Proliferation and evolution of lactococcal bacteriophages in cheese fermentations

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dc.check.chapterOfThesisNon applicableen
dc.check.date2035-09-30
dc.contributor.advisorvan Sinderen, Douwe
dc.contributor.advisorMahony, Jennifer
dc.contributor.advisorexternalNauta, Arjen
dc.contributor.authorYu, Jun-Hyeok
dc.contributor.funderFrieslandCampina
dc.contributor.funderIrish Research Council
dc.date.accessioned2025-02-13T12:49:17Z
dc.date.available2025-02-13T12:49:17Z
dc.date.issued2024
dc.date.submitted2024
dc.description.abstractLactococcus is a widely exploited genus of the lactic acid bacteria in global dairy fermentations. However, bacterial strains that are routinely applied in these fermentations are susceptible to (bacterio)phage infection during the fermentation process which in certain case may have a significant negative economic impact. Although various strategies to prevent phage proliferation during fermentations have been developed, phages remain a major industrial challenge. In this thesis, an undefined mesophilic starter culture employed in the production of Dutch-type cheeses was dissected to elucidate its complex microbial community. Various lactococcal strains were isolated from this undefined mesophilic starter and used as potential bacterial hosts of phages present in associated samples in order to assess the presence and prevalence of phages during various stages of the cheese production process. Metagenome- and cultivation-based analysis of the starter culture revealed the dominant presence of Lactococcus species, particularly Lactococcus cremoris and Lactococcus lactis, in addition to minor populations of Leuconostoc mesenteroides. The intraspecies differentiation of L. cremoris/lactis isolates revealed a substantial strain-level diversity with regards to cell wall polysaccharide (CWPS) types, phage sensitivity profiles, and plasmid content. Additionally, the potential of isolated Lactococcus laudensis strains for future applications in the dairy industry was assessed by examining their genomic and phenotypic characteristics. DNA-based methods were used to track Lactococcus throughout the cheese production process using the corresponding starter culture, demonstrating significant shifts in both the relative abundance of the different component cwps genotypes and the overall bacterial population. In parallel, the proliferation and diversity of lactococcal phages during this process was analysed. Virome analysis of 17 dairy production samples identified Skunavirus-associated genome contigs containing distinct receptor binding proteins (RBPs). These RBPs are responsible for specific recognition and binding to the host cell surface. Phylogenetic comparison of the identified RBP amino acid sequences facilitated the prediction of their corresponding host cwps genotypes. These predictions were partially validated through host range analysis of isolated skunaviruses using the host panel isolated from the corresponding starter culture. Throughout the process, an increase in the absolute abundance of phages as well as phage compositional changes were observed. Furthermore, the isolated skunaviruses were shown to exhibit increased thermal resistance, suggesting a growing resilience against anti-phage measures employed in the production facility. Comparative genomic analysis of 18 newly and 71 previously isolated skunaviruses associated to Dutch dairy fermentation facilities revealed a correlation between their genome diversity and starter culture applied. Furthermore, a specific focus was placed on the HNH endonuclease (HNHE)-encoding genes in Skunavirus genomes, whose gene product is presumed to function as a homing endonuclease that facilitates phage evolution. The presence, diversity, and insertion location of HNHE-encoding genes were elucidated. Also, their protein sequence/structure features and enzymatic activities were elucidated. The distinct features of HNHEs at each insertion location in the structure of their C-terminal region were demonstrated. Furthermore, the correlation between used starter cultures and HNHEs were elucidated, suggesting that HNHEs in Skunavirus likely contribute to phage evolution. Among these identified HNHEs in Skunavirus genomes, the essential function of a conserved HNHE-encoding gene in phage DNA packaging was investigated. This conserved HNHE-encoding gene is located within the phage DNA packaging module in proximity to the terminase and portal protein-encoding genes. Deletion of the HNHE-encoding gene resulted in the production of defective phages, without disrupting other critical stages of the phage life cycle, such as DNA replication and structural protein assembly. This conserved HNHE possesses a typical active motif (ββα-metal fold), but exhibited non-specific endonuclease activity, highlighting the need for further studies.en
dc.description.statusNot peer revieweden
dc.description.versionAccepted Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationYu, J-H. 2024. Proliferation and evolution of lactococcal bacteriophages in cheese fermentations. PhD Thesis, University College Cork.
dc.identifier.endpage213
dc.identifier.urihttps://hdl.handle.net/10468/17036
dc.language.isoenen
dc.publisherUniversity College Corken
dc.relation.projectFrieslandCampina (Enterprise Partnership Scheme (Postgraduate))
dc.rights© 2024, Jun-Hyeok Yu.
dc.rights.urihttps://creativecommons.org/licenses/by-nc-sa/4.0/
dc.subjectBacteriophageen
dc.subjectLactococcusen
dc.subjectFermentationen
dc.subjectDairy industryen
dc.subjectMetagenomeen
dc.subjectEvolutionen
dc.titleProliferation and evolution of lactococcal bacteriophages in cheese fermentations
dc.typeDoctoral thesisen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD - Doctor of Philosophyen
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