Title Page
Abstract
Contents
List of Abbreviations 18
CHAPTER Ⅰ. General Introduction 19
1. Bacteriophage 20
1.1. General Introduction 20
1.2. Classification 22
1.3. Host Specificity 24
1.4. Genetic Diversity 24
1.5. Lytic Life Cycle 25
1.6. Phage Diversity 26
1.7. Challenges in Phage Applications 27
2. Objectives of This Study 30
2.1. Limitations of Bacteriophage research 30
2.2. Purposes of This Study 30
CHAPTER Ⅱ. Macromonas nakdongensis sp. nov., Isolated from Freshwater and Characterization of Bacteriophage BK-30P-the First Phage That Infects Genus Macromonas 32
1. Abstract 33
2. Introduction 34
3. Materials and Methods 36
3.1. Isolation of Bacterial Strain 36
3.2. 16S rRNA Gene Phylogeny 36
3.3. Polyphasic Taxonomy 37
3.4. Genome Sequencing, Assembly, and Annotation 38
3.5. Phage Isolation and Genomic DNA Preparation 39
3.6. Morphological and Genomic Characterization of Macromonasphage 40
4. Results 42
4.1. 16S rRNA Gene Phylogeny 42
4.2. Polyphasic Taxonomic Characterization 44
4.3. Genome Characterization 48
4.4. Macromonasphage Analysis 52
5. Discussion 60
6. Conclusions 61
7. Protologue 62
CHAPTER Ⅲ. Research on Bacteriophages that Infect Multi-Drug Resistant Bacteria 64
1. Abstract 65
2. Introduction 67
2.1. Background of the Research Project 67
2.2. Domestic and International Research Trends 69
2.3. Necessity of Research Project 71
2.4. The Project Involves Several Key Steps 71
3. Material and Methods 73
3.1. Selection of Study Site and Sample Collection 73
3.2. Isolation of Antibiotic-Resistant Bacteria 74
3.3. Bacteriophage Isolation 81
4. Results 86
4.1. Sample Collection and Environmental Factor Analysis 86
4.2. Isolation of Antibiotic-Resistant Bacteria 87
4.3. Isolated of Multi-Drug Resistant Bacteria Erwinia sp. 96
4.4. Bacteriophage Isolation and Genomic Analysis 107
5. Discussion 124
6. Appendix 126
CHAPTER Ⅳ. Research on Bacteriophages Infecting Roseobacter clade, Associated with Harmful Microalgae 134
1. Abstract 135
2. Introduction 137
3. Material and Methods 139
3.1. The Harmful Microalgae Aquatic Environment Survey 139
3.2. Isolation of Symbiotic Microorganisms 141
3.3. Isolation and Characterization of Bacteriophages 142
3.4. Confirming the Effect of Symbiotic Microorganisms and Bacteriophages on Harmful Microalgae 145
4. Results 147
4.1. Analysis of Microbial Communities in the Seomjin River 147
4.2. Selection of harmful microalgae symbiotic bacteria that cause red tide 149
4.3. Isolation and characterization of bacteriophages related to the Roseobacter clade 162
4.4. Symbiotic Bacteria and the Impact of Bacteriophages 166
5. Discussion 169
CHAPTER Ⅴ. Conclusion 172
References 174
국문초록 183
APPENDICES Ⅰ. Abstracts of the Paper 186
Table 2.1. Major characteristics that differentiate between strain BK-30T and Macromonas bipunctata DSM 12705T[이미지참조] 46
Table 2.2. Fatty acid profiles of strains BK-30T and Macromonas bipunctata DSM12705T[이미지참조] 47
Table 2.3. Summary of the draft genomes of strain BK-30T and Macromonas bipunctata DSM 12705T.[이미지참조] 50
Table 2.4. Genome statistics of Macromonasphage BK-30P 53
Table 2.5. Functional annotation of the ORFs predicted in the Macromonasphage BK-30P genome 56
Table 3.1. Antibiotics used in media preparation 75
Table 3.2. Antibiotics used in media preparation 77
Table 3.3. Field investigation point information and environmental factor 86
Table 3.4. No. of AR strains screened antibiotic medium. 88
Table 3.5. Multi-drug resistant bacteria showing sensitivity to more than 8 types of antibiotics 95
Table 3.6. The main transcriptome GO categories of Erwinian sp. AnSW2-5 100
Table 3.7. Secondary metabolism biosynthesis gene cluster of Erwinian sp. AnSW2-5 104
Table 3.8. Annotation summary for the genome sequence of phage AnSW2-5-P-S 116
Table 3.9. Annotation summary for the genome sequence of phage AnSW2-5-P-K 119
Table 3.10. List of antibiotic-resistant bacterial isolates 126
Table 4.1. Roseobacter clade and their habitat and distribution 150
Table 4.2. Subsystem functions of Roseobacter sp. BK-777 161
Table 4.3. Statistical analysis of co-culture of Alexandrium tamarense with commensal microorganisms and bacteriophages 168
Figure 1.1. Mechanism of interaction between phage and bacteria 21
Figure 1.2. Phage life cycle 23
Figure 1.3. Phage in the Biosphere 26
Figure 1.4. Phage therapy trial launched following FDA approval(UCSanDiegoHealthO) 29
Figure 1.5. Schematic overview of this study 31
Figure 2.1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationship between strain BK-30T and other representative type species of the family Comamonadaceae.[이미지참조] 43
Figure 2.2. Transmission electron micrograph of BK-30T cells. 45
Figure 2.3. Subsystem category distribution of strain BK-30T based on the RAST annotation server (https://rast.nmpdr.org/) (accessed on 24 August 2023)[이미지참조] 51
Figure 2.4. Phylogenomic tree based on concatenated multiple alignment of 84 genes showing the relationship between BK-30T and members of the family Comamonadaceae.[이미지참조] 51
Figure 2.5. Transmission electron micrograph of Macromonasphage BK-30P. 54
Figure 2.6. Genome map of Macromonasphage BK-30P. 54
Figure 2.7. The relationship between Macromonasphage BK-30P (shown in bold) and other phages is depicted in a phylogenetic tree. 55
Figure 3.1. Advances in antibiotics and the emergence of antibiotic-resistant bacteria 68
Figure 3.2. Global response strategy and report on antibiotic-resistant bacteria 70
Figure 3.3. Antibiotic-resistant microorganisms and bacteriophage research trends 70
Figure 3.4. Field survey points 73
Figure 3.5. Taxonomic distribution of antibiotic-resistant bacteria at the Phylum and class-level 90
Figure 3.6. Taxonomic distribution of antibiotic-resistant bacteria at the order and family level 90
Figure 3.7. Antibiotic disc assay 93
Figure 3.8. Susceptibility to each antibiotic 94
Figure 3.9. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationship between strain AnSW2-5 and other representative type species... 97
Figure 3.10. Transmission electron micrograph of Erwinia sp. AnSW2-5 97
Figure 3.11. Genoem map of Erwinian ps. AnSW2-5 99
Figure 3.12. RAST analysis results of Erwinian ps. AnSW2-5 99
Figure 3.13. OrthoANI values between Erwinian sp. AnSW2-5 and comparison strains 102
Figure 3.14. The bacteriocin biosynthetic gene cluster of Erwinian sp. AnSW2-5 105
Figure 3.15. The NRPS biosynthetic gene cluster of Erwinian sp. AnSW2-5 105
Figure 3.16. The Thiopeptide biosynthetic gene cluster of Erwinian sp. AnSW2-5 106
Figure 3.17. The Aryl Polyene biosynthetic gene cluster of Erwinian sp. AnSW2-5 106
Figure 3.18. Result of antibiotic-resistant bacteria lysis plaque test 108
Figure 3.19. TEM image of AnSW2-5-P-S 109
Figure 3.20. TEM image of AnSW2-5-P-K 109
Figure 3.21. Phylogenetic tree of AnW2-5-P-S infection Erwinian sp. AnSW2-5 111
Figure 3.22. Phylogenetic tree of AnW2-5-P-K infection Erwinian sp. AnSW2-5 112
Figure 3.23. Co-culture Experiment of Host and Phages 113
Figure 3.24. Gene Schematic Diagram of Bacteriophage AnSW2-5-P-S 115
Figure 3.25. Genome map of Bacteriophage AnSW2-5-P-K 115
Figure 4.1. Seomjingang River downstream and seawater field survey site 140
Figure 4.2. Example of co-culture experiment of microalgae, symbiotic microorganisms, and bacteriophages 146
Figure 4.3. Taxonomic distribution of bacteria along the Seomjin River at the phylum, class, and order levels 148
Figure 4.4. Distribution map of microbial communities and Rhodobacterales (Roseobacter clade) in the Seomjin River-coastal area 148
Figure 4.5. Neighbor-joining phylogenetic tree of Celeribacter marius SJW1-15 and related species 153
Figure 4.6. Neighbor-joining phylogenetic tree of Planktotalea arctica SJW4-3 and related species 154
Figure 4.7. Neighbor-joining phylogenetic tree of Confluentimicrobium sp. SJO10 and its related species 155
Figure 4.8. TEM image of Roseobacter sp. BK-777 156
Figure 4.9. Neighbor-joining phylogenetic tree of Roseobacter sp. BK-777 and its related species 157
Figure 4.10. Genome map of Roseobacter sp. BK-777 160
Figure 4.11. RAST analysis results of Roseobacter sp. BK-777 160
Figure 4.12. TEM image of Celeribacter phage SJW1-15P 164
Figure 4.13. TEM image of Planktotalea phage SJW4-3P 164
Figure 4.14. TEM image of Confluentimicrobium phage SJO-10P 165
Figure 4.15. TEM image of Roseobacter phage BK-777P 165
Figure 4.16. Day 4 of co-culture of Alexandrium tamarense and Roseobacter sp. BK-777 (left), death of microorganisms and microalgae 4 days after injection of Roseobacter phage BK-777P (right) 167
Figure 4.17. Effects of Co-Culture with Roseobacter sp. BK-777 and their phage BK-777P on the Growth of Microalgae. 168