Isolation and molecular characterization of new bacteriophages infecting Bacillus cereus and Acidovorax citrulli
- Abstract
- Abstract
Bacteriophages or phages in short are the viruses infecting bacteria which have been of interest as agents for the control of many diseases caused by bacteria since their discovery in of early twenty century. However, poor understanding of them and huge development of antibiotics delayed they development of the bacteriophages as therapeutic agents. Recent appearance of antibiotics resistant bacteria and public concern on the contamination of food and environment by antibiotics revived the interest on bacteriophages
In a search for bacteriophages that infect Bukholderia glumae, the causal agent of bacterial panicle blight on rice, we have isolated many Bacillus subtilis that showed antifungal activity against plant pathogenic fungi Rhizoctonia solani, and virus that infect B. subtilis. We have characterized one of the bacteriophage strain named as PK16 that infect B. cereus strain named as BC1.
PK16 had an icosahedral head of 100±5 nm and tail of 200±5 nm, indicating that it belonged to family Myoviridae. Latent period of 30±3 mins and burst size of 100±3 PFU/ml. Four strains out of 6 strains available in this study can be lysed by PK16. Analysis of the complete linear dsDNA genome revealed a 158,127-bp and it is similar to other B. cereus phages JBP901 and BCP8-2.
As an initial step of phage therapy of bacterial fruit blotch which is one of the most economically important diseases in Cucurbitaceae that caused by Acidovorax citrulli, we have isolated bacteriophages infecting this bacterium, which is named as ACP17. Totally 39 watermelon, pumpkin and cucumber leaf samples were collected from different parts of Korea and tested against 18 A. citrulli strains. ACP17 belongs to Myoviridae family with head diameter 100±5nm and a tail length 150±5 nm. ACP17 can lyse all A. citrulli strains belong to group I available in this study. Genome analysis also indicated that ACP17 is dsDNA viruses, linear form with a genome size of 156,972 bp. One step growth curve shows ACP17 has a latent period of 50±5 mins, burst size of 120 PFU/ml and eclise period of 25±5 min. During Initial in planta assay on watermelon seeds, the germination rate of seed coated phages sown in soil with Acidovorax citrulli presence, reached up to 100%. Results of this investigation show that both PK16 and ACP17 have the potential for phage therapy development.
- Issued Date
- 2017
- Awarded Date
- 2017. 8
- Type
- Dissertation
- Publisher
- Pukyong National University
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 미생물학과
- Advisor
- Tae-Jin Choi
- Table Of Contents
- Abstract vi
General introduction 1
Chapter1: Isolation and characterization of a bacteriophage preying an antifungal bacterium 3
1.1. Introduction: 3
1.2. Materials and Methods 4
1.2.1. Culture conditions for bacterium 4
1.2.2. Phage Isolation 4
1.2.3. Bacteriophage host range 5
1.2.4. Phage purification and virus DNA extraction 5
1.2.5. Transmission Electron Microscope 5
1.2.6. Antifungal activity of B. cereus 6
1.2.7. One-step growth curve of phage 6
1.2.8. Isolation of phage DNA sequencing 6
1.3. Results 7
1.3.1. Bacteria isolation 7
1.3.2. Bacteriophage Isolation 7
1.3.3. Transmission Electron Microscopy 8
1.3.4. Host range 10
1.3.5. One step growth curve 12
1.3.6. Genome analysis 14
1.3.7. Antifungal activity 20
1.4. Discussion: 22
1.5. Conclusion 25
Chapter2: Isolation and molecular characterization of new bacteriophage preying Acivorax citrulli, the causal agent of bacterial fruit blotch 26
2.1. Introduction 26
2.2. Materials and methods 28
2.2.1. Bacterial strains and culture conditions: 28
2.2.2. Isolation ofbacteriophage 28
2.2.3. Propagation and Purification ofBacteriophages 30
2.2.4. Temprature and pH sensitivity of ACP17
2.2.5. Host range determination 30
2.2.6. Library preparation for massively 31
2.2.7. Genome assembly and annotation 31
2.2.8. One step growth curve 32
2.2.8. Phage stability 32
2.2.9. In planta assay 32
2.3. Results 35
2.3.1. Isolation of bacteriophage 35
2.3.2. Bacterial characterization 36
2.3.3. One-step growth curve 38
2.3.4. Temprature and Ph sensitivity of ACP17 40
2.3.5. Phage Genome analysis 42
2.3.6. Phylogenic tree: 51
2.3.7. In planta Assay 53
2.4. Discussion 54
2.6. Conclusions 56
References 57
LIST OF FIGUERS
Figure 1. Transmission electron Microscopy bacteriophage PK16 9
Figure 2. One step growth curve of PK16 on exponential culture of B. cereus. 13
Figure 3. Circular genome map of PK16 15
Figure 4. Comparative genomic analysis of phage PK16 18
Figure 5. Phylogenetic tree based 19
Figure 6. Antifungal activity of Bacillus cereus isolated from a rice field. B. cereus BC1. 21
Figure 7. Locations and plants from which phage isolation was attempted 29
Figure 8. Electron microscopic pictures showing that ACP17 35
Figure 9 . One step growth curve of ACP17. 39
Figure 10. Stability of ACP17 at various PH (top) and temperatures (bottom). 41
Figure 11. Characterization of ACP17 genome. 43
Figure 12. Circular map of ACP17 genome 44
Figure 13. Comparative analysis of phage ACP17 45
Figure 14. Phylogenic tree based on the amino acid sequences . 52
. Figure 15. Comparison results of in planta assay with ACP17 and A .citrulli. 53
LIST OF TABLES
Table 1. Host range of phage 11
Table 2. Distributions of protein 17
Table 3. In planta Assay treatment 34
Table 4. Host range of phage ACP17. 37
Table 5. Functional classification of genes encoded by ACP17 genome 46
- Degree
- Master
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