Diversity and evolution of bacteriophages infecting lactic acid bacteria used for commercial dairy fermentations

Strains of Lactococcus lactis are widely employed in the dairy industry as starter cultures for cheese fermentations. This continuous and widespread use brings with it the constant threat of (bacterio)phage attack. Indeed, phage predation of starter culture strains is the primary cause of the majority of commercial dairy fermentation disruption. Despite extensive prevention efforts, phages are still ubiquitous in dairy processing environments and, as a result, lactococcal phages have become one of the most intensively studied phage groups. In this thesis, a multi-year biodiversity study of the phage population of a number of Dutch dairy facilities was performed in order to monitor the population dynamics and evolution of phages in these facilities. An extensive screening of whey samples gathered from a variety of sources and processes throughout the facilities between 2015 and 2018 was performed. Results were combined with phage screenings of the same facilities between 2009 and 2013 to provide an almost decade-long overview of the population dynamics of phages in these facilities. A total of 31 novel phages were isolated and their genomes sequenced, none of which were completely identical to those isolated between 2009 and 2013, indicating a constantly evolving population. Consistent with other studies, phages of the 936 group were found to predominate, while c2 group phages were occasionally encountered in these facilities. The cell wall polysaccharide genotypes of the representative starter culture strains used as hosts for phage screening were determined, while novel phages were incorporated into previously established Receptor Binding Protein (RBP) groups. This data, when combined, revealed a strict correlation between the RBP group of a phage and the Cell Wall PolySaccharide (CWPS) genotype of its host. Sanitisation and disinfection using purpose-made biocidal solutions is a critical step in controlling phage contamination in dairy processing plants. The susceptibility of thirty-six 936 group phages to biocidal treatments was examined using fourteen biocides and commercially available sanitisers. The targets of a number of these biocides were investigated by electron microscopic and structural protein analyses. The results from this study highlight significant variations in phage resistance to biocides among 936 phages. A number of phage structural proteins were identified as targets of biocidal action, highlighting the distinct mode of action of these compounds. Furthermore, rather than possessing resistance to specific biocides or biocide types, biocide-resistant phages tend to possess a broad tolerance to multiple classes of antimicrobial compounds. Thus it is likely that, rather than being the result of small, precise differences between phages, resistance to biocides is due to an accumulation of features and differences between resistant and more susceptible phages. The ever increasing number of available 3D structures of bacteriophage components, combined with constantly improving in silico predictive tools, has made possible to decipher the structural assembly and associated functionality of phage adhesion devices. Using bioinformatic analysis, a conserved Carbohydrate Binding Module (CBM) was identified within the Dit (Distal tail) protein of a large number of 936 group phages, a central component of the so-called baseplate, which also contains the RBP with its own CBM. In these so-called ‘evolved’ Dit proteins, the identified CBMs have structurally conserved folds, yet can be grouped into four distinct classes which correspond yet are unrelated to the RBP group of the phage. A number of CBM-encoding Dits were expressed in fusion with GFP, and their host-binding capability was confirmed using fluorescent binding assays imaged via confocal microscopy. Evolved Dits were also detected via bioinformatic analysis in several phages infecting various Gram-positive bacterial species, including lactobacilli and mycobacteria. Research into 936 group phage-host binding interactions was then broadened beyond the RBP and Dit proteins, allowing the identification of a number of CBMs in the Neck Passage Structure and Major Tail Protein. Several of these modules were produced in fusion with GFP, and the binding capability of an example of each was demonstrated. Bioinformatic analysis of these CBMs, along with a detailed bioinformatic survey of 936 group receptor binding proteins, reveals that these phages incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups, and even to phages as disparate as the Escherichia coli phage T4, indicating that phages may utilise common structural “bricks” to enhance host binding capabilities. The omnipresence of CBM domains in Siphophages indicates their beneficial role, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their continued success in their respective ecological niches. Phages of the 936 group possess a discrete baseplate at the tip of their tail - a complex harbouring the RBP, which is primarily responsible for host recognition and attachment. The baseplate-encoding region is highly conserved amongst 936 phages, with 112 of 115 examined phages exhibiting complete gene synteny. Three exceptions were identified (phages Phi4.2, Phi4R15L, and Phi4R16L) which differ from this genomic architecture by possessing an apparent second RBP-encoding gene upstream of the ‘classical’ rbp gene. The newly identified RBP protein possesses an elongated neck region relative to currently defined 936 phage RBPs and is genetically distinct from defined 936 group RBPs. Through detailed characterisation of the representative phage Phi4.2 using a range of complementary techniques, it was demonstrated that it possesses a complex and atypical baseplate structure. Furthermore, the presence of both RBPs in the tail tip of the mature virion was confirmed, while the host-binding capabilities of both proteins were also verified. The research presented in this thesis significantly adds to the already extensive knowledge of the predominant 936 group of phages. Through a decade-long biodiversity study, new insights into phage population dynamics and their ever evolving nature have been gained. Patterns in phage resistance to frequently employed biocides have been identified, and for the first time a number of structural targets of biocidal action have been determined. Initially, it was hypothesised that RBPs were the sole facilitators of 936 phage-host interactions. However, through bioinformatic analysis and experimental verification, the work in this thesis has demonstrated that these phages have evolved to incorporate a number of binding domains throughout their structure, and that phages from a diverse range of hosts incorporate similar binding machinery. Finally, the study of the unique baseplate of Phi4.2 highlights that adaptations are still to be unearthed, even amongst well characterised members of the 936 phage group