High-throughput protein characterization by complementation using DNA barcoded fragment libraries.

Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation.

Decomposing a San Francisco estuary microbiome using long-read metagenomics reveals species- and strain-level dominance from picoeukaryotes to viruses.

Unlabelled: Although long-read sequencing has enabled obtaining high-quality and complete genomes from metagenomes, many challenges still remain to completely decompose a metagenome into its constituent prokaryotic and viral genomes. This study focuses on decomposing an estuarine metagenome to obtain a more accurate estimate of microbial diversity. To achieve this, we developed a new bead-based DNA extraction method, a novel bin refinement method, and obtained 150 Gbp of Nanopore sequencing.

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 suboptimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, and nitrate or metal concentrations are underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimens of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse levels of pH (3.

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.

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.

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.

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.

Mixed nitrate and metal contamination influences operational speciation of toxic and essential elements.

Environmental contamination constrains microbial communities impacting diversity and total metabolic activity. The former S-3 Ponds contamination site at Oak Ridge Reservation (ORR), TN, has elevated concentrations of nitric acid and multiple metals from decades of processing nuclear material. To determine the nature of the metal contamination in the sediment, a three-step sequential chemical extraction (BCR) was performed on sediment segments from a core located upgradient (EB271, non-contaminated) and one downgradient (EB106, contaminated) of the S-3 Ponds.

Distinct Depth-Discrete Profiles of Microbial Communities and Geochemical Insights in the Subsurface Critical Zone.

Microbial assembly and metabolic potential in the subsurface critical zone (SCZ) are substantially impacted by subsurface geochemistry and hydrogeology, selecting for microbes distinct from those in surficial soils. In this study, we integrated metagenomics and geochemistry to elucidate how microbial composition and metabolic potential are shaped and impacted by vertical variations in geochemistry and hydrogeology in terrestrial subsurface sediment. A sediment core from an uncontaminated, pristine well at Oak Ridge Field Research Center in Oak Ridge, Tennessee, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone, was used in this study.

Ecophysiological and genomic analyses of a representative isolate of highly abundant Bacillus cereus strains in contaminated subsurface sediments.

Bacillus cereus strain CPT56D-587-MTF (CPTF) was isolated from the highly contaminated Oak Ridge Reservation (ORR) subsurface. This site is contaminated with high levels of nitric acid and multiple heavy metals. Amplicon sequencing of the 16S rRNA genes (V4 region) in sediment from this area revealed an amplicon sequence variant (ASV) with 100% identity to the CPTF 16S rRNA sequence.

Complete Genome Sequence of Bacillus cereus Strain CPT56D-587-MTF, Isolated from a Nitrate- and Metal-Contaminated Subsurface Environment.

Bacillus cereus strain CPT56D-587-MTF was isolated from nitrate- and toxic metal-contaminated subsurface sediment at the Oak Ridge Reservation (ORR) (Oak Ridge, TN, USA). Here, we report the complete genome sequence of this strain to provide genomic insight into its strategies for survival at this mixed-waste site.

Genotype to ecotype in niche environments: adaptation of Arthrobacter to carbon availability and environmental conditions.

Niche environmental conditions influence both the structure and function of microbial communities and the cellular function of individual strains. The terrestrial subsurface is a dynamic and diverse environment that exhibits specific biogeochemical conditions associated with depth, resulting in distinct environmental niches. Here, we present the characterization of seven distinct strains belonging to the genus Arthrobacter isolated from varying depths of a single sediment core and associated groundwater from an adjacent well.

Genomic Features and Pervasive Negative Selection in Strains Isolated from Nitrate and Heavy Metal Contaminated Aquifer.

species dominate in the Oak Ridge Reservation (ORR) subsurface environments contaminated with acids, nitrate, metal radionuclides, and other heavy metals. To uncover the genomic features underlying adaptations to these mixed-waste environments and to guide genetic tool development, we sequenced the whole genomes of eight strains isolated from the ORR site. The genome sizes ranged from 3.

The genetic basis of phage susceptibility, cross-resistance and host-range in .

Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms.

Deciphering Microbial Metal Toxicity Responses via Random Bar Code Transposon Site Sequencing and Activity-Based Metabolomics.

To uncover metal toxicity targets and defense mechanisms of the facultative anaerobe sp. strain MT58 (MT58), we used a multiomic strategy combining two global techniques, random bar code transposon site sequencing (RB-TnSeq) and activity-based metabolomics. MT58 is a metal-tolerant Oak Ridge Reservation (ORR) environmental isolate that was enriched in the presence of metals at concentrations measured in contaminated groundwater at an ORR nuclear waste site.

A method for achieving complete microbial genomes and improving bins from metagenomics data.

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete genomes from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Some studies have attempted to extract complete bacterial, archaeal, and viral genomes and often focus on species with circular genomes so they can help confirm completeness with circularity.

Mechanism Across Scales: A Holistic Modeling Framework Integrating Laboratory and Field Studies for Microbial Ecology.

Over the last century, leaps in technology for imaging, sampling, detection, high-throughput sequencing, and -omics analyses have revolutionized microbial ecology to enable rapid acquisition of extensive datasets for microbial communities across the ever-increasing temporal and spatial scales. The present challenge is capitalizing on our enhanced abilities of observation and integrating diverse data types from different scales, resolutions, and disciplines to reach a causal and mechanistic understanding of how microbial communities transform and respond to perturbations in the environment. This type of causal and mechanistic understanding will make predictions of microbial community behavior more robust and actionable in addressing microbially mediated global problems.

Systematic discovery of pseudomonad genetic factors involved in sensitivity to tailocins.

Tailocins are bactericidal protein complexes produced by a wide variety of bacteria that kill closely related strains and may play a role in microbial community structure. Thanks to their high specificity, tailocins have been proposed as precision antibacterial agents for therapeutic applications. Compared to tailed phages, with whom they share an evolutionary and morphological relationship, bacterially produced tailocins kill their host upon production but producing strains display resistance to self-intoxication.

Alignment of Biological Sequences with Jalview.

In this chapter, we introduce core functionality of the Jalview interactive platform for the creation, analysis, and publication of multiple sequence alignments. A workflow is described based on Jalview's core functions: from data import to figure generation, including import of alignment reliability scores from T-Coffee and use of Jalview from the command line. The accompanying notes provide background information on the underlying methods and discuss additional options for working with Jalview to perform multiple sequence alignment, functional site analysis, and publication of alignments on the web.

Characterization of a Metal-Resistant Strain With a High Molybdate Affinity ModA From Contaminated Sediments at the Oak Ridge Reservation.

A nitrate- and metal-contaminated site at the Oak Ridge Reservation (ORR) was previously shown to contain the metal molybdenum (Mo) at picomolar concentrations. This potentially limits microbial nitrate reduction, as Mo is required by the enzyme nitrate reductase, which catalyzes the first step of nitrate removal. Enrichment for anaerobic nitrate-reducing microbes from contaminated sediment at the ORR yielded strain EB106-08-02-XG196.

Selective carbon sources influence the end products of microbial nitrate respiration.

Respiratory and catabolic genes are differentially distributed across microbial genomes. Thus, specific carbon sources may favor different respiratory processes. We profiled the influence of 94 carbon sources on the end products of nitrate respiration in microbial enrichment cultures from diverse terrestrial environments.