Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast
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59 BASIC BIOLOGICAL SCIENCES↗
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Quantifying the Spatiotemporal Dynamics of Plant Root Colonization by Beneficial Bacteria in a Microfluidic Habitat
Plant–microbe interactions underpin processes related to soil ecology, plant function, and global carbon cycling. However, quantifying the spatial dynamics of these interactions has proven challenging in natural systems. Currently, microfluidic platforms are at the forefront of innovation for culturing, imaging, and manipulating plants in controlled environments. Using a microfluidic platform to culture plants with beneficial bacteria, visualization and quantification of the spatial dynamics of these interactions during the early stages of plant development is possible. For two plant growth–promoting bacterial isolates, the population of bacterial cells reaches a coverage density of 1–2% of the root's surface at the end of a 4 d observation period regardless of bacterial species or inoculum concentration. The two bacterial species form distinct associations with root tissue through a mechanism that appears to be independent of the presence of the other bacterial species, despite evidence for their competition. Root development changes associated with these bacterial treatments depend on the initial concentrations and species of the bacterial population present. Finally, this microfluidic approach provides context for understanding plant–microbe interactions during the early stages of plant development and can be used to generate new hypotheses about physical and biochemical exchanges between plants and their associated microbial communities.
59 BASIC BIOLOGICAL SCIENCES↗
Biogeochemical controls on iron speciation and cycling across upland to shoreline gradients in freshwater and estuarine coastal soils (Lake Erie and Chesapeake Bay, United States)
Coastal environments are dynamic interfaces that mediate carbon and nutrient exchanges between terrestrial landscapes and open waters, and understanding the biogeochemical factors controlling these exchanges, particularly iron (Fe) redox transformations, is crucial for predicting coastal ecosystem functions. Here, we investigated the mechanisms controlling Fe speciation changes across upland-to-shoreline gradients in freshwater and estuarine soils using Fe K-edge X-ray absorption spectroscopy, solid and porewater composition analysis, and 16S rRNA sequencing analysis. We show that Fe transformations depend primarily on inundation patterns. In unsaturated uplands, Fe occurs as Fe(III) oxyhydroxides, mainly goethite (9–35 %), Fe(II,III)-phyllosilicates (39–89 %), and Fe(III)-organic species (0–61 %). Soils influenced by estuarine waters exhibit porewater sulfide concentrations reaching up to 221 μM, Fe- and S-cycling bacteria, and up to 81 % pyrite (FeS 2 ), indicating that sulfur-driven redox dynamics control Fe transformations. In lacustrine wetlands, Fe(III) reduction is indicated by porewater Fe(II) concentrations increasing to 1.0–2.1 mM, and ~10–15 % of Fe as Fe(II,III)-(hydr)oxides (green rust), vivianite (Fe 3 (PO 4 ) 2 ·8H 2 O), and/or adsorbed Fe(II) species. EXAFS data also indicate reduction of structural Fe(III) to Fe(II) in phyllosilicates. The presence of Fe- and S-cycling bacteria, as well as sulfide (0–10 μM), suggests that Fe-cycling is microbially driven and potentially coupled with cryptic S-cycling. Fe(II) oxidation was indicated above/near the water table by the presence of Fe(III) oxyhydroxides (ferrihydrite, lepidocrocite). Furthermore, negligible Fe(III) or sulfate reduction was observed at some water-saturated sites located at the upland-wetland transition, likely due to oxic (sub-)surface water inputs. Overall, our results highlight the importance of considering both Fe-speciation and hydro-biogeochemical dynamics when predicting Fe-cycling at coastal interfaces.
54 ENVIRONMENTAL SCIENCES↗
Old Woman Creek Wetland Sediment and Electrochemical Sensor Microbial Community, 2023
We are developing a technique to monitor microbiological activities referred to as zero resistance ammetry, which entails the deployment of graphite electrodes in sediments. Measurement of current between electrodes of contrasting redox regimes and/or predominant terminal electron accepting processes can be used as an indicator of the extents of microbiological activity. We deployed an electrode array at depths of 2 mm, 4 mm, 76 mm, 78 mm, 152 mm, 154 mm, 227 mm, and 229 mm below the wetland sediment water interface in the Old Woman Creek National Estuarine Research Center, Huron, OH, USA (Lat. = 41.380833, Long. = -82.508889). A core was collected from adjacent sediment and subsamples were collected from depth intervals of 0 – 25 mm, 25 – 127 mm, 127 – 128 mm, and below 178 mm. To determine if the microbial communities attached to the electrodes were reflective of the adjacent sediment-associated microbial community, we conducted a 16S rRNA gene-based (V4 region) survey of these respective materials. This data package contains the results of these surveys, including metadata on the depths from which samples were collected (samples.csv), DNA extraction and sequencing information (OWC_DEPTH_AMPLICON_SEQUENCING_METADATA), sequence processing information (OWC_DEPTH_BIOINFORMATIC_METADATA.csv), an operational taxonomic unit (OTU) table (OWC_DEPTH_97OTUS_TABLE.csv), and nucleotide sequences of OTUs (OWC_DEPTH_97OTUS_SEQS.fasta). All files can be opened using a text-editing application. The fasta file is compatible with bioinformatics applications.
54 ENVIRONMENTAL SCIENCES↗
Assessing electrogenetic activation via a network model of biological signal propagation
Introduction: Molecular communication is the transfer of information encoded by molecular structure and activity. We examine molecular communication within bacterial consortia as cells with diverse biosynthetic capabilities can be assembled for enhanced function. Their coordination, both in terms of engineered genetic circuits within individual cells as well as their population-scale functions, is needed to ensure robust performance. We have suggested that “electrogenetics,” the use of electronics to activate specific genetic circuits, is a means by which electronic devices can mediate molecular communication, ultimately enabling programmable control. Methods: Here, we have developed a graphical network model for dynamically assessing electronic and molecular signal propagation schemes wherein nodes represent individual cells, and their edges represent communication channels by which signaling molecules are transferred. We utilize graph properties such as edge dynamics and graph topology to interrogate the signaling dynamics of specific engineered bacterial consortia. Results: We were able to recapitulate previous experimental systems with our model. In addition, we found that networks with more distinct subpopulations (high network modularity) propagated signals more slowly than randomized networks, while strategic arrangement of subpopulations with respect to the inducer source (an electrode) can increase signal output and outperform otherwise homogeneous networks. Discussion: We developed this model to better understand our previous experimental results, but also to enable future designs wherein subpopulation composition, genetic circuits, and spatial configurations can be varied to tune performance. We suggest that this work may provide insight into the signaling which occurs in synthetically assembled systems as well as native microbial communities.
Chun, Kayla↗
Nonlinear CO 2 flux response to 7 years of experimentally induced permafrost thaw
Rapid Arctic warming is expected to increase global greenhouse gas concentrations as permafrost thaw exposes immense stores of frozen carbon (C) to microbial decomposition. Permafrost thaw also stimulates plant growth, which could offset C loss. Using data from 7 years of experimental Air and Soil warming in moist acidic tundra, we show that Soil warming had a much stronger effect on CO 2 flux than Air warming. Soil warming caused rapid permafrost thaw and increased ecosystem respiration (R eco ), gross primary productivity (GPP), and net summer CO 2 storage (NEE). Over 7 years R eco , GPP, and NEE also increased in Control (i.e., ambient plots), but this change could be explained by slow thaw in Control areas. In the initial stages of thaw, R eco , GPP, and NEE increased linearly with thaw across all treatments, despite different rates of thaw. As thaw in Soil warming continued to increase linearly, ground surface subsidence created saturated microsites and suppressed R eco , GPP, and NEE. However R eco and GPP remained high in areas with large Eriophorum vaginatum biomass. In general NEE increased with thaw, but was more strongly correlated with plant biomass than thaw, indicating that higher R eco in deeply thawed areas during summer months was balanced by GPP. Summer CO 2 flux across treatments fit a single quadratic relationship that captured the functional response of CO 2 flux to thaw, water table depth, and plant biomass. These results demonstrate the importance of indirect thaw effects on CO 2 flux: plant growth and water table dynamics. Nonsummer R eco models estimated that the area was an annual CO 2 source during all years of observation. As a result, nonsummer CO 2 loss in warmer, more deeply thawed soils exceeded the increases in summer GPP, and thawed tundra was a net annual CO 2 source.
54 ENVIRONMENTAL SCIENCES↗
The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones
Microbial competition within plant tissues affects invading pathogens’ fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce’s disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography–mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.
60 APPLIED LIFE SCIENCES↗
Rewiring Aromatic Compound Consumption: Chromosomal Amplification and Evolution of a Foreign Pathway in Acinetobacter baylyi ADP1
Rational engineering strategies that seek to harness the remarkable diversity of microbial metabolism can be limited by incomplete biological knowledge. As described here, a novel approach to address this challenge involved replacing a native pathway for degrading lignin-derived aromatic compounds via ortho cleavage of protocatechuate in Acinetobacter baylyi ADP1 with a foreign meta-cleavage pathway that uses different enzymes, metabolites, and redox carriers. This alteration may improve lignin valorization and coordinate catabolism with bioproduction strategies. When a 14-kbp region of foreign DNA was inserted in the chromosome, the heterologous genes failed to confer growth on target substrates. Regional gene dosage was increased using a synthetic DNA fragment to promote recombination, and higher copy number enabled growth. During adaptive laboratory evolution, compensatory mutations arose that permit growth with one copy of the foreign genes. This complex metabolic remodeling was accomplished without assumptions about the impediments that initially prevented growth. To understand the changes that emerged, a novel transformation assay identified a combination of mutations sufficient for the new phenotype. Three unexpected changes were revealed: loss of one foreign enzyme, loss of one native enzyme, and loss of a two-component transcriptional regulatory system. This study establishes that large multicopy tandem arrays of poorly adapted pathway genes can confer new functions and improve understanding of metabolism.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-DOPA production
Abstract Optimizing microbial hosts for the large-scale production of valuable metabolites often requires multiple mutations and modifications to the host’s genome. We describe a three-round screen for increased L-DOPA production in S. cerevisiae using FACS enrichment of an enzyme-coupled biosensor for L-DOPA. Multiple rounds of screening were enabled by a single build of a barcoded in vitro transposon-mediated disruption library. New background strains for screening were built for each iteration using results from previous iterations. The same in vitro transposon-mediated disruption library was integrated by homologous recombination into new background strains in each round of screening. Compared with creating new transposon insertions in each round, this method takes less time and saves the cost of additional sequencing to characterize transposon insertion sites. In the first two rounds of screening, we identified deletions that improved biosensor compartmentalization and, consequently, improved our ability to screen for L-DOPA production. In a final round, we discovered that deletion of heme oxygenase (HMX1) increases total heme concentration and increases L-DOPA production, using dopamine measurement as a proxy. We further demonstrated that deleting HMX1 may represent a general strategy for P450 function improvement by improving activity of a second P450 enzyme, BM3, which performs a distinct reaction.
59 BASIC BIOLOGICAL SCIENCES↗
Insights into the single cell draft genome of “Candidatus Achromatium palustre”
Abstract “ Candidatus Achromatium palustre” was recently described as the first marine representative of the Achromatium spp. in the Thiotrichaceae - a sister lineage to the Chromatiaceae in the Gammaproteobacteria. Achromatium spp. belong to the group of large sulfur bacteria as they can grow to nearly 100 μm in size and store elemental sulfur (S 0 ) intracellularly. As a unique feature, Achromatium spp. can accumulate colloidal calcite (CaCO 3 ) inclusions in great amounts. Currently, both process and function of calcite accumulation in bacteria is unknown, and all Achromatium spp. are uncultured. Recently, three single-cell draft genomes of Achromatium spp. from a brackish mineral spring were published, and here we present the first draft genome of a single “ Candidatus Achromatium palustre” cell collected in the sediments of the Sippewissett Salt Marsh, Cape Cod, MA. Our draft dataset consists of 3.6 Mbp, has a G + C content of 38.1 % and is nearly complete (83 %). The next closest relative to the Achromatium spp. genomes is Thiorhodovibrio sp. 907 of the family Chromatiaceae , containing phototrophic sulfide-oxidizing bacteria.
"Candidatus Achromatium palustre"↗
High-Throughput Functional Genomics for Energy Production
Functional genomics remains a foundational field for establishing genotype-phenotype relationships that enable strain engineering. High-throughput (HTP) methods accelerate the Design-Build-Test-Learn cycle that currently drives synthetic biology towards a forward engineering future. Trackable mutagenesis techniques including transposon insertion sequencing and CRISPR-Cas-mediated genome editing allow for rapid fitness profiling of a collection, or library, of mutants to discover beneficial mutations. Due to the relative speed of these experiments compared to adaptive evolution experiments, iterative rounds of mutagenesis can be implemented for next-generation metabolic engineering efforts to design complex production and tolerance phenotypes. Further, the expansion of these mutagenesis techniques to novel bacteria are opening up industrial microbes that show promise for establishing a bio-based economy.
59 BASIC BIOLOGICAL SCIENCES↗
Hicks Pries, Sulman, et al 2018. In situ incubation of 13C-labeled litter in three soil pits at Blodgett Forest, CA from 2013 to 2016
Even though over half of the world's soil organic carbon (SOC) is stored in subsoils (>20 cm deep), and the old ages of subsoil OC indicate its cycling differs from surface SOC, there are few studies examining in situ decomposition processes in deep soils. The purpose of this dataset is to elucidate these processes. In this study we added 13C-labeled fine roots to 15, 55, and 95 cm depths of a well-characterized coniferous forest Alfisol and monitored the amount of root-derived C remaining over 6, 12, and 30 months. We recovered the root-derived C in microbial phospholipid fatty acids (PLFAs) after 6 months and in coarse (>2 mm) particulate, fine (<2 mm) particulate, and dense, mineral-associated pools after 6, 12, and 30 months. Overall, root decomposition in the first 6 months was similar among all depths but significantly diverged at 30 months with faster decomposition at 15 cm than at 95 cm. There were more fungal and Gram negative-associated PLFAs at 15 cm than at 95 cm, and 13C analysis revealed those microbial groups preferred the added root carbon to native SOC. Mineral-associations were not the cause of slower decomposition at depth because similar amounts of applied root C was recovered in the dense fraction at all depths. The largest difference among depths was in the amount of root C recovered in the coarse particulate fraction, which was greater at 95 cm (50%) than at 15 cm (15%). Slower decomposition of the particulate pool at depth likely contributed to the increase in C:N ratios and depletion of δ13C values below 60 cm depth in our soil profiles. Simulations of these soils using the CORPSE model, which incorporates microbial priming effects and mineral stabilization of SOC, reproduced patterns of particulate and mineral-associated SOC over both time and depth and suggested that a lack of priming by root exudates at depth could account for the slower decomposition rate of particulate root material. Decomposition of deep particulate SOC may increase if root exudation or dissolved OC transport to depth increases.This dataset includes (1) characterization of the soil pits prior to litter addition, (2) characterization of how the litter changed after 6, 12, and 30 months, and (3) the CORPSE model structure, parameterization, and output.
54 ENVIRONMENTAL SCIENCES↗
Temperature and injection water source influence microbial community structure in four Alaskan North Slope hydrocarbon reservoirs
A fundamental knowledge of microbial community structure in petroleum reservoirs can improve predictive modeling of these environments. We used hydrocarbon profiles, stable isotopes, and high-density DNA microarray analysis to characterize microbial communities in produced water from four Alaskan North Slope hydrocarbon reservoirs. Produced fluids from Schrader Bluff (24–27°C), Kuparuk (47–70°C), Sag River (80°C), and Ivishak (80–83°C) reservoirs were collected, with paired soured/non-soured wells sampled from Kuparuk and Ivishak. Chemical and stable isotope data suggested Schrader Bluff had substantial biogenic methane, whereas methane was mostly thermogenic in deeper reservoirs. Acetoclastic methanogens (Methanosaeta) were most prominent in Schrader Bluff samples, and the combined δD and δ 13 C values of methane also indicated acetoclastic methanogenesis could be a primary route for biogenic methane. Conversely, hydrogenotrophic methanogens (e.g., Methanobacteriaceae) and sulfide-producing Archaeoglobus and Thermococcus were more prominent in Kuparuk samples. Sulfide-producing microbes were detected in all reservoirs, uncoupled from souring status (e.g., the non-soured Kuparuk samples had higher relative abundances of many sulfate-reducers compared to the soured sample, suggesting sulfate-reducers may be living fermentatively/syntrophically when sulfate is limited). Sulfate abundance via long-term seawater injection resulted in greater relative abundances of Desulfonauticus, Desulfomicrobium, and Desulfuromonas in the soured Ivishak well compared to the non-soured well. In the non-soured Ivishak sample, several taxa affiliated with Thermoanaerobacter and Halomonas predominated. Archaea were not detected in the deepest reservoirs. Functional group taxa differed in relative abundance among reservoirs, likely reflecting differing thermal and/or geochemical influences.
59 BASIC BIOLOGICAL SCIENCES↗
Tracking microbial colonization in fecal microbiota transplantation experiments via genome-resolved metagenomics
Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection and shows promise for treating other medical conditions associated with intestinal dysbioses. However, we lack a sufficient understanding of which microbial populations successfully colonize the recipient gut, and the widely used approaches to study the microbial ecology of FMT experiments fail to provide enough resolution to identify populations that are likely responsible for FMT-derived benefits. Here, we used shotgun metagenomics together with assembly and binning strategies to reconstruct metagenome-assembled genomes (MAGs) from fecal samples of a single FMT donor. We then used metagenomic mapping to track the occurrence and distribution patterns of donor MAGs in two FMT recipients. Our analyses revealed that 22% of the 92 highly complete bacterial MAGs that we identified from the donor successfully colonized and remained abundant in two recipients for at least 8 weeks. Most MAGs with a high colonization rate belonged to the order Bacteroidales. The vast majority of those that lacked evidence of colonization belonged to the order Clostridiales, and colonization success was negatively correlated with the number of genes related to sporulation. Our analysis of 151 publicly available gut metagenomes showed that the donor MAGs that colonized both recipients were prevalent, and the ones that colonized neither were rare across the participants of the Human Microbiome Project. Although our dataset showed a link between taxonomy and the colonization ability of a given MAG, we also identified MAGs that belong to the same taxon with different colonization properties, highlighting the importance of an appropriate level of resolution to explore the functional basis of colonization and to identify targets for cultivation, hypothesis generation, and testing in model systems. Lastly, the analytical strategy adopted in our study can provide genomic insights into bacterial populations that may be critical to the efficacy of FMT due to their success in gut colonization and metabolic properties, and guide cultivation efforts to investigate mechanistic underpinnings of this procedure beyond associations.
59 BASIC BIOLOGICAL SCIENCES↗
Hyporheic Oxygen Dynamics in the East River, Colorado: Insights From an In–Situ, High Frequency Time Series During Two Distinct Flow Seasons
Dissolved oxygen (DO) is critical for aquatic ecosystems, however, few studies have focused on the long-term DO dynamics in hyporheic zones, which are a function of both transport (hydrologic exchange between river and hyporheic zone) and uptake by biogeochemical reactions or respiration. We explore the dynamics of temperature and DO at 10, 20, and 35 cm depth beneath the East River, Colorado, from July–October 2017 (relatively normal water year) and April to October 2018 (comparatively low flow year), enabled by distinctive, in-situ, high frequency (Δt = 5 min) sensors that provided continuous time-series from the undisturbed study site over 14 months. We expect that hyporheic DO, which has a regular daily fluctuation pattern, is supplied by the surface water (at all times we estimate downwelling) and that diurnal hyporheic DO temporal patterns should be aligned with diurnal hyporheic temperature patterns. However, this was not found to be the case. Hyporheic DO becomes depleted briefly at 20 and 35 cm depths in 2017, and at all three hyporheic depths for extended periods in 2018. Whereas diurnal temperature fluctuations have consistent timings of maxima and minima, hyporheic DO rarely has as regular a pattern, and daily ranges are inconsistent. Rainfall events caused some of these changes to diurnal hyporheic DO patterns without repeatable effects. Antecedent snowpack conditions influence streamflow dynamics and therefore hyporheic DO dynamics in this alpine river. These results also point to the strong and variable influence of hyporheic microbial communities regulating hyporheic DO.
54 ENVIRONMENTAL SCIENCES↗
Diverse Bacterial Groups Contribute to the Alkane Degradation Potential of Chronically Polluted Subantarctic Coastal Sediments
We aimed to gain insight into the alkane degradation potential of microbial communities from chronically polluted sediments of a subantarctic coastal environment using a combination of metagenomic approaches. A total of 6178 sequences annotated as alkane-1-monooxygenases (EC 1.14.15.3) were retrieved from a shotgun metagenomic dataset that included two sites analyzed in triplicate. The majority of the sequences binned with AlkB described in Bacteroidetes (32 ± 13 %) or Proteobacteria (29 ± 7 %), although a large proportion remained unclassified at the phylum level. Operational taxonomic unit (OTU)-based analyses showed small differences in AlkB distribution among samples that could be correlated with alkane concentrations, as well as with site-specific variations in pH and salinity. A number of low-abundance OTUs, mostly affiliated with Actinobacterial sequences, were found to be only present in the most contaminated samples. On the other hand, the molecular screening of a large-insert metagenomic library of intertidal sediments from one of the sampling sites identified two genomic fragments containing novel alkB gene sequences, as well as various contiguous genes related to lipid metabolism. Both genomic fragments were affiliated with the phylum Planctomycetes, and one could be further assigned to the genus Rhodopirellula due to the presence of a partial sequence of the 23S ribosomal RNA (rRNA) gene. This work highlights the diversity of bacterial groups contributing to the alkane degradation potential and reveals patterns of functional diversity in relation with environmental stressors in a chronically polluted, high-latitude coastal environment. In addition, alkane biodegradation genes are described for the first time in members of Planctomycetes.
59 BASIC BIOLOGICAL SCIENCES↗
Physiological and Comparative Genomic Analysis of Arthrobacter sp. SRS-W-1-2016 Provides Insights on Niche Adaptation for Survival in Uraniferous Soils
Arthrobacter sp. strain SRS-W-1-2016 was isolated on high concentrations of uranium (U) from the Savannah River Site (SRS) that remains co-contaminated by radionuclides, heavy metals, and organics. SRS is located on the northeast bank of the Savannah River (South Carolina, USA), which is a U.S. Department of Energy (DOE) managed ecosystem left historically contaminated from decades of nuclear weapons production activities. Predominant contaminants within the impacted SRS environment include U and Nickel (Ni), both of which can be transformed microbially into less toxic forms via metal complexation mechanisms. Strain SRS-W-1-2016 was isolated from the uraniferous SRS soils on high concentrations of U (4200 μM) and Ni (8500 μM), but rapid growth was observed at much lower concentrations of 500 μM U and 1000 μM Ni, respectively. Microcosm studies established with strain SRS-W-1-2016 revealed a rapid decline in the concentration of spiked U such that it was almost undetectable in the supernatant by 72 h of incubation. Conversely, Ni concentrations remained unchanged, suggesting that the strain removed U but not Ni under the tested conditions. To obtain a deeper understanding of the metabolic potential, a draft genome sequence of strain SRS-W-1-2016 was obtained at a coverage of 90×, assembling into 93 contigs with an N50 contig length of 92,788 bases. The genomic size of strain SRS-W-1-2016 was found to be 4,564,701 bases with a total number of 4327 putative genes. An in-depth, genome-wide comparison between strain SRS-W-1-2016 and its four closest taxonomic relatives revealed 1159 distinct genes, representing 26.7% of its total genome; many associating with metal resistance proteins (e.g., for cadmium, cobalt, and zinc), transporter proteins, stress proteins, cytochromes, and drug resistance functions. Additionally, several gene homologues coding for resistance to metals were identified in the strain, such as outer membrane efflux pump proteins, peptide/nickel transport substrate and ATP-binding proteins, a high-affinity nickel-transport protein, and the spoT gene, which was recently implicated in bacterial resistance towards U. Detailed genome mining analysis of strain SRS-W-1-2016 also revealed the presence of a plethora of secondary metabolite biosynthetic gene clusters likely facilitating resistance to antibiotics, biocides, and metals. Additionally, several gene homologous for the well-known oxygenase enzyme system were also identified, potentially functioning to generate energy via the breakdown of organic compounds and thus enabling the successful colonization and natural attenuation of contaminants by Arthrobacter sp. SRS-W-1-2016 at the SRS site.
59 BASIC BIOLOGICAL SCIENCES↗
Multiple Lines of Evidences Reveal Mechanisms Underpinning Mercury Resistance and Volatilization by Stenotrophomonas sp. MA5 Isolated from the Savannah River Site (SRS), USA
A largely understudied microbially mediated mercury (Hg) bioremediative pathway includes the volatilization of Hg 2+ to Hg 0 . Therefore, studies on Hg resistant bacteria (HgR), isolated from historically long-term contaminated environments, can serve as models to understand mechanisms underpinning Hg cycling. Towards this end, a mercury resistant bacterial strain, identified as Stenotrophomonas sp., strain MA5, was isolated from Mill Branch on the Savannah River Site (SRS); an Hg-impacted ecosystem. Minimum inhibitory concentration (MIC) analysis showed Hg resistance of up to 20 µg/mL by MA5 with 95% of cells retaining viability. Microcosm studies showed that the strain depleted more than 90% of spiked Hg 2+ within the first 24 h of growth and the detection of volatilized mercury indicated that the strain was able to reduce Hg 2+ to Hg 0 . To understand molecular mechanisms of Hg volatilization, a draft whole genome sequence was obtained, annotated and analyzed, which revealed the presence of a transposon-derived mer operon (merRTPADE) in MA5, known to transport and reduce Hg 2+ into Hg 0 . Based on the whole genome sequence of strain MA5, qRT-PCR assays were designed on merRTPADE, we found a ~40-fold higher transcription of mer T, P, A, D and E when cells were exposed to 5 µg/mL Hg 2+ . Interestingly, strain MA5 increased cellular size as a function of increasing Hg concentrations, which is likely an evolutionary response mechanism to cope with Hg stress. Moreover, metal contaminated environments are shown to co-select for antibiotic resistance. When MA5 was screened for antibiotic resistance, broad resistance against penicillin, streptomycin, tetracycline, ampicillin, rifampicin, and erythromycin was found; this correlated with the presence of multiple gene determinants for antibiotic resistance within the whole genome sequence of MA5. Overall, this study provides an in-depth understanding of the underpinnings of Stenotrophomonas-mercury interactions that facilitate cellular survival in a contaminated soil habitat.
59 BASIC BIOLOGICAL SCIENCES↗