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.

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.

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.

Cooperation and spatial self-organization determine rate and efficiency of particulate organic matter degradation in marine bacteria.

The recycling of particulate organic matter (POM) by microbes is a key part of the global carbon cycle. This process is mediated by the extracellular hydrolysis of polysaccharides, which can trigger social behaviors in bacteria resulting from the production of public goods. Despite the potential importance of public good-mediated interactions, their relevance in the environment remains unclear. In this study, we developed a computational and experimental model system to address this challenge and studied how the POM depolymerization rate and its uptake efficiency (2 main ecosystem function parameters) depended on social interactions and spatial self-organization on particle surfaces. We found an emergent trade-off between rate and efficiency resulting from the competition between oligosaccharide diffusion and cellular uptake, with low rate and high efficiency being achieved through cell-to-cell cooperation between degraders. Bacteria cooperated by aggregating in cell clusters of ∼10 to 20 µm, in which cells were able to share public goods. This phenomenon, which was independent of any explicit group-level regulation, led to the emergence of critical cell concentrations below which degradation did not occur, despite all resources being available in excess. In contrast, when particles were labile and turnover rates were high, aggregation promoted competition and decreased the efficiency of carbon use. Our study shows how social interactions and cell aggregation determine the rate and efficiency of particulate carbon turnover in environmentally relevant scenarios.

Illuminating microbial species-specific effects on organic matter remineralization in marine sediments.

Marine microorganisms play a fundamental role in the global carbon cycle by mediating the sequestration of organic matter in ocean waters and sediments. A better understanding of how biological factors, such as microbial community composition, influence the lability and fate of organic matter is needed. Here, we explored the extent to which organic matter remineralization is influenced by species-specific metabolic capabilities. We carried out aerobic time-series incubations of Guaymas Basin sediments to quantify the dynamics of carbon utilization by two different heterotrophic marine isolates (Vibrio splendidus 1A01; Pseudoalteromonas sp. 3D05). Continuous measurement of respiratory CO2 production and its carbon isotopic compositions (13 C and 14 C) shows species-specific differences in the rate, quantity and type of organic matter remineralized. Each species was incubated with hydrothermally-influenced versus unimpacted sediments, resulting in a ~2-fold difference in respiratory CO2 yield across the experiments. Genomic analysis indicated that the observed carbon utilization patterns may be attributed in part to the number of gene copies encoding for extracellular hydrolytic enzymes. Our results demonstrate that the lability and remineralization of organic matter in marine environments is not only a function of chemical composition and/or environmental conditions, but also a function of the microorganisms that are present and active.

Physics-based prediction of biopolymer degradation.

In the natural environment, insoluble biomatter provides a preeminent source of carbon for bacteria. Its degradation by microbial communities thus plays a major role in the global carbon-cycle. The prediction of degradation processes and their sensitivity to changes in environmental conditions can therefore provide critical insights into globally occurring environmental adaptations. To elucidate and quantify this macro-scale phenomenon, we conduct micro-scale experiments that examine the degradation of isolated biopolymer particles and observe highly nonlinear degradation kinetics. Since conventional scaling arguments fail to explain these observations, it is inferred that the coupled influence of both the physical and biochemical processes must be considered. Hence, we develop a theoretical model that accounts for the bio-chemo-mechanically coupled kinetics of polymer degradation, by considering the production of bio-degraders and their ability to both dissociate the material from its external boundaries and to penetrate it to degrade its internal mechanical properties. This change in mechanical properties combined with the intake of solvent or moisture from the environment leads to chemo-mechanically coupled swelling of the material and, in-turn, influences the degradation kinetics. We show that the model quantitatively captures our experimental results and reveals distinct signatures of different bacteria that are independent of the specific experimental conditions (i.e. particle volume and initial concentrations). Finally, after validating our model against the experimental data we extend our predictions for degradation processes across various length and time scales that are inaccessible in a laboratory setting.

Endemic cyanophages and the puzzle of phage-bacteria coevolution.

A recent genomic analysis of Synechococcus cyanophages sampled for over 15 years reveals a remarkable pattern of stable phage population structure, highly reminiscent of the ecotype structure observed in bacteria and archaeal ecotypes. In this highlight I discuss the importance of this finding and the questions and opportunities it opens to learn more about the nature of phage-bacterial coevolution in the environment.

Ecology drives a global network of gene exchange connecting the human microbiome.

Horizontal gene transfer (HGT), the acquisition of genetic material from non-parental lineages, is known to be important in bacterial evolution. In particular, HGT provides rapid access to genetic innovations, allowing traits such as virulence, antibiotic resistance and xenobiotic metabolism to spread through the human microbiome. Recent anecdotal studies providing snapshots of active gene flow on the human body have highlighted the need to determine the frequency of such recent transfers and the forces that govern these events. Here we report the discovery and characterization of a vast, human-associated network of gene exchange, large enough to directly compare the principal forces shaping HGT. We show that this network of 10,770 unique, recently transferred (more than 99% nucleotide identity) genes found in 2,235 full bacterial genomes, is shaped principally by ecology rather than geography or phylogeny, with most gene exchange occurring between isolates from ecologically similar, but geographically separated, environments. For example, we observe 25-fold more HGT between human-associated bacteria than among ecologically diverse non-human isolates (P = 3.0 × 10(-270)). We show that within the human microbiome this ecological architecture continues across multiple spatial scales, functional classes and ecological niches with transfer further enriched among bacteria that inhabit the same body site, have the same oxygen tolerance or have the same ability to cause disease. This structure offers a window into the molecular traits that define ecological niches, insight that we use to uncover sources of antibiotic resistance and identify genes associated with the pathology of meningitis and other diseases.

Competition-dispersal tradeoff ecologically differentiates recently speciated marine bacterioplankton populations.

Although competition-dispersal tradeoffs are commonly invoked to explain species coexistence for animals and plants in spatially structured environments, such mechanisms for coexistence remain unknown for microorganisms. Here we show that two recently speciated marine bacterioplankton populations pursue different behavioral strategies to exploit nutrient particles in adaptation to the landscape of ephemeral nutrient patches characteristic of ocean water. These differences are mediated primarily by differential colonization of and dispersal among particles. Whereas one population is specialized to colonize particles by attaching and growing biofilms, the other is specialized to disperse among particles by rapidly detecting and swimming toward new particles, implying that it can better exploit short-lived patches. Because the two populations are very similar in their genomic composition, metabolic abilities, chemotactic sensitivity, and swimming speed, this fine-scale behavioral adaptation may have been responsible for the onset of the ecological differentiation between them. These results demonstrate that the principles of spatial ecology, traditionally applied at macroscales, can be extended to the ocean's microscale to understand how the rich spatiotemporal structure of the resource landscape contributes to the fine-scale ecological differentiation and species coexistence among marine bacteria.

Gene-specific selective sweeps in bacteria and archaea caused by negative frequency-dependent selection.

BACKGROUND: Fixation of beneficial genes in bacteria and archaea (collectively, prokaryotes) is often believed to erase pre-existing genomic diversity through the hitchhiking effect, a phenomenon known as genome-wide selective sweep. Recent studies, however, indicate that beneficial genes spread through a prokaryotic population via recombination without causing genome-wide selective sweeps. These gene-specific selective sweeps seem to be at odds with the existing estimates of recombination rates in prokaryotes, which appear far too low to explain such phenomena. RESULTS: We use mathematical modeling to investigate potential solutions to this apparent paradox. Most microbes in nature evolve in heterogeneous, dynamic communities, in which ecological interactions can substantially impact evolution. Here, we focus on the effect of negative frequency-dependent selection (NFDS) such as caused by viral predation (kill-the-winner dynamics). The NFDS maintains multiple genotypes within a population, so that a gene beneficial to every individual would have to spread via recombination, hence a gene-specific selective sweep. However, gene loci affected by NFDS often are located in variable regions of microbial genomes that contain genes involved in the mobility of selfish genetic elements, such as integrases or transposases. Thus, the NFDS-affected loci are likely to experience elevated rates of recombination compared with the other loci. Consequently, these loci might be effectively unlinked from the rest of the genome, so that NFDS would be unable to prevent genome-wide selective sweeps. To address this problem, we analyzed population genetic models of selective sweeps in prokaryotes under NFDS. The results indicate that NFDS can cause gene-specific selective sweeps despite the effect of locally elevated recombination rates, provided NFDS affects more than one locus and the basal rate of recombination is sufficiently low. Although these conditions might seem to contradict the intuition that gene-specific selective sweeps require high recombination rates, they actually decrease the effective rate of recombination at loci affected by NFDS relative to the per-locus basal level, so that NFDS can cause gene-specific selective sweeps. CONCLUSION: Because many free-living prokaryotes are likely to evolve under NFDS caused by ubiquitous viruses, gene-specific selective sweeps driven by NFDS are expected to be a major, general phenomenon in prokaryotic populations.

Public good dynamics drive evolution of iron acquisition strategies in natural bacterioplankton populations.

A common strategy among microbes living in iron-limited environments is the secretion of siderophores, which can bind poorly soluble iron and make it available to cells via active transport mechanisms. Such siderophore-iron complexes can be thought of as public goods that can be exploited by local communities and drive diversification, for example by the evolution of "cheating." However, it is unclear whether bacterial populations in the environment form stable enough communities such that social interactions significantly impact evolutionary dynamics. Here we show that public good games drive the evolution of iron acquisition strategies in wild populations of marine bacteria. We found that within nonclonal but ecologically cohesive genotypic clusters of closely related Vibrionaceae, only an intermediate percentage of genotypes are able to produce siderophores. Nonproducers within these clusters exhibited selective loss of siderophore biosynthetic pathways, whereas siderophore transport mechanisms were retained, suggesting that these nonproducers can act as cheaters that benefit from siderophore producers in their local environment. In support of this hypothesis, these nonproducers in iron-limited media suffer a significant decrease in growth, which can be alleviated by siderophores, presumably owing to the retention of transport mechanisms. Moreover, using ecological data of resource partitioning, we found that cheating coevolves with the ecological specialization toward association with larger particles in the water column, suggesting that these can harbor stable enough communities for dependencies among organisms to evolve.

Distinct dissolved organic matter sources induce rapid transcriptional responses in coexisting populations of Prochlorococcus, Pelagibacter and the OM60 clade.

A considerable fraction of the Earth's organic carbon exists in dissolved form in seawater. To investigate the roles of planktonic marine microbes in the biogeochemical cycling of this dissolved organic matter (DOM), we performed controlled seawater incubation experiments and followed the responses of an oligotrophic surface water microbial assemblage to perturbations with DOM derived from an axenic culture of Prochlorococcus, or high-molecular weight DOM concentrated from nearby surface waters. The rapid transcriptional responses of both Prochlorococcus and Pelagibacter populations suggested the utilization of organic nitrogen compounds common to both DOM treatments. Along with these responses, both populations demonstrated decreases in gene transcripts associated with nitrogen stress, including those involved in ammonium acquisition. In contrast, responses from low abundance organisms of the NOR5/OM60 gammaproteobacteria were observed later in the experiment, and included elevated levels of gene transcripts associated with polysaccharide uptake and oxidation. In total, these results suggest that numerically dominant oligotrophic microbes rapidly acquire nitrogen from commonly available organic sources, and also point to an important role for carbohydrates found within the DOM pool for sustaining the less abundant microorganisms in these oligotrophic systems.

DNA phosphorothioation is widespread and quantized in bacterial genomes.

Phosphorothioate (PT) modification of DNA, with sulfur replacing a nonbridging phosphate oxygen, was recently discovered as a product of the dnd genes found in bacteria and archaea. Given our limited understanding of the biological function of PT modifications, including sequence context, genomic frequencies, and relationships to the diversity of dnd gene clusters, we undertook a quantitative study of PT modifications in prokaryotic genomes using a liquid chromatography-coupled tandem quadrupole mass spectrometry approach. The results revealed a diversity of unique PT sequence contexts and three discrete genomic frequencies in a wide range of bacteria. Metagenomic analyses of PT modifications revealed unique ecological distributions, and a phylogenetic comparison of dnd genes and PT sequence contexts strongly supports the horizontal transfer of dnd genes. These results are consistent with the involvement of PT modifications in a type of restriction-modification system with wide distribution in prokaryotes.

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.

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.

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.

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.

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.

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.