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ABSTRACT Anaerolineae, particularly uncultured representatives, are one of the most abundant microbial groups in coastal salt marshes, dominating the belowground rhizosphere, where over half of plant biomass production occurs. However, this class generally remains poorly understood, particularly in a salt marsh context. Here, novelAnaerolineaemetagenome-assembled genomes (MAGs) were generated from the salt marsh rhizosphere representingAnaerolineales,Promineifilales, JAAYZQ01, B4-G1, JAFGEY01, UCB3, andCaldilinealesorders. Metagenome and metatranscriptome reads were mapped to annotated MAGs, revealing nearly allAnaerolineaeencoded and transcribed genes required for oxidation of carbon compounds ranging from simple sugars to complex polysaccharides, fermentation, and carbon fixation. Furthermore, the majority ofAnaerolineaeexpressed genes involved in anaerobic and aerobic respiration and secondary metabolite production. The data revealed that the belowground salt marshAnaerolineaein the rhizosphere are important players in carbon cycling, including degradation of simple carbon compounds and more recalcitrant plant material, such as cellulose, using a diversity of electron acceptors and represent an unexplored reservoir of novel secondary metabolites.IMPORTANCEGiven that coastal salt marshes are recognized as biogeochemical hotspots, it is fundamentally important to understand the functional role of the microbiome in this ecosystem. In particular,Anaerolineaeare abundant members of the salt marsh rhizosphere and have been identified as core microbes, suggesting they play an important functional role. Yet, little is known about the metabolic pathways encoded and expressed in this abundant salt marsh clade. Using an ‘omics-based approach, we determined thatAnaerolineaeare capable of oxidizing a range of carbon compounds, including simple sugars to complex carbon compounds, while also encoding fermentation and carbon fixation. Surprisingly,Anaerolineaeencoded and transcribed genes involved in aerobic respiration, which was unexpected given the reduced nature of the salt marsh rhizosphere. Finally, the majority ofAnaerolineaeappear to be involved in secondary metabolite production, suggesting that this group represents an unexplored reservoir of novel and important secondary metabolites. 

ABSTRACT Sulfur-cycling microbial communities in salt marsh rhizosphere sediments mediate a recycling and detoxification system central to plant productivity. Despite the importance of sulfur-cycling microbes, their biogeographic, phylogenetic, and functional diversity remain poorly understood. Here, we use metagenomic data sets from Massachusetts (MA) and Alabama (AL) salt marshes to examine the distribution and genomic diversity of sulfur-cycling plant-associated microbes. Samples were collected from sediments underSporobolus alterniflorusandSporobolus pumilusin separate MA vegetation zones, and underS. alterniflorusandJuncus roemerianusco-occuring in AL. We grouped metagenomic data by plant species and site and identified 38 MAGs that included pathways for sulfate reduction or sulfur oxidation. Phylogenetic analyses indicated that 29 of the 38 were affiliated with uncultivated lineages. We showed differentiation in the distribution of MAGs between AL and MA, betweenS. alterniflorusandS. pumilusvegetation zones in MA, but no differentiation betweenS. alterniflorusandJ. roemerianusin AL. Pangenomic analyses of eight ubiquitous MAGs also detected site- and vegetation-specific genomic features, including varied sulfur-cycling operons, carbon fixation pathways, fixed single-nucleotide variants, and active diversity-generating retroelements. This genetic diversity, detected at multiple scales, suggests evolutionary relationships affected by distance and local environment, and demonstrates differential microbial capacities for sulfur and carbon cycling in salt marsh sediments. IMPORTANCESalt marshes are known for their significant carbon storage capacity, and sulfur cycling is closely linked with the ecosystem-scale carbon cycling in these ecosystems. Sulfate reducers are key for the decomposition of organic matter, and sulfur oxidizers remove toxic sulfide, supporting the productivity of marsh plants. To date, the complexity of coastal environments, heterogeneity of the rhizosphere, high microbial diversity, and uncultured majority hindered our understanding of the genomic diversity of sulfur-cycling microbes in salt marshes. Here, we use comparative genomics to overcome these challenges and provide an in-depth characterization of sulfur-cycling microbial diversity in salt marshes. We characterize communities across distinct sites and plant species and uncover extensive genomic diversity at the taxon level and specific genomic features present in MAGs affiliated with uncultivated sulfur-cycling lineages. Our work provides insights into the partnerships in salt marshes and a roadmap for multiscale analyses of diversity in complex biological systems. 

Abstract The spatial distribution of predators can affect both the distribution and diversity of their prey. Therefore, differences in predator dispersal ability that affect their spatial distribution, could also affect prey communities. Here, we use the microbial communities within pitcher plant leaves as a model system to test the relationship between predator (protozoa) dispersal ability and distribution, and its consequences for prey (bacteria) diversity and composition. We hypothesized that limited predator dispersal results in clustered distributions and heterogeneous patches for prey species, whereas wide predator dispersal and distribution could homogenize prey metacommunities. We analyzed the distribution of two prominent bacterivore protozoans from a 2‐year survey of an intact field ofSarracenia purpureapitcher plants, and found a clustered distribution ofTetrahymenaand homogeneous distribution ofPoterioochromonas. We manipulated the sources of protozoan colonists and recorded protozoan recruitment and bacterial diversity in target leaves in a field experiment. We found the large ciliate,Tetrahymena, was dispersal limited and occupied few leaves, whereas the small flagellatePoterioochromonaswas widely dispersed. However, the bacterial communities these protozoans feed on was unaffected by clustering ofTetrahymena, but likely influenced byPoterioochromonasand other bacterivores dispersing in the field. We propose that bacterial communities in this system are structured by a combination of well dispersed bacterivores, bacterial dispersal, and bottom‐up mechanisms. Clustered predators could become strong drivers of prey communities if they were specialists or keystone predators, or if they exerted a dominant influence on other predators in top‐down controlled systems. Linking dispersal ability within trophic levels and its consequences for trophic dynamics can lead to a more robust perspective on trophic metacommunities. 

ABSTRACT Two-component sensory (TCS) systems link microbial physiology to the environment and thus may play key roles in biogeochemical cycles. In this study, we surveyed the TCS systems of 328 diverse marine bacterial species. We identified lifestyle traits such as copiotrophy and diazotrophy that are associated with larger numbers of TCS system genes within the genome. We compared marine bacterial species with 1,152 reference bacterial species from a variety of habitats and found evidence of extra response regulators in marine genomes. Examining the location of TCS genes along the circular bacterial genome, we also found that marine bacteria have a large number of “orphan” genes, as well as many hybrid histidine kinases. The prevalence of “extra” response regulators, orphan genes, and hybrid TCS systems suggests that marine bacteria break with traditional understanding of how TCS systems operate. These trends suggest prevalent regulatory networking, which may allow coordinated physiological responses to multiple environmental signals and may represent a specific adaptation to the marine environment. We examine phylogenetic and lifestyle traits that influence the number and structure of two-component systems in the genome, finding, for example, that a lack of two-component systems is a hallmark of oligotrophy. Finally, in an effort to demonstrate the importance of TCS systems to marine biogeochemistry, we examined the distribution of Prochlorococcus/Synechococcus response regulator PMT9312_0717 in metaproteomes of the tropical South Pacific. We found that this protein’s abundance is related to phosphate concentrations, consistent with a putative role in phosphate regulation. IMPORTANCE Marine microbes must manage variation in their chemical, physical, and biological surroundings. Because they directly link bacterial physiology to environmental changes, TCS systems are crucial to the bacterial cell. This study surveyed TCS systems in a large number of marine bacteria and identified key phylogenetic and lifestyle patterns in environmental sensing. We found evidence that, in comparison with bacteria as a whole, marine organisms have irregular TCS system constructs which might represent an adaptation specific to the marine environment. Additionally, we demonstrate the biogeochemical relevance of TCS systems by correlating the presence of the PMT9312_0717 response regulator protein to phosphate concentrations in the South Pacific. We highlight that despite their potential ecological and biogeochemical relevance, TCS systems have been understudied in the marine ecosystem. This report expands our understanding of the breadth of bacterial TCS systems and how marine bacteria have adapted to survive in their unique environment. 

Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyze 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical, and gene neighborhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter.