Molecular and Genomic Study of Bacteriophages Targeting Salmonella enterica serovar Typhimurium and Bacillus cereus : Salmonella enterica serovar Typhimurium과 Bacillus cereus에 특이적인 박테리오파지에 대한 분자적/유전체적 연구

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농업생명과학대학 식품공학과
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서울대학교 대학원
BacteriophageSalmonella TyphimuirumBacillus cereusPhage-host interactionPhage genomics
학위논문 (박사)-- 서울대학교 대학원 : 식품공학과, 2013. 8. 유상렬.
Salmonella enterica subspecies enterica serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as alternative biocontrol agents to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated. Twenty-five Salmonella phages were isolated and their receptors were identified by screening several mutant strains of S. Typhimurium SL1344. Among them, only three types of receptors were identified: flagella, vitamin B12 uptake outer membrane protein, BtuB and lipopolysaccharide-related O-antigen. TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L) belong to Podoviridae family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella.
To understand phage infection and host lysis mechanisms with pathogenic Salmonella, a novel Salmonella Typhimurium-targeting bacteriophage SPN9CC, belonging to the Podoviridae family, was isolated and characterized. The phage infects S. Typhimurium via the O-antigen of lipopolysaccharide (LPS) and forms unique clear plaques with cloudy centers due to lysogen formation. Phylogenetic analysis of phage major capsid proteins (MCPs) revealed that this phage is a member of lysogen-forming P22-like phage group. However, comparative genomic analysis of SPN9CC with P22-like phages indicated that their lysogeny control regions and host lysis gene clusters share very low identities, suggesting that lysogen formation and host lysis mechanisms may be diverse among phages in this group. Analysis of the expression of SPN9CC host cell lysis genes encoding holin, endolysin, and Rz/Rz1-like proteins individually or in combinations in S. Typhimurium and E. coli hosts revealed that collaboration of these lysis proteins is important for both host lysis, and holin is a key protein. To further investigate the role of the lysogeny control region in phage SPN9CC, a ΔcI mutant (SPN9CCM) of phage SPN9CC was constructed. The mutant does not produce a cloudy center in the plaques, suggesting that this mutant phage is virulent and no longer temperate. Subsequent comparative one-step growth analysis and challenge assays revealed that SPN9CCM has shorter eclipse/latent periods and a larger burst size as well as higher host lysis activity than SPN9CC. The present work indicates the possibility of engineering temperate phages as promising biocontrol agents similar to virulent phages.
The Bacillus cereus group phages infecting B. cereus, B. anthracis, and B. thurigiensis (Bt) have been studied at a molecular level and recently at a genomic level to control pathogens of B. cereus and B. anthracis and to prevent phage contamination of the natural insect pesticide Bt. A comparative phylogenetic analyses revealed three different phage groups with different morphologies (Myoviridae for group I, Siphoviridae for group II, and Tectiviridae for group III), genome size (group I > group II > group III), and lifestyle (virulent for group I and temperate for group II and III). A subsequent phage genome comparison using a dot plot analysis showed that phages in each group are highly homologous, substantiating the grouping of B. cereus phages. Endolysin is a host lysis protein that contains two conserved domains such as a cell wall binding domain (CBD) and an enzymatic activity domain (EAD). In B. cereus sensu lato phage group I, four different endolysin groups were detected, according to combinations of two types of CBD and four types of EAD. Whereas group I phages share two copies of tail lysins and one copy of endolysin, the functions of the tail lysins are still unknown. In the B. cereus sensu lato phage group II, the B. anthracis phages have been studied and applied for typing and rapid detection of the pathogenic host strains. In the B. cereus sensu lato phage group III, the B. thuringiensis phages, such as Bam35 and GIL01, were studied to understand phage entry and lytic switch regulation mechanisms. In this study, I suggest that further study of the B. cereus group phages would be useful for various phage applications, such as biocontrol, typing, and rapid detection of the pathogens B. cereus and B. anthracis and for the prevention of phage contamination of the natural insect pesticide Bt.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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