustainable strategy for mitigating the toxic cyanobacterial blooms and elucidate the nutritional stress mechanism of two amino acids in M. aeruginosa cells. This provides foundational knowledge and insights for the development of technologies to control harmful cyanobacterial blooms.

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초록/요약

전세계 담수 유역에서 발생하는 환경문제 중 하나인 유해 남세균 대발생은 우점종인 Microcystis aeruginosa가 생산하는 독소 및 세균 군집 층의 빠른 용존산소량 소모로 인한 수질오염 및 수생태계 내 악영향을 유발한다. M. aeruginosa의 생장을 제어하는 기술은 이러한 유해 남세균 대발생을 관리하는데 필수적이지만, 현재 주로 사용되는 황토 살포 및 과산화수소 처리법 같은 비 특이적 제어 방법은 2차 환경오염을 유발한다는 단점을 지니고있다. 이러한 부수적인 환경오염 및 생태계 유해성을 피하는 실질적인 남세균 제어방법을 개발하기 위하여, 아멘토플라본과 같은 M. aeruginosa 특이적 사멸 물질을 다량 함유한 Selginella tamariscina 식물 유래 추출물을 활용하였다. 식물 유래 추출물은 담수 세균, 조류, 어류 등에 대한 낮은 환경독성 수준을 보였으며, 세균 군집분석 결과 M. aeruginosa 특이적 생장제어에 효과적이었다. 그럼에도 불구하고 부영양화 및 비특이적 반응 등의 문제점들을 더욱 줄이기 위하여, 분자적인 수준에서의 M. aeruginosa 균주 세포사멸 현상을 이해할 필요성이 있지만, 단일 균주 분리가 어렵고 유전자 조작이 제한되는 M. aeruginosa균주의 특성 상 광합성 저해 및 활성산소종 스트레스와 같은 일반적인 세포사멸 기작을 제외한 연구는 거의 진행되지 않은 실정이다. 따라서 이 연구논문에서는 기존에 밝혀지지 않았던 M aeruginosa의 세포벽 수준, 전사체 및 대사체 수준, 단백질 수준에서의 사멸기작을 구명하여 유해남세균 대발생 억제를 위한 기초지식과 통찰을 제공하고자 하였다. 환경에 분리 및 배양된 M. aeruginosa 군집체의 메타유전체 분석 결과, 특이하게 M. aeruginosa의 유전체 내에 라이신 디카복실레이스와 같은 아미노산 분해 유전자가 부재했으며 연관 세균들의 유전체에는 분해 유전자들이 존재하였다. 이런 특징으로 인하여 일반적인 영양배지에서는 M. aeruginosa 세포가 생장하지 못하는 모습을 확인하였고, 특히 아미노산 중 라이신과 트레오닌에 의해 높은 생장 저해를 받는 것을 확인하였다. 라이신을 분해하지 못하는 M. aeruginosa는 생장속도가 느려 고농도의 라이신을 충분히 해소하지 못하고 세포벽 합성 과정에 문제가 생겨 사멸하는 기작을 방사성동위원소가 표지된 L-라이신과 형광 표지된 D-아미노산을 활용하여 규명하였다. 또한 트레오닌은 세포질 내 함입 후 트레오닌 신테이스의 새로운 기능에 의한 케토산 생성을 유도하고, 동시에 아미노산 불균형을 유도하여 세포막 및 세포벽의 무결성에 부정적인 영향을 줘 빠른 세포사멸이 진행됨을 보였다. 본 연구는 일반적인 세균에 영양분으로 쓰일 수 있는 단일 아미노산이 M. aeruginosa의 세포사멸을 유도하는 기작을 밝힘으로써 유해 남세균 대발생을 제어하기 위한 기술 개발을 위한 초석과 발판을 제공한다.

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목차

ABSTRACT .......................................................................................................................... i
국문 초록 ....................................................................................................................... iv
PREFACE ...................................................................................................................... viii
ACKNOWLEDGMENTS .................................................................................................... ix
TABLE OF CONTENTS ...................................................................................................... x
LIST OF TABLES ............................................................................................................. xiii
LIST OF FIGURES ............................................................................................................ xiv
CHAPTER 1. General introduction ....................................................................................... 1
CHAPTER 2. Biological and chemical approaches for controlling harmful Microcystis blooms ............................................................................................................... 6
2.1 Introduction............................................................................................................ 8
2.2 Characteristics of the freshwater microbiomes associated with Microcystis blooms ................................................................................................................. 15
2.3 Killing mechanisms of antagonistic bacteria against M. aeruginosa .................. 15
2.4 Bacteria-derived chemical weapons against M. aeruginosa ................................ 24
2.5 Natural and biosynthetic compounds for the control of Microcystis blooms ..... 27
2.6 Conclusion .......................................................................................................... 32
2.7 References .......................................................................................................... 35
CHAPTER 3. Sustainable control of Microcystis aeruginosa, a harmful cyanobacterium, using Selaginella tamariscina extracts ............................................................ 51
3.1 Introduction.......................................................................................................... 53
3.2 Materials and methods ......................................................................................... 56
3.2.1 Cyanobacterial strains and culture conditions ......................................... 56
3.2.2 Preparation and identification of compounds active against M.
aeruginosa in Selaginella tamariscina extracts (STEs) .......................... 57
3.2.3 Morphological analysis using scanning electron microscopy ................. 58
3.2.4 Quantification of microcystin-LR released from M. aeruginosa cultures ......................................................................................................................... 59
3.2.5 Collection of cyanobacterial bloom samples and bacterial community
analysis ................................................................................................... 59
3.2.6 Toxicity assessment of STE for not-target aquatic organisms ................ 61
3.3 Results and discussion ......................................................................................... 62
3.3.1 STE as an inhibitor of M. aeruginosa ..................................................... 62
3.3.2 Identification of anticyanobacterial compounds in S. tamariscina ......... 74
3.3.3 Bacterial community analysis of xenic Microcystis and cyanobacterial bloom samples under STE conditions .............................................................. 82
3.3.4 Non-toxic effect of STE on freshwater bacteria and algae ..................... 91
3.3.5 Ecotoxicological assessment of STE ...................................................... 61
3.4 Conclusion ........................................................................................................ 102
3.5 References ........................................................................................................ 103
CHAPTER 4. Unlocking the mystery of lysine toxicity on Microcystis aeruginosa ........ 114
4.1 Introduction........................................................................................................ 116
4.2 Materials and methods ....................................................................................... 121
4.2.1 Bacterial strains and culture media ....................................................... 121
4.2.2 Metagenome analysis using Illumina and Oxford Nanopore sequencing tools ................................................................................................................ 121
4.2.3 Quantitative real-time PCR and transcriptomic data analysis ............... 123
4.2.4 Culture conditions of M. aeruginosa and microscopic analyses ........... 124
4.2.5 Autoradiography analysis using 14C-L-lysine ....................................... 126
4.2.6 Peptidoglycan purification .................................................................... 127
4.2.7 Mass spectrometry analyses .................................................................. 128
4.2.8 Quantitative measurement of 8-hydroxydeoxyguanosine as a marker for oxidative stress ............................................................................................... 129
4.2.9 Alteration of detergent sensitivity and membrane depolarization ........ 131
4.2.10 Collection of freshwater samples and bacterial community analysis . 132
4.2.11 Data availability .................................................................................. 133
4.3 Results................................................................................................................ 134
4.3.1 Lack of lysine degradation pathway in the genome of Microcystis aeruginosa...................................................................................................... 134
4.3.2 Lysine-mediated disruption of peptidoglycan network ......................... 146
4.3.3 Failure of cross-linking in peptidoglycan structures ............................. 155
4.3.4 Widespread lysine toxicity to freshwater bacteria ................................ 165
4.4 Discussion ......................................................................................................... 170
4.5 Conclusion ........................................................................................................ 177
4.6 References ........................................................................................................ 179
CHAPTER 5. Threonine is not one-size-fits-all for freshwater cyanobacterial growth .... 188
5.1 Introduction........................................................................................................ 190
5.2 Materials and methods ....................................................................................... 194
5.2.1 Culture conditions for M. aeruginosa ................................................... 194
5.2.2 Microscopic analyses ............................................................................ 195
5.2.3 Transcriptomic analysis and quantitative real-time PCR (qRT-PCR) .. 196
5.2.4 Proteomics analysis ............................................................................... 197
5.2.5 Analyses of cell viability ....................................................................... 199
5.2.6 Phylogenetic tree and 3D homology analyses of protein ...................... 200
5.2.7 Overexpression and purification of His-tagged protein ........................ 201
5.2.8 Detection of cellular and enzymatic formation of α-ketoacid ............... 202
5.2.9 Metabolomic analysis of amino acid derivatives in Microcystis .......... 203
5.3 Results................................................................................................................ 206
5.3.1 Snapshot of threonine-triggered cyanobacterial death .......................... 206
5.3.2 Moonlighting function of threonine synthase as threonine deaminase . 219
5.3.3 Distinctive lineage of ThrC in the threonine synthase family ............... 230
5.3.4 Metabolomic analysis for amino acid profiles ...................................... 236
5.3.5 Impact on α-ketobutyrate production and bacterial cell envelope integrity ....................................................................................................................... 246
5.4 Discussion .......................................................................................................... 253
5.5 References ........................................................................................................ 258
CHAPTER 6. General conclusion ..................................................................................... 269

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