Horizontal Gene Transfer (HGT) is the transfer of bacterial genes from one bacteria to another and is a vital tool that allows various microbes to adapt rapidly to new environments by exchanging potentially beneficial genes. Previously, our lab identified a large mobile genetic region putatively involved in iron acquisition (termed RUSTI for iRon Update Siderophore Transport Island) which we believe is horizontally transferred in the cheese community. The RUSTI region is on a much larger mobile genetic region called an integrative and conjugative element (ICE). We termed the RUSTI-containing ICE for Proteobacteria the ProteoRUSTI ICE. To test if RUSTI is indeed horizontally transferred we co-cultured a Vibrio casei carrying the ProteoRUSTI ICE with three cheese-rind isolated Proteobacteria: Psycrobacter, Vibrio sp., and Pseudoalteromonas. These bacteria were found to not carry the ProteoRUSTI ICE. We unfortunately did not observe an HGT event between Vibrio casei and the ProteoRUSTI ICE negative bacteria. However, this masters laid the ground work on how to design and organize HGT experiments in the cheese rind. These findings are important for the development of our model because they highlight the characteristics of RUSTI in various ways. Creating a better understanding of how mobile genetic elements such as RUSTI behave in species-species interactions provides us insight into how the microbes or their genes in more complex microbial communities may interact with one another.

Many aged cheeses consistently develop a bloom of microbes at their surface, called a rind. Proper development of the rind and the underlying cheese’s defining characteristics depends on the specific assembly of a community of microbes at the rind. Oftentimes, the assembly process of rind microbiomes as well as other relevant microbiomes is quite dynamic and results in a series of different dominant community members, called a pattern of ecological succession. The specific factors that shape a specific pattern of succession are only superficially understood. While microbial communities serve as useful study models because of their rapid development in comparison to, for example, a forest, the drivers of their successional patterns are primarily studied observationally or theoretically using sequencing datasets. This dissertation explores the drivers underlying the pattern of succession that occurs during the aging of a natural rind cheese, through both metagenomic investigations of rind microbiome development as well as in vitro experimentation using a model community assembled from the cave-aged rind. We first find that the overarching pattern of succession is driven primarily by two fungal species who modulate the pH of the environment or inhibit population size. I further explore the mechanisms underlying these activities and identify potentially novel mechanisms involved in both stimulation and inhibition of community neighbors. We also show how segregated spatial organization of the rind biofilm may explain why the effects of fungal species are stronger than those of bacteria. And finally, we start to consider how bacteriophage might contribute to rind assembly dynamics, by contributing genetic functions or by generating dynamic patterns at the strain level. I conclude these studies with advice on how our findings can be followed up to further explore and identify mechanisms involved in community assembly both of a cheese rind and potentially of other medicinally-, environmentally-, and industrially-relevant microbiomes.

Diverse and widespread populations of bacteriophages infect and co-evolve with their bacterial hosts. Although the process of host recognition and infection occurs within microbiomes, the molecular mechanisms underlying host-phage interactions within the context of a community remain poorly studied. The biofilms, or rinds, of cheese, contain taxonomically diverse microbial communities that follow reproducible growth patterns and can be manipulated under laboratory conditions. Our lab has previously used a model Brie community to demonstrate specific microbe-microbe interactions occurring within a community. Using this system, we are investigating how host-phage interactions and co-evolution influence other community members, and vice versa. The work outlined here described our efforts to use cheese as a model for studying phage-microbe interactions by identifying and characterizing a tractable host-phage pair co-occurring within this system. We successfully isolated lytic bacteriophage TS33 that kills Hafnia strain JB232, a member of a model Brie community. TS33 is easily propagated in the lab and naturally co-occurs in the cheese with its Hafnia host and other members of the Brie community, rendering it a prime candidate for the study of host-phage interactions. We used Random Barcode Transposon Sequencing (RB-TnSeq) experiments to identify candidate host factors that contribute to TS33 infectivity, many of which are critical to the integrity of the lipopolysaccharide (LPS) layer of the host cell. Notably, disruption of these genes results in decreased susceptibility to infection by phage TS33, while simultaneously exhibiting a significant negative effect on the fitness of Hafnia strain JB232 in the presence of its fungal partners, Geotrichum candidum and Penicillium camemberti. Therefore, LPS mutations may have pleiotropic effects on the interactions between JB232 and the rest of the Brie community. Ongoing and future studies aim to unearth the molecular mechanisms by which the LPS of JB232 mediates its interactions with its viral and fungal partners.

Despite having traditionally been studied in isolation, the natural context of microbial growth is in the presence of other species. Research on microbes in interactive contexts has previously been challenging due to the complexity of natural microbiomes and an inability to culture non-model organisms. To address these challenges, a model microbiome based on the cheese rind has been developed. This system is of intermediate diversity and contains genetically tractable and culturable organisms, enabling mechanistic study of microbial interactions. Using species from this model microbiome, we apply cutting-edge genetic, metabolomic, and imaging techniques to investigate mechanisms of intermicrobial interactions. Chapter 1 reviews recent findings regarding intermicrobial interactions. Chapter 2 reports on the development of an RB-TnSeq library in cheese-associated bacterium Pseudomonas psychrophila and the use of this library in a three-member cheese rind model community to identify intermicrobial interactions. Chapter 3 presents the application of RB-TnSeq to the study of mechanisms related to bacterial-fungal interactions. This chapter also introduces a custom analysis pipeline developed for cross-condition RB-TnSeq comparisons. Chapter 4 describes three projects stemming from Chapter 3,which relate to an E. coli gene of unknown function, a fungal protein with potential antibiotic activity, and the application of RB-TnSeq to the study of bacterial-bacterial interactions, including the creation and characterization of an RB-TnSeq library in another cheese-associated bacterium Hafnia sp. str. JB232. Finally, Chapter 5 discusses potential future directions for this work and provides concluding remarks.

The physiological processes associated with microbiomes represent the biology of individual microbial cells and their interactions with other cells. Moreover, the interactions between microbes are not static, but represent dynamic processes that are subject to ecological and evolutionary changes. One of the main drivers of rapid evolution in microbial species is the process of horizontal gene transfer, often mediated by mobile genetic elements. Few systems or tools exist that allow the study of this process in microbiomes. In this thesis, we review experimental methods to study horizontal gene transfer, as well as catalog computational methods to sequence and filter for mobile elements, including integrative and conjugative elements, plasmids, and viruses. We next leverage long read DNA sequencing to lay the groundwork for a complex in vitro washed cheese rind microbiome model. With our microbiome model and computational methods, we are able to 1) probe natural microbiomes in kefirs and cheese rinds to better understand the breadth and novelty of mobile elements and 2) begin to understand how different community members play a role in the evolution of species and mobility of plasmids within a genus. Our work in the identification, characterization, and tracking of mobile elements in microbiomes enhances our understanding of the unique and niche-adaptive genes carried by these fermenting microbes. Finally, we explore the potential clinical and industrial applications of long read and proximity ligation. 

The majority of the deep ocean is an extreme environment characterized by low temperature and high pressure, which drives bacteria to evolve unique adaptations in the deep sea. Analyses of cold-adapted microbes, psychrophiles, have suggested that their adaptations, which increase fluidity in membrane composition are similar to the adaptations of high-pressure-adapted microbes, piezophiles. To determine the adaptation mechanics of Antarctic seawater microbes, I performed adaptive laboratory evolution experiments using microbes from Antarctic surface seawater samples, by gradually increasing the incubation pressure of the microbes. ALE experiments were carried out with both the original microbial communities and bacterial strain isolates from the samples, by gradually increasing the incubation pressure of the microbes. This paper presents the evaluations of changes in community structures through the ALEs and characterizations of the strains isolated at high pressure.

In all the ALEs, I observed decreased growth rates at high pressure for both microbial communities and isolates, as well as decreased diversities within microbial communities at high pressure, suggesting that high pressure hinders microbial growth. However, further growth analysis of Psychrobacter isolate ALE at 50 MPa after 6 rounds of transfers, surviving bacteria were capable of growing better at a higher pressure compared to the ancestral strains. Genome resequencing of the Psychrobacter strains suggested that the bacteria could have possibly adapted to growing at a higher pressure through mutations of proteins coding for transmembrane transport, membrane proteins and peptidoglycan production. In conclusion, this thesis project presents primary analysis that shows psychrophilic bacteria were capable of adapting to higher pressure environments, which might suggest that bacteria adapted to both low temperature and high pressure environment, such as the deep sea, could have first evolved to become psychrophilic which in turn enabled them to adapt to high pressure.