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ABSTRACT Bacteria and archaea employ a rudimentary immune system, CRISPR-Cas, to protect against foreign genetic elements such as bacteriophage. CRISPR-Cas systems are found inBombella apis.B. apisis an important honey bee symbiont, found primarily in larvae, queens, and hive compartments.B. apisis found in the worker bee gut but is not considered a core member of the bee microbiome and has therefore been understudied with regard to its importance in the honey bee colony. However,B. apisappears to play beneficial roles in the colony, by protecting developing brood from fungal pathogens and by bolstering their development under nutritional stress. Previously, we identified CRISPR-Cas systems as being acquired byB. apisin its transition to bee association, as they are absent in a sister clade. Here, we assess the variation and distribution of CRISPR-Cas types acrossB. apisstrains. We found multiple CRISPR-Cas types, some of which have multiple arrays, within the sameB. apisgenomes and also in the honey bee queen gut metagenomes. We analyzed the spacers between strains to identify the history of mobile element interaction for eachB. apisstrain. Finally, we predict interactions between viral sequences and CRISPR systems from different honey bee microbiome members. Our analyses show that theB. apisCRISPR-Cas systems are dynamic; that microbes in the same niche have unique spacers, which supports the functionality of these CRISPR-Cas systems; and that acquisition of new spacers may be occurring in multiple locations in the genome, allowing for a flexible antiviral arsenal for the microbe. IMPORTANCEHoney bee worker gut microbes have been implicated in everything from protection from pathogens to breakdown of complex polysaccharides in the diet. However, there are multiple niches within a honey bee colony that host different groups of microbes, including the acetic acid bacteriumBombella apis.B. apisis found in the colony food stores, in association with brood, in worker hypopharyngeal glands, and in the queen’s digestive tract. The roles thatB. apismay serve in these environments are just beginning to be discovered and include the production of a potent antifungal that protects developing bees and supplementation of dietary lysine to young larvae, bolstering their nutrition. Niche specificity inB. apismay be affected by the pressures of bacteriophage and other mobile elements, which may target different strains in each specific bee environment. Studying the interplay betweenB. apisand its mobile genetic elements (MGEs) may help us better understand microbial community dynamics within the colony and the potential ramifications for the honey bee host. 

ABSTRACT Climate change is inducing wide-scale permafrost thaw in the Arctic and subarctic, triggering concerns that long-dormant pathogens could reemerge from the thawing ground and initiate epidemics or pandemics. Viruses, as opposed to bacterial pathogens, garner particular interest because outbreaks cannot be controlled with antibiotics, though the effects can be mitigated by vaccines and newer antiviral drugs. To evaluate the potential hazards posed by viral pathogens emerging from thawing permafrost, we review information from a diverse range of disciplines. This includes efforts to recover infectious virus from human remains, studies on disease occurrence in polar animal populations, investigations into viral persistence and infectivity in permafrost, and assessments of human exposure to the enormous viral diversity present in the environment. Based on currently available knowledge, we conclude that the risk posed by viruses from thawing permafrost is no greater than viruses in other environments such as temperate soils and aquatic systems. 

Colpoda, one of the most widespread ciliated protozoa in soil, are poorly understood in regard to their genetics and evolution. Our research revealed extreme mitochondrial gene rearrangements dominated by gene loss events, potentially leading to the streamlining ofColpodamitogenomes. Surprisingly, while interspecific rearrangements abound, our population-level mitogenomic study revealed a conserved gene order within species, offering a potential new identification criterion. Phylogenomic analysis traced their lineage over 326 million years, revealing two distinct groups. Substantial genomic divergence might be associated with the lack of extended collinear blocks and relaxed purifying selection. This study systematically revealsColpodaciliate mitogenome structures and evolution, providing insights into the survival and evolution of these vital soil microorganisms. 

ABSTRACT Targeted amplicon sequencing is widely used in microbial ecology studies. However, sequencing artifacts and amplification biases are of great concern. To identify sources of these artifacts, a systematic analysis was performed using mock communities comprised of 16S rRNA genes from 33 bacterial strains. Our results indicated that while sequencing errors were generally isolated to low-abundance operational taxonomic units, chimeric sequences were a major source of artifacts. Singleton and doubleton sequences were primarily chimeras. Formation of chimeric sequences was significantly correlated with the GC content of the targeted sequences. Low-GC-content mock community members exhibited lower rates of chimeric sequence formation. GC content also had a large impact on sequence recovery. The quantitative capacity was notably limited, with substantial recovery variations and weak correlation between anticipated and observed strain abundances. The mock community strains with higher GC content had higher recovery rates than strains with lower GC content. Amplification bias was also observed due to the differences in primer affinity. A two-step PCR strategy reduced the number of chimeric sequences by half. In addition, comparative analyses based on the mock communities showed that several widely used sequence processing pipelines/methods, including DADA2, Deblur, UCLUST, UNOISE, and UPARSE, had different advantages and disadvantages in artifact removal and rare species detection. These results are important for improving sequencing quality and reliability and developing new algorithms to process targeted amplicon sequences. IMPORTANCEAmplicon sequencing of targeted genes is the predominant approach to estimate the membership and structure of microbial communities. However, accurate reconstruction of community composition is difficult due to sequencing errors, and other methodological biases and effective approaches to overcome these challenges are essential. Using a mock community of 33 phylogenetically diverse strains, this study evaluated the effect of GC content on sequencing results and tested different approaches to improve overall sequencing accuracy while characterizing the pros and cons of popular amplicon sequence data processing approaches. The sequencing results from this study can serve as a benchmarking data set for future algorithmic improvements. Furthermore, the new insights on sequencing error, chimera formation, and GC bias from this study will help enhance the quality of amplicon sequencing studies and support the development of new data analysis approaches. 

ABSTRACT The euphotic zone of the surface ocean contains distinct physical-chemical regimes that vary in light and nutrient concentrations as an inverse function of depth. The most numerous phytoplankter of the mid- and low-latitude ocean is the picocyanobacteriumProchlorococcus, which consists of ecologically distinct subpopulations (i.e., “ecotypes”). Ecotypes have different temperature, light, and nutrient optima and display distinct relative abundances along gradients of these niche dimensions. As a primary producer,Prochlorococcusfixes and releases organic carbon to neighboring microbes as part of the microbial loop. However, little is known about the specific moleculesProchlorococcusaccumulates and releases or how these processes vary among its ecotypes. Here, we characterize the metabolite diversity ofProchlorococcusby profiling three ecologically distinct cultured strains: MIT9301, representing a high-light-adapted ecotype dominating shallow tropical and sub-tropical waters; MIT0801, representing a low-light-adapted ecotype found throughout the euphotic zone; and MIT9313, representing a low-light-adapted ecotype relatively most abundant at the base of the euphotic zone. In both intracellular and extracellular metabolite profiles, we observe striking differences across strains in the accumulation and release of molecules, such as the DNA methylating agent S-adenosyl-methionine (intracellular) and the branched-chain amino acids (intracellular) and their precursors (extracellular). While some differences reflect variable genome content across the strains, others likely reflect variable regulation of conserved pathways. In the extracellular profiles, we identify molecules such as pantothenic acid and aromatic amino acids that may serve as currencies inProchlorococcus’ interactions with neighboring microbes and, therefore, merit further investigation. IMPORTANCEApproximately half of the annual carbon fixation on Earth occurs in the surface ocean through the photosynthetic activities of phytoplankton such as the ubiquitous picocyanobacteriumProchlorococcus. Ecologically distinct subpopulations (or ecotypes) ofProchlorococcusare central conduits of organic substrates into the ocean microbiome, thus playing important roles in surface ocean production. We measured the chemical profile of three cultured ecotype strains, observing striking differences among them that have implications for the likely chemical impact ofProchlorococcussubpopulations on their surroundings in the wild. Subpopulations differ in abundance along gradients of temperature, light, and nutrient concentrations, suggesting that these chemical differences could affect carbon cycling in different ocean strata and should be considered in models ofProchlorococcusphysiology and marine carbon dynamics. 

ABSTRACT Biological soil crusts (biocrusts) are widespread in drylands and deserts. At the microhabitat scale, they also host hypolithic communities that live under semitranslucent stones. Both environmental niches experience exposure to extreme conditions such as high UV radiation, desiccation, temperature fluctuations, and resource limitation. However, hypolithic communities are somewhat protected from extremes relative to biocrust communities. Conditions are otherwise similar, so comparing them can answer outstanding questions regarding adaptations to environmental extremes. Using metagenomic sequencing, we assessed the functional potential of dryland soil communities and identified the functional underpinnings of ecological niche differentiation in biocrusts versus hypoliths. We also determined the effect of the anchoring photoautotroph (moss or cyanobacteria). Genes and pathways differing in abundance between biocrusts and hypoliths indicate that biocrust communities adapt to the higher levels of UV radiation, desiccation, and temperature extremes through an increased ability to repair damaged DNA, sense and respond to environmental stimuli, and interact with other community members and the environment. Intracellular competition appears to be crucial to both communities, with biocrust communities using the Type VI Secretion System (T6SS) and hypoliths favoring a diversity of antibiotics. The dominant primary producer had a reduced effect on community functional potential compared with niche, but an abundance of genes related to monosaccharide, amino acid, and osmoprotectant uptake in moss-dominated communities indicates reliance on resources provided to heterotrophs by mosses. Our findings indicate that functional traits in dryland communities are driven by adaptations to extremes and we identify strategies that likely enable survival in dryland ecosystems. IMPORTANCE Biocrusts serve as a keystone element of desert and dryland ecosystems, stabilizing soils, retaining moisture, and serving as a carbon and nitrogen source in oligotrophic environments. Biocrusts cover approximately 12% of the Earth’s terrestrial surface but are threatened by climate change and anthropogenic disturbance. Given their keystone role in ecosystem functioning, loss will have wide-spread consequences. Biocrust microbial constituents must withstand polyextreme environmental conditions including high UV exposure, desiccation, oligotrophic conditions, and temperature fluctuations over short time scales. By comparing biocrust communities with co-occurring hypolithic communities (which inhabit the ventral sides of semitranslucent stones and are buffered from environmental extremes), we identified traits that are likely key adaptations to extreme conditions. These include DNA damage repair, environmental sensing and response, and intracellular competition. Comparison of the two niches, which differ primarily in exposure levels to extreme conditions, makes this system ideal for understanding how functional traits are structured by the environment. 

ABSTRACT Host association—the selective adaptation of pathogens to specific host species—evolves through constant interactions between host and pathogens, leaving a lot yet to be discovered on immunological mechanisms and genomic determinants. The causative agents of Lyme disease (LD) are spirochete bacteria composed of multiple species of the Borrelia burgdorferi sensu lato complex, including B. burgdorferi ( Bb ), the main LD pathogen in North America—a useful model for the study of mechanisms underlying host-pathogen association. Host adaptation requires pathogens’ ability to evade host immune responses, such as complement, the first-line innate immune defense mechanism. We tested the hypothesis that different host-adapted phenotypes among Bb strains are linked to polymorphic loci that confer complement evasion traits in a host-specific manner. We first examined the survivability of 20 Bb strains in sera in vitro and/or bloodstream and tissues in vivo from rodent and avian LD models. Three groups of complement-dependent host-association phenotypes emerged. We analyzed complement-evasion genes, identified a priori among all strains and sequenced and compared genomes for individual strains representing each phenotype. The evolutionary history of ospC loci is correlated with host-specific complement-evasion phenotypes, while comparative genomics suggests that several gene families and loci are potentially involved in host association. This multidisciplinary work provides novel insights into the functional evolution of host-adapted phenotypes, building a foundation for further investigation of the immunological and genomic determinants of host association. IMPORTANCE Host association is the phenotype that is commonly found in many pathogens that preferential survive in particular hosts. The Lyme disease (LD)-causing agent, B. burgdorferi ( Bb ), is an ideal model to study host association, as Bb is mainly maintained in nature through rodent and avian hosts. A widespread yet untested concept posits that host association in Bb strains is linked to Bb functional genetic variation conferring evasion to complement, an innate defense mechanism in vertebrate sera. Here, we tested this concept by grouping 20 Bb strains into three complement-dependent host-association phenotypes based on their survivability in sera and/or bloodstream and distal tissues in rodent and avian LD models. Phylogenomic analysis of these strains further correlated several gene families and loci, including ospC , with host-specific complement-evasion phenotypes. Such multifaceted studies thus pave the road to further identify the determinants of host association, providing mechanistic insights into host-pathogen interaction. 

ABSTRACT Whether a microbe is free-living or associated with a host from across the tree of life, its existence depends on a limited number of elements and electron donors and acceptors. Yet divergent approaches have been used by investigators from different fields. The “environment first” research tradition emphasizes thermodynamics and biogeochemical principles, including the quantification of redox environments and elemental stoichiometry to identify transformations and thus an underlying microbe. The increasingly common “microbe first” research approach benefits from culturing and/or DNA sequencing methods to first identify a microbe and encoded metabolic functions. Here, the microbe itself serves as an indicator for environmental conditions and transformations. We illustrate the application of both approaches to the study of microbiomes and emphasize how both can reveal the selection of microbial metabolisms across diverse environments, anticipate alterations to microbiomes in host health, and understand the implications of a changing climate for microbial function. 

ABSTRACT Humans are inextricably linked to each other and our natural world, and microorganisms lie at the nexus of those interactions. Microorganisms form genetically flexible, taxonomically diverse, and biochemically rich communities, i.e., microbiomes that are integral to the health and development of macroorganisms, societies, and ecosystems. Yet engagement with beneficial microbiomes is dictated by access to public resources, such as nutritious food, clean water and air, safe shelter, social interactions, and effective medicine. In this way, microbiomes have sociopolitical contexts that must be considered. The Microbes and Social Equity (MSE) Working Group connects microbiology with social equity research, education, policy, and practice to understand the interplay of microorganisms, individuals, societies, and ecosystems. Here, we outline opportunities for integrating microbiology and social equity work through broadening education and training; diversifying research topics, methods, and perspectives; and advocating for evidence-based public policy that supports sustainable, equitable, and microbial wealth for all. 

ABSTRACT Small subunit rRNA (SSU rRNA) amplicon sequencing can quantitatively and comprehensively profile natural microbiomes, representing a critically important tool for studying diverse global ecosystems. However, results will only be accurate if PCR primers perfectly match the rRNA of all organisms present. To evaluate how well marine microorganisms across all 3 domains are detected by this method, we compared commonly used primers with >300 million rRNA gene sequences retrieved from globally distributed marine metagenomes. The best-performing primers compared to 16S rRNA of bacteria and archaea were 515Y/926R and 515Y/806RB, which perfectly matched over 96% of all sequences. Considering cyanobacterial and chloroplast 16S rRNA, 515Y/926R had the highest coverage (99%), making this set ideal for quantifying marine primary producers. For eukaryotic 18S rRNA sequences, 515Y/926R also performed best (88%), followed by V4R/V4RB (18S rRNA specific; 82%)—demonstrating that the 515Y/926R combination performs best overall for all 3 domains. Using Atlantic and Pacific Ocean samples, we demonstrate high correspondence between 515Y/926R amplicon abundances (generated for this study) and metagenomic 16S rRNA (median R 2 = 0.98, n  = 272), indicating amplicons can produce equally accurate community composition data compared with shotgun metagenomics. Our analysis also revealed that expected performance of all primer sets could be improved with minor modifications, pointing toward a nearly completely universal primer set that could accurately quantify biogeochemically important taxa in ecosystems ranging from the deep sea to the surface. In addition, our reproducible bioinformatic workflow can guide microbiome researchers studying different ecosystems or human health to similarly improve existing primers and generate more accurate quantitative amplicon data. IMPORTANCE PCR amplification and sequencing of marker genes is a low-cost technique for monitoring prokaryotic and eukaryotic microbial communities across space and time but will work optimally only if environmental organisms match PCR primer sequences exactly. In this study, we evaluated how well primers match globally distributed short-read oceanic metagenomes. Our results demonstrate that primer sets vary widely in performance, and that at least for marine systems, rRNA amplicon data from some primers lack significant biases compared to metagenomes. We also show that it is theoretically possible to create a nearly universal primer set for diverse saline environments by defining a specific mixture of a few dozen oligonucleotides, and present a software pipeline that can guide rational design of primers for any environment with available meta’omic data.