Metaproteome plasticity sheds light on the ecology of the rumen microbiome and its connection to host traits.

The arsenal of genes that microbes express reflect the way in which they sense their environment. We have previously reported that the rumen microbiome composition and its coding capacity are different in animals having distinct feed efficiency states, even when fed an identical diet. Here, we reveal that many microbial populations belonging to the bacteria and archaea domains show divergent proteome production in function of the feed efficiency state. Thus, proteomic data serve as a strong indicator of host feed efficiency state phenotype, overpowering predictions based on genomic and taxonomic information. We highlight protein production of specific phylogenies associated with each of the feed efficiency states. We also find remarkable plasticity of the proteome both in the individual population and at the community level, driven by niche partitioning and competition. These mechanisms result in protein production patterns that exhibit functional redundancy and checkerboard distribution that are tightly linked to the host feed efficiency phenotype. By linking microbial protein production and the ecological mechanisms that act within the microbiome feed efficiency states, our present work reveals a layer of complexity that bears immense importance to the current global challenges of food security and sustainability.

Cytokinin-microbiome interactions regulate developmental functions.

BACKGROUND: The interaction of plants with the complex microbial networks that inhabit them is important for plant health. While the reliance of plants on their microbial inhabitants for defense against invading pathogens is well documented, the acquisition of data concerning the relationships between plant developmental stage or aging, and microbiome assembly, is still underway. The plant hormone cytokinin (CK) regulates various plant growth and developmental processes. Here, examining the relationships between plant development and microbiome assembly, we observed developmental-age dependent changes in the phyllopshere microbiome. We show that age-related shifts in microbiome content vary based on content of, or sensitivity to, CK. RESULTS: We found a developmental age associated decline in microbial richness and diversity, accompanied by a decline in the presence of growth promoting and resistance inducing Bacilli in the phyllosphere. This decline was absent from CK-rich or CK-hypersensitive genotypes. Bacillus isolates we obtained from CK rich genotypes were found to alter the expression of developmental genes to support morphogenesis and alter the leaf developmental program when applied to seedlings, and enhance yield and agricultural productivity when applied to mature plants. CONCLUSIONS: Our results support the notion that CK supports developmental functions in part via the bacterial community.

Cytokinin drives assembly of the phyllosphere microbiome and promotes disease resistance through structural and chemical cues.

The plant hormone cytokinin (CK) is an important developmental regulator, promoting morphogenesis and delaying differentiation and senescence. From developmental processes, to growth, to stress tolerance, CKs are central in plant life. CKs are also known to mediate plant immunity and disease resistance, and several classes of microbes can also produce CKs, affecting the interaction with their plant hosts. While host species and genotype can be a driving force in shaping the plant microbiome, how plant developmental hormones such as CK can shape the microbiome is largely uninvestigated. Here, we examined the relationship between CK and the phyllosphere microbiome, finding that CK acts as a selective force in microbiome assembly, increasing richness, and promoting the presence of Firmicutes. CK-mediated immunity was found to partially depend on the microbial community, and bacilli isolated from previously described CK-rich plant genotypes, which overexpress a CK biosynthesis gene or have increased CK sensitivity, induced plant immunity, and promoted disease resistance. Using a biomimetic system, we investigated the relationship between the leaf microstructure, which is differentially patterned upon changes in CK content or signaling, and the growth of different phyllosphere microbes. We found that leaf structures derived from CK-rich plant genotypes support bacilli in the biomimetic system. CK was able to promote the growth, swarming, and biofilm formation of immunity inducing bacillus isolates in vitro. Overall, our results indicate that host genotype and hormonal profiles can act as a strong selective force in microbiome assembly, underlying differential immunity profiles, and pathogen resistance as a result.

Concepts and Consequences of a Core Gut Microbiota for Animal Growth and Development.

Animal microbiomes are occasionally considered as an extension of host anatomy, physiology, and even their genomic architecture. Their compositions encompass variable and constant portions when examined across multiple hosts. The latter, termed the core microbiome, is viewed as more accommodated to its host environment and suggested to benefit host fitness. Nevertheless, discrepancies in its definitions, characteristics, and importance to its hosts exist across studies. We survey studies that characterize the core microbiome, detail its current definitions and available methods to identify it, and emphasize the crucial need to upgrade and standardize the methodologies among studies. We highlight ruminants as a case study and discussthe link between the core microbiome and host physiology and genetics, as well as potential factors that shape it. We conclude with main directives of action to better understand the host-core microbiome axis and acquire the necessary insights into its controlled modulation.

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem.

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community-either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.

A method to the madness: Disentangling the individual forces that shape the rumen microbiome.

The rumen microbiome - a remarkable example of obligatory symbiosis with high ecological and social relevance.

Currency, Exchange, and Inheritance in the Evolution of Symbiosis.

Symbiotic interactions between eukaryotes and prokaryotes are widespread in nature. Here we offer a conceptual framework to study the evolutionary origins and ecological circumstances of species in beneficial symbiosis. We posit that mutual symbiotic interactions are well described by three elements: a currency, the mechanism of currency exchange, and mechanisms of symbiont inheritance. Each of these elements may be at the origin of symbiosis, with the other elements developing with time. The identity of currency in symbiosis depends on the ecological context of the symbiosis, while the specificity of the exchange mechanism underlies molecular adaptations for the symbiosis. The inheritance regime determines the degree of partner dependency and the symbiosis evolutionary trajectory. Focusing on these three elements, we review examples and open questions in the research on symbiosis.

Exploring the Prokaryotic Community Associated With the Rumen Ciliate Protozoa Population.

Ciliate protozoa are an integral part of the rumen microbiome and were found to exert a large effect on the rumen ecosystem itself as well as their host animal physiology. Part of these effects have been attributed to their ability to harbor a diverse ecto- and endo-symbiotic community of prokaryotic cells. Studies on the relationship between the protozoa population and their associated prokaryotic community in the rumen mainly focused on the methanogens, revealing that protozoa play a major role in enhancing methanogenesis potential. In contrast, little is known about the composition and function of the bacteria associated with rumen protozoa and the extent of this association. In this study, we characterize the prokaryotic communities associated with different protozoa populations and compare their structure to the free-living prokaryotic population residing in the cow rumen. We show that the overall protozoa associated prokaryotic community structure differs significantly compared to the free-living community in terms of richness and composition. The methanogens proportion was significantly higher in all protozoa populations compared to the free-living fraction, while the Lachnospiraceae was the most prevalent bacterial family in the protozoa associated bacterial communities. Several taxa not detected or detected in extremely low abundance in the free-living community were enriched in the protozoa associated bacterial community. These include members of the Endomicrobia class, previously identified as protozoa symbionts in the termite gut. Our results show that rumen protozoa harbor prokaryotic communities that are compositionally different from their surroundings, which may be the result of specific tropism between the prokaryotic community and protozoa.

Compositional and functional dynamics of the bovine rumen methanogenic community across different developmental stages.

Methanogenic archaea in the bovine rumen are responsible for the reduction of carbon molecules to methane, using various electron donors and driving the electron flow across the microbial food webs. Thus, methanogens play a key role in sustaining rumen metabolism and function. Research of rumen methanogenic archaea typically focuses on their composition and function in mature animals, while studies of early colonization and functional establishment remain scarce. Here, we investigated the metabolic potential and taxonomic composition of the methanogenic communities across different rumen developmental stages. We discovered that the methanogenesis process changes with age and that the early methanogenic community is characterized by a high activity of methylotrophic methanogenesis, likely performed by members of the order Methanosarcinales, exclusively found in young rumen. In contrast, higher hydrogenotrophic activity was observed in the mature rumen, where a higher proportion of exclusively hydrogenotrophic taxa are found. These findings 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. This study provides a better understanding of the compositional and metabolic changes that occur in the rumen microbiome from its initial stages of colonization and throughout the animals' life.

Evaluation of automated ribosomal intergenic spacer analysis for bacterial fingerprinting of rumen microbiome compared to pyrosequencing technology.

The mammalian gut houses a complex microbial community which is believed to play a significant role in host physiology. In recent years, several microbial community analysis methods have been implemented to study the whole gut microbial environment, in contrast to classical microbiological methods focusing on bacteria which can be cultivated. One of these is automated ribosomal intergenic spacer analysis (ARISA), an inexpensive and popular way of analyzing bacterial diversity and community fingerprinting in ecological samples. ARISA uses the natural variability in length of the DNA fragment found between the 16S and 23S genes in different bacterial lineages to infer diversity. This method is now being supplanted by affordable next-generation sequencing technologies that can also simultaneously annotate operational taxonomic units for taxonomic identification. We compared ARISA and pyrosequencing of samples from the rumen microbiome of cows, previously sampled at different stages of development and varying in microbial complexity using several ecological parameters. We revealed close agreement between ARISA and pyrosequencing outputs, especially in their ability to discriminate samples from different ecological niches. In contrast, the ARISA method seemed to underestimate sample richness. The good performance of the relatively inexpensive ARISA makes it relevant for straightforward use in bacterial fingerprinting analysis as well as for quick cross-validation of pyrosequencing data.

Potential role of the bovine rumen microbiome in modulating milk composition and feed efficiency.

Ruminants are completely dependent on their microbiota for feed digestion and consequently, their viability. It is therefore tempting to hypothesize a connection between the composition and abundance of resident rumen bacterial taxa and the physiological parameters of the host. Using a pyrosequencing approach, we characterized the rumen bacterial community composition in 15 dairy cows and their physiological parameters. We analyzed the degree of divergence between the different animals and found that some physiological parameters, such as milk yield and composition, are highly correlated with the abundance of various bacterial members of the rumen microbiome. One apparent finding was a strong correlation between the ratio of the phyla Firmicutes to Bacteroidetes and milk-fat yield. These findings paralleled human studies showing similar trends of increased adiposity with an increase in Bacteroidetes. This correlation remained evident at the genus level, where several genera showed correlations with the animals' physiological parameters. This suggests that the bacterial community has a role in shaping host physiological parameters. A deeper understanding of this process may allow us to modulate the rumen microbiome for better agricultural yield through bacterial community design.

Involvement of dietary salt in shaping bacterial communities in European sea bass (Dicentrarchus labrax).

Bacteria associated with the digestive tract of multicellular organisms have been shown to play a major role in their hosts' functioning. In fish, it has been proposed that food fermentation occurs inside the pyloric ceca, pouch like organs found in their digestive tract. However, this notion remains controversial. Furthermore, changes in pyloric cecal bacterial populations under different diets have yet to be demonstrated in fish. In this study, we explore the changes occurring in the bacterial community residing in the pyloric ceca of carnivorous fish fed different diets, which were shown to induce different growth rates. Our results revealed that different diets do indeed induce distinct bacterial compositions within the pyloric ceca. We found that, when salt was added to a low fish meal diet, the bacterial changes were accompanied by a significant enhancement in weight gain, hinting at a possible involvement of the bacterial community in energy harvest.

Exploring the bovine rumen bacterial community from birth to adulthood.

The mammalian gut microbiota is essential in shaping many of its host's functional attributes. One such microbiota resides in the bovine digestive tract in a compartment termed as the rumen. The rumen microbiota is necessary for the proper physiological development of the rumen and for the animal's ability to digest and convert plant mass into food products, making it highly significant to humans. The establishment of this microbial population and the changes occurring with the host's age are important for understanding this key microbial community. Despite its importance, little information about colonization of the microbial populations in newborn animals, and the gradual changes occurring thereafter, exists. Here, we characterized the overall bovine ruminal bacterial populations of five age groups, from 1-day-old calves to 2-year-old cows. We describe the changes occurring in the rumen ecosystem after birth, reflected by a decline in aerobic and facultative anaerobic taxa and an increase in anaerobic ones. Some rumen bacteria that are essential for mature rumen function could be detected as early as 1 day after birth, long before the rumen is active or even before ingestion of plant material occurs. The diversity and within-group similarity increased with age, suggesting a more diverse but homogeneous and specific mature community, compared with the more heterogeneous and less diverse primary community. In addition, a convergence toward a mature bacterial arrangement with age was observed. These findings have also been reported for human gut microbiota, suggesting that similar forces drive the establishment of gut microbiotas in these two distinct mammalian digestive systems.

Similarity of the ruminal bacteria across individual lactating cows.

Dairy cattle hold enormous significance for man as a source of milk and meat. Their remarkable ability to convert indigestible plant mass into these digestible food products resides in the rumen - an anaerobic chambered compartment - in the bovine digestive system. The rumen houses a complex microbiota which is responsible for the degradation of plant material, consequently enabling the conversion of plant fibers into milk and meat and determining their quality and quantity. Hence, an understanding of this complex ecosystem has major economic implications. One important question that is yet to be addressed is the degree of conservation of rumen microbial composition across individual animals. Here we quantified the degree of similarity between rumen bacterial populations of 16 individual cows. We used real-time PCR to determine the variance of specific ruminal bacterial species with different metabolic functions, revealing that while some bacterial strains vary greatly across animals, others show only very low variability. This variance could not be linked to the metabolic traits of these bacteria. We examined the degree of similarity in the dominant bacterial populations across all animals using automated ribosomal intergenic spacer analysis (ARISA), and identified a bacterial community consisting of 32% operational taxonomic units (OTUs) shared by at least 90% of the animals and 19% OTUs shared by 100% of the animals. Looking only at the presence or absence of each OTU gave an average similarity of 75% between each cow pair. When abundance of each OTU was added to the analysis, this similarity decreased to an average of less than 60%. Thus, as suggested in similar recent studies of the human gut, a bovine rumen core microbiome does exist, but taxa abundance may vary greatly across animals.

Insights into the bovine rumen plasmidome.

Plasmids are self-replicating genetic elements capable of mobilization between different hosts. Plasmids often serve as mediators of lateral gene transfer, a process considered to be a strong and sculpting evolutionary force in microbial environments. Our aim was to characterize the overall plasmid population in the environment of the bovine rumen, which houses a complex and dense microbiota that holds enormous significance for humans. We developed a procedure for the isolation of total rumen plasmid DNA, termed rumen plasmidome, and subjected it to deep sequencing using the Illumina paired-end protocol and analysis using public and custom-made bioinformatics tools. A large number of plasmidome contigs aligned with plasmids of rumen bacteria isolated from different locations and at various time points, suggesting that not only the bacterial taxa, but also their plasmids, are defined by the ecological niche. The bacterial phylum distribution of the plasmidome was different from that of the rumen bacterial taxa. Nevertheless, both shared a dominance of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. Evidently, the rumen plasmidome is of a highly mosaic nature that can cross phyla. Interestingly, when we compared the functional profile of the rumen plasmidome to two plasmid databases and two recently published rumen metagenomes, it became apparent that the rumen plasmidome codes for functions, which are enriched in the rumen ecological niche and could confer advantages to their hosts, suggesting that the functional profiles of mobile genetic elements are associated with their environment, as has been previously implied for viruses.

Composition and similarity of bovine rumen microbiota across individual animals.

The bovine rumen houses a complex microbiota which is responsible for cattle's remarkable ability to convert indigestible plant mass into food products. Despite this ecosystem's enormous significance for humans, the composition and similarity of bacterial communities across different animals and the possible presence of some bacterial taxa in all animals' rumens have yet to be determined. We characterized the rumen bacterial populations of 16 individual lactating cows using tag amplicon pyrosequencing. Our data showed 51% similarity in bacterial taxa across samples when abundance and occurrence were analyzed using the Bray-Curtis metric. By adding taxon phylogeny to the analysis using a weighted UniFrac metric, the similarity increased to 82%. We also counted 32 genera that are shared by all samples, exhibiting high variability in abundance across samples. Taken together, our results suggest a core microbiome in the bovine rumen. Furthermore, although the bacterial taxa may vary considerably between cow rumens, they appear to be phylogenetically related. This suggests that the functional requirement imposed by the rumen ecological niche selects taxa that potentially share similar genetic features.