Structure-informed microbial population genetics elucidate selective pressures that shape protein evolution.

Comprehensive sampling of natural genetic diversity with metagenomics enables highly resolved insights into the interplay between ecology and evolution. However, resolving adaptive, neutral, or purifying processes of evolution from intrapopulation genomic variation remains a challenge, partly due to the sole reliance on gene sequences to interpret variants. Here, we describe an approach to analyze genetic variation in the context of predicted protein structures and apply it to a marine microbial population within the SAR11 subclade 1a.3.V, which dominates low-latitude surface oceans. Our analyses reveal a tight association between genetic variation and protein structure. In a central gene in nitrogen metabolism, we observe decreased occurrence of nonsynonymous variants from ligand-binding sites as a function of nitrate concentrations, revealing genetic targets of distinct evolutionary pressures maintained by nutrient availability. Our work yields insights into the governing principles of evolution and enables structure-aware investigations of microbial population genetics.

FORENSIC: an Online Platform for Fecal Source Identification.

Sewage overflows, agricultural runoff, and stormwater discharges introduce fecal pollution into surface waters. Distinguishing these sources is critical for evaluating water quality and formulating remediation strategies. With the falling costs of sequencing, microbial community-based water quality assessment tools are under development. However, their application is limited by the need to build reference libraries, which requires extensive sampling of sources and bioinformatic expertise. Here, we introduce FORest Enteric Source IdentifiCation (FORENSIC; https://forensic.sfs.uwm.edu/), an online, library-independent source tracking platform based on random forest classification and 16S rRNA gene amplicon sequences to identify in environmental samples common fecal contamination sources, including humans, domestic pets, and agricultural animals. FORENSIC relies on a broad reference signature database of Bacteroidales and Clostridiales, two predominant bacterial groups that have coevolved with their hosts. As a result, these groups demonstrate cohesive and reliable assemblage patterns within mammalian species or among species sharing the same diet/physiology. We created a scalable and extensible platform that we tested for global applicability using samples collected in distant geographic locations. This Web application offers a fast and intuitive approach for fecal source identification, particularly in sewage-contaminated waters.IMPORTANCE FORENSIC is an online platform to identify sources of fecal pollution without the need to create reference libraries. FORENSIC is based on the ability of random forest classification to extract cohesive source microbial signatures to create classifiers despite individual variability and to detect the signatures in environmental samples. We primarily focused on defining sewage signals, which are associated with a high human health risk in polluted waters. To test for fecal contamination sources, the platform only requires paired-end reads targeting the V4 or V6 regions of the 16S rRNA gene. We demonstrated that we could use V4V5 reads trimmed to the V4 positions to generate the reference signature. The systematic workflow we describe to create and validate the signatures could be applied to many disciplines. With the increasing gap between advancing technology and practical applications, this platform makes sequence-based water quality assessments accessible to the public health and water resource communities.

Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome.

INTRODUCTION: Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and intriguing patterns of site specificity amidst the lack of dispersal limitation. However, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among taxa that belong to recently discovered branches of microbial life. RESULTS: Here, we assemble metagenomes from tongue and dental plaque samples from multiple individuals and reconstruct 790 non-redundant genomes, 43 of which resolve to TM7, a member of the Candidate Phyla Radiation, forming six monophyletic clades that distinctly associate with either plaque or tongue. Both pangenomic and phylogenomic analyses group tongue-specific clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 group with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and mobilome of cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of microbes. Additionally, we report that prophages are widespread among oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment. CONCLUSIONS: Our data illuminate niche partitioning of enigmatic members of the oral cavity, including TM7, SR1, and GN02, and provide genomes for poorly characterized yet prevalent members of this biome, such as uncultivated Flavobacteriaceae.

Single-amino acid variants reveal evolutionary processes that shape the biogeography of a global SAR11 subclade.

Members of the SAR11 order Pelagibacterales dominate the surface oceans. Their extensive diversity challenges emerging operational boundaries defined for microbial 'species' and complicates efforts of population genetics to study their evolution. Here, we employed single-amino acid variants (SAAVs) to investigate ecological and evolutionary forces that maintain the genomic heterogeneity within ubiquitous SAR11 populations we accessed through metagenomic read recruitment using a single isolate genome. Integrating amino acid and protein biochemistry with metagenomics revealed that systematic purifying selection against deleterious variants governs non-synonymous variation among very closely related populations of SAR11. SAAVs partitioned metagenomes into two main groups matching large-scale oceanic current temperatures, and six finer proteotypes that connect distant oceanic regions. These findings suggest that environmentally-mediated selection plays a critical role in the journey of cosmopolitan surface ocean microbial populations, and the idea 'everything is everywhere but the environment selects' has credence even at the finest resolutions.

The Wolbachia mobilome in Culex pipiens includes a putative plasmid.

Wolbachia is a genus of obligate intracellular bacteria found in nematodes and arthropods worldwide, including insect vectors that transmit dengue, West Nile, and Zika viruses. Wolbachia's unique ability to alter host reproductive behavior through its temperate bacteriophage WO has enabled the development of new vector control strategies. However, our understanding of Wolbachia's mobilome beyond its bacteriophages is incomplete. Here, we reconstruct near-complete Wolbachia genomes from individual ovary metagenomes of four wild Culex pipiens mosquitoes captured in France. In addition to viral genes missing from the Wolbachia reference genome, we identify a putative plasmid (pWCP), consisting of a 9.23-kbp circular element with 14 genes. We validate its presence in additional Culex pipiens mosquitoes using PCR, long-read sequencing, and screening of existing metagenomes. The discovery of this previously unrecognized extrachromosomal element opens additional possibilities for genetic manipulation of Wolbachia.

Author Correction: The Wolbachia mobilome in Culex pipiens includes a putative plasmid.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-resolved insights into a novel Spiroplasma symbiont of the Wheat Stem Sawfly (Cephus cinctus).

Arthropods often have obligate relationships with symbiotic microbes, and recent investigations have demonstrated that such host-microbe relationships could be exploited to suppress natural populations of vector carrying mosquitos. Strategies that target the interplay between agricultural pests and their symbionts could decrease the burden caused by agricultural pests; however, the lack of comprehensive genomic insights into naturally occurring microbial symbionts presents a significant bottleneck. Here we employed amplicon surveys, genome-resolved metagenomics, and scanning electron microscopy to investigate symbionts of the wheat stem sawfly (Cephus cinctus), a major pest that causes an estimated $350 million dollars or more in wheat yield losses in the northwestern United States annually. Through 16S rRNA gene sequencing of two major haplotypes and life stages of wheat stem sawfly, we show a novel Spiroplasma species is ever-present and predominant, with phylogenomic analyses placing it as a member of the ixodetis clade of mollicutes. Using state-of-the-art metagenomic assembly and binning strategies we were able to reconstruct a 714 Kb, 72.7%-complete Spiroplasma genome, which represents just the second draft genome from the ixodetis clade of mollicutes. Functional annotation of the Spiroplasma genome indicated carbohydrate-metabolism involved PTS-mediated import of glucose and fructose followed by glycolysis to lactate, acetate, and propionoate. The bacterium also encoded biosynthetic pathways for essential vitamins B2, B3, and B9. We identified putative Spiroplasma virulence genes: cardiolipin and chitinase. These results identify a previously undescribed symbiosis between wheat stem sawfly and a novel Spiroplasma sp., availing insight into their molecular relationship, and may yield new opportunities for microbially-mediated pest control strategies.

Nitrogen-fixing populations of Planctomycetes and Proteobacteria are abundant in surface ocean metagenomes.

Nitrogen fixation in the surface ocean impacts global marine nitrogen bioavailability and thus microbial primary productivity. Until now, cyanobacterial populations have been viewed as the main suppliers of bioavailable nitrogen in this habitat. Although PCR amplicon surveys targeting the nitrogenase reductase gene have revealed the existence of diverse non-cyanobacterial diazotrophic populations, subsequent quantitative PCR surveys suggest that they generally occur in low abundance. Here, we use state-of-the-art metagenomic assembly and binning strategies to recover nearly one thousand non-redundant microbial population genomes from the TARA Oceans metagenomes. Among these, we provide the first genomic evidence for non-cyanobacterial diazotrophs inhabiting surface waters of the open ocean, which correspond to lineages within the Proteobacteria and, most strikingly, the Planctomycetes. Members of the latter phylum are prevalent in aquatic systems, but have never been linked to nitrogen fixation previously. Moreover, using genome-wide quantitative read recruitment, we demonstrate that the discovered diazotrophs were not only widespread but also remarkably abundant (up to 0.3% of metagenomic reads for a single population) in both the Pacific Ocean and the Atlantic Ocean northwest. Our results extend decades of PCR-based gene surveys, and substantiate the importance of heterotrophic bacteria in the fixation of nitrogen in the surface ocean.

Author Correction: Nitrogen-fixing populations of Planctomycetes and Proteobacteria are abundant in surface ocean metagenomes.

In the version of this Article originally published, the surname of author Sandra M. McLellan was spelt incorrectly as 'MacLellan'. This has now been corrected. In addition, Fig. 2 was mistakenly duplicated in the Supplementary Information as Supplementary Fig. 2. This has now been replaced with the correct supplementary figure (shown below).

Tracking microbial colonization in fecal microbiota transplantation experiments via genome-resolved metagenomics.

BACKGROUND: Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection and shows promise for treating other medical conditions associated with intestinal dysbioses. However, we lack a sufficient understanding of which microbial populations successfully colonize the recipient gut, and the widely used approaches to study the microbial ecology of FMT experiments fail to provide enough resolution to identify populations that are likely responsible for FMT-derived benefits. METHODS: We used shotgun metagenomics together with assembly and binning strategies to reconstruct metagenome-assembled genomes (MAGs) from fecal samples of a single FMT donor. We then used metagenomic mapping to track the occurrence and distribution patterns of donor MAGs in two FMT recipients. RESULTS: Our analyses revealed that 22% of the 92 highly complete bacterial MAGs that we identified from the donor successfully colonized and remained abundant in two recipients for at least 8 weeks. Most MAGs with a high colonization rate belonged to the order Bacteroidales. The vast majority of those that lacked evidence of colonization belonged to the order Clostridiales, and colonization success was negatively correlated with the number of genes related to sporulation. Our analysis of 151 publicly available gut metagenomes showed that the donor MAGs that colonized both recipients were prevalent, and the ones that colonized neither were rare across the participants of the Human Microbiome Project. Although our dataset showed a link between taxonomy and the colonization ability of a given MAG, we also identified MAGs that belong to the same taxon with different colonization properties, highlighting the importance of an appropriate level of resolution to explore the functional basis of colonization and to identify targets for cultivation, hypothesis generation, and testing in model systems. CONCLUSIONS: The analytical strategy adopted in our study can provide genomic insights into bacterial populations that may be critical to the efficacy of FMT due to their success in gut colonization and metabolic properties, and guide cultivation efforts to investigate mechanistic underpinnings of this procedure beyond associations.