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The Plasmidome: a Driving Force of Rumen Microbial Evolution from Birth to Adulthood

Periodic Reporting for period 3 - RuMicroPlas (The Plasmidome: a Driving Force of Rumen Microbial Evolution from Birth to Adulthood)

Reporting period: 2019-01-01 to 2020-06-30

The goal of my project is to understand the community structure of the microbiome, its drivers and the role(s) of mobile genetic elements (plasmids) within it.
In recent years, the mammalian gut, including the ruminant gut, has emerged as a fundamentally important microbial environment. The intricate relationships between mammalian hosts and their microbial communities have been shown to play a central role in the host's well-being The rumen environment is an anaerobic compartment in the ruminant digestive system that accommodates heterogeneous microbial communities. This complex microbiome is comprised of Protozoa, Archaea and Bacteria co-residing at a density greater than 1010 ml-1. The rumen, together with its microbial symbionts, is responsible for the ruminant's remarkable ability to convert indigestible plant mass into digestible food products. These microorganisms are entirely responsible for the degradation and fermentation of the plant material, consisting mainly of indigestible sugar polymers such as cellulose and hemicellulose, consequently enabling the conversion of plant fibers into chemical compounds that can be digested by the animal. In this sense, ruminants are completely dependent on their rumen microbiome for their existence. This cooperative relationship between the ruminant and its resident microbiome has evolved over millions of years and has implications for our everyday lives with respect to food sustainability, environment, renewable energy, and economics. Ruminants hold enormous significance for man, as they convert the energy stored in plant biomass polymers, which are indigestible for humans, to digestible food products. Humans domesticated these animals for this purpose in the Neolithic era and have been farming them ever since for the production and consumption of animal protein in the form of meat and milk. In today's extensive production regimes, ruminants consume 30% of the crops grown on earth and occupy another 30% of the earth's land mass. These animals also emit methane—a highly potent greenhouse gas—to the atmosphere and are considered to be responsible for a considerable portion of its emission because of anthropogenic activities. Hence, an understanding of this complex microbial ecosystem and the evolutionary rules that govern it is of major interest. The complexity of the rumen microbial environment and its key role in animal physiology raises intriguing questions regarding the genetics and mobility of its functions among its microbial members. Lateral gene transfer (LGT)—the process by which microbial species donate and receive genetic material—is a major determinant of genetic novelty and genome evolution in prokaryotes. Mobile genetic elements serve as DNA vehicles for the communal gene pool. Plasmids are self-replicating, extrachromosomal, mobile genetic elements that operate as “gene ferries”, transferring genes from one host to another. Plasmids have been recognized as key vectors of genetic exchange between microbial chromosomes. Their high abundance in microbial populations sampled from various habitats indicates that they have an important ecological role. Plasmids are composed of a conserved DNA backbone that includes replication and mobilization genes, which are important for plasmid maintenance within the host and transfer among hosts. They also carry a variable assortment of accessory genes, which often contribute to the phenotypic diversity of their host. Plasmids isolated from different ecological niches encode a versatile array of accessory functions, ranging from antibiotic resistance to nitrogen fixation. These plasmid-borne functions may confer an advantage to their host in its niche, making the burden of carrying the plasmid worthwhile. An understanding of plasmid biology and biodiversity is expected to greatly contribute to our understanding of microbial ecology and evolution in diverse environments.
An ecological and mechanistic understanding of the rumen microbiome, which we aim with this project, could lead to an increase in available food resources and environmentally friendly livestock agriculture. Therefore, this funding is instrumental for our ability to make several important breakthroughs in the field. So far (mid-term), the project outcomes have resulted in ten publications, and three currently under review in high-impact journals and several others in preparation.
The main results achieved so far showed several fundamental and global aspects connected to the functioning of the rumen ecosystem, host-microbiome interactions , its plasmid composition and key components such as individual microbes and metabolites.
1. Ecosystem functions and composition
We identified several fundamental aspects that are connected to the functioning of the rumen ecosystem (publications 8, 9, 10), providing support to findings from microbial communities worldwide. Within the rumen ecosystem, we explored the microbial interactions as a response to diet and feeding cycle (fibre degradation and other extracellular matrices).
Using dietary intervention experiments, we revealed that diet affects the most abundant taxa within the microbiome and that a specific group of methanogenic archaea of the order Methanomicrobiales is highly sensitive to changes. Using metabolomic analyses together with in vitro microbiology approaches and whole-genome sequencing of Methanomicrobium mobile, a key species within this group, we identified that redox potential changes with diet, is the main factor that causes these dietary-induced alternations in this taxon’s abundance. Our genomic analysis suggests that the redox potential effect stems from a reduced number of anti-reactive oxygen species proteins coded in this taxon's genome. Our study highlighted redox potential as a pivotal factor that could serve as a sculpturing force of community assembly within anaerobic gut microbial communities (publication 8). Moreover, we investigated the metabolic potential and taxonomic composition of the rumen methanogens that play a key role in sustaining host metabolism and function. We discovered that the methanogenesis process changes with age and that the early methanogenic community is characterized by a high activity of methylotrophic methanogenesis. These findings were highlighted by science journal as they suggest that environmental filtering acts on the archaeal communities and select for different methanogenic lineages during different growth stages, affecting the functionality of this ecosystem (publication 9).
We next studied the postprandial diurnal community oscillatory patterns of the rumen microbiome, in order to understand what affects the community composition during the feeding cycle. We showed that metabolites produced by the rumen microbiome serve to condition its environment and lead to dramatic diurnal changes in community composition and function. These changes in community composition were accompanied by changes in pH and methane partial pressure, suggesting a strong functional connection. Our experiments showed that the metabolites released by microbes are sufficient to reproduce changes in community function comparable to those observed in vivo. These findings highlighted microbiome niche modification as a deterministic process that drives diurnal community assembly via environmental filtering (publication 10)

2. Host-microbiome axis
In order to understand the associations between the rumen host and its essential microbiome, we aimed to link the rumen microbial components to the cow’s ability to extract energy from their feed, termed as feed efficiency (publications 1, 3, 4). We discovered that rumen microbiome components are tightly linked to the cows' feed efficiency as well as methane emission. This work is seminal and of high importance in the field and has high relevance to our every life therefore is also highly cited (publication 1). Moreover, we further aimed to understand the role of rumen genetics on the microbiome composition. We found that genetics and physiology are correlated with microbiome structure and that host genetics may shape the microbiome landscape by enriching for phylogenetically related taxa (publication 3). We found that the gut microbiomes of hosts that were genetically selected are different ones that were not selected, although subjected to the same environmental conditions. Moreover, microbiomes of hosts that were selected show higher resilience to environmental stress, in agreement with their host response. These findings indicate that host selection shapes the gut microbiome composition and modulates its acclimatization. These findings lead to a new paradigm in host-microbe interactions, which highlight connections between host adaptivity and its microbiome response to environmental stress. These findings are of great importance to our understanding of the evolution and ecology of the halobiont unit. And were chosen by elife journal for to be highlighted in their article digest to be released to the press . Such notions could be potentially applied broadly in agriculture and clinical sciences. Based on these findings, we were invited to write several review articles, one of which is already published (publication 4). Furthermore, our research has shed light on various ways that we can utilize the microbiome for more applied approaches. We have assembled synthetic functional cellulosomal structures onto a potent member of the gut using mimicry of natural cellulosome elaborated architectures, thus exploiting the exponential features of their ‘Lego-like’ combinations. Using this approach, we produced several bacterial consortia of potent gut microbes which provide a very robust framework for degradation of lignocellulosic biomass, containing an unprecedented number of catalytic subunits all produced in vivo by the cell consortia. (publication 5).

3. Plasmids within the microbiome
We have developed a set of molecular and bioinformatic tools that enable the study of mobile genetic elements (plasmids) in natural microbial ecosystems and we were able to describe such elements in the rumen ecosystem (publication 2). This avenue continues to be highly productive, and currently, three additional manuscripts describing the forces that drive plasmid dispersal as well as mechanisms that enable their maintenance in the rumen and other gut systems are in review or in preparation.
Deep sequencing techniques used in metagenomic approaches have greatly advanced the study of microbial communities in various environments. We studied the rumen metagenomic plasmid population using a newly developed procedure that successfully overcomes many molecular obstacles. We introduced the Recycler, the first tool that can extract complete circular contigs from sequence data of isolated microbial genomes, plasmidomes and metagenome sequence data (publication 2). We further reviewed our results to plasmids in gut ecosystems. We recently found that plasmid dispersal across gut ecosystems fits a random dispersal model, in contradiction to the accessory functions carried by plasmids that show deterministic patterns associated with ecosystem state. Moreover, we found enriched plasmid recombination events between ecosystems that share disease states or geography (this manuscript is currently under preparation). A unique look at the plasmidome's (total plasmid content) variation in its natural environment and its ecological role as an independent entity is the subject of another manuscript, which is currently under review. In this study, we found that the rumen plasmidome is highly diverse compared to the host microbiome, across either similar or different rumen habitats. Our analysis demonstrates that its structure is shaped more by stochasticity than selection. We studied the role of plasmids as antibiotic resistance vectors in wild and urban environments to understand the changes in the level of dispersal and persistence (manuscript is currently under review).

4. Cultivation of the rumen microbiome
Finally, we have taken a step forward and have commenced cultivation of numerous microbial components of the rumen microbiome to be able to mechanically understand plasmid mobility and community dynamics in this ecosystem by perturbing it to identify the individual effect(s) of its component parts. Our cultivation experiment captured 23% of all operational taxonomic units (OTUs) found in the rumen microbiome in this study. The use of different media increased the number of cultured OTUs by up to 40%. Sample dilution had the strongest effect on increasing richness on the plates, while abundance and phylogeny were the main factors determining cultivability of rumen microbes. Our findings from phylogenetic analysis of cultured OTUs on the lower branches of the phylogenetic tree suggest that multifactorial traits govern cultivability. Interestingly, most of our cultured OTUs belonged to the rare rumen biosphere. These cultured OTUs could not be detected in the rumen microbiome, even when we surveyed it across a 38 rumen microbiome samples (publication 6).
We have developed two novel methodologies that enables us to detect plasmids in their natural environments – the Recycler - as well as a new FISH (Fluorescence In-Situ Hybridization) method, combining single molecule FISH (smFISH), used to target mRNA molecules with single fluorophores per probe, together with Catalyzed Reporter Deposition FISH (CARD FISH), which amplifies fluorescent signals. Moreover, we have developed a method to predict the microbial community temporal dynamics based on the community composition at previous time stamps (MTV-LMM).