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Diarrheal diseases are still a significant problem for humankind, causing approximately half a million deaths annually. To cause diarrhea, enteric bacterial pathogens must first colonize the gut, which is a niche occupied by the normal bacterial microbiota. Therefore, the ability of pathogenic bacteria to inhibit the growth of other bacteria can facilitate the colonization process. Although enterotoxigenic Escherichia coli (ETEC) is one of the major causative agents of diarrheal diseases, little is known about the competition systems found in and used by ETEC and how they contribute to the ability of ETEC to colonize a host. Here, we collected a set of 94 fully assembled ETEC genomes by performing whole-genome sequencing and mining the NCBI RefSeq database. Using this set, we performed a comprehensive search for delivered bacterial toxins and investi-gated how these toxins contribute to ETEC competitiveness in vitro. We found that type VI secretion systems (T6SS) were widespread among ETEC (n = 47). In addition, several closely related ETEC strains were found to encode Colicin Ia and T6SS (n = 8). These toxins provide ETEC compe-titive advantages during in vitro competition against other E. coli, suggesting that the role of T6SS as well as colicins in ETEC biology has until now been underappreciated.

Bacteria are prokaryotic microorganisms that can be found in all niches of the biosphere. Bacteria must constantly evolve in order to survive and adapt to their environments. Defence systems and competition systems play a vital role in ensuring the survival of bacteria. The projects included in this thesis aim to further understand how these systems function, as well as adapting them in order to design probiotic strains with built-in biocontainment mechanisms. In paper I, we investigate how internal expression of Rearrangement hotspot (Rhs) toxins allows protection against Rhs toxin delivery in S. Typhimurium. In paper II, we examine bacterial competition systems found in enterotoxigenic E. coli (ETEC) strains and explore the evolution of toxin arsenals within ETEC family lineages. In paper III, we develop a fluorescence flow cytometry-based high-throughput screening method and apply it to isolate a highly competitive commensal E. coli strain that possesses a diverse arsenal of competition systems. In paper IV, we design and construct a synthetic CRISPR system that is able to protect E. coli from acquisition of antibiotic resistance genes through horizontal gene transfer. The work presented in this thesis contribute to our understanding of the functions of bacterial defence and competition systems, at the same time, laying the groundwork for how these systems can be studied in a high-throughput manner as well as how they can be adapted in the future to design synthetic microbial strains with clinical and environmental applications.

Rhs elements are some of the most positively selected genes known and were recently found to function as type II TA-systems in addition to their previous role as delivered toxins. Antitoxins of type II TA systems are often inherently unstable proteins. Here we discover that the RhsI immunity proteins in Salmonella enterica serovar Typhimurium are also unstable on their own, but stabilized in the presence of their cognate toxins. This raises interesting questions regarding how such unstable immunity proteins can protect against incoming toxins delivered by neighboring attackers. In this study, we observed that one Rhs immunity protein can protect against more than one toxin. Therefore, the internal expression of RhsCT-I could be important for protection against delivered Rhs toxins in addition to regulating Salmonellagrowth in macrophages. The internal transcripts of rhsCT-I are upregulated in InSPI-2 and within macrophages by the combined action of RpoS, PhoP/Q and H-NS. These are the same regulators that control the expression of the type 6 secretion system known to deliver Rhs effectors in Salmonella. This suggests that expression of rhsCT-I is elevated under conditions that favor delivery, which further supports a role for the internal expression of Rhs proteins in protection against toxin delivery.

Background Wine is a complex beverage, comprising hundreds of metabolites produced through the action of yeasts and bacteria in fermenting grape must. Commercially, there is now a growing trend away from using wine yeast (Saccharomyces) starter cultures, towards the historic practice of uninoculated or "wild" fermentation, where the yeasts and bacteria associated with the grapes and/or winery perform the fermentation. It is the varied metabolic contributions of these numerous non-Saccharomyces species that are thought to impart complexity and desirable taste and aroma attributes to wild ferments in comparison to their inoculated counterparts. Results To map the microflora of spontaneous fermentation, metagenomic techniques were employed to characterize and monitor the progression of fungal species in five different wild fermentations. Both amplicon-based ribosomal DNA internal transcribed spacer )ITS) phylotyping and shotgun metagenomics were used to assess community structure across different stages of fermentation. While providing a sensitive and highly accurate means of characterizing the wine microbiome, the shotgun metagenomic data also uncovered a significant over-abundance bias in the ITS phylotyping abundance estimations for the common non-Saccharomyces wine yeast genus Metschnikowia. Conclusions By identifying biases such as that observed for Metschnikowia, abundance mesurements from future ITS-phylotyping datasets can corrected to provide more accurate species representation. Ulitmtaely, as more shotgun metagenomic and single-strain de novo assemblies for key wine species become available, the accuracy of both ITS-amplicon and shotgun studies will greatly increase, providing a powerful methodology for deciphering the influence of the microbial community on the wine flavor and aroma.

Wine is a complex beverage, comprising hundreds of metabolites produced through the action of yeasts and bacteria in fermenting grape must. To ensure a robust and reliable fermentation, most commercial wines are produced via inoculation with commercial strains of the major wine yeast, Saccharomyces cerevisiae. However, there is a growing trend towards the use of uninoculated or "wild" fermentations, in which the yeasts and bacteria that are naturally associated with the vineyard and winery, perform the fermentation. In doing so, the varied metabolic contributions of the numerous non-Saccharomyces species in this microbial community are thought to impart complexity and desirable taste and aroma attributes to wild ferments in comparison to their inoculated counterparts. In order the map the microflora of spontaneous fermentation, metagenomic techniques were used to characterize and monitor the progression of fungal species in several wild fermentations. Both amplicon-based ITS phylotyping (meta-barcoding) and shotgun metagenomics were used to assess community structure. While providing a sensitive and highly accurate means of characterizing the wine microbiome, the shotgun metagenomic data also uncovered a significant over-abundance bias in the ITS phylotyping abundance estimations for the common non-Saccharomyces wine yeast genus Metschnikowia.

Members of the genus Hanseniaspora represent a significant proportion of the normal flora of grape berries and play a significant role in wine fermentation. Here, we present genome sequences for three species of Hanseniaspora, H. opuntiae, H. osmophila, and H. uvarum, which were isolated from spontaneous Chardonnay wine fermentation.

Members of the genus Hanseniaspora represent a significant proportion of the normal flora of grape berries and play a significant role in wine fermentation. Here, we present genome sequences for three species of Hanseniaspora, H. opuntiae, H. osmophila, and H. uvarum, which were isolated from spontaneous Chardonnay wine fermentation.

Rhs elements are some of the most positively selected genes known. We recently discovered that the two rhs elements in Salmonella enterica serovar Typhimurium encode functional type-II TA-systems that are expressed and regulate bacterial growth during macrophage infections. However, whether these proteins have an effect on the bacterial cell when not inside the macrophage is not known. Here we investigate how expression of the rhs toxins in Salmonella enterica serovar Typhimurium is regulated and whether they can regulate growth also in laboratory media. We find that, even though expression of both rhs modules is higher in macrophages, Salmonella Typhimurium lacking both rhs toxins only grows faster compared to wildtype in LB media but not in macrophage mimicking media (InSpi-2). Thus, expression levels of the TA modules do not correlate with their effects on growth. The internal transcripts of rhsCT-I are upregulated in InSPI-2 by the combined action of RpoS, PhoP/Q and H-NS. These are the same conditions and regulators that enable expression of the type 6 secretion system, known to deliver Rhs effectors. In addition, we find that the RhsI immunity proteins are highly unstable on their own, but stabilized in the presence of their cognate toxin, suggesting that internal expression of the RhsCT-I could be important for protection against delivered Rhs toxin.

Diarrheal diseases still present a large problem for human kind, causing roughly half a million deaths annually. To cause infection, bacterial pathogens must first colonize the gut, a niche that is already occupied by the normal bacterial flora. The ability for a pathogenic strain to inhibit the growth of other bacteria can facilitate the colonization process. Although Enterotoxigenic E. coli (ETEC) is one of the major causative agents of diarrheal disease, very little is known about the competition systems found in and used by ETEC. Here we perform a comprehensive search for bacteriocins, type 6 secretion and contact-dependent growth inhibition systems in all ETEC strains found in the NCBI database. We discover a set of closely related strains, isolated from different geographical regions over several decades, all contain colicin Ia. Using at least one representative strain from each of the 8 ETEC families commonly associated with childhood diarrhea, we demonstrate that colicin Ia is expressed in these strains, allowing them to inhibit the growth of laboratory strains as well as commensal normal flora E. coli in vitro. In addition, we identify T6SS in 50% of all ETEC isolates found in the NCBI database. We observe that he most prevalent T6SS is active in the presence of bile salts, suggesting a role of this system within the host. We discovered that T6SS are under strong positive selection and we find evidence of frequent horizontal gene transfer of the T6SS loci. In summary, we demonstrate that bacterial toxin delivery systems and the ability to compete with other bacteria is important for ETEC during some parts of its lifecycle.