High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen.

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.

Barcoded overexpression screens in gut Bacteroidales identify genes with roles in carbon utilization and stress resistance.

A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to-phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions. To address these challenges, we develop Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron. From hundreds of experiments, we identify new functions and phenotypes for 29 genes important for carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a D-glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to ceftriaxone and bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assays.

Molecular mechanisms and environmental adaptations of flagellar loss and biofilm growth of Rhodanobacter under environmental stress.

Biofilms aid bacterial adhesion to surfaces via direct and indirect mechanisms, and formation of biofilms is considered as an important strategy for adaptation and survival in sub-optimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, nitrate or metal concentrations is underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimes of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse pH (3.5 to 5) and nitrate levels (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptation to survive and grow at low pH. Biofilms intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation warranting further investigation. Through RB-TnSeq, proteomics, use of specific mutants and transmission electron microscopy analysis, we discovered flagellar loss under aluminum stress, indicating a potential relationship between motility, metal tolerance, and biofilm growth. Comparative genomic analyses revealed absence of flagella and chemotaxis genes, and presence of putative Type VI secretion system in the high biofilm-forming strain FW021-MT20. This study identifies genetic determinants associated with biofilm growth in a predominant environmental genus, Rhodanobacter, under metal stress and identifies traits aiding survival and adaptation to contaminated subsurface environments.

Mixed waste contamination selects for a mobile genetic element population enriched in multiple heavy metal resistance genes.

Mobile genetic elements (MGEs) like plasmids, viruses, and transposable elements can provide fitness benefits to their hosts for survival in the presence of environmental stressors. Heavy metal resistance genes (HMRGs) are frequently observed on MGEs, suggesting that MGEs may be an important driver of adaptive evolution in environments contaminated with heavy metals. Here, we report the meta-mobilome of the heavy metal-contaminated regions of the Oak Ridge Reservation subsurface. This meta-mobilome was compared with one derived from samples collected from unimpacted regions of the Oak Ridge Reservation subsurface. We assembled 1615 unique circularized DNA elements that we propose to be MGEs. The circular elements from the highly contaminated subsurface were enriched in HMRG clusters relative to those from the nearby unimpacted regions. Additionally, we found that these HMRGs were associated with Gamma and Betaproteobacteria hosts in the contaminated subsurface and potentially facilitate the persistence and dominance of these taxa in this region. Finally, the HMRGs were associated with conjugative elements, suggesting their potential for future lateral transfer. We demonstrate how our understanding of MGE ecology, evolution, and function can be enhanced through the genomic context provided by completed MGE assemblies.

A fast comparative genome browser for diverse bacteria and archaea.

Genome sequencing has revealed an incredible diversity of bacteria and archaea, but there are no fast and convenient tools for browsing across these genomes. It is cumbersome to view the prevalence of homologs for a protein of interest, or the gene neighborhoods of those homologs, across the diversity of the prokaryotes. We developed a web-based tool, fast.genomics, that uses two strategies to support fast browsing across the diversity of prokaryotes. First, the database of genomes is split up. The main database contains one representative from each of the 6,377 genera that have a high-quality genome, and additional databases for each taxonomic order contain up to 10 representatives of each species. Second, homologs of proteins of interest are identified quickly by using accelerated searches, usually in a few seconds. Once homologs are identified, fast.genomics can quickly show their prevalence across taxa, view their neighboring genes, or compare the prevalence of two different proteins. Fast.genomics is available at https://fast.genomics.lbl.gov.

Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide.

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface.

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

Environmental stress mediates groundwater microbial community assembly.

Community assembly describes how different ecological processes shape microbial community composition and structure. How environmental factors impact community assembly remains elusive. Here we sampled microbial communities and >200 biogeochemical variables in groundwater at the Oak Ridge Field Research Center, a former nuclear waste disposal site, and developed a theoretical framework to conceptualize the relationships between community assembly processes and environmental stresses. We found that stochastic assembly processes were critical (>60% on average) in shaping community structure, but their relative importance decreased as stress increased. Dispersal limitation and 'drift' related to random birth and death had negative correlations with stresses, whereas the selection processes leading to dissimilar communities increased with stresses, primarily related to pH, cobalt and molybdenum. Assembly mechanisms also varied greatly among different phylogenetic groups. Our findings highlight the importance of microbial dispersal limitation and environmental heterogeneity in ecosystem restoration and management.