Microbiology - Doctoral Theses
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Item Discovery and characterisation of bacteriocins from microbiomes(University College Cork, 2024) Hourigan, David; Ross, R. Paul; Hill, Colin; Science Foundation IrelandBacteria are social microorganisms that inhabit every corner of the world. They rarely live in isolation and are key members of the microbiome along with viruses, archaea, protozoa and algae. Microbiome research has exploded over the past 20 years, but we are only starting to disentangle the complexity and dynamic nature it holds. Bacteriocins are antimicrobial peptides produced by bacteria across the bacterial kingdom. They have gained attention as a suitable alternative or adjunct to traditional antibiotics due to low levels of documented resistance. However, the roles they play in shaping the microbiome or acting as selfish genetic elements are often overlooked when studying them through such a narrow scope. While it is widely accepted that these peptides can be exploited to target specific bacterial species, their abundance and diversity is often under explored. These factors will have a huge impetus on their suitability to be used as antimicrobials. In Chapter 1, we perform a literature review describing recent advancements in enterococcal genomics and how it’s advancements can aid in therapeutic strategy. Clinically problematic Enterococcus faecium are promiscuous with respect to horizontal gene transfer and are becoming a global healthcare issue. The ability to expand its own genome and rapidly mutate has made this bacterium resistant to multiple antibiotics. Therefore we explore non-antibiotic interventions, including bacteriocins, to tackle these pathogenic bacteria. However, in Chapter 2 we show that E. faecium APC1031 rapidly becomes resistant to the potent class IIa bacteriocin avicin A. We generated mutants at a frequency of approximately 1e-06 suggesting that class IIa bacteriocins alone may not be suitable to target such a “hardy” and adaptable bacterium. Over the past century bacteriocin research has been skewed towards the Bacillota. However, the Actinomycetota are gathering interest as an underexploited bacteriocin-producing phylum. In Chapter 3 we discover novel aureocin A53-like bacteriocins found within this phylum. We also show they are found in microbiomes. We then synthetise the first aureocin A53-like bacteriocins from Actinomycetota and show that they have antimicrobial activity. Chapter 4 explores the rumen microbiome as a source of novel bacteriocins by genome mining the Hungate1000 culture collection of rumen strains. We discover that between 30-70% of bacteria found in the rumen produce at least one bacteriocin which is more than double previous estimates of the bacteria found in mammalian gastrointestinal tract. Nisin is the most well studied bacteriocin. It is gathering attention as a suitable alternative to antibiotics. However, little is known of how widespread the genes responsible for its production are. In Chapter 5 we discover widespread nisin-like bacteriocin production genes and show they are on mobile genetic elements and present in bacteria of aetiological concern. We also show that some of these peptides can be heterologously expressed and that they have antimicrobial activity. We then show that nisin VP is a novel nisin variant from an anaerobic bacterium isolated from the pig gut. Chapter 6 shows that two Streptococcus devriesei strains have the genetic capability to produce a conserved novel circular bacteriocin. This novel bacteriocin streptocyclin BTW is an amylocyclicin-like bacteriocin found within the genus streptococcus. Both strains with the genetic capacity to produce this bacteriocin are found in the oral microbiota. The role of bacteriocins as antimicrobial peptides is a well-studied trait. However, little is known of the genetic systems that localise near them within genomes. In Chapter 7, we search for genes encoding protein families that have co-localised near lanthipeptide biosynthetic gene clusters and discover an enrichment of anti-phage defence systems. This suggests lanthipeptide production may be attribute to bacteriophage-bacteria interactions. This thesis explored the diversity of bacteriocin gene clusters that are found in microbiomes. We have shown that these peptides have antimicrobial activity in vitro. However, their abundance and diversity implies they play multiple roles in complex microbiomes.Item Effects of the biotic and abiotic environment on ecology and evolution within novel in vitro and ex vivo models of the human gut microbiome(University College Cork, 2024) Pennycook, Joseph; Scanlan, Pauline; Claesson, Marcus; Science Foundation IrelandLeveraging the precision and replicability of in vitro and ex vivo models, we investigated the ecology and evolution of the human gut microbiota in a variety of environmental conditions. We developed a novel system for culturing microbiota communities and first used it to study the effects of the biotic and abiotic environment on the relationship between a gut-derived bacteriophage and its host. We found that an interaction between the nutrient medium and the broader community of bacteria led to the extinction of phage populations via competitive exclusion of the host and acidification of the medium. After reviewing the existing body of literature regarding the effects of antibiotic treatment on human gut microbiota, we tested the effects of sub-clinical antibiotic exposure on ex vivo faecal communities. Exposure to sub-clinical concentrations of clindamycin led to less diverse communities, more prevalent antibiotic resistance, and a greatly reduced presence of the Actinobacteria phylum, while exposure to amoxicillin and ciprofloxacin had less clear effects. Metabolic assays revealed that exposure to sub-inhibitory antibiotics promoted faster metabolism in Bifidobacterium, although not a faster growth rate, and also that the presence of an antibiotic often prevented the inhibition of metabolism by other environmental stressors. This work has demonstrated the value of laboratory models in the study of the human gut microbiota, while highlighting the importance of environmental context in understanding the system's ecology and evolution.Item Proliferation and evolution of lactococcal bacteriophages in cheese fermentations(University College Cork, 2024) Yu, Jun-Hyeok; van Sinderen, Douwe; Mahony, Jennifer; Nauta, Arjen; FrieslandCampina; Irish Research CouncilLactococcus 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.Item Outsmarting phages to generate better dairy products(University College Cork, 2024) White, Kelsey; van Sinderen, Douwe; Mahony, Jennifer; Irish Research CouncilThe dairy fermentation industry relies on the activity of various lactic acid bacteria in starter cultures to acidify milk and produce a variety of fermented dairy products. Undefined mesophilic starter cultures are predominantly composed of many strains (in an unknown composition) belonging to species such as Lactococcus lactis and Lactococcus cremoris. Bacteriophage infection of the bacterial strains within these starter cultures are one of the most significant and persistent threats to successful dairy fermentations. Delayed or disrupted fermentations, as a result of phage attacks, may cause costly disruptions resulting in unnecessary waste and a loss of product. One of the most problematic and abundant lactococcal phage genera is Skunavirus. Most lactococcal phages, including skunaviruses, commence infection with the binding of a phage-encoded receptor binding protein (RBP) to a surface-exposed host receptor, which in the case of skunaviruses is a cell wall polysaccharide (CWPS). To ensure the manufacture of consistent and high-quality fermentation end products, it is essential that the composition and integrity of starter cultures is maintained. Therefore, this thesis aims to investigate phage-host interactions between skunaviruses and their lactococcal hosts pertinent to dairy fermentation facilities utilising undefined mesophilic starter cultures. Chapter 2 of this thesis outlines a combined culture-based analysis and metagenomics investigation to evaluate the composition of two mesophilic undefined starter cultures. A novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes, was devised and used to examine compositional changes of an undefined starter culture. These analytical approaches facilitated a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important quality control aspect due to increasing demand for reliable and high-quality dairy products. In Chapter 3, a phageome analysis of whey samples originating from dairy fermentation factories employing mesophilic undefined starter cultures was conducted to investigate the phage prevalence and diversity in the samples. Particular focus was placed on investigating the range of Skunavirus RBP genotypes and associated RBP-CWPS binding abilities present in these whey phageomes. These findings allowed for the refinement and expansion of the Skunavirus RBP grouping systems thereby vastly expanding our knowledge on lactococcal Skunavirus RBP diversity and CWPS specificity. Chapter 4 of this thesis details the diversity of skunaviruses present in the same whey samples used for the phageome analysis described in Chapter 3 through culture-dependent phage screening, targeted isolation based on RBP specificity, and phage RBP-activated cell sorting (PhRACS). Through this investigation, the genomes of 26 newly isolated Skunavirus members were sequenced and analysed, with a specific focus placed on surveying genes encoding auxiliary carbohydrate binding module-containing proteins. Host range analysis of the skunaviruses revealed a clear correlation between the encoded RBP phylogroups and cwps genotypes of corresponding host strains. In addition, phageome-derived Skunavirus contigs were analysed to further determine the diversity of phages present in the dairy fermentation facilities. Chapter 5 assesses the strain-level diversity of lactococcal strains originating from mesophilic undefined starter cultures. Culture-dependent approaches were used to assess the technological attributes, such as acidification, proteolytic activity, and phage sensitivity, of these isolates. Genomic analysis of sequenced lactococcal strains was focused on industrially relevant genomic elements such as cwps biosynthesis gene clusters, plasmids, and phage-defense systems. In addition, a genomic analysis of strains belonging to the emerging dairy-associated species Lactococcus laudensis was carried out. In summation, both the bacterial composition of starter cultures as well as the phage diversity of corresponding whey samples were thoroughly investigated, generating comprehensive insights into the complex microbial community of these mesophilic fermentation systems. Understanding the intricate phage-host interactions occurring in the environment in which mesophilic fermentation systems are extensively applied will aid in efforts to development and maintain robust dairy starter culture systems for sustainable food production practices.Item Discovery and characterization of antiphage systems in the lactococcal plasmidome(University College Cork, 2024) Grafakou, Andriana; van Sinderen, Douwe; Mahony, Jennifer; Science Foundation IrelandLactococcus lactis and Lactococcus cremoris are important bacterial species that are widely used in the production of fermented dairy products such as buttermilk and cheese. However, the non-sterile dairy environment exposes them to (bacterio)phage infections, which can cause substantial economic losses. To counteract phage threats, bacteria have evolved various defence mechanisms, including abortive infection (Abi) systems. Abi systems are activated upon phage infection, and they typically interfere with essential cellular functions, thereby significantly limiting phage propagation. In Chapter 1, we review the existing literature of 34 lactococcal Abi-like systems. Using the latest structural and functional prediction tools, we categorize certain Abi-like systems through structural superimposition and hypothesize their potential mechanisms based on predicted domain information. We also examine previously established mechanisms of action of certain Abi-like systems. In Chapter 2, we use Lactococcus as a model to identify novel plasmid-encoded antiphage systems, leveraging the fact that many of the first Abi systems were discovered in these bacteria. A systematic evaluation of candidate defence systems led to the discovery of seven new plasmid-encoded antiphage systems (Rhea, Aristaios, Kamadhenu, Fliodhais, Audmula, Rugutis, Hesat), which seem to be widely distributed across bacteria, as well as five systems (PARIS, type I and II CBASS, Lamassu, Septu), homologues of which had previously been identified as antiphage systems in other bacteria. These systems confer resistance against the most prevalent lactococcal phages, and all but one were shown to exhibit characteristics of abortive infection systems. Insights into their mechanisms of action were gained through structural and domain predictions. In Chapter 3, functional insights into three novel plasmid-encoded lactococcal Abi-like antiphage systems are presented. Their effectiveness in milk-based media was established, supporting their application potential to enhance the reliability of dairy fermentations. Our findings indicate that two of these systems do not directly impede phage genome replication, transcription, or translation, while one was found to interfere specifically with phage transcription. Additionally, the plasmid carrying one of these systems was successfully transferred via conjugation to various lactococcal strains, each of which showed a significant increase in phage resistance. In Chapter 4, mechanistic insights into the identified antiphage systems were established by isolating phage escape mutants that had overcome these systems. Genome analysis of these phage escape mutants revealed mutations in various genes, some of which encode proteins that appear to activate specific antiphage systems (terminase large subunit, major capsid protein, hypothetical proteins and major tail proteins), while others might act as targets (ssDNA annealing proteins). The cross-resistance of some phage escape mutants to different antiphage systems, as well as the activation of some systems by phage proteins with similar functions, indicates mechanistic commonalities among various antiphage systems. Additionally, we propose a novel mode of action for one antiphage system. Our findings demonstrate that the co-evolution of Lactococcus species with their phages, spurred by their widespread use in dairy fermentation, has driven the acquisition of a diverse range of phage defence mechanisms, with plasmids serving as a rich source of such antiphage systems. Expanding our understanding of the lactococcal phage resistome not only offers practical solutions to the persistent phage challenges in industrial food fermentations but also holds potential for broader applications in biotechnology and biomedicine, while providing deeper insights into the dynamics of phage-host interactions.