Protocol for isolating single species of bacteria with swarming ability from human feces.

Understanding the specific movements of bacteria isolated from human feces can serve as a novel diagnostic and therapeutic tool for inflammatory bowel disease. Here, we present a protocol for a microbial swarming assay and to isolate the bacteria responsible for swarming activity. We describe steps for identifying bacteria using MALDI-TOF mass spectrometry and whole-genome sequencing. We then detail procedures for validating findings by observing the same swarming phenotype upon reperforming the swarming assay. For complete details on the use and execution of this protocol, please refer to De et al.1.

Retention of an Endosymbiont for the Production of a Single Molecule.

Sap-feeding insects often maintain two or more nutritional endosymbionts that act in concert to produce compounds essential for insect survival. Many mealybugs have endosymbionts in a nested configuration: one or two bacterial species reside within the cytoplasm of another bacterium, and together, these bacteria have genomes that encode interdependent sets of genes needed to produce key nutritional molecules. Here, we show that the mealybug Pseudococcus viburni has three endosymbionts, one of which contributes only two unique genes that produce the host nutrition-related molecule chorismate. All three bacterial endosymbionts have tiny genomes, suggesting that they have been coevolving inside their insect host for millions of years.

How do bacterial endosymbionts work with so few genes?

The move from a free-living environment to a long-term residence inside a host eukaryotic cell has profound effects on bacterial function. While endosymbioses are found in many eukaryotes, from protists to plants to animals, the bacteria that form these host-beneficial relationships are even more diverse. Endosymbiont genomes can become radically smaller than their free-living relatives, and their few remaining genes show extreme compositional biases. The details of how these reduced and divergent gene sets work, and how they interact with their host cell, remain mysterious. This Unsolved Mystery reviews how genome reduction alters endosymbiont biology and highlights a "tipping point" where the loss of the ability to build a cell envelope coincides with a marked erosion of translation-related genes.

Bacterial aerobic methane cycling by the marine sponge-associated microbiome.

BACKGROUND: Methanotrophy by the sponge-hosted microbiome has been mainly reported in the ecological context of deep-sea hydrocarbon seep niches where methane is either produced geothermically or via anaerobic methanogenic archaea inhabiting the sulfate-depleted sediments. However, methane-oxidizing bacteria from the candidate phylum Binatota have recently been described and shown to be present in oxic shallow-water marine sponges, where sources of methane remain undescribed. RESULTS: Here, using an integrative -omics approach, we provide evidence for sponge-hosted bacterial methane synthesis occurring in fully oxygenated shallow-water habitats. Specifically, we suggest methane generation occurs via at least two independent pathways involving methylamine and methylphosphonate transformations that, concomitantly to aerobic methane production, generate bioavailable nitrogen and phosphate, respectively. Methylphosphonate may be sourced from seawater continuously filtered by the sponge host. Methylamines may also be externally sourced or, alternatively, generated by a multi-step metabolic process where carnitine, derived from sponge cell debris, is transformed to methylamine by different sponge-hosted microbial lineages. Finally, methanotrophs specialized in pigment production, affiliated to the phylum Binatota, may provide a photoprotective function, closing a previously undescribed C1-metabolic loop that involves both the sponge host and specific members of the associated microbial community. CONCLUSION: Given the global distribution of this ancient animal lineage and their remarkable water filtration activity, sponge-hosted methane cycling may affect methane supersaturation in oxic coastal environments. Depending on the net balance between methane production and consumption, sponges may serve as marine sources or sinks of this potent greenhouse gas. Video Abstract.

Cecal Microbial Hydrogen Cycling Potential Is Linked to Feed Efficiency Phenotypes in Chickens.

In chickens, early life exposure to environmental microbes has long-lasting impacts on gastrointestinal (GI) microbiome development and host health and growth, via mechanisms that remain uncharacterized. In this study, we demonstrated that administrating a fecal microbiome transplant (FMT) from adults to day-of-hatch chicks results in significantly higher body mass of birds and decreased residual feed intake (RFI), implying enhanced feed efficiency, at 6 weeks of age. To assess the potential mechanisms through which FMT affects adult bird phenotype, we combined 16 S rRNA gene amplification, metagenomic, and comparative genomic approaches to survey the composition and predicted activities of the resident microbiome of various GI tract segments. Early life FMT exposure had a long-lasting significant effect on the microbial community composition and function of the ceca but not on other GI segments. Within the ceca of 6-week-old FMT birds, hydrogenotrophic microbial lineages and genes were most differentially enriched. The results suggest that thermodynamic regulation in the cecum, in this case via hydrogenotrophic methanogenic and sulfur-cycling lineages, potentially serving as hydrogen sinks, may enhance fermentative efficiency and dietary energy harvest capacity. Our study provides a specific mechanism of action through which early-life microbiome transplants modulate market-relevant phenotypes in poultry and, thereby, may represent a significant advance toward microbiome-focused sustainable agriculture.

Pseudofinder: Detection of Pseudogenes in Prokaryotic Genomes.

Prokaryotic genomes are usually densely packed with intact and functional genes. However, in certain contexts, such as after recent ecological shifts or extreme population bottlenecks, broken and nonfunctional gene fragments can quickly accumulate and form a substantial fraction of the genome. Identification of these broken genes, called pseudogenes, is a critical step for understanding the evolutionary forces acting upon, and the functional potential encoded within, prokaryotic genomes. Here, we present Pseudofinder, an open-source software dedicated to pseudogene identification and analysis in bacterial and archaeal genomes. We demonstrate that Pseudofinder's multi-pronged, reference-based approach can detect a wide variety of pseudogenes, including those that are highly degraded and typically missed by gene-calling pipelines, as well newly formed pseudogenes containing only one or a few inactivating mutations. Additionally, Pseudofinder can detect genes that lack inactivating substitutions but experiencing relaxed selection. Implementation of Pseudofinder in annotation pipelines will allow more precise estimations of the functional potential of sequenced microbes, while also generating new hypotheses related to the evolutionary dynamics of bacterial and archaeal genomes.

SprayNPray: user-friendly taxonomic profiling of genome and metagenome contigs.

BACKGROUND: Shotgun sequencing of cultured microbial isolates/individual eukaryotes (whole-genome sequencing) and microbial communities (metagenomics) has become commonplace in biology. Very often, sequenced samples encompass organisms spanning multiple domains of life, necessitating increasingly elaborate software for accurate taxonomic classification of assembled sequences. RESULTS: While many software tools for taxonomic classification exist, SprayNPray offers a quick and user-friendly, semi-automated approach, allowing users to separate contigs by taxonomy (and other metrics) of interest. Easy installation, usage, and intuitive output, which is amenable to visual inspection and/or further computational parsing, will reduce barriers for biologists beginning to analyze genomes and metagenomes. This approach can be used for broad-level overviews, preliminary analyses, or as a supplement to other taxonomic classification or binning software. SprayNPray profiles contigs using multiple metrics, including closest homologs from a user-specified reference database, gene density, read coverage, GC content, tetranucleotide frequency, and codon-usage bias. CONCLUSIONS: The output from this software is designed to allow users to spot-check metagenome-assembled genomes, identify, and remove contigs from putative contaminants in isolate assemblies, identify bacteria in eukaryotic assemblies (and vice-versa), and identify possible horizontal gene transfer events.

Genomic and Functional Variation of the Chlorophyll d-Producing Cyanobacterium Acaryochloris marina.

The Chlorophyll d-producing cyanobacterium Acaryochloris marina is widely distributed in marine environments enriched in far-red light, but our understanding of its genomic and functional diversity is limited. Here, we take an integrative approach to investigate A. marina diversity for 37 strains, which includes twelve newly isolated strains from previously unsampled locations in Europe and the Pacific Northwest of North America. A genome-wide phylogeny revealed both that closely related A. marina have migrated within geographic regions and that distantly related A. marina lineages can co-occur. The distribution of traits mapped onto the phylogeny provided evidence of a dynamic evolutionary history of gene gain and loss during A. marina diversification. Ancestral genes that were differentially retained or lost by strains include plasmid-encoded sodium-transporting ATPase and bidirectional NiFe-hydrogenase genes that may be involved in salt tolerance and redox balance under fermentative conditions, respectively. The acquisition of genes by horizontal transfer has also played an important role in the evolution of new functions, such as nitrogen fixation. Together, our results resolve examples in which genome content and ecotypic variation for nutrient metabolism and environmental tolerance have diversified during the evolutionary history of this unusual photosynthetic bacterium.

Metagenomics of Antarctic Marine Sediment Reveals Potential for Diverse Chemolithoautotrophy.

The microbial biogeochemical processes occurring in marine sediment in Antarctica remain underexplored due to limited access. Further, these polar habitats are unique, as they are being exposed to significant changes in their climate. To explore how microbes drive biogeochemistry in these sediments, we performed a shotgun metagenomic survey of marine surficial sediment (0 to 3 cm of the seafloor) collected from 13 locations in western Antarctica and assembled 16 high-quality metagenome assembled genomes for focused interrogation of the lifestyles of some abundant lineages. We observe an abundance of genes from pathways for the utilization of reduced carbon, sulfur, and nitrogen sources. Although organotrophy is pervasive, nitrification and sulfide oxidation are the dominant lithotrophic pathways and likely fuel carbon fixation via the reverse tricarboxylic acid and Calvin cycles. Oxygen-dependent terminal oxidases are common, and genes for reduction of oxidized nitrogen are sporadically present in our samples. Our results suggest that the underlying benthic communities are well primed for the utilization of settling organic matter, which is consistent with findings from highly productive surface water. Despite the genetic potential for nitrate reduction, the net catabolic pathway in our samples remains aerobic respiration, likely coupled to the oxidation of sulfur and nitrogen imported from the highly productive Antarctic water column above. IMPORTANCE The impacts of climate change in polar regions, like Antarctica, have the potential to alter numerous ecosystems and biogeochemical cycles. Increasing temperature and freshwater runoff from melting ice can have profound impacts on the cycling of organic and inorganic nutrients between the pelagic and benthic ecosystems. Within the benthos, sediment microbial communities play a critical role in carbon mineralization and the cycles of essential nutrients like nitrogen and sulfur. Metagenomic data collected from sediment samples from the continental shelf of western Antarctica help to examine this unique system and document the metagenomic potential for lithotrophic metabolisms and the cycles of both nitrogen and sulfur, which support not only benthic microbes but also life in the pelagic zone.

Bacterial Adaptation by a Transposition Burst of an Invading IS Element.

The general importance of transposable elements (TEs) for adaptive evolution remains unclear. This in part reflects a poor understanding of the role of TEs for adaptation in nonmodel systems. Here, we investigated whether insertion sequence (IS) elements are a major source of beneficial mutations during 400 generations of laboratory evolution of the cyanobacterium Acaryochloris marina strain CCMEE 5410, which has experienced a recent or on-going IS element expansion and has among the highest transposase gene contents for a bacterial genome. Most mutations detected in the eight independent experimental populations were IS transposition events. Surprisingly, however, the majority of these involved the copy-and-paste activity of only a single copy of an unclassified element (ISAm1) that has recently invaded the strain CCMEE 5410 genome. ISAm1 transposition was largely responsible for the highly repeatable evolutionary dynamics observed among populations. Notably, this included mutations in multiple targets involved in the acquisition of inorganic carbon for photosynthesis that were exclusively due to ISAm1 activity. These mutations were associated with an increase in linear growth rate under conditions of reduced carbon availability but did not appear to impact fitness when carbon was readily available. Our study reveals that the activity of a single transposase can fuel adaptation for at least several hundred generations but may also potentially limit the rate of adaptation through clonal interference.

Adaptation and genomic erosion in fragmented Pseudomonas aeruginosa populations in the sinuses of people with cystic fibrosis.

Pseudomonas aeruginosa notoriously adapts to the airways of people with cystic fibrosis (CF), yet how infection-site biogeography and associated evolutionary processes vary as lifelong infections progress remains unclear. Here we test the hypothesis that early adaptations promoting aggregation influence evolutionary-genetic trajectories by examining longitudinal P. aeruginosa from the sinuses of six adults with CF. Highly host-adapted lineages harbored mutator genotypes displaying signatures of early genome degradation associated with recent host restriction. Using an advanced imaging technique (MiPACT-HCR [microbial identification after passive clarity technique]), we find population structure tracks with genome degradation, with the most host-adapted, genome-degraded P. aeruginosa (the mutators) residing in small, sparse aggregates. We propose that following initial adaptive evolution in larger populations under strong selection for aggregation, P. aeruginosa persists in small, fragmented populations that experience stronger effects of genetic drift. These conditions enrich for mutators and promote degenerative genome evolution. Our findings underscore the importance of infection-site biogeography to pathogen evolution.

Metagenomic Insights Into the Microbial Iron Cycle of Subseafloor Habitats.

Microbial iron cycling influences the flux of major nutrients in the environment (e.g., through the adsorptive capacity of iron oxides) and includes biotically induced iron oxidation and reduction processes. The ecological extent of microbial iron cycling is not well understood, even with increased sequencing efforts, in part due to limitations in gene annotation pipelines and limitations in experimental studies linking phenotype to genotype. This is particularly true for the marine subseafloor, which remains undersampled, but represents the largest contiguous habitat on Earth. To address this limitation, we used FeGenie, a database and bioinformatics tool that identifies microbial iron cycling genes and enables the development of testable hypotheses on the biogeochemical cycling of iron. Herein, we survey the microbial iron cycle in diverse subseafloor habitats, including sediment-buried crustal aquifers, as well as surficial and deep sediments. We inferred the genetic potential for iron redox cycling in 32 of the 46 metagenomes included in our analysis, demonstrating the prevalence of these activities across underexplored subseafloor ecosystems. We show that while some processes (e.g., iron uptake and storage, siderophore transport potential, and iron gene regulation) are near-universal, others (e.g., iron reduction/oxidation, siderophore synthesis, and magnetosome formation) are dependent on local redox and nutrient status. Additionally, we detected niche-specific differences in strategies used for dissimilatory iron reduction, suggesting that geochemical constraints likely play an important role in dictating the dominant mechanisms for iron cycling. Overall, our survey advances the known distribution, magnitude, and potential ecological impact of microbe-mediated iron cycling and utilization in sub-benthic ecosystems.

Iron Oxidation by a Fused Cytochrome-Porin Common to Diverse Iron-Oxidizing Bacteria.

Iron (Fe) oxidation is one of Earth's major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism. IMPORTANCE Iron is practically ubiquitous across Earth's environments, central to both life and geochemical processes, which depend heavily on the redox state of iron. Although iron oxidation, or "rusting," can occur abiotically at near-neutral pH, we find neutrophilic iron-oxidizing bacteria (FeOB) are widespread, including in aquifers, sediments, hydrothermal vents, pipes, and water treatment systems. FeOB produce highly reactive Fe(III) oxyhydroxides that bind a variety of nutrients and toxins; thus, these microbes are likely a controlling force in iron and other biogeochemical cycles. There has been mounting evidence that Cyc2 functions as an iron oxidase in neutrophiles, but definitive proof of its function has long eluded us. This work provides conclusive biochemical evidence of iron oxidation by Cyc2 from neutrophiles. Cyc2 is common to a wide variety of iron oxidizers, including acidophilic and phototrophic iron oxidizers, suggesting that this fused cytochrome-porin structure is especially well adapted for iron oxidation.

Cryptic metabolisms in anoxic subseafloor sediment.

Microbial gene expression in anoxic subseafloor sediment was recently explored in the Baltic Sea and the Peru Margin. Our analysis of these data reveals diverse transcripts encoding proteins associated with neutralization of reactive oxygen species, including catalase, which may provide an in situ source of oxygen. We also detect transcripts associated with oxidation of iron and sulfur, and with reduction of arsenate, selenate and nitrate. Given limited input of electron acceptors from outside the system, these results suggest that the microbial communities use an unexpectedly diverse variety of electron acceptors. Products of water radiolysis and their interactions with sediment continuously provide diverse electron acceptors and hydrogen. Cryptic microbial utilization of these oxidized substrates and H2 may be an important mechanism for multi-million-year survival under the extreme energy limitation in subseafloor sediment.

The Evolution of Interdependence in a Four-Way Mealybug Symbiosis.

Mealybugs are insects that maintain intracellular bacterial symbionts to supplement their nutrient-poor plant sap diets. Some mealybugs have a single betaproteobacterial endosymbiont, a Candidatus Tremblaya species (hereafter Tremblaya) that alone provides the insect with its required nutrients. Other mealybugs have two nutritional endosymbionts that together provision these same nutrients, where Tremblaya has gained a gammaproteobacterial partner that resides in its cytoplasm. Previous work had established that Pseudococcus longispinus mealybugs maintain not one but two species of gammaproteobacterial endosymbionts along with Tremblaya. Preliminary genomic analyses suggested that these two gammaproteobacterial endosymbionts have large genomes with features consistent with a relatively recent origin as insect endosymbionts, but the patterns of genomic complementarity between members of the symbiosis and their relative cellular locations were unknown. Here, using long-read sequencing and various types of microscopy, we show that the two gammaproteobacterial symbionts of P. longispinus are mixed together within Tremblaya cells, and that their genomes are somewhat reduced in size compared with their closest nonendosymbiotic relatives. Both gammaproteobacterial genomes contain thousands of pseudogenes, consistent with a relatively recent shift from a free-living to an endosymbiotic lifestyle. Biosynthetic pathways of key metabolites are partitioned in complex interdependent patterns among the two gammaproteobacterial genomes, the Tremblaya genome, and horizontally acquired bacterial genes that are encoded on the mealybug nuclear genome. Although these two gammaproteobacterial endosymbionts have been acquired recently in evolutionary time, they have already evolved codependencies with each other, Tremblaya, and their insect host.

FeGenie: A Comprehensive Tool for the Identification of Iron Genes and Iron Gene Neighborhoods in Genome and Metagenome Assemblies.

Iron is a micronutrient for nearly all life on Earth. It can be used as an electron donor and electron acceptor by iron-oxidizing and iron-reducing microorganisms and is used in a variety of biological processes, including photosynthesis and respiration. While it is the fourth most abundant metal in the Earth's crust, iron is often limiting for growth in oxic environments because it is readily oxidized and precipitated. Much of our understanding of how microorganisms compete for and utilize iron is based on laboratory experiments. However, the advent of next-generation sequencing and surge in publicly available sequence data has made it possible to probe the structure and function of microbial communities in the environment. To bridge the gap between our understanding of iron acquisition, iron redox cycling, iron storage, and magnetosome formation in model microorganisms and the plethora of sequence data available from environmental studies, we have created a comprehensive database of hidden Markov models (HMMs) based on genes related to iron acquisition, storage, and reduction/oxidation in Bacteria and Archaea. Along with this database, we present FeGenie, a bioinformatics tool that accepts genome and metagenome assemblies as input and uses our comprehensive HMM database to annotate provided datasets with respect to iron-related genes and gene neighborhood. An important contribution of this tool is the efficient identification of genes involved in iron oxidation and dissimilatory iron reduction, which have been largely overlooked by standard annotation pipelines. We validated FeGenie against a selected set of 28 isolate genomes and showcase its utility in exploring iron genes present in 27 metagenomes, 4 isolate genomes from human oral biofilms, and 17 genomes from candidate organisms, including members of the candidate phyla radiation. We show that FeGenie accurately identifies iron genes in isolates. Furthermore, analysis of metagenomes using FeGenie demonstrates that the iron gene repertoire and abundance of each environment is correlated with iron richness. While this tool will not replace the reliability of culture-dependent analyses of microbial physiology, it provides reliable predictions derived from the most up-to-date genetic markers. FeGenie's database will be maintained and continually updated as new genes are discovered. FeGenie is freely available: https://github.com/Arkadiy-Garber/FeGenie.

Peptidoglycan Production by an Insect-Bacterial Mosaic.

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

Ecology of Subseafloor Crustal Biofilms.

The crustal subseafloor is the least explored and largest biome on Earth. Interrogating crustal life is difficult due to habitat inaccessibility, low-biomass and contamination challenges. Subseafloor observatories have facilitated the study of planktonic life in crustal aquifers, however, studies of life in crust-attached biofilms are rare. Here, we investigate biofilms grown on various minerals at different temperatures over 1-6 years at subseafloor observatories in the Eastern Pacific. To mitigate potential sequence contamination, we developed a new bioinformatics tool - TaxonSluice. We explore ecological factors driving community structure and potential function of biofilms by comparing our sequence data to previous amplicon and metagenomic surveys of this habitat. We reveal that biofilm community structure is driven by temperature rather than minerology, and that rare planktonic lineages colonize the crustal biofilms. Based on 16S rRNA gene overlap, we partition metagenome assembled genomes into planktonic and biofilm fractions and suggest that there are functional differences between these community types, emphasizing the need to separately examine each to accurately describe subseafloor microbe-rock-fluid processes. Lastly, we report that some rare lineages present in our warm and anoxic study site are also found in cold and oxic crustal fluids in the Mid-Atlantic Ridge, suggesting global crustal biogeography patterns.