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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Detection of anaerobic and aerobic bacteria from commercial tattoo and permanent makeup inks

ABSTRACT Tattooing and use of permanent makeup (PMU) have dramatically increased over the last decade, with a concomitant increase in ink-related infections. Studies have shown evidence that commercial tattoo and PMU inks are frequently contaminated with pathogenic microorganisms. Considering that tattoo inks are placed into the dermal layer of the skin where anaerobic bacteria can thrive and cause infections in low-oxygen environments, the prevalence of anaerobic and aerobic bacteria should be assessed in tattoo and PMU inks. In this study, we tested 75 tattoo and PMU inks using the analytical methods described in the FDA Bacteriological Analytical Manual Chapter 23 for the detection of both aerobic and anaerobic bacterial contamination, followed by 16S rRNA gene sequencing for microbial identification. Of 75 ink samples, we found 26 contaminated samples with 34 bacterial isolates taxonomically classified into 14 genera and 22 species. Among the 34 bacterial isolates, 19 were identified as possibly pathogenic bacterial strains. Two species, namelyCutibacterium acnes(four strains) andStaphylococcus epidermidis(two strains) were isolated under anaerobic conditions. Two possibly pathogenic bacterial strains,Staphylococcus saprophyticusandC. acnes, were isolated together from the same ink samples (n= 2), indicating that tattoo and PMU inks can contain both aerobic (S. saprophyticus) and anaerobic bacteria (C. acnes). No significant association was found between sterility claims on the ink label and the absence of bacterial contamination. The results indicate that tattoo and PMU inks can also contain anaerobic bacteria. IMPORTANCE The rising popularity of tattooing and permanent makeup (PMU) has led to increased reports of ink-related infections. This study is the first to investigate the presence of both aerobic and anaerobic bacteria in commercial tattoo and PMU inks under aerobic and anaerobic conditions. Our findings reveal that unopened and sealed tattoo inks can harbor anaerobic bacteria, known to thrive in low-oxygen environments, such as the dermal layer of the skin, alongside aerobic bacteria. This suggests that contaminated tattoo inks could be a source of infection from both types of bacteria. The results emphasize the importance of monitoring these products for both aerobic and anaerobic bacteria, including possibly pathogenic microorganisms.

Biotechnology & Applied Microbiology↗
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 OSTI2024

Nitrogen-Fixing Gamma Proteobacteria Azotobacter vinelandii—A Blueprint for Nitrogen-Fixing Plants?

The availability of fixed nitrogen limits overall agricultural crop production worldwide. The so-called modern “green revolution” catalyzed by the widespread application of nitrogenous fertilizer has propelled global population growth. It has led to imbalances in global biogeochemical nitrogen cycling, resulting in a “nitrogen problem” that is growing at a similar trajectory to the “carbon problem”. As a result of the increasing imbalances in nitrogen cycling and additional environmental problems such as soil acidification, there is renewed and increasing interest in increasing the contributions of biological nitrogen fixation to reduce the inputs of nitrogenous fertilizers in agriculture. Interestingly, biological nitrogen fixation, or life’s ability to convert atmospheric dinitrogen to ammonia, is restricted to microbial life and not associated with any known eukaryotes. It is not clear why plants never evolved the ability to fix nitrogen and rather form associations with nitrogen-fixing microorganisms. Perhaps it is because of the large energy demand of the process, the oxygen sensitivity of the enzymatic apparatus, or simply failure to encounter the appropriate selective pressure. Whatever the reason, it is clear that this ability of crop plants, especially cereals, would transform modern agriculture once again. Successfully engineering plants will require creating an oxygen-free niche that can supply ample energy in a tightly regulated manner to minimize energy waste and ensure the ammonia produced is assimilated. Nitrogen-fixing aerobic bacteria can perhaps provide a blueprint for engineering nitrogen-fixing plants. This short review discusses the key features of robust nitrogen fixation in the model nitrogen-fixing aerobe, gamma proteobacteria Azotobacter vinelandii, in the context of the basic requirements for engineering nitrogen-fixing plants.

Microbiology↗
DOE OSTI2021

Four families of folate-independent methionine synthases

Although most organisms synthesize methionine from homocysteine and methyl folates, some have “core” methionine synthases that lack folate-binding domains and use other methyl donors. In vitro , the characterized core synthases use methylcobalamin as a methyl donor, but in vivo , they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Impact of sanitizer application on Salmonella mitigation and microbiome shift on diced tomato during washing and storage

Here, this study examined the shift in Salmonella and indigenous microbiota on diced tomato after washing with three different sanitizers and during post-wash cold storage. Roma tomatoes were inoculated with a Salmonella cocktail (initial level ~ 5.6 CFU g –1 ), diced and washed along with uninoculated diced tomato in simulated flume wash water with sanitizers, including 10 mg L –1 free chlorine (FC10), 90 mg L –1 peracetic acid (PA90), PA90 in combination with a proprietary acidified surfactant blend (PS90), and unsanitized control (CK). Salmonella, total mesophilic aerobic bacteria (MAB), and yeast and mold (YM) populations on both inoculated and uninoculated samples were measured before and after washing, and during storage at 4 °C. 16S rRNA amplicon sequencing was also performed to determine the shift in bacterial microbiome. Washing with all tested sanitizers, especially PS90 (> 4 log Salmonella reduction), reduced Salmonella and MAB populations on inoculated diced tomatoes. Additionally, application of sanitizers mitigated Salmonella cross-contamination onto uninoculated samples. PS90 treatment inhibited the proliferation of most dominant bacteria on diced tomatoes during storage, including Erwiniaceae, Curtobacterium, Pantoea, Erwinia and Enterobacterales, which may benefit product quality and safety.

16S rRNA amplicon sequencing↗
DOE OSTI2022

Methylphosphonate Degradation and Salt-Tolerance Genes of Two Novel Halophilic Marivita Metagenome-Assembled Genomes from Unrestored Solar Salterns

Aerobic bacteria that degrade methylphosphonates and produce methane as a byproduct have emerged as key players in marine carbon and phosphorus cycles. Here, we present two new draft genome sequences of the genus Marivita that were assembled from metagenomes from hypersaline former industrial salterns and compare them to five other Marivita reference genomes. Phylogenetic analyses suggest that both of these metagenome-assembled genomes (MAGs) represent new species in the genus. Average nucleotide identities to the closest taxon were <85%. The MAGs were assembled with SPAdes, binned with MetaBAT, and curated with scaffold extension and reassembly. Both genomes contained the phnCDEGHIJLMP suite of genes encoding the full C-P lyase pathway of methylphosphonate degradation and were significantly more abundant in two former industrial salterns than in nearby reference and restored wetlands, which have lower salinity levels and lower methane emissions than the salterns. These organisms contain a variety of compatible solute biosynthesis and transporter genes to cope with high salinity levels but harbor only slightly acidic proteomes (mean isoelectric point of 6.48).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Soil Candidate Phyla Radiation Bacteria Encode Components of Aerobic Metabolism and Co-occur with Nanoarchaea in the Rare Biosphere of Rhizosphere Grassland Communities

Here, we investigated overlooked microbes in soil, candidate phyla radiation (CPR) bacteria and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) archaea, by size fractionating small particles from soil, an approach typically used for the recovery of viral metagenomes. Concentration of these small cells (<0.2 μm) allowed us to identify these organisms as part of the rare soil biosphere and to sample genomes that were absent from non-size-fractionated metagenomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Plant-Nitrifier Interactions in Topsoil and Subsoil

Plants can influence soil microbes through resource acquisition and interference competition, with consequences for ecosystem function such as nitrification. However, how plants alter soil conditions to influence nitrifiers and nitrification rates remains poorly understood, especially in the subsoil. Here, coupling the 15N isotopic pool dilution technique, high throughput sequencing and in situ soil O2 monitoring, we investigated how a deep-rooted perennial grass, miscanthus, versus an adjacent shallow-rooted turfgrass reference shapes nitrifier assembly and function along 1 m soil profiles. In topsoil, the suppression of ammonia (NH3) oxidizing archaea (AOA) and gross nitrification rates in miscanthus relative to the reference likely resulted from nitrifiers being outcompeted by plant roots and heterotrophic bacteria for ammonium (NH4+). The stronger tripartite competition under miscanthus may have been caused in part by the lower soil organic matter (SOM) content, which supported lower gross nitrogen (N) mineralization, the major soil process that produces NH4+. In contrast, below 10 cm soil depth, significantly greater gross nitrification rates were observed in miscanthus compared to the reference. This was likely driven by the significantly lower oxygen (O2) in miscanthus than reference subsoil, which selected against aerobic heterotrophic bacteria but in favor of AOA. Overall, we found that plants can regulate AOA community structure and function through different mechanisms in topsoil and subsoil, with suppression of nitrification in topsoil and enhancement of nitrification in subsoil.

Field Data↗
DOE OSTI2021

Development of an inexpensive matrix-assisted laser desorption—time of flight mass spectrometry method for the identification of endophytes and rhizobacteria cultured from the microbiome associated with maize

Many endophytes and rhizobacteria associated with plants support the growth and health of their hosts. The vast majority of these potentially beneficial bacteria have yet to be characterized, in part because of the cost of identifying bacterial isolates. Matrix-assisted laser desorption-time of flight (MALDI-TOF) has enabled culturomic studies of host-associated microbiomes but analysis of mass spectra generated from plant-associated bacteria requires optimization. In this study, we aligned mass spectra generated from endophytes and rhizobacteria isolated from heritage and sweet varieties of Zea mays. Multiple iterations of alignment attempts identified a set of parameters that sorted 114 isolates into 60 coherent MALDI-TOF taxonomic units (MTUs). These MTUs corresponded to strains with practically identical (>99%) 16S rRNA gene sequences. Mass spectra were used to train a machine learning algorithm that classified 100% of the isolates into 60 MTUs. These MTUs provided >70% coverage of aerobic, heterotrophic bacteria readily cultured with nutrient rich media from the maize microbiome and allowed prediction of the total diversity recoverable with that particular cultivation method. Acidovorax sp., Pseudomonas sp. and Cellulosimicrobium sp. dominated the library generated from the rhizoplane. Relative to the sweet variety, the heritage variety contained a high number of MTUs. The ability to detect these differences in libraries, suggests a rapid and inexpensive method of describing the diversity of bacteria cultured from the endosphere and rhizosphere of maize.

dereplication↗
DOE OSTI2022

Biofilm disruption enhances growth rate and carbohydrate-active enzyme production in anaerobic fungi

Anaerobic gut fungi (AGF) are lignocellulose degraders that naturally form biofilms in the rumen of large herbivores and in standard culture techniques. While biofilm formation enhances biomass degradation and carbohydrate-active enzyme (CAZyme) production in some bacteria and aerobic fungi, gene expression and metabolism in AGF biofilms have not been compared to non-biofilm cultures. Here, using the tunable morphology of the non-rhizoidal AGF, Caecomyces churrovis, the impacts of biofilm formation on AGF gene expression, metabolic flux, growth rate, and xylan degradation rate are quantified to inform future industrial scale-up efforts. Contrary to previous findings, C. churrovis upregulated catabolic CAZymes in stirred culture relative to biofilm culture. Using a de novo transcriptome, 197 new transcripts with predicted CAZyme function were identified. Stirred cultures grew and degraded xylan significantly faster than biofilm-forming cultures with negligible differences in primary metabolic flux, offering a way to accelerate AGF biomass valorization without altering the fermentation product profile. The rhizoidal AGF, Neocallimastix lanati, also grew faster with stirring on a solid plant substrate, suggesting that the advantages of stirred C. churrovis cultures may apply broadly to other AGF.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Systems Level Understanding of Interconnected Methane and Nitrogen Cycles Using Complex Synthetic Communities

The main goal of this project was to follow up on prior discovery of the communal nature of methane utilization by the aerobic methanotrophic bacteria, resulting in carbon transfer along a food chain. This goal was approached via manipulation of specially designed, synthetic bacterial communities, fed exclusively by methane, under a variety of simulated environmental conditions. Using (meta)genomics, metatranscriptomics, stable isotope probing and mutant generation, we obtained solid evidence for the nature of the core microbiota involved in collaborative methane consumption, the major players (true methanotrophs) being the members of the family Methylococcaceae, their major partners (non-methanotrophic methylotrophs) being members of the family Methylophilaceae, and minor but persistent partners in the communities being members of the orders Burkholderiales and Bacteroidetes. Metabolic reconstruction from single genomes and metagenome-assembled genomes (MAGs), along with metatranscriptomics results delineated major pathways for carbon metabolism, suggesting that the methanotrophs share different organic compounds with different functional guilds: methanol with Methylophilaceae, acetate with Burkholderiales, and polymeric substances with Bacteroidetes. Overall, the results of this project expand our understanding of microbial metabolism of methane and, thus, provide basis for strategies for microbial methane mitigation, with obvious benefits to the society of the United States and worldwide.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Physiological and genomic evidence of cysteine degradation and aerobic hydrogen sulfide production in freshwater bacteria

The sulfur-containing amino acid cysteine is abundant in the environment, including in freshwater lakes. Biological cysteine degradation can result in hydrogen sulfide (H 2 S), a toxic and ecologically relevant compound that is a central player in biogeochemical cycling in aquatic environments. Here, we investigated the ecological significance of cysteine in oxic freshwater, using isolated cultures, controlled experiments, and multiomics. We screened bacterial isolates enriched from natural lake water for their ability to produce H 2 S when provided cysteine. We identified 29 isolates (Bacteroidota, Proteobacteria, and Actinobacteria) that produced H 2 S. To understand the genomic and genetic basis for cysteine degradation and H 2 S production, we further characterized three isolates using whole-genome sequencing (using a combination of short-read and long-read sequencing) and tracked cysteine and H 2 S levels over their growth ranges: Stenotrophomonas maltophilia (Gammaproteobacteria), S. bentonitica (Gammaproteobacteria), and Chryseobacterium piscium (Bacteroidota). Cysteine decreased and H 2 S increased, and all three genomes had genes involved in cysteine degradation. Finally, to assess the presence of these organisms and genes in the environment, we surveyed a 5-year time series of metagenomic data from the same isolation source (Lake Mendota, Madison, WI, USA) and identified their presence throughout the time series. Overall, our study shows that diverse isolated bacterial strains can use cysteine and produce H 2 S under oxic conditions, and we show evidence using metagenomic data that this process may occur more broadly in natural freshwater lakes. Future considerations of sulfur cycling and biogeochemistry in oxic environments should account for H 2 S production from the degradation of organosulfur compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Spectroscopic and computational investigations of organometallic complexation of group 12 transition metals by methanobactins from Methylocystis sp. SB2

Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as “methanobactins,” which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. In this study, the complexation of Zn, Cd, and Hg by methanobactin from Methylocystis sp. strain SB2 was studied using a combination of absorbance, fluorescence, extended x-ray absorption fine structure (EXAFS) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. We report changes in sample absorbance and fluorescence spectral dynamics, which occur on a wide range of experimental timescales and characterize a clear stoichiometric complexation dependence. Mercury L3-edge EXAFS and TD-DFT calculations suggest a linear model for Hg--S coordination, and TD-DFT suggests a tetrahedral model for Zn 2+ and Cd 2+ . We observed an enhancement in the fluorescence of methanobactin upon interaction with transition metals and propose a mechanism of complexation-hindered isomerization drawing inspiration from the wild-type Green Fluorescent Protein active site. Collectively, our results represent the first combined computational and experimental spectroscopy study of methanobactins and shed new light on molecular interactions and dynamics that characterize complexes of methanobactins with Group 12 transition metals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Microbial Communities in Model Seawater-Compensated Fuel Ballast Tanks: Biodegradation and Biocorrosion Stimulated by Marine Sediments

Some naval vessels add seawater to carbon steel fuel ballast tanks to maintain stability during fuel consumption. Marine sediments often contaminate ballast tank fluids and have been implicated in stimulating fuel biodegradation and enhancing biocorrosion. The impact of the marine sediment was evaluated in model ballast tank reactors containing seawater, fuel (petroleum-F76, Fischer–Tropsch F76, or a 1:1 mixture), and carbon steel coupons. Control reactors did not receive fuel. The marine sediment was added to the reactors after 400 days and incubated for another year. Sediment addition produced higher estimated bacterial numbers and enhanced sulfate reduction. Ferrous sulfides were detected on all coupons, but pitting corrosion was only identified on coupons exposed to FT-F76. Aerobic hydrocarbon-degrading bacteria increased, and the level of dissolved iron decreased, consistent with the stimulation of aerobic hydrocarbon degradation by iron. We propose that sediments provide an inoculum of hydrocarbon-degrading microbes that are stimulated by dissolved iron released during steel corrosion. Hydrocarbon degradation provides intermediates for use by sulfate-reducing bacteria and reduces the level of fuel components inhibitory to anaerobic bacteria. The synergistic effect of dissolved iron produced by corrosion, biodegradable fuels, and iron-stimulated hydrocarbon-degrading microbes is a poorly recognized but potentially significant biocorrosion mechanism.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Biochemistry of aerobic biological methane oxidation

Methanotrophic bacteria represent a potential route to methane utilization and mitigation of methane emissions. In the first step of their metabolic pathway, aerobic methanotrophs use methane monooxygenases (MMOs) to activate methane, oxidizing it to methanol. There are two types of MMOs: a particulate, membrane-bound enzyme (pMMO) and a soluble, cytoplasmic enzyme (sMMO). The two MMOs are completely unrelated, with different architectures, metal cofactors, and mechanisms. The more prevalent of the two, pMMO, is copper-dependent, but the identity of its copper active site remains unclear. By contrast, sMMO uses a diiron active site, the catalytic cycle of which is well understood. Here we review the current state of knowledge for both MMOs, with an emphasis on recent developments and emerging hypotheses. In addition, we discuss obstacles to developing expression systems, which are needed to address outstanding questions and to facilitate future protein engineering efforts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Molecular basis of the pleiotropic effects by the antibiotic amikacin on the ribosome

Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N 1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3’-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rnf and Fix Have Specific Roles during Aerobic Nitrogen Fixation in Azotobacter vinelandii

Biological nitrogen fixation requires large amounts of energy in the form of ATP and low potential electrons to overcome the high activation barrier for cleavage of the dinitrogen triple bond. The model aerobic nitrogen-fixing bacteria, Azotobacter vinelandii, generates low potential electrons in the form of reduced ferredoxin (Fd) and flavodoxin (Fld) using two distinct mechanisms via the enzyme complexes Rnf and Fix. Both Rnf and Fix are expressed during nitrogen fixation, but deleting either rnf1 or fix genes has little effect on diazotrophic growth. However, deleting both rnf1 and fix eliminates the ability to grow diazotrophically. Rnf and Fix both use NADH as a source of electrons, but overcoming the energetics of NADH's endergonic reduction of Fd/Fld is accomplished through different mechanisms. Rnf harnesses free energy from the chemiosmotic potential, whereas Fix uses electron bifurcation to effectively couple the endergonic reduction of Fd/Fld to the exergonic reduction of quinone. Different reaction stoichiometries and condition-specific differential gene expression indicate specific roles for the two reactions. This work's complementary physiological studies and thermodynamic modeling reveal how Rnf and Fix balance redox homeostasis in various conditions. Specifically, the Fix complex is required for efficient growth under low oxygen concentrations, while Rnf is presumed to maintain reduced Fd/Fld production for nitrogenase under standard conditions. This work provides a framework for understanding how the production of low potential electrons sustains robust nitrogen fixation in various conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Variable Inhibition of Nitrous Oxide Reduction in Denitrifying Bacteria by Different Forms of Methanobactin

Aerobic methanotrophic activity is highly dependent on copper availability, and methanotrophs have developed multiple strategies to collect copper. Specifically, when copper is limiting (ambient concentrations < 1 μM), some methanotrophs produce and secret a small modified peptide (< 1,300 Da) termed methanobactin (MB) that binds copper with high affinity. As MB is secreted into the environment, other microbes that require copper for their metabolism may be inhibited as MB may make copper unavailable; e.g., inhibition of denitrifiers as complete conversion nitrate to dinitrogen involves multiple enzymes, some of which are copper-dependent. Of key concern is inhibition of the copper-dependent nitrous oxide reductase (NosZ), the only known enzyme capable of converting nitrous oxide (N 2 O) to dinitrogen. Herein, we show that different forms of MB differentially affect copper uptake and N2O reduction by Pseudomonas stutzeri strain DCP-Ps1 (that expresses clade I NosZ) and Dechloromonas aromatica strain RCB (that expresses clade II NosZ). Specifically, in the presence of MB from Methylocystis sp. strain SB2 (SB2-MB), copper uptake and nosZ expression were more significantly reduced than in the presence of MB from Methylosinus trichosporium OB3b (OB3b-MB). Further, N2O accumulation increased more significantly for both P. stutzeri strain DCP-Ps1 and D. aromatica strain RCB in the presence of SB2-MB versus OB3b-MB. These data illustrate that copper competition between methanotrophs and denitrifying bacteria can be significant and that the extent of such competition is dependent on the form of MB that methanotrophs produce.

54 ENVIRONMENTAL SCIENCES↗