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DOE OSTI2024

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB

ABSTRACT Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase. IMPORTANCE Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Assessment of Bacterial Inoculant Delivery Methods for Cereal Crops

Despite growing evidence that plant growth-promoting bacteria can be used to improve crop vigor, a comparison of the different methods of delivery to determine which is optimal has not been published. An optimal inoculation method ensures that the inoculant colonizes the host plant so that its potential for plant growth-promotion is fully evaluated. The objective of this study was to compare the efficacy of three seed coating methods, seedling priming, and soil drench for delivering three bacterial inoculants to the sorghum rhizosphere and root endosphere. The methods were compared across multiple time points under axenic conditions and colonization efficiency was determined by quantitative polymerase chain reaction (qPCR). Two seed coating methods were also assessed in the field to test the reproducibility of the greenhouse results under non-sterile conditions. In the greenhouse seed coating methods were more successful in delivering the Gram-positive inoculant (Terrabacter sp.) while better colonization from the Gram-negative bacteria (Chitinophaga pinensis and Caulobacter rhizosphaerae) was observed with seedling priming and soil drench. This suggested that Gram-positive bacteria may be more suitable for the seed coating methods possibly because of their thick peptidoglycan cell wall. We also demonstrated that prolonged seed coating for 12 h could effectively enhance the colonization of C. pinensis, an endophytic bacterium, but not the rhizosphere colonizing C. rhizosphaerae. In the field only a small amount of inoculant was detected in the rhizosphere. This comparison demonstrates the importance of using the appropriate inoculation method for testing different types of bacteria for their plant growth-promotion potential.

Terrabacter↗

DOE OSTI2025

A widespread family of molecular chaperones promotes the intracellular stability of type VIIb secretion system– exported toxins

To survive in highly competitive environments, bacteria use specialized secretion systems to deliver antibacterial toxins into neighboring cells, thereby inhibiting their growth. In many Gram-positive bacteria, the export of such toxins requires a membrane-bound molecular apparatus known as the type VIIb secretion system (T7SSb). Recently, it was shown that toxin recruitment to the T7SSb requires a physical interaction between a toxin and two or more so-called targeting factors, which harbor key residues required for T7SS-dependent protein export. However, in addition to these targeting factors, some toxins additionally require a protein belonging to the DUF4176 protein family. Here, by examining two toxin–DUF4176 protein pairs, we demonstrate that DUF4176 constitutes a family of toxin-specific molecular chaperones. In addition to being required for toxin stability in producing cells, we find that DUF4176 proteins facilitate toxin export by specifically interacting with a previously uncharacterized intrinsically disordered region found in many T7SS toxins. Using X-ray crystallography, we determine structures of several DUF4176 chaperones in their unbound state, and of a DUF4176 chaperone in complex with the binding site of its cognate toxin. These structures reveal that this binding site consists of a disordered amphipathic α-helix that requires interaction with its cognate chaperone for proper folding. Overall, we have identified a family of secretion system associated molecular chaperones found throughout T7SSb-containing Gram-positive bacteria.

Gkragkopoulou, Polyniki↗

DOE OSTI2025

Characterization of Tank 9H Annulus Sample in Support of Residual Material Inventory Determinations

The Savannah River National Laboratory (SRNL) was requested by Savannah River Mission Completion (SRMC) to provide sample preparation and characterization of the Tank 9H annulus sample in support of Residual Material Inventory Determinations. One Tank 9H sample in three vials [HTF-9-25-13, HTF-9-25-14 and HTF-9-25-15], with each vial containing approximately 200 mL of the Tank 9H annulus salt solution, were delivered to the SRNL Shielded Cells for sample preparation and characterizations in February 2025. The density of the “as-received” solution contained in each of the three Tank 9H annulus sample vials were determined followed by a solid-liquid separation on each one using 0.45-micron Nalgene® nylon filter membranes. The resulting filtrates were combined to form the Tank 9H annulus sample with a total volume of about 600 mL. The combined wet solid fractions, about a total of 4.8 grams of salt material, remaining on the filter membranes were air-dried in the Shielded Cells for 72 hours. The total weight of the air-dried solids was 2.1 grams. These air-dried solids were washed with deionized water (DI water) at a phase ratio of 60 mL DI water/gram of solids to recover insoluble solids, if any. No visible or measurable quantity of insoluble solids were recovered after DI water washing of the air-dried solids because the air-dried solids completely dissolved in the DI water. The solid fraction-wash water was not combined with the 600 mL of the filtrate solution, and the resulting solution was not screened or analyzed for radionuclides. Aliquot sample volumes of the undiluted Tank 9H annulus sample were sent to the SRNL analytical services groups for radionuclides, elementals, anions and total mercury analysis by various methods including radiochemical separations/counting methods, inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) and special preparations. All sample analyses were performed in triplicate. This report presents the analytical characterization results for the Tank 9H annulus sample. The results are also reported where analytical methods yielded additional analytes, other than those requested by SRMC. In the characterization of the Tank 9H annulus sample, the detection limits for all the analytes, as specified in the Technical Task Request (TTR) and Task Technical and Quality Assurance Plan (TTQAP), were met.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2020

Scaling Trivalent Actinide and Lanthanide Recovery by Diglycolamide Resin from Savannah River Site’s Mark-18A Targets

The Savannah River National Laboratory is implementing a separation flowsheet to recover rare isotopes and transplutonium elements from irradiated 242 Pu targets known as Mark-18A targets. The Mark-18A targets contain the United States’ supply of nonseparated 244 Pu, which has a wide range of applications from nuclear forensics to production of superheavy elements such as flerovium. The targets also contain hundreds of grams of heavy curium ( 246–248 Cm), which is used as a target material for 252 Cf production. This work investigates the use of diglycolomide resin (DGA Resin) to recover valuable trivalent actinides from the Mark-18A targets. Batch contact experiments were performed on a representative simulant to determine mass loadings. Furthermore, the resin showed an overall capacity of 11 mg/mL for a mixed metal matrix (Zr and La–Gd). Column experiments showed chromatographic separation with transition-metal breakthroughs occurring first followed by the lanthanide series La–Gd. The experiments showed that lanthanide breakthrough occurred after 11 mg/mL mass loading was reached on the column with the mixed metal matrix. A radiological column experiment with an in-line UV/vis cell was able to detect Nd breakthrough just prior to 241 Am breakthrough. Implementing an in-line UV/vis cell into full-scale Mark-18A target processing will be used to limit breakthrough of trivalent actinides recovered from the targets.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

The Impact of Sorbent Amendments for Mercury Remediation on the Viability of Soil Microorganisms

Mercury (Hg) remediation across contaminated environments in the United States is an ongoing project. As part of the Hg cleanup strategy at East Fork Poplar Creek (EFPC), located in Oak Ridge, TN, the deployment of sorbents is considered. However, the impact of sorbents on soil microorganisms is poorly understood. In this study, we investigated the effect of sorbents on soil microorganism viability and biofilm formation to assess soil health during sorbent application for Hg remediation. We specifically investigated the effect of two engineered sorbents, Organoclay PM-199 and Organoclay MRM (which are manufactured from clay minerals formulated for various remediation applications), on two gram-negative organisms ( Serratia marcescens and Burkholderia thailandensis ) isolated from the Hg-contaminated EFPC bank soil. Pure cultures of S. marcescens or B. thailandensis were amended with 5% (w/v) and 25% (w/v) PM-199 and MRM, respectively, for 9 days. The samples were harvested, and bacterial cell viability was determined using a BacLight staining kit. Results showed that the growth of sorbent-amended S. marcescens was inhibited in contrast to that of unamended control. Furthermore, biochemical assays were used to analyze bacterial biofilm formation and integral biofilm components. Our results suggest that biofilm formation by sorbent-amended S. marcescens was negatively affected. In contrast, B. thailandensis amended with low concentrations of MRM showed enhanced growth and notable differences in biofilm morphology. These results suggest that the use of organoclay PM-199 and MRM at higher concentrations in field studies may hinder the growth of specific soil microorganisms.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

‘ Candidatus Liberibacter asiaticus’-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress In Planta

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic ‘Candidatus Liberibacter’ spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of ‘Ca. L. asiaticus’ bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that ‘Ca. L. asiaticus’ BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. ‘Ca. L. asiaticus’ BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of ‘Ca. L. asiaticus’ acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Development of a microwave-assisted downdraft moving-bed gasifier for continuous processing of lignite and biomass chars

This research illustrates a microwave-assisted downdraft moving-bed gasifier for the first time. Such design enables continuous solid gasification process. An adjustable auger was applied to control the solid removal rate and the gas-solid interaction time. Both lignite and biomass chars were investigated to determine the capability of the current system for low-tar feedstocks with different densities. Here, the presented reactor design was able to operate continuously for 3 hours and 20 minutes under 700 ℃ and atmospheric pressure, with air as the gasifying agent. For yellow pine char, the processing rate could reach 34.1 grams per hour with decent syngas production. The downdraft moving-bed design shows better cold gas and syngas production efficiencies compared to the common fixed-bed design, due to controllable residence time and more homogeneous microwave heating. The limitations of the current design and the direction of novel microwave-assisted chemical reactor design were discussed. This novel reactor design provides a way to improve the efficiency of microwave-assisted gasification process and shows its potential to be incorporated into other established chemical reaction processes as a modular add-on.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Novel Extracellular Electron Transfer Channels in a Gram-Positive Thermophilic Bacterium

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium Carboxydothermus ferrireducens that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by C. ferrireducens . The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive c -type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Partitioning of Seven Different Classes of Antibiotics into LPS Monolayers Supports Three Different Permeation Mechanisms through the Outer Bacterial Membrane

The outer membrane (OM) of Gram-negative (G-) bacteria presents a barrier for x classes of antibacterial agents. Lipopolysaccharide (LPS), present in the outer leaflet of the OM, is stabilized by divalent cations and is considered to be the major impediment for antibacterial agent permeation. However, the actual affinities of major antibiotic classes toward LPS have not yet been determined. In the present work, we use Langmuir monolayers formed from E. coli Re and Rd types of LPS to record pressure-area isotherms in the presence of antimicrobial agents. Our observations suggest three general types of interactions. Firstly, some antimicrobials demonstrated no measurable interactions with LPS. This lack of interaction in the case of cefsulodin, a third-generation cephalosporin antibiotic, correlates with its low efficacy against G- bacteria. Ampicillin and ciprofloxacin also show no interactions with LPS, but in contrast to cefsulodin, both exhibit good efficacy against G- bacteria, indicating permeation through common porins. Secondly, we observe substantial intercalation of the more hydrophobic antibiotics novobiocin, rifampicin, azithromycin, and telithromycin into relaxed LPS monolayers. These largely re-partition back to the subphase with monolayer compression. We find that the hydrophobic area, charge, and dipole all show correlations with both the mole fraction of antibiotic retained in the monolayer at the monolayer-bilayer equivalence pressure and the efficacies of these antibiotics against G- bacteria. Thirdly, amine-rich gentamicin and the cationic antimicrobial peptides polymyxin B and colistin show no hydrophobic insertion but are instead strongly driven into the polar LPS layer by electrostatic interactions in a pressure-independent manner. Their intercalation stably increases the area per molecule (by up to 20%), which indicates massive formation of defects in the LPS layer. These defects support a self-promoted permeation mechanism of these antibiotics through the OM, which explains the high efficacy and specificity of these antimicrobials against G- bacteria.

lateral pressure↗

DOE OSTI2022

Co-flow injection for serial crystallography at X-ray free-electron lasers

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

3D printing↗

DOE OSTI2025

Data from: Short-Term Inhibition and Long-Term Enhancement of IrreversibleTrace Metal Binding to Goethite in Multi-Metal Systems

These files include the data used in all figures from the following paper: Short-Term Inhibition and Long-Term Enhancement of Irreversible Trace Metal Binding to Goethite in Multi-Metal Systems Below is a brief description of each of the figures and associated data. Fig 1. Ni adsorption isotherms to goethite in the presence and absence of Zn and Cd Fig 2. % of Ni, Zn, and Cd exchanged as a function of time in isotope exchange experiments conducted after 2 days, 30 days, and 60 days aging. Fig. 3. This shows the % of Ni, Cd, and Zn in single metal and mixed metal systems that is non-exchangeable, both in units of % non-exchangeable and in micromoles of metal per gram of goethite. Fig. 4. Particle size distributions of Ni-Zn and Ni-Cd systems after aging. Fig 5. Ni and Zn XANES fits for Ni-Cd and Ni-Zn systems after 30 and 60 days aging. Linear combination fits were performed between freshly adsorbed and incorporated phases (synthesized as Ni- or Zn-substituted goethite) to determine the proportions of each. Figure S1. Dissolved Metal Concentrations at the Start of Isotope Exchange Figure S2. Reactor pH with Aging Figure S3. Dissolved Cd and Ni Concentrations during Isotope Exchange Figure S4. Dissolved Zn and Ni Concentrations during Isotope Exchange Figure S5. Tracer Mole Fractions in Ni-Zn Isotope Exchange Experiments Figure S6. Tracer Mole Fractions in Ni-Cd Isotope Exchange Experiments

58 GEOSCIENCES↗

DOE OSTI2023

First measurement of the nuclear-recoil ionization yield in silicon at 100 eV

We measured the nuclear--recoil ionization yield in silicon with a cryogenic phonon-sensitive gram-scale detector. Neutrons from a mono-energetic beam scatter off of the silicon nuclei at angles corresponding to energy depositions from 4 keV down to 100 eV, the lowest energy probed so far. The results show no sign of an ionization production threshold above 100 eV. These results call for further investigation of the ionization yield theory and a comprehensive determination of the detector response function at energies below the keV scale.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2023

First Measurement of the Nuclear-Recoil Ionization Yield in Silicon at 100 eV

We measured the nuclear-recoil ionization yield in silicon with a cryogenic phonon-sensitive gram-scale detector. Neutrons from a mono-energetic beam scatter off of the silicon nuclei at angles corresponding to energy depositions from 4 keV down to 100 eV, the lowest energy probed so far. The results show no sign of an ionization production threshold above 100 eV. In conclusion, these results call for further investigation of the ionization yield theory and a comprehensive determination of the detector response function at energies below the keV scale.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

DOE OSTI2023

Aculeaxanthones A–E, new xanthones from the marine-derived fungus Aspergillus aculeatinus WHUF0198

Introduction Dimeric natural products are widespread in plants and microorganisms, which usually have complex structures and exhibit greater bioactivities than their corresponding monomers. In this study, we report five new dimeric tetrahydroxanthones, aculeaxanthones A−E ( 4−8 ), along with the homodimeric tetrahydroxanthone secalonic acid D ( 1 ), chrysoxanthones B and C ( 2 and 3 ), and 4−4’-secalonic acid D ( 9 ), from different fermentation batches of the title fungus. Methods A part of the culture was added to a total of 60 flasks containing 300 ml each of number II fungus liquid medium and culture 4 weeks in a static state at 28˚C. The liquid phase (18 L) and mycelia was separated from the fungal culture by filtering. A crude extract was obtained from the mycelia by ultrasound using acetone. To obtain a dry extract (18 g), the liquid phase combined with the crude extract were further extracted by EtOAc and concentrated in vacuo. The MIC of anaerobic bacteria was examined by a broth microdilution assay. To obtain MICs for aerobic bacteria, the agar dilution streak method recommended in Clinical and Laboratory Standards Institute document (CLSI) M07-A10 was used. Compounds 1−9 was tested against the Bel-7402, A-549 and HCT-116 cell lines according to MTT assay. Results and Discussion The structures of these compounds were elucidated on the base of 1D and 2D NMR and HR-ESIMS data, and the absolute configurations of the new xanthones 4−8 were determined by conformational analysis and time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) calculations. Compounds 1–9 were tested for cytotoxicity against the Bel-7402, A549, and HCT-116 cancer cell lines. Of the dimeric tetrahydroxanthone derivatives, only compound 6 provided cytotoxicity effect against Bel-7402 cell line (IC50, 1.96 µM). Additionally, antimicrobial activity was evaluated for all dimeric tetrahydroxanthones, including four Gram-positive bacteria including Enterococcus faecium ATCC 19434, Bacillus subtilis 168, Staphylococcus aureus ATCC 25923 and MRSA USA300; four Gram-negative bacteria, including Helicobacter pylori 129, G27, as well as 26,695, and multi drug-resistant strain H. pylori 159, and one Mycobacterium M. smegmatis ATCC 607. However, only compound 1 performed activities against H. pylori G27, H. pylori 26695, H. pylori 129, H. pylori 159, S. aureus USA300, and B. subtilis 168 with MIC values of 4.0, 4.0, 2.0, 2.0, 2.0 and 1.0 μg/mL, respectively.

Wu, Jun↗

DOE OSTI2023

Automatic Determination of the Weak-Beam Condition in Dark Field X-ray Microscopy

Mechanical properties in crystals are strongly correlated to the arrangement of 1D line defects, termed dislocations. Recently, Dark field X-ray Microscopy (DFXM) has emerged as a new tool to image and interpret dislocations within crystals using multidimensional scans. However, the methods required to reconstruct meaningful dislocation information from high-dimensional DFXM scans are still nascent and require significant manual oversight (i.e. supervision). In this work, we present a new relatively unsupervised method that extracts dislocation-specific information (features) from a 3D dataset (x, y, Φ) using Gram-Schmidt orthogonalization to represent the large dataset as an array of 3-component feature vectors for each position, corresponding to the weak-beam conditions and the strong-beam condition. Finally, this method offers key opportunities to significantly reduce dataset size while preserving only the crystallographic information that is important for data reconstruction.

36 MATERIALS SCIENCE↗

DOE OSTI2020

Paraburkholderia solitsugae sp. nov. and Paraburkholderia elongata sp. nov., phenolic acid-degrading bacteria isolated from forest soil and emended description of Paraburkholderia madseniana

Two bacterial strains, 1N T and 5N T , were isolated from hemlock forest soil using a soluble organic matter enrichment. Cells of 1N T (0.65×1.85 µm) and 5N T (0.6×1.85 µm) are Gram-stain-negative, aerobic, motile, non-sporulating and exist as single rods, diplobacilli or in chains of varying length. During growth in dilute media (≤0.1× tryptic soy broth; TSB), cells are primarily motile with flagella. At higher concentrations (≥0.3× TSB), cells of both strains increasingly form non-motile chains, and cells of 5N T elongate (0.57×~7 µm) and form especially long filaments. Optimum growth of 1N T and 5N T occurred at 25–30 °C, pH 6.5–7.0 and <0.5% salinity. Results of comparative chemotaxonomic, genomic and phylogenetic analyses revealed that 1N T and 5N T were distinct from one another and their closest related type strains: Paraburkholderia madseniana RP11 T , Paraburkholderia aspalathi LMG 27731 T and Paraburkholderia caffeinilytica CF1 T . The genomes of 1N T and 5N T had an average nucleotide identity (91.6 and 91.3%) and in silico DNA–DNA hybridization values (45.8%±2.6 and 45.5%±2.5) and differed in functional gene content from their closest related type strains. The composition of fatty acids and patterns of substrate use, including the catabolism of phenolic acids, also differentiated strains 1N T and 5N T from each other and their closest relatives. The only ubiquinone present in strains 1N T and 5N T was Q-8. The major cellular fatty acids were C 16 : 0 , 3OH-C 16 : 0 , C 17 : 0 cyclo, C 19 : 0 cyclo ω8 c and summed features 2 (3OH-C 14 : 0 / C 16 : 1 iso I), 3 (C 16 : 1 ω6 c /ω7 c ) and 8 (C 18 : 1 ω7 c /ω6 c ). A third bacterium, strain RL16-012-BIC-B, was isolated from soil associated with shallow roots and was determined to be a strain of P. madseniana (ANI, 98.8%; 16S rRNA gene similarity, 100%). Characterizations of strain RL16-012-BIC-B (DSM 110723=LMG 31706) led to proposed emendments to the species description of P. madseniana . Our polyphasic approach demonstrated that strains 1N T and 5N T represent novel species from the genus Paraburkholderia for which the names Paraburkholderia solitsugae sp. nov. (type strain 1N T =DSM 110721 T =LMG 31704 T ) and Paraburkholderia elongata sp. nov. (type strain 5N T =DSM 110722 T =LMG 31705 T ) are proposed.

Microbiology↗

DOE OSTI2022

Structure-guided functional studies of plasmid-encoded dihydrofolate reductases reveal a common mechanism of trimethoprim resistance in Gram-negative pathogens

Two plasmid-encoded dihydrofolate reductase (DHFR) isoforms, DfrA1 and DfrA5, that give rise to high levels of resistance in Gram-negative bacteria were structurally and biochemically characterized to reveal the mechanism of TMP resistance and to support phylogenic groupings for drug development against antibiotic resistant pathogens. Preliminary screening of novel antifolates revealed related chemotypes that showed high levels of inhibitory potency against Escherichia coli chromosomal DHFR (EcDHFR), DfrA1, and DfrA5. Kinetics and biophysical analysis, coupled with crystal structures of trimethoprim bound to EcDHFR, DfrA1 and DfrA5, and two propargyl-linked antifolates (PLA) complexed with EcDHFR, DfrA1 and DfrA5, were determined to define structural features of the substrate binding pocket and guide synthesis of pan-DHFR inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗