Spatially structured microbial consortia and their role in food fermentations.

Microbial consortia are important for the fermentation of foods. They bring combined functionalities to the fermented product, but stability and product consistency of fermentations with complex consortia can be hard to control. Some of these consortia, such as water- and milk-kefir and kombucha, grow as multispecies aggregates or biofilms, in which micro-organisms taking part in a fermentation cascade are spatially organized. The spatial organization of micro-organisms in these aggregates can impact what metabolic interactions are realized in the consortia, ultimately affecting the growth dynamics and evolution of microbes. A better understanding of such spatially structured communities is of interest from the perspective of microbial ecology and biotechnology, as multispecies aggregates can be used to valorize energy-rich substrates, such as plant-based substrates or side streams from the food industry.

Targeted hypermutation of putative antigen sensors in multicellular bacteria.

Diversity-generating retroelements (DGRs) are used by bacteria, archaea, and viruses as a targeted mutagenesis tool. Through error-prone reverse transcription, DGRs introduce random mutations at specific genomic loci, enabling rapid evolution of these targeted genes. However, the function and benefits of DGR-diversified proteins in cellular hosts remain elusive. We find that 82% of DGRs from one of the major monophyletic lineages of DGR reverse transcriptases are encoded by multicellular bacteria, which often have two or more DGR loci in their genomes. Using the multicellular purple sulfur bacterium Thiohalocapsa sp. PB-PSB1 as an example, we characterized nine distinct DGR loci capable of generating 10282 different combinations of target proteins. With environmental metagenomes from individual Thiohalocapsa aggregates, we show that most of PB-PSB1's DGR target genes are diversified across its biogeographic range, with spatial heterogeneity in the diversity of each locus. In Thiohalocapsa PB-PSB1 and other bacteria hosting this lineage of cellular DGRs, the diversified target genes are associated with NACHT-domain anti-phage defenses and putative ternary conflict systems previously shown to be enriched in multicellular bacteria. We propose that these DGR-diversified targets act as antigen sensors that confer a form of adaptive immunity to their multicellular consortia, though this remains to be experimentally tested. These findings could have implications for understanding the evolution of multicellularity, as the NACHT-domain anti-phage systems and ternary systems share both domain homology and conceptual similarities with the innate immune and programmed cell death pathways of plants and metazoans.

Interspecies interactions determine growth dynamics of biopolymer-degrading populations in microbial communities.

Microbial communities perform essential ecosystem functions such as the remineralization of organic carbon that exists as biopolymers. The first step in mineralization is performed by biopolymer degraders, which harbor enzymes that can break down polymers into constituent oligo- or monomeric forms. The released nutrients not only allow degraders to grow, but also promote growth of cells that either consume the degradation products, i.e., exploiters, or consume metabolites released by the degraders or exploiters, i.e., scavengers. It is currently not clear how such remineralizing communities assemble at the microscale-how interactions between the different guilds influence their growth and spatial distribution, and hence the development and dynamics of the community. Here, we address this knowledge gap by studying marine microbial communities that grow on the abundant marine biopolymer alginate. We used batch growth assays and microfluidics coupled to time-lapse microscopy to quantitatively investigate growth and spatial distribution of single cells. We found that the presence of exploiters or scavengers alters the spatial distribution of degrader cells. In general, exploiters and scavengers-which we collectively refer to as cross-feeder cells-slowed down the growth of degrader cells. In addition, coexistence with cross-feeders altered the production of the extracellular enzymes that break down polymers by degrader cells. Our findings reveal that ecological interactions by nondegrading community members have a profound impact on the functions of microbial communities that remineralize carbon biopolymers in nature.

Genome content predicts the carbon catabolic preferences of heterotrophic bacteria.

Heterotrophic bacteria-bacteria that utilize organic carbon sources-are taxonomically and functionally diverse across environments. It is challenging to map metabolic interactions and niches within microbial communities due to the large number of metabolites that could serve as potential carbon and energy sources for heterotrophs. Whether their metabolic niches can be understood using general principles, such as a small number of simplified metabolic categories, is unclear. Here we perform high-throughput metabolic profiling of 186 marine heterotrophic bacterial strains cultured in media containing one of 135 carbon substrates to determine growth rates, lag times and yields. We show that, despite high variability at all levels of taxonomy, the catabolic niches of heterotrophic bacteria can be understood in terms of their preference for either glycolytic (sugars) or gluconeogenic (amino and organic acids) carbon sources. This preference is encoded by the total number of genes found in pathways that feed into the two modes of carbon utilization and can be predicted using a simple linear model based on gene counts. This allows for coarse-grained descriptions of microbial communities in terms of prevalent modes of carbon catabolism. The sugar-acid preference is also associated with genomic GC content and thus with the carbon-nitrogen requirements of their encoded proteome. Our work reveals how the evolution of bacterial genomes is structured by fundamental constraints rooted in metabolism.

Chitin utilization by marine picocyanobacteria and the evolution of a planktonic lifestyle.

Marine picocyanobacteria Prochlorococcus and Synechococcus, the most abundant photosynthetic cells in the oceans, are generally thought to have a primarily single-celled and free-living lifestyle. However, while studying the ability of picocyanobacteria to supplement photosynthetic carbon fixation with the use of exogenous organic carbon, we found the widespread occurrence of genes for breaking down chitin, an abundant source of organic carbon that exists primarily as particles. We show that cells that encode a chitin degradation pathway display chitin degradation activity, attach to chitin particles, and show enhanced growth under low light conditions when exposed to chitosan, a partially deacetylated soluble form of chitin. Marine chitin is largely derived from arthropods, which underwent major diversifications 520 to 535 Mya, close to when marine picocyanobacteria are inferred to have appeared in the ocean. Phylogenetic analyses confirm that the chitin utilization trait was acquired at the root of marine picocyanobacteria. Together this leads us to postulate that attachment to chitin particles allowed benthic cyanobacteria to emulate their mat-based lifestyle in the water column, initiating their expansion into the open ocean, seeding the rise of modern marine ecosystems. Subsequently, transitioning to a constitutive planktonic life without chitin associations led to cellular and genomic streamlining along a major early branch within Prochlorococcus. Our work highlights how the emergence of associations between organisms from different trophic levels, and their coevolution, creates opportunities for colonizing new environments. In this view, the rise of ecological complexity and the expansion of the biosphere are deeply intertwined processes.

Stress-induced metabolic exchanges between complementary bacterial types underly a dynamic mechanism of inter-species stress resistance.

Metabolic cross-feeding plays vital roles in promoting ecological diversity. While some microbes depend on exchanges of essential nutrients for growth, the forces driving the extensive cross-feeding needed to support the coexistence of free-living microbes are poorly understood. Here we characterize bacterial physiology under self-acidification and establish that extensive excretion of key metabolites following growth arrest provides a collaborative, inter-species mechanism of stress resistance. This collaboration occurs not only between species isolated from the same community, but also between unrelated species with complementary (glycolytic vs. gluconeogenic) modes of metabolism. Cultures of such communities progress through distinct phases of growth-dilution cycles, comprising of exponential growth, acidification-triggered growth arrest, collaborative deacidification, and growth recovery, with each phase involving different combinations of physiological states of individual species. Our findings challenge the steady-state view of ecosystems commonly portrayed in ecological models, offering an alternative dynamical view based on growth advantages of complementary species in different phases.

Mutation-induced infections of phage-plasmids.

Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages, whose eco-evolutionary dynamics remain poorly constrained. Here, we show that segregational drift and loss-of-function mutations play key roles in the infection dynamics of a cosmopolitan phage-plasmid, allowing it to create continuous productive infections in a population of marine Roseobacter. Recurrent loss-of-function mutations in the phage repressor that controls prophage induction leads to constitutively lytic phage-plasmids that spread rapidly throughout the population. The entire phage-plasmid genome is packaged into virions, which were horizontally transferred by re-infecting lysogenized cells, leading to an increase in phage-plasmid copy number and to heterozygosity in a phage repressor locus in re-infected cells. However, the uneven distribution of phage-plasmids after cell division (i.e., segregational drift) leads to the production of offspring carrying only the constitutively lytic phage-plasmid, thus restarting the lysis-reinfection-segregation life cycle. Mathematical models and experiments show that these dynamics lead to a continuous productive infection of the bacterial population, in which lytic and lysogenic phage-plasmids coexist. Furthermore, analyses of marine bacterial genome sequences indicate that the plasmid backbone here can carry different phages and disseminates trans-continentally. Our study highlights how the interplay between phage infection and plasmid genetics provides a unique eco-evolutionary strategy for phage-plasmids.

Annotation-free discovery of functional groups in microbial communities.

Recent studies have shown that microbial communities are composed of groups of functionally cohesive taxa whose abundance is more stable and better-associated with metabolic fluxes than that of any individual taxon. However, identifying these functional groups in a manner that is independent of error-prone functional gene annotations remains a major open problem. Here we tackle this structure-function problem by developing a novel unsupervised approach that coarse-grains taxa into functional groups, solely on the basis of the patterns of statistical variation in species abundances and functional read-outs. We demonstrate the power of this approach on three distinct datasets. On data of replicate microcosms with heterotrophic soil bacteria, our unsupervised algorithm recovered experimentally validated functional groups that divide metabolic labour and remain stable despite large variation in species composition. When leveraged against the ocean microbiome data, our approach discovered a functional group that combines aerobic and anaerobic ammonia oxidizers whose summed abundance tracks closely with nitrate concentrations in the water column. Finally, we show that our framework can enable the detection of species groups that are probably responsible for the production or consumption of metabolites abundant in animal gut microbiomes, serving as a hypothesis-generating tool for mechanistic studies. Overall, this work advances our understanding of structure-function relationships in complex microbiomes and provides a powerful approach to discover functional groups in an objective and systematic manner.

A Genome-Scale Metabolic Model of Marine Heterotroph Vibrio splendidus Strain 1A01.

While Vibrio splendidus is best known as an opportunistic pathogen in oysters, Vibrio splendidus strain 1A01 was first identified as an early colonizer of synthetic chitin particles incubated in seawater. To gain a better understanding of its metabolism, a genome-scale metabolic model (GSMM) of V. splendidus 1A01 was reconstructed. GSMMs enable us to simulate all metabolic reactions in a bacterial cell using flux balance analysis. A draft model was built using an automated pipeline from BioCyc. Manual curation was then performed based on experimental data, in part by gap-filling metabolic pathways and tailoring the model's biomass reaction to V. splendidus 1A01. The challenges of building a metabolic model for a marine microorganism like V. splendidus 1A01 are described. IMPORTANCE A genome-scale metabolic model of V. splendidus 1A01 was reconstructed in this work. We offer solutions to the technical problems associated with model reconstruction for a marine bacterial strain like V. splendidus 1A01, which arise largely from the high salt concentration found in both seawater and culture media that simulate seawater.

Viral infection switches the balance between bacterial and eukaryotic recyclers of organic matter during coccolithophore blooms.

Algal blooms are hotspots of marine primary production and play central roles in microbial ecology and global elemental cycling. Upon demise of the bloom, organic carbon is partly respired and partly transferred to either higher trophic levels, bacterial biomass production or sinking. Viral infection can lead to bloom termination, but its impact on the fate of carbon remains largely unquantified. Here, we characterize the interplay between viral infection and the composition of a bloom-associated microbiome and consequently the evolving biogeochemical landscape, by conducting a large-scale mesocosm experiment where we monitor seven induced coccolithophore blooms. The blooms show different degrees of viral infection and reveal that only high levels of viral infection are followed by significant shifts in the composition of free-living bacterial and eukaryotic assemblages. Intriguingly, upon viral infection the biomass of eukaryotic heterotrophs (thraustochytrids) rivals that of bacteria as potential recyclers of organic matter. By combining modeling and quantification of active viral infection at a single-cell resolution, we estimate that viral infection causes a 2-4 fold increase in per-cell rates of extracellular carbon release in the form of acidic polysaccharides and particulate inorganic carbon, two major contributors to carbon sinking into the deep ocean. These results reveal the impact of viral infection on the fate of carbon through microbial recyclers of organic matter in large-scale coccolithophore blooms.

Historical contingencies and phage induction diversify bacterioplankton communities at the microscale.

In many natural environments, microorganisms decompose microscale resource patches made of complex organic matter. The growth and collapse of populations on these resource patches unfold within spatial ranges of a few hundred micrometers or less, making such microscale ecosystems hotspots of heterotrophic metabolism. Despite the potential importance of patch-level dynamics for the large-scale functioning of heterotrophic microbial communities, we have not yet been able to delineate the ecological processes that control natural populations at the microscale. Here, we address this challenge by characterizing the natural marine communities that assembled on over 1,000 individual microscale particles of chitin, the most abundant marine polysaccharide. Using low-template shotgun metagenomics and imaging, we find significant variation in microscale community composition despite the similarity in initial species pools across replicates. Chitin-degrading taxa that were rare in seawater established large populations on a subset of particles, resulting in a wide range of predicted chitinolytic abilities and biomass at the level of individual particles. We show, through a mathematical model, that this variability can be attributed to stochastic colonization and historical contingencies affecting the tempo of growth on particles. We find evidence that one biological process leading to such noisy growth across particles is differential predation by temperate bacteriophages of chitin-degrading strains, the keystone members of the community. Thus, initial stochasticity in assembly states on individual particles, amplified through ecological interactions, may have significant consequences for the diversity and functionality of systems of microscale patches.

Bacterial growth in multicellular aggregates leads to the emergence of complex life cycles.

Facultative multicellular behaviors expand the metabolic capacity and physiological resilience of bacteria. Despite their ubiquity in nature, we lack an understanding of how these behaviors emerge from cellular-scale phenomena. Here, we show how the coupling between growth and resource gradient formation leads to the emergence of multicellular lifecycles in a marine bacterium. Under otherwise carbon-limited growth conditions, Vibrio splendidus 12B01 forms clonal multicellular groups to collectively harvest carbon from soluble polymers of the brown-algal polysaccharide alginate. As they grow, groups phenotypically differentiate into two spatially distinct sub-populations: a static "shell" surrounding a motile, carbon-storing "core." Differentiation of these two sub-populations coincides with the formation of a gradient in nitrogen-source availability within clusters. Additionally, we find that populations of cells containing a high proportion of carbon-storing individuals propagate and form new clusters more readily on alginate than do populations with few carbon-storing cells. Together, these results suggest that local metabolic activity and differential partitioning of resources leads to the emergence of reproductive cycles in a facultatively multicellular bacterium.

Turnover in Life-Strategies Recapitulates Marine Microbial Succession Colonizing Model Particles.

Particulate organic matter (POM) in the ocean sustains diverse communities of bacteria that mediate the remineralization of organic complex matter. However, the variability of these particles and of the environmental conditions surrounding them present a challenge to the study of the ecological processes shaping particle-associated communities and their function. In this work, we utilize data from experiments in which coastal water communities are grown on synthetic particles to ask which are the most important ecological drivers of their assembly and associated traits. Combining 16S rRNA amplicon sequencing with shotgun metagenomics, together with an analysis of the full genomes of a subset of isolated strains, we were able to identify two-to-three distinct community classes, corresponding to early vs. late colonizers. We show that these classes are shaped by environmental selection (early colonizers) and facilitation (late colonizers) and find distinctive traits associated with each class. While early colonizers have a larger proportion of genes related to the uptake of nutrients, motility, and environmental sensing with few pathways enriched for metabolism, late colonizers devote a higher proportion of genes for metabolism, comprising a wide array of different pathways including the metabolism of carbohydrates, amino acids, and xenobiotics. Analysis of selected pathways suggests the existence of a trophic-chain topology connecting both classes for nitrogen metabolism, potential exchange of branched chain amino acids for late colonizers, and differences in bacterial doubling times throughout the succession. The interpretation of these traits suggests a distinction between early and late colonizers analogous to other classifications found in the literature, and we discuss connections with the classical distinction between r- and K-strategists.

Microbes contribute to setting the ocean carbon flux by altering the fate of sinking particulates.

Sinking particulate organic carbon out of the surface ocean sequesters carbon on decadal to millennial timescales. Predicting the particulate carbon flux is therefore critical for understanding both global carbon cycling and the future climate. Microbes play a crucial role in particulate organic carbon degradation, but the impact of depth-dependent microbial dynamics on ocean-scale particulate carbon fluxes is poorly understood. Here we scale-up essential features of particle-associated microbial dynamics to understand the large-scale vertical carbon flux in the ocean. Our model provides mechanistic insight into the microbial contribution to the particulate organic carbon flux profile. We show that the enhanced transfer of carbon to depth can result from populations struggling to establish colonies on sinking particles due to diffusive nutrient loss, cell detachment, and mortality. These dynamics are controlled by the interaction between multiple biotic and abiotic factors. Accurately capturing particle-microbe interactions is essential for predicting variability in large-scale carbon cycling.

Particle foraging strategies promote microbial diversity in marine environments.

Microbial foraging in patchy environments, where resources are fragmented into particles or pockets embedded in a large matrix, plays a key role in natural environments. In the oceans and freshwater systems, particle-associated bacteria can interact with particle surfaces in different ways: some colonize only during short transients, while others form long-lived, stable colonies. We do not yet understand the ecological mechanisms by which both short- and long-term colonizers can coexist. Here, we address this problem with a mathematical model that explains how marine populations with different detachment rates from particles can stably coexist. In our model, populations grow only while on particles, but also face the increased risk of mortality by predation and sinking. Key to coexistence is the idea that detachment from particles modulates both net growth and mortality, but in opposite directions, creating a trade-off between them. While slow-detaching populations show the highest growth return (i.e., produce more net offspring), they are more susceptible to suffer higher rates of mortality than fast-detaching populations. Surprisingly, fluctuating environments, manifesting as blooms of particles (favoring growth) and predators (favoring mortality) significantly expand the likelihood that populations with different detachment rates can coexist. Our study shows how the spatial ecology of microbes in the ocean can lead to a predictable diversification of foraging strategies and the coexistence of multiple taxa on a single growth-limiting resource.

Interactions between strains govern the eco-evolutionary dynamics of microbial communities.

Genomic data has revealed that genotypic variants of the same species, that is, strains, coexist and are abundant in natural microbial communities. However, it is not clear if strains are ecologically equivalent, and at what characteristic genetic distance they might exhibit distinct interactions and dynamics. Here, we address this problem by tracking 10 taxonomically diverse microbial communities from the pitcher plant Sarracenia purpurea in the laboratory for more than 300 generations. Using metagenomic sequencing, we reconstruct their dynamics over time and across scales, from distant phyla to closely related genotypes. We find that most strains are not ecologically equivalent and exhibit distinct dynamical patterns, often being significantly more correlated with strains from another species than their own. Although even a single mutation can affect laboratory strains, on average, natural strains typically decouple in their dynamics beyond a genetic distance of 100 base pairs. Using mathematical consumer-resource models, we show that these taxonomic patterns emerge naturally from ecological interactions between community members, but only if the interactions are coarse-grained at the level of strains, not species. Finally, by analyzing genomic differences between strains, we identify major functional hubs such as transporters, regulators, and carbohydrate-catabolizing enzymes, which might be the basis for strain-specific interactions. Our work suggests that fine-scale genetic differences in natural communities could be created and stabilized via the rapid diversification of ecological interactions between strains.

Metabolic cross-feeding structures the assembly of polysaccharide degrading communities.

Metabolic processes that fuel the growth of heterotrophic microbial communities are initiated by specialized biopolymer degraders that decompose complex forms of organic matter. It is unclear, however, to what extent degraders structure the downstream assembly of the community that follows polymer breakdown. Investigating a model marine microbial community that degrades chitin, we show that chitinases secreted by different degraders produce oligomers of specific chain lengths that not only select for specialized consumers but also influence the metabolites secreted by these consumers into a shared resource pool. Each species participating in the breakdown cascade exhibits unique hierarchical preferences for substrates, which underlies the sequential colonization of metabolically distinct groups as resource availability changes over time. By identifying the metabolic underpinnings of microbial community assembly, we reveal a hierarchical cross-feeding structure that allows biopolymer degraders to shape the dynamics of community assembly.

Evolutionary Ecology of Natural Comammox Nitrospira Populations.

Microbes commonly exist in diverse and complex communities where species interact, and their genomic repertoires evolve over time. Our understanding of species interaction and evolution has increased during the last decades, but most studies of evolutionary dynamics are based on single species in isolation or in experimental systems composed of few interacting species. Here, we use the microbial ecosystem found in groundwater-fed sand filter as a model to avoid this limitation. In these open systems, diverse microbial communities experience relatively stable conditions, and the coupling between chemical and biological processes is generally well defined. Metagenomic analysis of 12 sand filters communities revealed systematic co-occurrence of at least five comammox Nitrospira species, likely promoted by low ammonium concentrations. These Nitrospira species showed intrapopulation sequence diversity, although possible clonal expansion was detected in a few abundant local comammox populations. Nitrospira species showed low homologous recombination and strong purifying selection, the latter process being especially strong in genes essential in energy metabolism. Positive selection was detected for genes related to resistance to foreign DNA and phages. We found that, compared to other habitats, groundwater-fed sand filters impose strong purifying selection and low recombination on comammox Nitrospira populations. These results suggest that evolutionary processes are more affected by habitat type than by species identity. Together, this study improves our understanding of species interaction and evolution in complex microbial communities and sheds light on the environmental dependency of evolutionary processes. IMPORTANCE Microbial species interact with each other and their environment (ecological processes) and undergo changes in their genomic repertoire over time (evolutionary processes). How these two classes of processes interact is largely unknown, especially for complex communities, as most studies of microbial evolutionary dynamics consider single species in isolation or a few interacting species in simplified experimental systems. In this study, these limitations are circumvented by examining the microbial communities found in stable and well-described groundwater-fed sand filters. Combining metagenomics and strain-level analyses, we identified the microbial interactions and evolutionary processes affecting comammox Nitrospira, a recently discovered bacterial type capable of performing the whole nitrification process. We found that abundant and co-occurrent Nitrospira populations in groundwater-fed sand filters are characterized by low recombination and strong purifying selection. In addition, by comparing these observations with those obtained from Nitrospira species inhabiting other environments, we revealed that evolutionary processes are more affected by habitat type than by species identity.

Polysaccharide-Bacteria Interactions From the Lens of Evolutionary Ecology.

Microbes have the unique ability to break down the complex polysaccharides that make up the bulk of organic matter, initiating a cascade of events that leads to their recycling. Traditionally, the rate of organic matter degradation is perceived to be limited by the chemical and physical structure of polymers. Recent advances in microbial ecology, however, suggest that polysaccharide persistence can result from non-linear growth dynamics created by the coexistence of alternate degradation strategies, metabolic roles as well as by ecological interactions between microbes. This complex "landscape" of degradation strategies and interspecific interactions present in natural microbial communities appears to be far from evolutionarily stable, as frequent gene gain and loss reshape enzymatic repertoires and metabolic roles. In this perspective, we discuss six challenges at the heart of this problem, ranging from the evolution of genetic repertoires, phenotypic heterogeneity in clonal populations, the development of a trait-based ecology, and the impact of metabolic interactions and microbial cooperation on degradation rates. We aim to reframe some of the key questions in the study of polysaccharide-bacteria interactions in the context of eco-evolutionary dynamics, highlighting possible research directions that, if pursued, would advance our understanding of polysaccharide degraders at the interface between biochemistry, ecology and evolution.

Nationwide genomic atlas of soil-dwelling Listeria reveals effects of selection and population ecology on pangenome evolution.

Natural bacterial populations can display enormous genomic diversity, primarily in the form of gene content variation caused by the frequent exchange of DNA with the local environment. However, the ecological drivers of genomic variability and the role of selection remain controversial. Here, we address this gap by developing a nationwide atlas of 1,854 Listeria isolates, collected systematically from soils across the contiguous United States. We found that Listeria was present across a wide range of environmental parameters, being mainly controlled by soil moisture, molybdenum and salinity concentrations. Whole-genome data from 594 representative strains allowed us to decompose Listeria diversity into 12 phylogroups, each with large differences in habitat breadth and endemism. 'Cosmopolitan' phylogroups, prevalent across many different habitats, had more open pangenomes and displayed weaker linkage disequilibrium, reflecting higher rates of gene gain and loss, and allele exchange than phylogroups with narrow habitat ranges. Cosmopolitan phylogroups also had a large fraction of genes affected by positive selection. The effect of positive selection was more pronounced in the phylogroup-specific core genome, suggesting that lineage-specific core genes are important drivers of adaptation. These results indicate that genome flexibility and recombination are the consequence of selection to survive in variable environments.