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Background: Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order.Results: We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis.Conclusion: The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic differences, coupled with unknown differences in gene expression, distinguish these organisms enough to allow for co-existence. Overall this study reveals shared features of organisms from the Thermoplasmatales lineage and provides new insights into the functioning of AMD communities. © 2013 Yelton et al.; licensee BioMed Central Ltd.

Phytoplankton and their associated microbiomes of heterotrophic bacteria are foundational to primary production, energy transfer, and biogeochemical cycling in aquatic systems. While it is known that these microbiomes are shaped by host-released dissolved organic matter (DOM), the extent to which dynamic phytoplankton-bacteria interactions shape bacterial community assembly remains to be examined. Here, we investigated the effects of two mechanisms in host-microbiome interactions on phytoplankton bacterial microbiome formation: (i) innate host selection and (ii) host-microbiome feedback. For the former, phytoplankton-produced DOM composition is based solely on the host's properties (species or physiological state); for the latter, the presence of the microbiome modifies host DOM production. The microbiome of Chlorella sorokiniana was extracted and exposed to six ratios of the two effects. We found that microbiome composition changed along with the six host-microbiome feedback versus innate host selection ratios, with the highest compositional distance between communities under the strongest and the weakest ratio of the two effects. This indicates that each mechanism selects for different bacterial species. In addition, our findings showed that when both selective forces were applied, it led to a higher community richness, while host-microbiome feedback alone reduces community evenness due to its strong species-specific selection., (© 2024 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Microcystis-dominated cyanobacterial harmful algal blooms (cyanoHABs) have a global impact on freshwater environments, affecting both wildlife and human health. Microcystis diversity and function in field samples and laboratory cultures can be determined by sequencing whole genomes of cultured isolates or natural populations, but these methods remain computationally and financially expensive. Amplicon sequencing of marker genes is a lower cost and higher throughput alternative to characterize strain composition and diversity in mixed samples. However, the selection of appropriate marker gene region(s) and primers requires prior understanding of the relationship between single gene genotype, whole genome content, and phenotype. To identify phylogenetic markers of Microcystis strain diversity, we compared phylogenetic trees built from each of 2,351 individual core genes to an established phylogeny and assessed the ability of these core genes to predict whole genome content and bioactive compound genotypes. We identified single-copy core genes better able to resolve Microcystis phylogenies than previously identified marker genes. We developed primers suitable for current Illumina-based amplicon sequencing with near-complete coverage of available Microcystis genomes and demonstrate that they outperform existing options for assessing Microcystis strain composition. Results showed that genetic markers can be used to infer Microcystis gene content and phenotypes such as potential production of bioactive compounds , although marker performance varies by bioactive compound gene and sequence similarity. Finally, we demonstrate that these markers can be used to characterize the Microcystis strain composition of laboratory or field samples like those collected for surveillance and modeling of Microcystis-dominated cyanobacterial harmful algal blooms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Freshwater harmful algal blooms are often dominated by Microcystis , a phylogenetically cohesive group of cyanobacteria marked by extensive genetic and physiological diversity. We have previously shown that this genetic diversity and the presence of a microbiome of heterotrophic bacteria influences competitive interactions with eukaryotic phytoplankton. In this study, we sought to explain these observations by characterizing Monod equation parameters for resource usage (maximum growth rate μ max , half-saturation value for growth K s, and quota) as a function of N and P levels for four strains (NIES-843, PCC 9701, PCC 7806 [WT], and PCC 7806 Δ mcyB ) in presence and absence of a microbiome derived from Microcystis isolated from Lake Erie. Results indicated limited differences in maximum growth rates but more pronounced differences in half-saturation values among Microcystis strains. The largest impact of the microbiome was reducing the minimal nitrogen concentration sustaining growth and reducing half saturation values, with variable results depending on the Microcystis strain. Microcystis strains also differed from each other in their N and P quotas and the extent to which microbiome presence affected them. Our data highlight the importance of the microbiome in altering Microcystis -intrinsic traits, strain competitive hierarchies, and thus bloom dynamics. As quota, μ max , and K s are commonly used in models for harmful algal blooms, our data suggest that model improvement may be possible by incorporating genotype dependencies of resource-use parameters., Competing Interests: The authors declare no conflict of interests., (© 2023 The Authors. mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.)

Cyanobacterial harmful algal blooms (cyanoHABs) dominated by Microcystis spp. have significant public health and economic implications in freshwater bodies around the world. These blooms are capable of producing a variety of cyanotoxins, including microcystins, that affect fishing and tourism industries, human and environmental health, and access to drinking water. In this study, we isolated and sequenced the genomes of 21 primarily unialgal Microcystis cultures collected from western Lake Erie between 2017 and 2019. While some cultures isolated in different years have a high degree of genetic similarity (genomic Average Nucleotide Identity >99%), genomic data show that these cultures also represent much of the breadth of known Microcystis diversity in natural populations. Only five isolates contained all the genes required for microcystin biosynthesis while two isolates contained a previously described partial mcy operon. Microcystin production within cultures was also assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and supported genomic results with high concentrations (up to 900 μg L⁻¹) in cultures with complete mcy operons and no or low toxin detected otherwise. These xenic cultures also contained a substantial diversity of bacteria associated with Microcystis, which has become increasingly recognized as an essential component of cyanoHAB community dynamics. These results highlight the genomic diversity among Microcystis strains and associated bacteria in Lake Erie, and their potential impacts on bloom development, toxin production, and toxin degradation. This culture collection significantly increases the availability of environmentally relevant Microcystis strains from temperate North America., Competing Interests: Declaration of Competing Interest The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Phytoplankton is fundamental to life on Earth. Their productivity is influenced by the microbial communities residing in the phycosphere surrounding algal cells. Expanding our knowledge on how algal-bacterial interactions affect algal growth to more hosts and bacteria can help elucidate general principles of algal-host interactions. Here, we isolated 368 bacterial strains from phycosphere communities, right after phycosphere recruitment from pond water and after a month of lab cultivation and examined their impacts on growth of five green algal species. We isolated both abundant and rare phycosphere members, representing 18.4% of the source communities. Positive and neutral effects predominated over negative effects on host growth. The proportion of each effect type and whether the day of isolation mattered varied by host species. Bacteria affected algal carrying capacity more than growth rate, suggesting that nutrient remineralization and toxic byproduct metabolism may be a dominant mechanism. Across-host algal fitness assays indicated host-specific growth effects of our isolates. We observed no phylogenetic conservation of the effect on host growth among bacterial isolates. Even isolates with the same ASV had divergent effects on host growth. Our results emphasize highly specific host-bacterial interactions in the phycosphere and raise questions as to which mechanisms mediate these interactions.

Bacteria represent most of the biodiversity and play key roles in virtually every ecosystem. In doing so, bacteria act as part of complex communities shaped by interactions across all domains of life. Here, we report on direct interactions between bacteria and dreissenid mussels, a group of invasive filter-feeders threatening global aquatic systems due to high filtration rates. Previous studies showed that dreissenids can impact bacterial community structure by changing trait distributions and abundances of specific taxa. However, studies on bacterial community effects were conducted using water from Lake Michigan (an oligotrophic lake) only, and it is unknown whether similar patterns are observed in systems with differing nutrient regimes. We conducted ten short-term dreissenid grazing experiments in 2019 using water from two eutrophic lake regions-the western basin of Lake Erie and Saginaw Bay in Lake Huron. Predation by dreissenids led to decline in overall bacterial abundance and diversity in both lakes. However, feeding on bacteria was not observed during every experiment. We also found that traits related to feeding resistance are less phylogenetically conserved than previously thought. Our results highlight the role of temporal, spatial, and genomic heterogeneity in bacterial response dynamics to a globally important invasive filter feeder., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Microcystis is a cyanobacterium that forms toxic blooms in freshwater ecosystems around the world. Biological variation among taxa within the genus is apparent through genetic and phenotypic differences between strains and via the spatial and temporal distribution of strains in the environment, and this fine-scale diversity exerts strong influence over bloom toxicity. Yet we do not know how varying traits of Microcystis strains govern their environmental distribution, the tradeoffs and links between these traits, or how they are encoded at the genomic level. Here we synthesize current knowledge on the importance of diversity within Microcystis and on the genes and traits that likely underpin ecological differentiation of taxa. We briefly review spatial and environmental patterns of Microcystis diversity in the field and genetic evidence for cohesive groups within Microcystis. We then compile data on strain-level diversity regarding growth responses to environmental conditions and explore evidence for variation of community interactions across Microcystis strains. Potential links and tradeoffs between traits are identified and discussed. The resulting picture, while incomplete, highlights key knowledge gaps that need to be filled to enable new models for predicting strain-level dynamics, which influence the development, toxicity and cosmopolitan nature of Microcystis blooms., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Insights into symbiosis between eukaryotic hosts and their microbiomes have shifted paradigms on what determines host fitness, ecology, and behavior. Questions remain regarding the roles of host versus environment in shaping microbiomes, and how microbiome composition affects host fitness. Using a model system in ecology, phytoplankton, we tested whether microbiomes are host-specific, confer fitness benefits that are host-specific, and remain conserved in time in their composition and fitness effects. We used an experimental approach in which hosts were cleaned of bacteria and then exposed to bacterial communities from natural environments to permit recruitment of microbiomes. We found that phytoplankton microbiomes consisted of a subset of taxa recruited from these natural environments. Microbiome recruitment was host-specific, with host species explaining more variation in microbiome composition than environment. While microbiome composition shifted and then stabilized over time, host specificity remained for dozens of generations. Microbiomes increased host fitness, but these fitness effects were host-specific for only two of the five species. The shifts in microbiome composition over time amplified fitness benefits to the hosts. Overall, this work solidifies the importance of host factors in shaping microbiomes and elucidates the temporal dynamics of microbiome compositional and fitness effects.

Cyanobacterial harmful algal blooms (cyanoHABs) continue to increase in frequency and magnitude, threatening global freshwater ecosystems and services. In north-temperate lakes cyanobacteria appear in early summer, succeeding green algae as the dominant phytoplankton group, a pattern thought to be mediated by changes in temperature and bioavailable nutrients. To understand additional drivers of this successional pattern our study used reciprocal invasion experiments to examine the competitive interaction between Microcystis aeruginosa, a dominant contributor to cyanoHABs, and the green alga Chlorella sorokiniana. We considered two factors that may impact these interactions: (1) strain variation, with a specific emphasis on the presence or absence of the gene for the hepatotoxin microcystin, and (2) host-associated bacteria. We used toxic M. aeruginosa PCC 7806 (microcystin producing strain), a non-toxic mutant of PCC 7806, non-toxic M. aeruginosa PCC 9701 (non-microcystin producing strain), and C. sorokiniana. Each organism was available free of all bacteria (i.e., axenic) and with a re-introduced defined bacterial community to generate their xenic counterparts. Competitive interactions were assessed with reciprocal invasion experiments between paired xenic and paired axenic populations of C. sorokiniana and one of the two Microcystis strains, each assessed separately. Flow cytometry and random forest models were used to rapidly discriminate and quantify phytoplankton population densities with 99% accuracy. We found that M. aeruginosa PCC 7806, but not strain PCC 9701, could proliferate from low abundance in a steady-state population of C. sorokiniana. Further, the presence of bacteria allowed M. aeruginosa PCC 7806 to grow to a higher population density into an established C. sorokiniana population than when grown axenic. Conversely, when M. aeruginosa was dominant, C. sorokiniana was only able to proliferate from low density into the PCC 9701 strain, and only when axenic. The mutant of PCC 7806 lacking the ability to produce microcystin behaved similarly to the toxic wild-type, implying microcystin is not responsible for the difference in competitive abilities observed between the two wild-type strains. Quantification of microcystins (MCs) when PCC 7806 M. aeruginosa was introduced into the C. sorokiniana culture showed two-fold more MCs per cell when host-associated bacteria were absent compared to present in both species cultures. Our results show that the ability of M. aeruginosa to compete with C. sorokiniana is determined by genomic differences beyond genes involved in microcystin toxin generation and indicate an important role of host-associated bacteria in mediating phytoplankton interspecies interactions. These results expand our understanding of the key drivers of phytoplankton succession and the establishment and persistence of freshwater harmful cyanobacterial blooms., (Copyright © 2020. Published by Elsevier B.V.)

Metagenome-assembled genomes (MAGs) expand our understanding of microbial diversity, evolution, and ecology. Concerns have been raised on how sequencing, assembly, binning, and quality assessment tools may result in MAGs that do not reflect single populations in nature. Here, we reflect on another issue, i.e., how to handle highly similar MAGs assembled from independent data sets. Obtaining multiple genomic representatives for a species is highly valuable, as it allows for population genomic analyses; however, when retaining genomes of closely related populations, it complicates MAG quality assessment and abundance inferences. We show that (i) published data sets contain a large fraction of MAGs sharing >99% average nucleotide identity, (ii) different software packages and parameters used to resolve this redundancy remove very different numbers of MAGs, and (iii) the removal of closely related genomes leads to losses of population-specific auxiliary genes. Finally, we highlight some approaches that can infer strain-specific dynamics across a sample series without dereplication., (Copyright © 2020 Evans and Denef.)

Most freshwater bacterial communities are characterized by a few dominant taxa that are often ubiquitous across freshwater biomes worldwide. Our understanding of the genomic diversity within these taxonomic groups is limited to a subset of taxa. Here, we investigated the genomic diversity that enables Limnohabitans , a freshwater genus key in funneling carbon from primary producers to higher trophic levels, to achieve abundance and ubiquity. We reconstructed eight putative Limnohabitans metagenome-assembled genomes (MAGs) from stations located along broad environmental gradients existing in Lake Michigan, part of Earth's largest surface freshwater system. De novo strain inference analysis resolved a total of 23 strains from these MAGs, which strongly partitioned into two habitat-specific clusters with cooccurring strains from different lineages. The largest number of strains belonged to the abundant LimB lineage, for which robust in situ strain delineation had not previously been achieved. Our data show that temperature and nutrient levels may be important environmental parameters associated with microdiversification within the Limnohabitans genus. In addition, strains predominant in low- and high-phosphorus conditions had larger genomic divergence than strains abundant under different temperatures. Comparative genomics and gene expression analysis yielded evidence for the ability of LimB populations to exhibit cellular motility and chemotaxis, a phenotype not yet associated with available Limnohabitans isolates. Our findings broaden historical marker gene-based surveys of Limnohabitans microdiversification and provide in situ evidence of genome diversity and its functional implications across freshwater gradients. IMPORTANCE Limnohabitans is an important bacterial taxonomic group for cycling carbon in freshwater ecosystems worldwide. Here, we examined the genomic diversity of different Limnohabitans lineages. We focused on the LimB lineage of this genus, which is globally distributed and often abundant, and its abundance has shown to be largely invariant to environmental change. Our data show that the LimB lineage is actually comprised of multiple cooccurring populations for which the composition and genomic characteristics are associated with variations in temperature and nutrient levels. The gene expression profiles of this lineage suggest the importance of chemotaxis and motility, traits that had not yet been associated with the Limnohabitans genus, in adapting to environmental conditions., (Copyright © 2020 American Society for Microbiology.)

Eukaryotic communities commonly display a positive relationship between biodiversity and ecosystem function (BEF) but the results have been mixed when assessed in bacterial communities. Habitat heterogeneity, a factor in eukaryotic BEFs, may explain these variable observations but it has not been thoroughly evaluated in bacterial communities. Here, we examined the impact of habitat on the relationship between diversity assessed based on the (phylogenetic) Hill diversity metrics and heterotrophic productivity. We sampled co-occurring free-living (more homogenous) and particle-associated (more heterogeneous) bacterial habitats in a freshwater, estuarine lake over three seasons: spring, summer and fall. There was a strong, positive, linear relationship between particle-associated bacterial richness and heterotrophic productivity that strengthened when considering dominant taxa. There were no observable BEF trends in free-living bacterial communities for any diversity metric. Biodiversity, richness and Inverse Simpson's index, were the best predictors of particle-associated production whereas pH was the best predictor of free-living production. Our findings show that heterotrophic productivity is positively correlated with the effective number of taxa and that BEF relationships are associated with microhabitats. These results add to the understanding of the highly distinct contributions to diversity and functioning contributed by bacteria in free-living and particle-associated habitats., (© FEMS 2020.)

Our current knowledge about nucleocytoplasmic large DNA viruses (NCLDVs) is largely derived from viral isolates that are co-cultivated with protists and algae. Here we reconstructed 2,074 NCLDV genomes from sampling sites across the globe by building on the rapidly increasing amount of publicly available metagenome data. This led to an 11-fold increase in phylogenetic diversity and a parallel 10-fold expansion in functional diversity. Analysis of 58,023 major capsid proteins from large and giant viruses using metagenomic data revealed the global distribution patterns and cosmopolitan nature of these viruses. The discovered viral genomes encoded a wide range of proteins with putative roles in photosynthesis and diverse substrate transport processes, indicating that host reprogramming is probably a common strategy in the NCLDVs. Furthermore, inferences of horizontal gene transfer connected viral lineages to diverse eukaryotic hosts. We anticipate that the global diversity of NCLDVs that we describe here will establish giant viruses-which are associated with most major eukaryotic lineages-as important players in ecosystems across Earth's biomes.

Bacteria associated with eukaryotic hosts can affect host fitness and trophic interactions between eukaryotes, but the extent to which bacteria influence the eukaryotic species interactions within trophic levels that modulate biodiversity and species coexistence is mostly unknown. Here, we used phytoplankton, which are a classic model for evaluating interactions between species, grown with and without associated bacteria to test whether the bacteria alter the strength and type of species interactions within a trophic level. We demonstrate that host-associated bacteria alter host growth rates and carrying capacity. This did not change the type but frequently changed the strength of host interspecific interactions by facilitating host growth in the presence of an established species. These findings indicate that microbiomes can regulate their host species' interspecific interactions. As between-species interaction strength impacts their ability to coexist, our findings show that microbiomes have the potential to modulate eukaryotic species diversity and community composition. IMPORTANCE Description of the Earth's microbiota has recently undergone a phenomenal expansion that has challenged basic assumptions in many areas of biology, including hominid evolution, human gastrointestinal and neurodevelopmental disorders, and plant adaptation to climate change. By using the classic model system of freshwater phytoplankton that has been drawn upon for numerous foundational theories in ecology, we show that microbiomes, by facilitating their host population, can also influence between-species interactions among their eukaryotic hosts. Between-species interactions, including competition for resources, has been a central tenet in the field of ecology because of its implications for the diversity and composition of communities and how this in turn shapes ecosystem functioning., (Copyright © 2020 Jackrel et al.)

High-nucleic-acid (HNA) and low-nucleic-acid (LNA) bacteria are two operational groups identified by flow cytometry (FCM) in aquatic systems. A number of reports have shown that HNA cell density correlates strongly with heterotrophic production, while LNA cell density does not. However, which taxa are specifically associated with these groups, and by extension, productivity has remained elusive. Here, we addressed this knowledge gap by using a machine learning-based variable selection approach that integrated FCM and 16S rRNA gene sequencing data collected from 14 freshwater lakes spanning a broad range in physicochemical conditions. There was a strong association between bacterial heterotrophic production and HNA absolute cell abundances ( R 2 = 0.65), but not with the more abundant LNA cells. This solidifies findings, mainly from marine systems, that HNA and LNA bacteria could be considered separate functional groups, the former contributing a disproportionately large share of carbon cycling. Taxa selected by the models could predict HNA and LNA absolute cell abundances at all taxonomic levels. Selected operational taxonomic units (OTUs) ranged from low to high relative abundance and were mostly lake system specific (89.5% to 99.2%). A subset of selected OTUs was associated with both LNA and HNA groups (12.5% to 33.3%), suggesting either phenotypic plasticity or within-OTU genetic and physiological heterogeneity. These findings may lead to the identification of system-specific putative ecological indicators for heterotrophic productivity. Generally, our approach allows for the association of OTUs with specific functional groups in diverse ecosystems in order to improve our understanding of (microbial) biodiversity-ecosystem functioning relationships. IMPORTANCE A major goal in microbial ecology is to understand how microbial community structure influences ecosystem functioning. Various methods to directly associate bacterial taxa to functional groups in the environment are being developed. In this study, we applied machine learning methods to relate taxonomic data obtained from marker gene surveys to functional groups identified by flow cytometry. This allowed us to identify the taxa that are associated with heterotrophic productivity in freshwater lakes and indicated that the key contributors were highly system specific, regularly rare members of the community, and that some could possibly switch between being low and high contributors. Our approach provides a promising framework to identify taxa that contribute to ecosystem functioning and can be further developed to explore microbial contributions beyond heterotrophic production., (Copyright © 2019 Rubbens et al.)

Intraspecific niche divergence is an important driver of species range, population abundance and impacts on ecosystem functions. Genetic changes are the primary focus when studying intraspecific divergence; however, the role of ecological interactions, particularly host-microbiome symbioses, is receiving increased attention. The relative importance of these evolutionary and ecological mechanisms has seen only limited evaluation. To address this question, we used Microcystis aeruginosa, the globally distributed cyanobacterium that dominates freshwater harmful algal blooms. These blooms have been increasing in occurrence and intensity worldwide, causing major economic and ecological damages. We evaluated 46 isolates of M. aeruginosa and their microbiomes, collected from 14 lakes in Michigan, USA, that vary over 20-fold in phosphorus levels, the primary limiting nutrient in freshwater systems. Genomes of M. aeruginosa diverged along this phosphorus gradient in genomic architecture and protein functions. Fitness in low-phosphorus lakes corresponded with additional shifts within M. aeruginosa including genome-wide reductions in nitrogen use, an expansion of phosphorus assimilation genes and an alternative life history strategy of nonclonal colony formation. In addition to host shifts, despite culturing in common-garden conditions, host-microbiomes diverged along the gradient in taxonomy, but converged in function with evidence of metabolic interdependence between the host and its microbiome. Divergence corresponded with a physiological trade-off between fitness in low-phosphorus environments and growth rate in phosphorus-rich conditions. Co-occurrence of genotypes adapted to different nutrient environments in phosphorus-rich lakes may have critical implications for understanding how M. aeruginosa blooms persist after initial nutrient depletion. Ultimately, we demonstrate that the intertwined effects of genome evolution, host life history strategy and ecological interactions between a host and its microbiome correspond with an intraspecific niche shift with important implications for whole ecosystem function., (© 2019 John Wiley & Sons Ltd.)

We examined the genomic adaptations of prevalent bacterial taxa in a highly nutrient- and ion-depleted freshwater environment located in the secondary cooling water system of a nuclear research reactor. Using genome-centric metagenomics, we found that none of the prevalent bacterial taxa were related to typical freshwater bacterial lineages. We also did not identify strong signatures of genome streamlining, which has been shown to be one of the ecoevolutionary forces shaping the genome characteristics of bacterial taxa in nutrient-depleted environments. Instead, focusing on the dominant taxon, a novel Ramlibacter sp. which we propose to name Ramlibacter aquaticus , we detected extensive positive selection on genes involved in phosphorus and carbon scavenging pathways. These genes were involved in the high-affinity phosphate uptake and storage into polyphosphate granules, metabolism of nitrogen-rich organic matter, and carbon/energy storage into polyhydroxyalkanoate. In parallel, comparative genomics revealed a high number of paralogs and an accessory genome significantly enriched in environmental sensing pathways (i.e., chemotaxis and motility), suggesting extensive gene expansions in R. aquaticus The type strain of R. aquaticus (LMG 30558 T ) displayed optimal growth kinetics and productivity at low nutrient concentrations, as well as substantial cell size plasticity. Our findings with R. aquaticus LMG 30558 T demonstrate that positive selection and gene expansions may represent successful adaptive strategies to oligotrophic environments that preserve high growth rates and cellular productivity. IMPORTANCE By combining a genome-centric metagenomic approach with a culture-based approach, we investigated the genomic adaptations of prevalent populations in an engineered oligotrophic freshwater system. We found evidence for widespread positive selection on genes involved in phosphorus and carbon scavenging pathways and for gene expansions in motility and environmental sensing to be important genomic adaptations of the abundant taxon in this system. In addition, microscopic and flow cytometric analysis of the first freshwater representative of this population ( Ramlibacter aquaticus LMG 30558 T ) demonstrated phenotypic plasticity, possibly due to the metabolic versatility granted by its larger genome, to be a strategy to cope with nutrient limitation. Our study clearly demonstrates the need for the use of a broad set of genomic tools combined with culture-based physiological characterization assays to investigate and validate genomic adaptations., (Copyright © 2019 Props et al.)

[This corrects the article DOI: 10.1371/journal.pone.0195112.].

Algal biofuels have the potential to curb the emissions of greenhouse gases from fossil fuels, but current growing methods fail to produce fuels that meet the multiple standards necessary for economical industrial use. For example, algae grown as monocultures for biofuel production have not simultaneously and economically achieved high yields of the high-quality lipid-rich biomass desired for the industrial-scale production of bio-oil. Decades of study in the field of ecology have demonstrated that simultaneous increases in multiple functions, such as the quantity and quality of biomass, can occur in natural ecosystems by increasing biological diversity. Here, we show that species consortia of algae can improve the production of bio-oil, which benefits from both a high biomass yield and a high quality of biomass rich in fatty acids. We explain the underlying causes of increased quantity and quality of algal biomass among species consortia by showing that, relative to monocultures, species consortia can differentially regulate lipid metabolism genes while growing to higher levels of biomass, in part due to a greater utilization of nutrient resources. We identify multiple genes involved in lipid biosynthesis that are frequently upregulated in bicultures and further show that these elevated levels of gene expression are highly predictive of the elevated levels in biculture relative to that in monoculture of multiple quality metrics of algal biomass. These results show that interactions between species can alter the expression of lipid metabolism genes and further demonstrate that our understanding of diversity-function relationships from natural ecosystems can be harnessed to improve the production of bio-oil. IMPORTANCE Algal biofuels are one of the more promising forms of renewable energy. In our study, we investigate whether ecological interactions between species of microalgae regulate two important factors in cultivation-the biomass of the crop produced and the quality of the biomass that is produced. We found that species interactions often improved production yields, especially the fatty acid content of the algal biomass, and that differentially expressed genes involved in fatty acid metabolism are predictive of improved quality metrics of bio-oil. Other studies have found that diversity often improves productivity and stability in agricultural and natural ecosystems. Our results provide further evidence that growing multispecies crops of microalgae may improve the production of high-quality biomass for bio-oil., (Copyright © 2018 American Society for Microbiology.)

The bacterial phylum Verrucomicrobia was formally described two decades ago and originally believed to be a minor member of many ecosystems; however, it is now recognized as ubiquitous and abundant in both soil and aquatic systems. Nevertheless, knowledge of the drivers of its relative abundance and within-phylum habitat preferences remains sparse, especially in lake systems. Here, we documented the distribution of Verrucomicrobia in 12 inland lakes in Southeastern Michigan, a Laurentian Great Lake (Lake Michigan), and a freshwater estuary, which span a gradient in lake sizes, depths, residence times, and trophic states. A wide range of physical and geochemical parameters was covered by sampling seasonally from the surface and bottom of each lake, and by separating samples into particle-associated and free-living fractions. On average, Verrucomicrobia was the 4th most abundant phylum (range 1.7-41.7%). Fraction, season, station, and depth explained up to 70% of the variance in Verrucomicrobia community composition and preference for these habitats was phylogenetically conserved at the class-level. When relative abundance was linearly modeled against environmental data, Verrucomicrobia and non-Verrucomicrobia bacterial community composition correlated to similar quantitative environmental parameters, although there were lake system-dependent differences and > 55% of the variance remained unexplained. A majority of the phylum exhibited preference for the particle-associated fraction and two classes (Opitutae and Verrucomicrobiae) were identified to be more abundant during the spring season. This study highlights the high relative abundance of Verrucomicrobia in north temperate lake systems and expands insights into drivers of within-phylum habitat preferences of the Verrucomicrobia.

Species invasion is an important disturbance to ecosystems worldwide, yet knowledge about the impacts of invasive species on bacterial communities remains sparse. Using a novel approach, we simultaneously detected phenotypic and derived taxonomic change in a natural bacterioplankton community when subjected to feeding pressure by quagga mussels, a widespread aquatic invasive species. We detected a significant decrease in diversity within 1 h of feeding and a total diversity loss of 11.6 ± 4.1% after 3 h. This loss of microbial diversity was caused by the selective removal of high nucleic acid populations (29 ± 5% after 3 h). We were able to track the community diversity at high temporal resolution by calculating phenotypic diversity estimates from flow cytometry (FCM) data of minute amounts of sample. Through parallel FCM and 16S rRNA gene amplicon sequencing analysis of environments spanning a broad diversity range, we showed that the two approaches resulted in highly correlated diversity measures and captured the same seasonal and lake-specific patterns in community composition. Based on our results, we predict that selective feeding by invasive dreissenid mussels directly impacts the microbial component of the carbon cycle, as it may drive bacterioplankton communities toward less diverse and potentially less productive states., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Blooms of the potentially toxic cyanobacterium Microcystis are increasing worldwide. In the Laurentian Great Lakes they pose major socioeconomic, ecological, and human health threats, particularly in western Lake Erie. However, the interpretation of "omics" data is constrained by the highly variable genome of Microcystis and the small number of reference genome sequences from strains isolated from the Great Lakes. To address this, we sequenced two Microcystis isolates from Lake Erie (Microcystis aeruginosa LE3 and M. wesenbergii LE013-01) and one from upstream Lake St. Clair (M. cf aeruginosa LSC13-02), and compared these data to the genomes of seventeen Microcystis spp. from across the globe as well as one metagenome and seven metatranscriptomes from a 2014 Lake Erie Microcystis bloom. For the publically available strains analyzed, the core genome is ~1900 genes, representing ~11% of total genes in the pan-genome and ~45% of each strain's genome. The flexible genome content was related to Microcystis subclades defined by phylogenetic analysis of both housekeeping genes and total core genes. To our knowledge this is the first evidence that the flexible genome is linked to the core genome of the Microcystis species complex. The majority of strain-specific genes were present and expressed in bloom communities in Lake Erie. Roughly 8% of these genes from the lower Great Lakes are involved in genome plasticity (rapid gain, loss, or rearrangement of genes) and resistance to foreign genetic elements (such as CRISPR-Cas systems). Intriguingly, strain-specific genes from Microcystis cultured from around the world were also present and expressed in the Lake Erie blooms, suggesting that the Microcystis pangenome is truly global. The presence and expression of flexible genes, including strain-specific genes, suggests that strain-level genomic diversity may be important in maintaining Microcystis abundance during bloom events.

One approach to improve forecasts of how global change will affect ecosystem processes is to better understand how anthropogenic disturbances alter bacterial assemblages that drive biogeochemical cycles. Species invasions are important contributors to global change, but their impacts on bacterial community ecology are rarely investigated. Here, we studied direct impacts of invasive dreissenid mussels (IDMs), one of many invasive filter feeders, on freshwater lake bacterioplankton. We demonstrated that direct effects of IDMs reduced bacterial abundance and altered assemblage composition by preferentially removing larger and particle-associated bacteria. While this increased the relative abundances of many free-living bacterial taxa, some were susceptible to filter feeding, in line with efficient removal of phytoplankton cells of <2 μm. This selective removal of particle-associated and larger bacteria by IDMs altered inferred bacterial functional group representation, defined by carbon and energy source utilization. Specifically, we inferred an increased relative abundance of chemoorganoheterotrophs predicted to be capable of rhodopsin-dependent energy generation. In contrast to the few previous studies that have focused on the longer-term combined direct and indirect effects of IDMs on bacterioplankton, our study showed that IDMs act directly as a biological disturbance to which freshwater bacterial assemblages are sensitive. The negative impacts on particle-associated bacteria, which have been shown to be more active than free-living bacteria, and the inferred shifts in functional group representation raise the possibility that IDMs may directly alter bacterially mediated ecosystem functions. IMPORTANCE Freshwater bacteria play fundamental roles in global elemental cycling and are an intrinsic part of local food webs. Human activities are altering freshwater environments, and much has been learned regarding the sensitivity of bacterial assemblages to a variety of these disturbances. Yet, relatively few studies have focused on how species invasion, which is one of the most important aspects of anthropogenic global change, affects freshwater bacterial assemblages. This study focuses on the impact of invasive dreissenid mussels (IDMs), a globally distributed group of invasive species with large impacts on freshwater phyto- and zooplankton assemblages. We show that IDMs have direct effects on lake bacterioplankton abundance, taxonomic composition, and inferred bacterial functional group representation.

Oligotyping is a computational method used to increase the resolution of marker gene microbiome studies. Although oligotyping can distinguish highly similar sequence variants, the resulting units are not necessarily phylogenetically and ecologically informative due to limitations of the selected marker gene. In this perspective, we examine how oligotyping data is interpreted in recent literature, and we illustrate some of the method's constraints with a case study of the harmful bloom-forming cyanobacterium Microcystis . We identified three Microcystis oligotypes from a western Lake Erie bacterial community 16S rRNA gene (V4 region) survey that had previously clustered into one OTU. We found the same three oligotypes and two additional sequence variants in 46 Microcystis cultures isolated from Michigan inland lakes spanning a trophic gradient. In Lake Erie, shifts in Microcystis oligotypes corresponded to spatial nutrient gradients and temporal transitions in bloom toxicity. In the cultures, Microcystis oligotypes showed preferential distributions for different trophic states, but genomic data revealed that the oligotypes identified in Lake Erie did not correspond to toxin gene presence. Thus, oligotypes could not be used for inferring toxic ecotypes. Most strikingly, Microcystis oligotypes were not monophyletic. Our study supports the utility of oligotyping for distinguishing sequence types along certain ecological features, while it stresses that 16S rRNA gene sequence types may not reflect ecologically or phylogenetically cohesive populations. Therefore, we recommend that studies employing oligotyping or related tools consider these caveats during data interpretation.

Human activities are causing a global proliferation of cyanobacterial harmful algal blooms (CHABs), yet we have limited understanding of how these events affect freshwater bacterial communities. Using weekly data from western Lake Erie in 2014, we investigated how the cyanobacterial community varied over space and time, and whether the bloom affected non-cyanobacterial (nc-bacterial) diversity and composition. Cyanobacterial community composition fluctuated dynamically during the bloom, but was dominated by Microcystis and Synechococcus OTUs. The bloom's progression revealed potential impacts to nc-bacterial diversity. Nc-bacterial evenness displayed linear, unimodal, or no response to algal pigment levels, depending on the taxonomic group. In addition, the bloom coincided with a large shift in nc-bacterial community composition. These shifts could be partitioned into components predicted by pH, chlorophyll a, temperature, and water mass movements. Actinobacteria OTUs showed particularly strong correlations to bloom dynamics. AcI-C OTUs became more abundant, while acI-A and acI-B OTUs declined during the bloom, providing evidence of niche partitioning at the sub-clade level. Thus, our observations in western Lake Erie support a link between CHABs and disturbances to bacterial community diversity and composition. Additionally, the short recovery of many taxa after the bloom indicates that bacterial communities may exhibit resilience to CHABs., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. However, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.

Despite their homogeneous appearance, aquatic systems harbour heterogeneous habitats resulting from nutrient gradients, suspended particulate matter and stratification. Recent reports suggest phylogenetically conserved habitat preferences among bacterioplankton, particularly for particle-associated (PA) and free-living (FL) habitats. Here, we show that independent of lake nutrient level and layer, PA and FL abundance-weighted bacterial community composition (BCC) differed and that inter-lake BCC varied more for PA than for FL fractions. In low-nutrient lakes, BCC differences between PA and FL fractions were larger than those between lake layers. The reverse was true for high-nutrient lakes. Nutrient level affected BCC more in hypolimnia than in epilimnia, likely due to hypolimnetic hypoxia in high-nutrient lakes. In line with previous reports, we observed within-phylum operational taxonomic unit (OTU) habitat preference conservation, although not for all phyla, including the phylum with the highest average relative abundance across all habitats (Bacteroidetes). Consistent phylum-level habitat preferences may indicate that the functional traits that underpin ecological adaptation of freshwater bacteria to lake habitats can be phylogenetically conserved, although the levels of conservation are phylum dependent. Resolving taxa preferences for freshwater habitats sets the stage for identification of traits that underpin habitat specialization and associated functional traits that influence differences in biogeochemical cycling across freshwater lake habitats., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The Chloroflexi CL500-11 clade contributes a large proportion of the bacterial biomass in the oxygenated hypolimnia of deep lakes worldwide, including the world's largest freshwater system, the Laurentian Great Lakes. Traits that allow CL500-11 to thrive and its biogeochemical role in these environments are currently unknown. Here, we found that a CL500-11 population was present mostly in offshore waters along a transect in ultraoligotrophic Lake Michigan (a Laurentian Great Lake). It occurred throughout the water column in spring and only in the hypolimnion during summer stratification, contributing up to 18.1% of all cells. Genome reconstruction from metagenomic data suggested an aerobic, motile, heterotrophic lifestyle, with additional energy being gained through carboxidovory and methylovory. Comparisons to other available streamlined freshwater genomes revealed that the CL500-11 genome contained a disproportionate number of cell wall/capsule biosynthesis genes and the most diverse spectrum of genes involved in the uptake of dissolved organic matter (DOM) substrates, particularly peptides. In situ expression patterns indicated the importance of DOM uptake and protein/peptide turnover, as well as type I and type II carbon monoxide dehydrogenase and flagellar motility. Its location in the water column influenced its gene expression patterns the most. We observed increased bacteriorhodopsin gene expression and a response to oxidative stress in surface waters compared to its response in deep waters. While CL500-11 carries multiple adaptations to an oligotrophic lifestyle, its investment in motility, its large cell size, and its distribution in both oligotrophic and mesotrophic lakes indicate its ability to thrive under conditions where resources are more plentiful. Our data indicate that CL500-11 plays an important role in nitrogen-rich DOM mineralization in the extensive deep-lake hypolimnion habitat., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and microbial biodegradation is an important means of remediation of PAH-contaminated soil. Delftia acidovorans Cs1-4 (formerly Delftia sp. Cs1-4) was isolated by using phenanthrene as the sole carbon source from PAH contaminated soil in Wisconsin. Its full genome sequence was determined to gain insights into a mechanisms underlying biodegradation of PAH. Three genomic libraries were constructed and sequenced: an Illumina GAii shotgun library (916,416,493 reads), a 454 Titanium standard library (770,171 reads) and one paired-end 454 library (average insert size of 8 kb, 508,092 reads). The initial assembly contained 40 contigs in two scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together and the consensus sequences were computationally shredded into 2 kb overlapping shreds. Illumina sequencing data was assembled, and the consensus sequence was computationally shredded into 1.5 kb overlapping shreds. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks. A total of 182 additional reactions were needed to close gaps and to raise the quality of the finished sequence. The final assembly is based on 253.3 Mb of 454 draft data (averaging 38.4 X coverage) and 590.2 Mb of Illumina draft data (averaging 89.4 X coverage). The genome of strain Cs1-4 consists of a single circular chromosome of 6,685,842 bp (66.7 %G+C) containing 6,028 predicted genes; 5,931 of these genes were protein-encoding and 4,425 gene products were assigned to a putative function. Genes encoding phenanthrene degradation were localized to a 232 kb genomic island (termed the phn island), which contained near its 3' end a bacteriophage P4-like integrase, an enzyme often associated with chromosomal integration of mobile genetic elements. Other biodegradation pathways reconstructed from the genome sequence included: benzoate (by the acetyl-CoA pathway), styrene, nicotinic acid (by the maleamate pathway) and the pesticides Dicamba and Fenitrothion. Determination of the complete genome sequence of D. acidovorans Cs1-4 has provided new insights the microbial mechanisms of PAH biodegradation that may shape the process in the environment.

Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples.

Few data are available on how quickly free-living microorganisms evolve. We analyzed biofilms collected from a well-defined acid mine drainage system over 9 years to investigate the processes and determine rates of bacterial evolution directly in the environment. Population metagenomic analyses of the dominant primary producer yielded the nucleotide substitution rate, which we used to show that proliferation of a series of recombinant bacterial strains occurred over the past few decades. The ecological success of hybrid bacterial types highlights the role of evolutionary processes in rapid adaptation within natural microbial communities.

Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This 'trailer-end conservation' occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. 'Trailer-end clonality' occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining 'trailer-end conservation,' we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.

Extensive genomic characterization of multi-species acid mine drainage microbial consortia combined with laboratory cultivation has enabled the application of quantitative proteomic analyses at the community level. In this study, quantitative proteomic comparisons were used to functionally characterize laboratory-cultivated acidophilic communities sustained in pH 1.45 or 0.85 conditions. The distributions of all proteins identified for individual organisms indicated biases for either high or low pH, and suggests pH-specific niche partitioning for low abundance bacteria and archaea. Although the proteome of the dominant bacterium, Leptospirillum group II, was largely unaffected by pH treatments, analysis of functional categories indicated proteins involved in amino acid and nucleotide metabolism, as well as cell membrane/envelope biogenesis were overrepresented at high pH. Comparison of specific protein abundances indicates higher pH conditions favor Leptospirillum group III, whereas low pH conditions promote the growth of certain archaea. Thus, quantitative proteomic comparisons revealed distinct differences in community composition and metabolic function of individual organisms during different pH treatments. Proteomic analysis revealed other aspects of community function. Different numbers of phage proteins were identified across biological replicates, indicating stochastic spatial heterogeneity of phage outbreaks. Additionally, proteomic data were used to identify a previously unknown genotypic variant of Leptospirillum group II, an indication of selection for a specific Leptospirillum group II population in laboratory communities. Our results confirm the importance of pH and related geochemical factors in fine-tuning acidophilic microbial community structure and function at the species and strain level, and demonstrate the broad utility of proteomics in laboratory community studies.

The intestinal microbiome is a critical determinant of human health. Alterations in its composition have been correlated with chronic disorders, such as obesity and inflammatory bowel disease in adults, and may be associated with neonatal necrotizing enterocolitis in premature infants. Increasing evidence suggests that strain-level genomic variation may underpin distinct ecological trajectories within mixed populations, yet there have been few strain-resolved analyses of genotype-phenotype connections in the context of the human ecosystem. Here, we document strain-level genomic divergence during the first 3 wk of life within the fecal microbiota of an infant born at 28-wk gestation. We observed three compositional phases during colonization, and reconstructed and intensively curated population genomic datasets from the third phase. The relative abundance of two Citrobacter strains sharing ~99% nucleotide identity changed significantly over time within a community dominated by a nearly clonal Serratia population and harboring a lower abundance Enterococcus population and multiple plasmids and bacteriophage. Modeling of Citrobacter strain abundance suggests differences in growth rates and host colonization patterns. We identified genotypic variation potentially responsible for divergent strain ecologies, including hotspots of sequence variation in regulatory genes and intergenic regions, and in genes involved in transport, flagellar biosynthesis, substrate metabolism, and host colonization, as well as differences in the complements of these genes. Our results demonstrate that a community genomic approach can elucidate gut microbial colonization at the resolution required to discern medically relevant strain and species population dynamics, and hence improve our ability to diagnose and treat microbial community-mediated disorders.

Tandem high-throughput proteomics and metabolomics were employed to functionally characterize natural microbial biofilm communities. Distinct molecular signatures exist for each analyzed sample. Deconvolution of the high-resolution molecular data demonstrates that identified proteins and detected metabolites exhibit organism-specific correlation patterns. These patterns are reflective of the functional differentiation of two bacterial species that share the same genus and that co-occur in the sampled microbial communities. Our analyses indicate that the two species have similar niche breadths and are not in strong competition with one another.

An important challenge in microbial ecology is developing methods that simultaneously examine the physiology of organisms at the molecular level and their ecosystem level interactions in complex natural systems. We integrated extensive proteomic, geochemical, and biological information from 28 microbial communities collected from an acid mine drainage environment and representing a range of biofilm development stages and geochemical conditions to evaluate how the physiologies of the dominant and less abundant organisms change along environmental gradients. The initial colonist dominates across all environments, but its proteome changes between two stable states as communities diversify, implying that interspecies interactions affect this organism's metabolism. Its overall physiology is robust to abiotic environmental factors, but strong correlations exist between these factors and certain subsets of proteins, possibly accounting for its wide environmental distribution. Lower abundance populations are patchier in their distribution, and proteomic data indicate that their environmental niches may be constrained by specific sets of abiotic environmental factors. This research establishes an effective strategy to investigate ecological relationships between microbial physiology and the environment for whole communities in situ.

Similar to virtually all components of natural environments, microbial systems are inherently complex and dynamic. Advances in cultivation-independent molecular methods have provided a route to study microbial consortia in their natural surroundings and to begin resolving the community structure, dominant metabolic processes and inter-organism interactions. However, the utility of these methods generally scales inversely with community complexity. By applying genomics-enabled methods to the study of natural microbial communities with reduced levels of species richness, a relatively comprehensive understanding of the metabolic networks and evolutionary processes within these communities can be attained. In such well-defined model systems, it is also possible to link emergent ecological patterns to their molecular and evolutionary underpinnings, facilitating construction of predictive ecosystem models. In this study, we review over a decade of research on one such system-acid mine drainage biofilm communities. We discuss the value and limitations of tractable model microbial communities in developing molecular methods for microbial ecology and in uncovering principles that may explain behavior in more complex systems.

Bacterial species concepts are controversial. More widely accepted is the need to understand how differences in gene content and sequence lead to ecological divergence. To address this relationship in ecosystem context, we investigated links between genotype and ecology of two genotypic groups of Leptospirillum group II bacteria in comprehensively characterized, natural acidophilic biofilm communities. These groups share 99.7% 16S rRNA gene sequence identity and 95% average amino acid identity between their orthologs. One genotypic group predominates during early colonization, and the other group typically proliferates in later successional stages, forming distinct patches tens to hundreds of micrometers in diameter. Among early colonizing populations, we observed dominance of five genotypes that differed from each other by the extent of recombination with the late colonizing type. Our analyses suggest that the specific recombinant variant within the early colonizing group is selected for by environmental parameters such as temperature, consistent with recombination as a mechanism for ecological fine tuning. Evolutionary signatures, and strain-resolved expression patterns measured via mass spectrometry-based proteomics, indicate increased cobalamin biosynthesis, (de)methylation, and glycine cleavage in the late colonizer. This may suggest environmental changes within the biofilm during development, accompanied by redirection of compatible solutes from osmoprotectants toward metabolism. Across 27 communities, comparative proteogenomic analyses show that differential regulation of shared genes and expression of a small subset of the approximately 15% of genes unique to each genotype are involved in niche partitioning. In summary, the results show how subtle genetic variations can lead to distinct ecological strategies.

The principal means for microbial degradation of polychlorinated biphenyls (PCBs) is through the biphenyl pathway. Although molecular aspects of the regulation of the biphenyl pathway have been studied, information on environmental facets such as the effect of alternative carbon sources on (polychlorinated) biphenyl degradation is limited. Here we explore the effect of environmental conditions (e.g., carbon source and growth phase) on the variation in PCB degradation profiles of Burkholderia xenovorans LB400. Genome-wide expression patterns reveal 25 genes commonly up-regulated during PCB degradation and growth on biphenyl to be upregulated in the transition to stationary phase (relative to growth on succinate) including two putative detoxification pathways. Quantitative reverse transcription PCR (Q-RT-PCR) analysis of the upper biphenyl pathway (bphA, bphD, and bphR1), and detoxification genes in response to environmental conditions suggest associated regulation of the biphenyl pathway and chloroacetaldehyde dehydrogenase. The response of genes in the upper biphenyl pathway to carbon source competition and growth phase reveals inhibition of the biphenyl pathway by PCBs. Although PCBs are not degraded during growth on succinate with PCBs, expression data indicate that the biphenyl pathway is induced, suggesting that post-transcriptional regulation or active transport of biphenyl maybe limiting PCB degradation. Identification of the involvement of peripheral pathways in degradation of PCBs is crucial to understanding PCB degradation in an environmental context as bacteria capable of biodegradation experience a range of carbon sources and growth phases.

We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.

We know very little about the metabolic functioning and evolutionary dynamics of microbial communities. Recent advances in comprehensive, sequencing-based methods, however, are laying a molecular foundation for new insights into how microbial communities shape the Earth's biosphere. Here we explore the convergence of microbial ecology, genomics, biological mass spectrometry and informatics that form the new field of microbial community proteogenomics. We discuss the first applications of proteogenomics and its potential for studying the physiology, ecology and evolution of microbial populations and communities.

Analyses of ecological and evolutionary processes that shape microbial consortia are facilitated by comprehensive studies of ecosystems with low species richness. In the current study we evaluated the role of recombination in altering the fitness of chemoautotrophic bacteria in their natural environment. Proteomics-inferred genome typing (PIGT) was used to genotype the dominant Leptospirillum group II populations in 27 biofilms sampled from six locations in the Richmond Mine acid mine drainage system (Iron Mountain, CA) over a 4-year period. We observed six distinct genotypes that are recombinants comprised of segments from two 'parental' genotypes. Community genomic analyses revealed additional low abundance recombinant variants. The dominance of some genotypes despite a larger available genome pool, and patterns of spatiotemporal distribution within the ecosystem, indicate selection for distinct recombinants. Genes involved in motility, signal transduction and transport were over-represented in the tens to hundreds of kilobase recombinant blocks, whereas core metabolic functions were significantly under-represented. Our findings demonstrate the power of PIGT and reveal that recombination is a mechanism for fine-scale adaptation in this system.

Community genomic data have revealed multiple levels of variation between and within microbial consortia. This variation includes large-scale differences in gene content between ecosystems as well as within-population sequence heterogeneity. In the present review, we focus specifically on how fine-scale variation within microbial and viral populations is apparent from community genomic data. A major unresolved question is how much of the observed variation is due to neutral vs. adaptive processes. Limited experimental data hint that some of this fine-scale variation may be in part functionally relevant, whereas sequence-based and modeling analyses suggest that much of it may be neutral. While methods for interpreting population genomic data are still in their infancy, we discuss current interpretations of existing datasets in the light of evolutionary processes and models. Finally, we highlight the importance of virus-host dynamics in generating and shaping within-population diversity.