Electrostatic coupling and water bridging in adsorption hierarchy of biomolecules at water-clay interfaces.

Clay minerals are implicated in the retention of biomolecules within organic matter in many soil environments. Spectroscopic studies have proposed several mechanisms for biomolecule adsorption on clays. Here, we employ molecular dynamics simulations to investigate these mechanisms in hydrated adsorbate conformations of montmorillonite, a smectite-type clay, with ten biomolecules of varying chemistry and structure, including sugars related to cellulose and hemicellulose, lignin-related phenolic acid, and amino acids with different functional groups. Our molecular modeling captures biomolecule-clay and biomolecule-biomolecule interactions that dictate selectivity and competition in adsorption retention and interlayer nanopore trapping, which we determine experimentally by NMR and X-ray diffraction, respectively. Specific adsorbate structures are important in facilitating the electrostatic attraction and Van der Waals energies underlying the hierarchy in biomolecule adsorption. Stabilized by a network of direct and water-bridged hydrogen bonds, favorable electrostatic interactions drive this hierarchy whereby amino acids with positively charged side chains are preferentially adsorbed on the negatively charged clay surface compared to the sugars and carboxylate-rich aromatics and amino acids. With divalent metal cations, our model adsorbate conformations illustrate hydrated metal cation bridging of carboxylate-containing biomolecules to the clay surface, thus explaining divalent cation-promoted adsorption from our experimental data. Adsorption experiments with a mixture of biomolecules reveal selective inhibition in biomolecule adsorption, which our molecular modeling attributes to electrostatic biomolecule-biomolecule pairing that is more energetically favorable than the biomolecule-clay complex. In sum, our findings highlight chemical and structural features that can inform hypotheses for predicting biomolecule adsorption at water-clay interfaces.

Microbially induced precipitation of silica by anaerobic methane-oxidizing consortia and implications for microbial fossil preservation.

Authigenic carbonate minerals can preserve biosignatures of microbial anaerobic oxidation of methane (AOM) in the rock record. It is not currently known whether the microorganisms that mediate sulfate-coupled AOM-often occurring as multicelled consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB)-are preserved as microfossils. Electron microscopy of ANME-SRB consortia in methane seep sediments has shown that these microorganisms can be associated with silicate minerals such as clays [Chen et al., Sci. Rep. 4, 1-9 (2014)], but the biogenicity of these phases, their geochemical composition, and their potential preservation in the rock record is poorly constrained. Long-term laboratory AOM enrichment cultures in sediment-free artificial seawater [Yu et al., Appl. Environ. Microbiol. 88, e02109-21 (2022)] resulted in precipitation of amorphous silicate particles (~200 nm) within clusters of exopolymer-rich AOM consortia from media undersaturated with respect to silica, suggestive of a microbially mediated process. The use of techniques like correlative fluorescence in situ hybridization (FISH), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and nanoscale secondary ion mass spectrometry (nanoSIMS) on AOM consortia from methane seep authigenic carbonates and sediments further revealed that they are enveloped in a silica-rich phase similar to the mineral phase on ANME-SRB consortia in enrichment cultures. Like in cyanobacteria [Moore et al., Geology 48, 862-866 (2020)], the Si-rich phases on ANME-SRB consortia identified here may enhance their preservation as microfossils. The morphology of these silica-rich precipitates, consistent with amorphous-type clay-like spheroids formed within organic assemblages, provides an additional mineralogical signature that may assist in the search for structural remnants of microbial consortia in rocks which formed in methane-rich environments from Earth and other planetary bodies.

Complex regulation in a Comamonas platform for diverse aromatic carbon metabolism.

Critical to a sustainable energy future are microbial platforms that can process aromatic carbons from the largely untapped reservoir of lignin and plastic feedstocks. Comamonas species present promising bacterial candidates for such platforms because they can use a range of natural and xenobiotic aromatic compounds and often possess innate genetic constraints that avoid competition with sugars. However, the metabolic reactions of these species are underexplored, and the regulatory mechanisms are unknown. Here we identify multilevel regulation in the conversion of lignin-related natural aromatic compounds, 4-hydroxybenzoate and vanillate, and the plastics-related xenobiotic aromatic compound, terephthalate, in Comamonas testosteroni KF-1. Transcription-level regulation controls initial catabolism and cleavage, but metabolite-level thermodynamic regulation governs fluxes in central carbon metabolism. Quantitative 13C mapping of tricarboxylic acid cycle and cataplerotic reactions elucidates key carbon routing not evident from enzyme abundance changes. This scheme of transcriptional activation coupled with metabolic fine-tuning challenges outcome predictions during metabolic manipulations.

Disproportionate Carbon Dioxide Efflux in Bacterial Metabolic Pathways for Different Organic Substrates Leads to Variable Contribution to Carbon-Use Efficiency.

Microbial organic matter turnover is an important contributor to the terrestrial carbon dioxide (CO2) budget. Partitioning of organic carbons into biomass relative to CO2 efflux, termed carbon-use efficiency (CUE), is widely used to characterize organic carbon cycling by soil microorganisms. Recent studies challenge proposals of CUE dependence on the oxidation state of the substrate carbon and implicate instead metabolic strategies. Still unknown are the metabolic mechanisms underlying variability in CUE. We performed a multiomics investigation of these mechanisms in Pseudomonas putida, a versatile soil bacterium of the Gammaproteobacteria, processing a mixture of plant matter derivatives. Our 13C-metabolomics data captured substrate carbons into different metabolic pathways: cellulose-derived sugar carbons in glycolytic and pentose-phosphate pathways; lignin-related aromatic carbons in the tricarboxylic acid cycle. Subsequent 13C-metabolic flux analysis revealed a 3-fold lower investment of sugar carbons in CO2 efflux compared to aromatic carbons, in agreement with reported substrate-dependent CUE. Proteomics analysis revealed enzyme-level regulation only for substrate uptake and initial catabolism, which dictated downstream fluxes through CO2-producing versus biomass-synthesizing reactions. Metabolic partitioning as shown here explained the substrate-dependent CUE calculated from reported metabolic flux analyses of other bacteria, further supporting a metabolism-guided perspective for predicting the microbial conversion of accessible organic matter to CO2 efflux.

Hierarchical routing in carbon metabolism favors iron-scavenging strategy in iron-deficient soil Pseudomonas species.

High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple 13C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soil Pseudomonas species reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated (Pseudomonas putida KT2440, Pseudomonas protegens Pf-5, and Pseudomonas putida S12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis-anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency.

Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases.

Among ubiquitous phosphorus (P) reserves in environmental matrices are ribonucleic acid (RNA) and polyphosphate (polyP), which are, respectively, organic and inorganic P-containing biopolymers. Relevant to P recycling from these biopolymers, much remains unknown about the kinetics and mechanisms of different acid phosphatases (APs) secreted by plants and soil microorganisms. Here we investigated RNA and polyP dephosphorylation by two common APs, a plant purple AP (PAP) from sweet potato and a fungal phytase from Aspergillus niger. Trends of δ18O values in released orthophosphate during each enzyme-catalyzed reaction in 18O-water implied a different extent of reactivity. Subsequent enzyme kinetics experiments revealed that A. niger phytase had 10-fold higher maximum rate for polyP dephosphorylation than the sweet potato PAP, whereas the sweet potato PAP dephosphorylated RNA at a 6-fold faster rate than A. niger phytase. Both enzymes had up to 3 orders of magnitude lower reactivity for RNA than for polyP. We determined a combined phosphodiesterase-monoesterase mechanism for RNA and terminal phosphatase mechanism for polyP using high-resolution mass spectrometry and 31P nuclear magnetic resonance, respectively. Molecular modeling with eight plant and fungal AP structures predicted substrate binding interactions consistent with the relative reactivity kinetics. Our findings implied a hierarchy in enzymatic P recycling from P-polymers by phosphatases from different biological origins, thereby influencing the relatively longer residence time of RNA versus polyP in environmental matrices. This research further sheds light on engineering strategies to enhance enzymatic recycling of biopolymer-derived P, in addition to advancing environmental predictions of this P recycling by plants and microorganisms.

Dynamic utilization of low-molecular-weight organic substrates across a microbial growth rate gradient.

AIM: Low-molecular-weight organic substances (LMWOSs) are at the nexus between micro-organisms, plant roots, detritus, and the soil mineral matrix. The nominal oxidation state of carbon (NOSC) has been suggested as a potential parameter for modelling microbial uptake rates of LMWOSs and the efficiency of carbon incorporation into new biomass. METHODS AND RESULTS: In this study, we assessed the role of compound class and oxidation state on uptake kinetics and substrate-specific carbon use efficiency (SUE) during the growth of three model soil micro-organisms, a fungal isolate (Penicillium spinulosum) and two bacterial isolates (Paraburkholderia solitsugae, and Ralstonia pickettii). Isolates were chosen that spanned a growth rate gradient (0.046-0.316 h-1 ) in media containing 34 common LMWOSs at realistically low initial concentrations (25 µM each). Clustered, co-utilization of LMWOSs occurred for all three organisms. Potential trends (p < 0.05) for early utilization of more oxidized substrates were present for the two bacterial isolates (P. solitsugae and R. pickettii), but high variability (R2 < 0.15) and a small effect of NOSC indicate these relationships are not useful for prediction. The SUEs of selected substrates ranged from 0.16 to 0.99 and there was no observed relationship between NOSC and SUE. CONCLUSION: Our results do not provide compelling population-level support for NOSC as a predictive tool for either uptake kinetics or the efficiency of use of LMWOS in soil solution. SIGNIFICANCE AND IMPACT OF THE STUDY: Metabolic strategies of organisms are likely more important than chemical identity in determining LMWOS cycling in soils. Previous community-level observations may be biased towards fast-responding bacterial community members.

Molecular Coordination, Structure, and Stability of Metal-Polyphosphate Complexes Resolved by Molecular Modeling and X-ray Scattering: Structural Insights on the Biological Fate of Polyphosphate.

Polyphosphate-accumulating organisms (PAOs), which can store high levels of phosphate (Pi) in the form of polyphosphate (polyP), are employed to engineer enhanced biological P removal (EBPR) from wastewaters. Co-localization of Mg and K in polyP granules of PAOs has been reported, and higher abundance of Mg-polyP granules relative to other metal complexes was correlated positively with EBPR performance stability. However, the underlying mechanism remains unknown. Here, we obtained molecular structural information of hydrated polyP complexes with four physiologically relevant metal cations (Na+, K+, Ca2+, and Mg2+) using computational and experimental techniques. Molecular dynamics simulations revealed that Mg-polyP and K-polyP complexes were the most and least stable of the complexes, respectively, suggesting that the co-occurrence of these complexes facilitates variable polyP bioavailability. The relative thermodynamic stability reflected the strength of metal chelation whereby the coordination distance between the polyP ligand O and the metal was 1.71-2.01 Å for Mg2+ but this distance was 2.64-2.70 Å for K+. Pair distribution function analysis of X-ray scattering data obtained with a Mg-polyP solution corroborated the theoretical Mg-polyP coordination geometry. These findings implied a possible mechanistic role of metal complexation in the P cycling traits of PAOs in engineered and natural systems.

Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida.

Pseudomonas species thrive in different nutritional environments and can catabolize divergent carbon substrates. These capabilities have important implications for the role of these species in natural and engineered carbon processing. However, the metabolic phenotypes enabling Pseudomonas to utilize mixed substrates remain poorly understood. Here, we employed a multi-omics approach involving stable isotope tracers, metabolomics, fluxomics, and proteomics in Pseudomonas putida KT2440 to investigate the constitutive metabolic network that achieves co-utilization of glucose and benzoate, respectively a monomer of carbohydrate polymers and a derivative of lignin monomers. Despite nearly equal consumption of both substrates, metabolite isotopologues revealed nonuniform assimilation throughout the metabolic network. Gluconeogenic flux of benzoate-derived carbons from the tricarboxylic acid cycle did not reach the upper Embden-Meyerhof-Parnas pathway nor the pentose-phosphate pathway. These latter two pathways were populated exclusively by glucose-derived carbons through a cyclic connection with the Entner-Doudoroff pathway. We integrated the 13C-metabolomics data with physiological parameters for quantitative flux analysis, demonstrating that the metabolic segregation of the substrate carbons optimally sustained biosynthetic flux demands and redox balance. Changes in protein abundance partially predicted the metabolic flux changes in cells grown on the glucose:benzoate mixture versus on glucose alone. Notably, flux magnitude and directionality were also maintained by metabolite levels and regulation of phosphorylation of key metabolic enzymes. These findings provide new insights into the metabolic architecture that affords adaptability of P. putida to divergent carbon substrates and highlight regulatory points at different metabolic nodes that may underlie the high nutritional flexibility of Pseudomonas species.

Ecophysiological Study of Paraburkholderia sp. Strain 1N under Soil Solution Conditions: Dynamic Substrate Preferences and Characterization of Carbon Use Efficiency.

We used time-resolved metabolic footprinting, an important technical approach used to monitor changes in extracellular compound concentrations during microbial growth, to study the order of substrate utilization (i.e., substrate preferences) and kinetics of a fast-growing soil isolate, Paraburkholderia sp. strain 1N. The growth of Paraburkholderia sp. 1N was monitored under aerobic conditions in a soil-extracted solubilized organic matter medium, representing a realistic diversity of available substrates and gradient of initial concentrations. We combined multiple analytical approaches to track over 150 compounds in the medium and complemented this with bulk carbon and nitrogen measurements, allowing estimates of carbon use efficiency throughout the growth curve. Targeted methods allowed the quantification of common low-molecular-weight substrates: glucose, 20 amino acids, and 9 organic acids. All targeted compounds were depleted from the medium, and depletion followed a sigmoidal curve where sufficient data were available. Substrates were utilized in at least three distinct temporal clusters as Paraburkholderia sp. 1N produced biomass at a cumulative carbon use efficiency of 0.43. The two substrates with highest initial concentrations, glucose and valine, exhibited longer usage windows, at higher biomass-normalized rates, and later in the growth curve. Contrary to hypotheses based on previous studies, we found no clear relationship between substrate nominal oxidation state of carbon (NOSC) or maximal growth rate and the order of substrate depletion. Under soil solution conditions, the growth of Paraburkholderia sp. 1N induced multiauxic substrate depletion patterns that could not be explained by the traditional paradigm of catabolite repression.IMPORTANCE Exometabolomic footprinting methods have the capability to provide time-resolved observations of the uptake and release of hundreds of compounds during microbial growth. Of particular interest is microbial phenotyping under environmentally relevant soil conditions, consisting of relatively low concentrations and modeling pulse input events. Here, we show that growth of a bacterial soil isolate, Paraburkholderia sp. 1N, on a dilute soil extract resulted in a multiauxic metabolic response, characterized by discrete temporal clusters of substrate depletion and metabolite production. Our data did not support the hypothesis that compounds with lower energy content are used preferentially, as each cluster contained compounds with a range of nominal oxidation states of carbon. These new findings with Paraburkholderia sp. 1N, which belongs to a metabolically diverse genus, provide insights on ecological strategies employed by aerobic heterotrophs competing for low-molecular-weight substrates in soil solution.

The Metabolome of Associations between Xylem-Feeding Insects and their Bacterial Symbionts.

Metabolomics has increasingly led to important insights in chemical ecology by identifying environmentally relevant small molecules that mediate inter-organismal interactions. Nevertheless, the application of metabolomics to investigate interactions between phytophagous insects and their microbial symbionts remains underutilized. Here, we investigated the metabolomes of the bacteriomes (organs bearing symbiotic bacteria) isolated from natural populations of five species of xylem-feeding insects. We identified three patterns. First, the metabolomes varied among the five species, likely influenced by insect phylogeny, food plant and taxonomic identity of the symbionts. Second, the ratio of glutamine: glutamate in the bacteriomes was 0.7-3.6 to 1, indicative of nitrogen-sufficient metabolism and raising the possibility that the insect sustains nitrogen-enriched status of the bacteriomes despite the nitrogen scarcity of the xylem diet. Finally, bacteriomes from insect species bearing genetically-similar symbionts displayed limited variation in their metabolomes, suggesting that the metabolic pattern of the bacteriome metabolic pools is correlated with the genetic repertoire of the symbionts. Altogether, these metabolomic patterns yield specific hypotheses of underlying processes that are testable by wider sampling of natural populations and experimental study.

A Cyclic Metabolic Network in Pseudomonas protegens Pf-5 Prioritizes the Entner-Doudoroff Pathway and Exhibits Substrate Hierarchy during Carbohydrate Co-Utilization.

The genetic characterization of Pseudomonas protegens Pf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here, we employed 13C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. In lieu of the direct phosphorylation of glucose by glucose kinase, glucose catabolism was characterized primarily by the oxidation of glucose to gluconate and 2-ketogluconate before the phosphorylation of these metabolites to feed the Entner-Doudoroff (ED) pathway. In the absence of phosphofructokinase activity, a cyclic flux from the ED pathway to the upper Embden-Meyerhof-Parnas (EMP) pathway was responsible for routing glucose-derived carbons to the non-oxidative pentose phosphate (PP) pathway. Consistent with the lack of annotated genes in P. protegens Pf-5 for the transport or initial catabolism of pentoses and galactose, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of these hexoses was evident, but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism to fructose-6-phosphate, metabolite labeling patterns revealed that mannose was assimilated into fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were also found to cycle backward through the upper EMP pathway toward the ED pathway. Therefore, the operational metabolic network for processing carbohydrates in P. protegens Pf-5 prioritizes flux through the ED pathway to channel carbons to EMP, PP, and downstream pathways.IMPORTANCE Species of the Pseudomonas genus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here, we investigated the metabolic network for carbohydrate metabolism in Pseudomonas protegens Pf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through a cyclic metabolic network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the annotation of a different pathway in the genome. Elucidation of the constitutive metabolic network in P. protegens is important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untapped Pseudomonas species.

Lead Solubility and Mineral Structures of Coprecipitated Lead/Calcium Oxalates.

Low-molecular-weight organic acids such as oxalate, which are ubiquitous in the environment, can control the solubility and bioavailability of toxic metals such as Pb in soils and water by influencing complexation and precipitation reactions. Here, we investigated Pb solubility in relation to Pb-oxalate precipitation at pH 5.0 in the absence and presence of calcium (Ca), a common cation in environmental matrices. At Pb mole fractions less than 0.10, sequestration of Pb into Ca oxalate to form a solid solution substantially lowered Pb solubility relative to that of pure Pb oxalate to an extent inversely proportional to the Pb mole fraction. Small Pb/Ca solid-solution distribution coefficients at these low mole ratios was largely attributed to the stronger complexation of Pb compared to Ca with oxalate to form soluble metal-oxalate complexes, which in turn limited Pb incorporation into the Ca-oxalate crystal lattice. Characterization of the Pb/Ca-oxalate coprecipitates by X-ray diffraction, optical microscopy, and Fourier transform infrared spectroscopy revealed that the whewellite (Ca-oxalate monohydrate) structure was destabilized by substitution of small amounts of Pb into the lattice, and thus, the formation of the Ca-oxalate dihydrate (weddellite) was favored over the monohydrate. At Pb mole fractions above 0.20, discrete crystallites of Pb oxalate were identified. These new findings imply that Pb/Ca-oxalate coprecipitates in the presence of Ca could reduce the solubility of Pb in Pb-contaminated acid soils.

Application of denitrifying bioreactors for the removal of atrazine in agricultural drainage water.

Atrazine and nitrate NO3-N are two agricultural pollutants that occur widely in surface and groundwater. One of the pathways by which these pollutants reach surface water is through subsurface drainage tile lines. Edge-of-field anaerobic denitrifying bioreactors apply organic substrates such as woodchips to stimulate the removal of NO3-N from the subsurface tile waters through denitrification. Here we investigated the co-removal of NO3-N and atrazine by these bioreactors. Laboratory experiments were conducted using 12-L woodchips-containing flow-through bioreactors, with and without the addition of biochar, to treat two concentrations of atrazine (20 and 50 µg L-1) and NO3-N (1.5 and 11.5 mg L-1), operated at four hydraulic retention time, HRT, (4 h, 8 h, 24 h, 72 h). Additionally, we examined the effect of aerating the bioreactors on atrazine removal. Furthermore, we tested atrazine removal by a field woodchip denitrifying bioreactor. The removal of both NO3-N and atrazine increased with increasing HRT in the laboratory bioreactors. At 4 h, the woodchip bioreactors removed 65% of NO3-N and 25% of atrazine but, at 72 h, the bioreactors eliminated all the NO3-N and 53% of atrazine. Biochar-amended bioreactors removed up to 90% of atrazine at 72-h retention time. We concluded that atrazine removal was primarily via adsorption because neither aeration nor NO3-N levels had an effect. At 4-h retention time, the field bioreactors achieved 2.5 times greater atrazine removal than the laboratory bioreactors. Our findings thus highlighted hydraulic retention time and biochar amendments as two important factors that may control the efficiency of atrazine removal by denitrifying bioreactors. In sum, laboratory and field data demonstrated that denitrifying bioreactors have the potential to decrease pesticide transport from agricultural lands to surface waters.

Profiling glucose-induced selective inhibition of disaccharide catabolism in Bacillus megaterium QM B1551 by stable isotope labelling.

We investigated the co-catabolism of carbohydrate mixtures in Bacillus megaterium QM B1551 using a 13C-assisted metabolomics profiling approach. Specifically, we monitored the ability of B. megaterium to achieve the simultaneous catabolism of glucose and a common disaccharide - cellobiose, maltose, or sucrose. Growth experiments indicated that each disaccharide alone can serve as a sole carbon source for B. megaterium, in accordance with the genetic analysis of this bacterium, which predicted diverse metabolic capabilities. However, following growth on 13C-labelled glucose and each unlabelled disaccharide, the labelling patterns of the intracellular metabolites in glycolysis and the pentose phosphate pathway revealed a hierarchy in disaccharide catabolism: (i) complete inhibition of cellobiose catabolism, (ii) minimal catabolism of maltose and (iii) unbiased catabolism of sucrose. The labelling of amino acids confirmed this selective assimilation of each substrate in biomass precursors. This study highlights the fact that B. megaterium exhibits a mixed-carbohydrate utilization that is different from that of B. subtilis, the most studied model Bacillus species.

Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study.

The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs.

Hierarchy in pentose sugar metabolism in Clostridium acetobutylicum.

Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that,as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.

Structural characterization of multiple pyoverdines secreted by two Pseudomonas strains using liquid chromatography-high resolution tandem mass spectrometry with varying dissociation energies.

High-affinity iron (Fe)-scavenging molecules, or siderophores, are secreted by microorganisms to acquire and compete for Fe. Pyoverdine (PVD), the primary siderophore produced by Pseudomonas, consists of a dihydroxyquinoline-type chromophore, a peptide chain of variable length and conformation, and a side chain composed of a dicarboxylic acid or its monoamide derivative. Elucidation of the PVD structures secreted by different Pseudomonas strains is an important step toward understanding their Fe-transport strategies. In this study, we characterized multiple PVDs secreted by Pseudomonas putida KT2440 and Pseudomonas fluorescens RA12 using ultra-high performance liquid chromatography coupled with high-resolution quadrupole-orbitrap tandem mass spectrometry. To avoid purification steps prior to characterizing the bacterial supernatants, PVD candidates were identified by extracting fragments of the dihydroxyquinoline component from the chromatographic peaks. Varying collisional dissociation energies were subsequently applied to achieve, with high mass accuracy, a broad coverage of fragments of the entire PVD. Our approach allowed us to discriminate between three different PVD structures in the secretion of each strain. The three PVDs of P. putida possess the same peptide chain of seven amino acids, Asp-Orn-OHAsp-Dab-Gly-Ser-cOHOrn, with a cyclicized portion present in two of the PVDs. For P. fluorescens, two of the PVDs had the same peptide chain of 13 amino acids, Ala-Lys-Gly-Gly-Ala-OHAsp-Gly-Ser-Ala-Ala-Ala-Ala-cOHOrn, whereas a third PVD had a Ser substituting for the first Ala. The side chain of the PVDs was either succinic acid or succinamide. The present approach can be employed for simultaneous structural characterization of several peptidic siderophores and related molecules in bacterial secretions. Graphical abstract Characterizing mutiple pyoverdine (PVD) structures in bacterial secretions without prepurification step.

Unraveling iron oxides as abiotic catalysts of organic phosphorus recycling in soil and sediment matrices.

In biogeochemical phosphorus cycling, iron oxide minerals are acknowledged as strong adsorbents of inorganic and organic phosphorus. Dephosphorylation of organic phosphorus is attributed only to biological processes, but iron oxides could also catalyze this reaction. Evidence of this abiotic catalysis has relied on monitoring products in solution, thereby ignoring iron oxides as both catalysts and adsorbents. Here we apply high-resolution mass spectrometry and X-ray absorption spectroscopy to characterize dissolved and particulate phosphorus species, respectively. In soil and sediment samples reacted with ribonucleotides, we uncover the abiotic production of particulate inorganic phosphate associated specifically with iron oxides. Reactions of various organic phosphorus compounds with the different minerals identified in the environmental samples reveal up to ten-fold greater catalytic reactivities with iron oxides than with silicate and aluminosilicate minerals. Importantly, accounting for inorganic phosphate both in solution and mineral-bound, the dephosphorylarion rates of iron oxides were within reported enzymatic rates in soils. Our findings thus imply a missing abiotic axiom for organic phosphorus mineralization in phosphorus cycling.