A Reverse Ecology Approach Based on a Biological Definition of Microbial Populations.

Delineating ecologically meaningful populations among microbes is important for identifying their roles in environmental and host-associated microbiomes. Here, we introduce a metric of recent gene flow, which when applied to co-existing microbes, identifies congruent genetic and ecological units separated by strong gene flow discontinuities from their next of kin. We then develop a pipeline to identify genome regions within these units that show differential adaptation and allow mapping of populations onto environmental variables or host associations. Using this reverse ecology approach, we show that the human commensal bacterium Ruminococcus gnavus breaks up into sharply delineated populations that show different associations with health and disease. Defining populations by recent gene flow in this way will facilitate the analysis of bacterial and archaeal genomes using ecological and evolutionary theory developed for plants and animals, thus allowing for testing unifying principles across all biology.

A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria.

The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses. In fact, non-tailed viruses often dominate ocean samples numerically, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria-virus interactions.

Conjugative plasmids interact with insertion sequences to shape the horizontal transfer of antimicrobial resistance genes.

It is well established that plasmids play an important role in the dissemination of antimicrobial resistance (AMR) genes; however, little is known about the role of the underlying interactions between different plasmid categories and other mobile genetic elements (MGEs) in shaping the promiscuous spread of AMR genes. Here, we developed a tool designed for plasmid classification, AMR gene annotation, and plasmid visualization and found that most plasmid-borne AMR genes, including those localized on class 1 integrons, are enriched in conjugative plasmids. Notably, we report the discovery and characterization of a massive insertion sequence (IS)-associated AMR gene transfer network (245 combinations covering 59 AMR gene subtypes and 53 ISs) linking conjugative plasmids and phylogenetically distant pathogens, suggesting a general evolutionary mechanism for the horizontal transfer of AMR genes mediated by the interaction between conjugative plasmids and ISs. Moreover, our experimental results confirmed the importance of the observed interactions in aiding the horizontal transfer and expanding the genetic range of AMR genes within complex microbial communities.

Culture-independent tracking of Vibrio cholerae lineages reveals complex spatiotemporal dynamics in a natural population.

Populations of the bacterium Vibrio cholerae consist of dozens of distinct lineages, with primarily (but not exclusively) members of the pandemic generating lineage capable of causing the diarrhoeal disease cholera. Assessing the composition and temporal dynamics of such populations requires extensive isolation efforts and thus only rarely covers large geographic areas or timeframes exhaustively. We developed a culture-independent amplicon sequencing strategy based on the protein-coding gene viuB (vibriobactin utilization) to study the structure of a V. cholerae population over the course of a summer. We show that the 26 co-occurring V. cholerae lineages continuously compete for limited space on nutrient-rich particles where only a few of them can grow to large numbers. Differential abundance of lineages between locations and size-fractions associated with a particle-attached or free-swimming lifestyle could reflect adaptation to various environmental niches. In particular, a major V. cholerae lineage occasionally grows to large numbers on particles but remain undetectable using isolation-based methods, indicating selective culturability for some members of the species. We thus demonstrate that isolation-based studies may not accurately reflect the structure and complex dynamics of V. cholerae populations and provide a scalable high-throughput method for both epidemiological and ecological approaches to studying this species.

Microbial community assembly and evolution in subseafloor sediment.

Bacterial and archaeal communities inhabiting the subsurface seabed live under strong energy limitation and have growth rates that are orders of magnitude slower than laboratory-grown cultures. It is not understood how subsurface microbial communities are assembled and whether populations undergo adaptive evolution or accumulate mutations as a result of impaired DNA repair under such energy-limited conditions. Here we use amplicon sequencing to explore changes of microbial communities during burial and isolation from the surface to the >5,000-y-old subsurface of marine sediment and identify a small core set of mostly uncultured bacteria and archaea that is present throughout the sediment column. These persisting populations constitute a small fraction of the entire community at the surface but become predominant in the subsurface. We followed patterns of genome diversity with depth in four dominant lineages of the persisting populations by mapping metagenomic sequence reads onto single-cell genomes. Nucleotide sequence diversity was uniformly low and did not change with age and depth of the sediment. Likewise, there was no detectable change in mutation rates and efficacy of selection. Our results indicate that subsurface microbial communities predominantly assemble by selective survival of taxa able to persist under extreme energy limitation.

Polyclonal symbiont populations in hydrothermal vent tubeworms and the environment.

Horizontally transmitted symbioses usually house multiple and variable symbiont genotypes that are acquired from a much more diverse environmental pool via partner choice mechanisms. However, in the deep-sea hydrothermal vent tubeworm Riftia pachyptila (Vestimentifera, Siboglinidae), it has been suggested that the Candidatus Endoriftia persephone symbiont is monoclonal. Here, we show with high-coverage metagenomics that adult R. pachyptila house a polyclonal symbiont population consisting of one dominant and several low-frequency variants. This dominance of one genotype is confirmed by multilocus gene sequencing of amplified housekeeping genes in a broad range of host individuals where three out of four loci ( atpA, uvrD and recA) revealed no genomic differences, while one locus ( gyrB) was more diverse in adults than in juveniles. We also analysed a metagenome of free-living Endoriftia and found that the free-living population showed greater sequence variability than the host-associated population. Most juveniles and adults shared a specific dominant genotype, while other genotypes can dominate in few individuals. We suggest that although generally permissive, partner choice is selective enough to restrict uptake of some genotypes present in the environment.

Evolution of a Vegetarian Vibrio: Metabolic Specialization of Vibrio breoganii to Macroalgal Substrates.

While most Vibrionaceae are considered generalists that thrive on diverse substrates, including animal-derived material, we show that Vibrio breoganii has specialized for the consumption of marine macroalga-derived substrates. Genomic and physiological comparisons of V. breoganii with other Vibrionaceae isolates revealed the ability to degrade alginate, laminarin, and additional glycans present in algal cell walls. Moreover, the widely conserved ability to hydrolyze animal-derived polymers, including chitin and glycogen, was lost, along with the ability to efficiently grow on a variety of amino acids. Ecological data showing associations with particulate algal material but not zooplankton further support this shift in niche preference, and the loss of motility appears to reflect a sessile macroalga-associated lifestyle. Together, these findings indicate that algal polysaccharides have become a major source of carbon and energy in V. breoganii, and these ecophysiological adaptations may facilitate transient commensal associations with marine invertebrates that feed on algae.IMPORTANCE Vibrios are often considered animal specialists or generalists. Here, we show that Vibrio breoganii has undergone massive genomic changes to become specialized on algal carbohydrates. Accompanying genomic changes include massive gene import and loss. These vibrios may help us better understand how algal biomass is degraded in the environment and may serve as a blueprint on how to optimize the conversion of algae to biofuels.

Exploiting fine-scale genetic and physiological variation of closely related microbes to reveal unknown enzyme functions.

Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium Vibrio splendidus One strain, V. splendidus 13B01, exhibited high extracellular alginate lyase activity compared with other V. splendidus strains. To identify the enzymes responsible for this high extracellular activity, we compared V. splendidus 13B01 with the previously characterized V. splendidus 12B01, which has low extracellular activity and lacks two alginate lyase genes present in V. splendidus 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to V. splendidus 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions.

Endosymbionts escape dead hydrothermal vent tubeworms to enrich the free-living population.

Theory predicts that horizontal acquisition of symbionts by plants and animals must be coupled to release and limited dispersal of symbionts for intergenerational persistence of mutualisms. For deep-sea hydrothermal vent tubeworms (Vestimentifera, Siboglinidae), it has been demonstrated that a few symbiotic bacteria infect aposymbiotic host larvae and grow in a newly formed organ, the trophosome. However, whether viable symbionts can be released to augment environmental populations has been doubtful, because (i) the adult worms lack obvious openings and (ii) the vast majority of symbionts has been regarded as terminally differentiated. Here we show experimentally that symbionts rapidly escape their hosts upon death and recruit to surfaces where they proliferate. Estimating symbiont release from our experiments taken together with well-known tubeworm density ranges, we suggest a few million to 1.5 billion symbionts seeding the environment upon death of a tubeworm clump. In situ observations show that such clumps have rapid turnover, suggesting that release of large numbers of symbionts may ensure effective dispersal to new sites followed by active larval colonization. Moreover, release of symbionts might enable adaptations that evolve within host individuals to spread within host populations and possibly to new environments.

Natural resource landscapes of a marine bacterium reveal distinct fitness-determining genes across the genome.

Heterotrophic bacteria exploit diverse microhabitats in the ocean, from particles to transient gradients. Yet the degree to which genes and pathways can contribute to an organism's fitness on such complex and variable natural resource landscapes remains poorly understood. Here, we determine the gene-by-gene fitness of a generalist saprophytic marine bacterium (Vibrio sp. F13 9CS106) on complex resources derived from its natural habitats - copepods (Apocyclops royi) and brown algae (Fucus vesiculosus) - and as reference substrates, glucose and the polysaccharide alginate, derived from brown algal cell walls. We find that resource complexity strongly buffers fitness costs of mutations, and that anabolic rather than catabolic pathways are more stringently required, likely due to functional redundancy in the latter. Moreover, while carbohydrate-rich algae requires several synthesis pathways, protein-rich Apocyclops does not, suggesting this ancestral habitat for Vibrios is a replete medium with metabolically redundant substrates. We also identify a candidate fitness trade-off for algal colonization: deletion of mshA increases mutant fitness. Our results demonstrate that gene fitness depends on habitat composition, and suggest that this generalist uses distinct resources in different natural habitats. The results further indicate that substrate replete conditions may lead to relatively relaxed selection on catabolic genes.

Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes.

Marine algae produce a variety of glycans, which fulfill diverse biological functions and fuel the carbon and energy demands of heterotrophic microbes. A common approach to analysis of marine organic matter uses acid to hydrolyze the glycans into measurable monosaccharides. The monosaccharides may be derived from different glycans that are built with the same monosaccharides, however, and this approach does not distinguish between glycans in natural samples. Here we use enzymes to digest selectively and thereby quantify laminarin in particulate organic matter. Environmental metaproteome data revealed carbohydrate-active enzymes from marine flavobacteria as tools for selective hydrolysis of the algal ß-glucan laminarin. The enzymes digested laminarin into glucose and oligosaccharides, which we measured with standard methods to establish the amounts of laminarin in the samples. We cloned, expressed, purified, and characterized three new glycoside hydrolases (GHs) of Formosa bacteria: two are endo-ß-1,3-glucanases, of the GH16 and GH17 families, and the other is a GH30 exo-ß-1,6-glucanase. Formosa sp. nov strain Hel1_33_131 GH30 (FbGH30) removed the ß-1,6-glucose side chains, and Formosa agariphila GH17A (FaGH17A) and FaGH16A hydrolyzed the ß-1,3-glucose backbone of laminarin. Specificity profiling with a library of glucan oligosaccharides and polysaccharides revealed that FaGH17A and FbGH30 were highly specific enzymes, while FaGH16A also hydrolyzed mixed-linked glucans with ß-1,4-glucose. Therefore, we chose the more specific FaGH17A and FbGH30 to quantify laminarin in two cultured diatoms, namely, Thalassiosira weissflogii and Thalassiosira pseudonana, and in seawater samples from the North Sea and the Arctic Ocean. Combined, these results demonstrate the potential of enzymes for faster, stereospecific, and sequence-specific analysis of select glycans in marine organic matter.IMPORTANCE Marine algae synthesize substantial amounts of the glucose polymer laminarin for energy and carbon storage. Its concentrations, rates of production by autotrophic organisms, and rates of digestion by heterotrophic organisms remain unknown. Here we present a method based on enzymes that hydrolyze laminarin and enable its quantification even in crude substrate mixtures, without purification. Compared to the commonly used acid hydrolysis, the enzymatic method presented here is faster and stereospecific and selectively cleaves laminarin in mixtures of glycans, releasing only glucose and oligosaccharides, which can be easily quantified with reducing sugar assays.

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.

Horizontal gene transfer and the evolution of bacterial and archaeal population structure.

Many bacterial and archaeal lineages have a history of extensive and ongoing horizontal gene transfer and loss, as evidenced by the large differences in genome content even among otherwise closely related isolates. How ecologically cohesive populations might evolve and be maintained under such conditions of rapid gene turnover has remained controversial. Here we synthesize recent literature demonstrating the importance of habitat and niche in structuring horizontal gene transfer. This leads to a model of ecological speciation via gradual genetic isolation triggered by differential habitat-association of nascent populations. Further, we hypothesize that subpopulations can evolve through local gene-exchange networks by tapping into a gene pool that is adaptive towards local, continuously changing organismic interactions and is, to a large degree, responsible for the observed rapid gene turnover. Overall, these insights help to explain how bacteria and archaea form populations that display both ecological cohesion and high genomic diversity.

Population genomics of the symbiotic plasmids of sympatric nitrogen-fixing Rhizobium species associated with Phaseolus vulgaris.

Cultivated common beans are the primary protein source for millions of people around the world who subsist on low-input agriculture, enabled by the symbiotic N2 -fixation these legumes perform in association with rhizobia. Within a single agricultural plot, multiple Rhizobium species can nodulate bean roots, but it is unclear how genetically isolated these species remain in sympatry. To better understand this issue, we sequenced and compared the genomes of 33 strains isolated from the rhizosphere and root nodules of a particular bean variety grown in the same agricultural plot. We found that the Rhizobium species we observed coexist with low genetic recombination across their core genomes. Accessory plasmids thought to be necessary for the saprophytic lifestyle in soil show similar levels of genetic isolation, but with higher rates of recombination than the chromosomes. However, the symbiotic plasmids are extremely similar, with high rates of recombination and do not appear to have co-evolved with the chromosome or accessory plasmids. Therefore, while Rhizobium species are genetically isolated units within the microbial community, a common symbiotic plasmid allows all Rhizobium species to engage in symbiosis with the same host in a single agricultural plot.

A Small Number of Phylogenetically Distinct Clonal Complexes Dominate a Coastal Vibrio cholerae Population.

UNLABELLED: Vibrio cholerae is a ubiquitous aquatic microbe in temperate and tropical coastal areas. It is a diverse species, with many isolates that are harmless to humans, while others are highly pathogenic. Most notable among them are strains belonging to the pandemic O1/O139 serogroup lineage, which contains the causative agents of cholera. The environmental selective regimes that led to this diversity are key to understanding how pathogens evolve in environmental reservoirs. A local population of V. cholerae and its close relative Vibrio metoecus from a coastal pond and lagoon system was extensively sampled during two consecutive months across four size fractions (480 isolates). In stark contrast to previous studies, the observed population was highly clonal, with 60% of V. cholerae isolates falling into one of five clonal complexes, which varied in abundance in the short temporal scale sampled. V. cholerae clonal complexes had significantly different distributions across size fractions and the two environments sampled, the pond and the lagoon. Sequencing the genomes of 20 isolates representing these five V. cholerae clonal complexes revealed different evolutionary trajectories, with considerable variations in gene content with potential ecological significance. Showing genotypic differentiation and differential spatial distribution, the dominant clonal complexes are likely ecologically divergent. Temporal variation in the relative abundance of these complexes suggests that transient blooms of specific clones could dominate local diversity. IMPORTANCE: Vibrio cholerae is commonly found in coastal areas worldwide, with only a single group of this bacterium capable of causing severe cholera outbreaks. However, the potential to evolve the ability to cause disease exists in many strains of this species in its aquatic reservoir. Understanding how pathogenic bacteria evolve requires the study of their natural environments. By extensive sampling in a geographically restricted location in the United States, we found that most cells of a V. cholerae population belong to only a small number of strains. Analysis of their genome composition and spatial distribution indicates differential environmental adaptations between these strains. Other strains exist in smaller numbers, and the population was found to be temporally varied. This suggests frequent bloom and collapse cycles on a time scale of weeks. These population dynamics make it possible that more virulent strains could stochastically rise to large numbers, allowing for infection to occur.

Microbial taxonomy in the post-genomic era: rebuilding from scratch?

Microbial taxonomy should provide adequate descriptions of bacterial, archaeal, and eukaryotic microbial diversity in ecological, clinical, and industrial environments. Its cornerstone, the prokaryote species has been re-evaluated twice. It is time to revisit polyphasic taxonomy, its principles, and its practice, including its underlying pragmatic species concept. Ultimately, we will be able to realize an old dream of our predecessor taxonomists and build a genomic-based microbial taxonomy, using standardized and automated curation of high-quality complete genome sequences as the new gold standard.

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.

Comparative biochemical characterization of three exolytic oligoalginate lyases from Vibrio splendidus reveals complementary substrate scope, temperature, and pH adaptations.

Marine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exo-type oligoalginate lyases (OalA, OalB, and OalC) from Vibrio splendidus 12B01 (12B01), a marine bacterioplankton species. OalA was most active at 16°C, had a pH optimum of 6.5, and displayed activities toward poly-ß-d-mannuronate [poly(M)] and poly-α-l-guluronate [poly(G)], indicating that it is a bifunctional enzyme. OalB and OalC were most active at 30 and 35°C, had pH optima of 7.0 and 7.5, and degraded poly(M·G) and poly(M), respectively. Detailed kinetic analyses of oligoalginate lyases with poly(G), poly(M), and poly(M·G) and sodium alginate as substrates demonstrated that OalA and OalC preferred poly(M), whereas OalB preferred poly(M·G). The catalytic efficiency (kcat/Km) of OalA against poly(M) increased with decreasing size of the substrate. OalA showed kcat/Km from 2,130 mg(-1) ml s(-1) for the trisaccharide to 224 mg(-1) ml s(-1) for larger oligomers of ∼50 residues, and 50.5 mg(-1) ml s(-1) for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.