Eric M. Bottos

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “Candidatus Pseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundance Acidobacteria were highly transcriptionally active, whereas bins corresponding to high-relative-abundance Verrucomicrobia were not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCE Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: An author video summary of this article is available.

Genomic and proteomic characterization of Gordonia sp. NB4-1Y in relation to 6 : 2 fluorotelomer sulfonate biodegradation

Gordonia sp. strain NB4-1Y was isolated from vermicompost using bis-(3-pentafluorophenylpropyl)-sulfide as the sole added sulfur source and was found to have a broad capacity for metabolizing organosulfur compounds. NB4-1Y is closely related to G. desulfuricans and was found to metabolize 6 : 2 fluorotelomer sulfonate (6 : 2 FTS) to 5 : 3 fluorotelomer acid (5 : 3 acid) via 6 : 2 fluorotelomer acid (6 : 2 FTCA), 6 : 2 unsaturated fluorotelomer acid (6 : 2 FTUCA) and 5 : 3 unsaturated fluorotelomer acid (5 : 3 Uacid). Given that the molecular and biochemical basis for the microbial metabolism of poly- and per-fluorinated compounds has yet to be examined, we undertook to investigate 6 : 2 FTS metabolism in NB4-1Y. To this end, a whole-genome shotgun sequence was prepared and two-dimensional differential in-gel electrophoresis was used to compare proteomes of MgSO4- and 6 : 2 FTS-grown cells. Of the three putative alkanesulfonate monooxygenases, four nitrilotriacetate monooxygenases and one taurine dioxygenase located in the draft genome, two nitrilotriacetate monooxygenases were differentially expressed in the presence of 6 : 2 FTS. It is hypothesized that these two enzymes may be responsible for 6 : 2 FTS desulfonation. In addition, a differentially expressed putative double bond reductase may be involved in the reduction of 5 : 3 Uacid to 5 : 3 acid. Other proteins differentially expressed during 6 : 2 FTS metabolism included a sulfate ABC transporter ATP-binding protein and two alkyl hydroperoxide reductases. This work establishes a foundation for future studies on the molecular biology and biochemistry of poly- and per-fluorinated compound metabolism in bacteria.

Draft Genome Sequence of Methylobacterium radiotolerans, a DDE-Degrading and Plant Growth-Promoting Strain Isolated from Cucurbita pepo.

ABSTRACT We announce the draft genome of Methylobacterium radiotolerans, a Gram-negative bacterium isolated from Cucurbita pepo roots. This strain shows 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (DDE)-degrading potential and plant growth-promoting capacities. Analyses of its 6.8-Mb genome will improve our understanding of DDE-degradation pathways and aid in the deployment of phytoremediation technologies to remediate DDE-contaminated soils.

Draft Genome Sequence of Enterobacter aerogenes, a DDE-Degrading and Plant Growth-Promoting Strain Isolated from Cucurbita pepo

ABSTRACT We report here the draft genome of Enterobacter aerogenes, a Gram-negative bacterium of the Enterobacteriaceae isolated from Cucurbita pepo root tissue. This bacterium shows 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (DDE)-degrading potential and plant growth-promoting capacity. An analysis of its 4.5-Mb draft genome will enhance the understanding of DDE degradation pathways and phytoremediation applications for DDE-contaminated soils.

Draft Genome Sequence of Arthrobacter sp. Strain SPG23, a Hydrocarbon-Degrading and Plant Growth-Promoting Soil Bacterium

ABSTRACT We report here the 4.7-Mb draft genome of Arthrobacter sp. SPG23, a hydrocarbonoclastic Gram-positive bacterium belonging to the Actinobacteria, isolated from diesel-contaminated soil at the Ford Motor Company site in Genk, Belgium. Strain SPG23 is a potent plant growth promoter useful for diesel fuel remediation applications based on plant-bacterium associations.

Sphingomonas taxi, Isolated from Cucurbita pepo, Proves to Be a DDE-Degrading and Plant Growth-Promoting Strain

ABSTRACT The draft genome of Sphingomonas taxi, a strain of the Sphingomonadaceae isolated from Cucurbita pepo root tissue, is presented. This Gram-negative bacterium shows 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (DDE)-degrading potential and plant growth-promoting capacities. An analysis of its 3.9-Mb draft genome will enhance the understanding of DDE-degradation pathways and phytoremediation applications for DDE-contaminated soils.

Dispersal limitation and thermodynamic constraints govern spatial structure of permafrost microbial communities

Understanding drivers of permafrost microbial community composition is critical for understanding permafrost microbiology and predicting ecosystem responses to thaw. We hypothesize that permafrost communities are shaped by physical constraints imposed by prolonged freezing, and exhibit spatial distributions that reflect dispersal limitation and selective pressures associated with these physical constraints. To test this, we characterized patterns of environmental variation and microbial community composition in permafrost across an Alaskan boreal forest landscape. We used null modeling to estimate the importance of selective and neutral assembly processes on community composition, and identified environmental factors influencing ecological selection through regression and structural equation modeling (SEM). Proportionally, the strongest process influencing community composition was dispersal limitation (0.36), exceeding the influence of homogenous selection (0.21), variable selection (0.16) and homogenizing dispersal (0.05). Fe(II) content was the most important factor explaining variable selection, and was significantly associated with total selection by univariate regression (R2 = 0.14, P = 0.003). SEM supported a model in which Fe(II) content mediated influences of the Gibbs free energy of the organic matter pool and organic acid concentration on total selection. These findings suggest that the dominant processes shaping microbial communities in permafrost result from the stability of the permafrost environment, which imposes dispersal and thermodynamic constraints.

Draft Genome Sequence of Pantoea ananatis GB1, a Plant-Growth-Promoting Hydrocarbonoclastic Root Endophyte, Isolated at a Diesel Fuel Phytoremediation Site Planted with Populus

ABSTRACT We report the 4.76-Mb draft genome of Pantoea ananatis GB1, a Gram-negative bacterium of the family Enterobacteriaceae, isolated from the roots of poplars planted for phytoremediation of a diesel-contaminated plume at the Ford Motor Company site in Genk, Belgium. Strain GB1 promotes plant growth in various hosts and metabolizes hydrocarbons.

Draft Genome Sequence of Acinetobacter calcoaceticus Strain GK1, a Hydrocarbon-Degrading Plant Growth-Promoting Rhizospheric Bacterium

ABSTRACT The 3.94-Mb draft genome of Acinetobacter calcoaceticus GK1, a hydrocarbonoclastic plant growth-promoting Gram-negative rhizospheric bacterium, is presented here. Isolated at the Ford Motor Company site in Genk, Belgium, from poplar trees planted on a diesel-contaminated plume, GK1 is useful for enhancing hydrocarbon phytoremediation.

Degradation and defluorination of 6:2 fluorotelomer sulfonamidoalkyl betaine and 6:2 fluorotelomer sulfonate by Gordonia sp. strain NB4-1Y under sulfur-limiting conditions

6:2 fluorotelomer sulfonamidoalkyl betaine (6:2 FTAB) is a major component of aqueous film-forming foams (AFFFs) used for firefighting and is frequently detected, along with one of its suspected transformation products, 6:2 fluorotelomer sulfonate (6:2 FTSA), in terrestrial and aquatic ecosystems impacted by AFFF usage. Biochemical processes underlying bacterial biodegradation of these compounds remain poorly understood due to a lack of pure culture studies. Here, we characterized the water-soluble and volatile breakdown products of 6:2 FTSA and 6:2 FTAB produced using Gordonia sp. strain NB4-1Y cultures over seven days under sulfur-limited conditions. After 168 h, 99.9% of 60 μM 6:2 FTSA was degraded into ten major breakdown products, with a mol% recovery of 88.2, while 70.4% of 60 μM 6:2 FTAB was degraded into ten major breakdown products, with a mol% recovery of 84.7. NB4-1Y uses two pathways for 6:2 FTSA metabolism, with 55 mol% of breakdown products assigned to a major pathway and <1.0 mol% assigned to a minor pathway. This work indicates that rapid transformation of 6:2 FTSA and 6:2 FTAB can be achieved under controlled conditions and improves the bacterial metabolism of these compounds.