Biology subjects

Metcalf, W. W.

Publications and source records attributed to Metcalf, W. W..

8 recordsLinked to original sources

bioRxiv2025

IDBac: an open-access web platform and compendium for the identification of bacteria by MALDI-TOF mass spectrometry.

The identification and analysis of bacteria is central to the microbiological sciences. While gene sequencing methods have been the standard to achieve this, use of MALDI-TOF mass spectrometry (MS), particularly in clinical microbiology, provides high-throughput identification to the subspecies level. However, biotyping has yet to be adopted outside of clinical settings due to the lack of a centralized public database of MS protein signatures that would facilitate isolate identification via spectral comparison. Further, current platforms lack meaningful ways to compare multiple properties from large numbers of bacterial isolates. Herein we present the IDBac web platform, a crowd-sourced central knowledgebase of protein MS signatures of >1400 strains spanning 6 bacterial phyla. Accompanying the knowledgebase is analysis infrastructure to identify unknown isolates, probe relationships within culture collections using metadata integration, and visualize specialized metabolite differences within groups of closely related bacteria. To highlight this utility and encourage wide community contribution, examples of each are presented.

microbiology↗

bioRxiv2021

Investigation of gating in outer membrane porins provides new perspectives on antibiotic resistance mechanisms

Gram-negative bacteria pose a serious public health concern, primarily due to a higher frequency of antibiotic resistance conferred to them as a result of low permeability of their outer membrane (OM). Antibiotics capable of traversing the OM typically permeate through OM porins; thus, understanding the permeation properties of these porins is instrumental to the development of new antibiotics. A common macroscopic feature of many OM porins is their ability to transition between functionally distinct open and closed states that regulate transport properties and rate. To obtain a molecular basis for these processes, we performed tens of microseconds of molecular dynamics simulations of E. coli OM porin, OmpF. We observed that large-scale motion of the internal loop, L3, leads to widening and narrowing of the pore, suggesting its potential role in gating. Furthermore, Markov state analysis revealed multiple energetically stable conformations of L3 corresponding to open and closed states of the porin. Dynamics between these functional states occurs on the time scale of tens of microseconds and are mediated by the movement of highly conserved acidic residues of L3 to form H-bonds with opposing sides of the barrel wall of the pore. To validate our mechanism, we mutated key residues involved in the gating process that alter the H-bond pattern in the open/closed states and performed additional simulations. These mutations shifted the dynamic equilibrium of the pore towards open or closed states. Complementarily, the mutations favoring the open/closed states lead to increased/decreased accumulation of multiple antibiotics in our whole-cell accumulation assays. Notably, porins containing one of the mutations favoring the closed state has previously been found in antibiotic resistant bacterial strains. Overall, our 180 {micro}s of simulation data (wild type and mutants) with concerted experiments suggests that regulation of the dynamic equilibrium between open and closed states of OM porins could be a mechanism by which Gram-negative bacteria acquire antibiotic resistance.

biophysics↗

bioRxiv2021

Rationalizing generation of broad spectrum antibiotics with the addition of a primary amine

Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their outer membrane (OM). Recently, we disclosed the eNTRy rules, a key lesson of which is that the introduction of a primary amine enhances OM permeation in certain contexts. To understand the molecular basis for this finding, we perform an extensive set of molecular dynamics (MD) simulations and free energy calculations comparing the permeation of aminated and amine-free antibiotic derivatives through the most abundant OM porin of E. coli, OmpF. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo and graph theory based algorithm to probe more efficiently the rotational and translational degrees of freedom visited during the permeation of the antibiotic molecule through OmpF. The resulting pathways were then used for free-energy calculations, revealing a lower barrier against the permeation of the aminated compound, substantiating its greater OM permeability. Further analysis revealed that the amine facilitates permeation by enabling the antibiotic to align its dipole to the luminal electric field of the porin and while forming favorable electrostatic interactions with specific, highly-conserved charged residues. The importance of these interactions in permeation was further validated with experimental mutagenesis and whole cell accumulation assays. Overall, this study provides insights on the importance of the primary amine for antibiotic permeation into Gram-negative pathogens that could help the design of future antibiotics. We also offer a new computational approach for calculating free-energy of processes where relevant molecular conformations cannot be efficiently captured.

biophysics↗

bioRxiv2019

Functional interactions between post-translationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans

Methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six post-translationally modified amino acids near the active site. Recent studies have identified genes that install two of these modifications (thioglycine and 5-(S)-methylarginine), yet little is known about the installation and function of the remaining post-translationally modified residues. Here, we provide in vivo evidence that a dedicated SAM-dependent methyltransferase encoded by a gene we designated mcmA is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue plays an important role in adaptation to a mesophilic lifestyle. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. Surprisingly, high-resolution crystal structures of the various unmodified MCRs were indistinguishable from the fully modified enzyme, suggesting that interactions between the post-translationally modified residues do not exert a major influence on the physical structure of the enzyme, but rather serve to fine-tune the activity and efficiency of MCR.

microbiology↗

bioRxiv2019

Biochemical characterization of the methylmercaptopropionate:cob(I)alamin methyltransferase from Methanosarcina acetivorans

Methanogenesis from methylated substrates is initiated by substrate specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate(MMPA):Coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s-1 and apparent Km for MPA of 12 M. CoM did not serve as a methyl acceptor, thus a second, unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, could also be demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were co-expressed. Biochemical characterization of this complex was not feasible because the corrinoid cofactor of this complex was in the inactive Co(II) state and could not be reactivated by incubation with strong reductants. The MtsF protein, comprised of both corrinoid and methyltransferase domains, co-purifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism. IMPORTANCEMMPA is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate methyl-CoM. Therefore, additional enzymes required for methanogenic MMPA catabolism await discovery.

microbiology↗

bioRxiv2018

Energy conservation via hydrogen cycling in the methanogenic archaeon Methanosarcina barkeri

Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H2 cycling, based on genetic data that suggest H2 is a preferred, but non-essential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H2 cycling. M. barkeri produces H2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions H2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature.\n\nImportanceAdenosine triphosphate (ATP) is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.

microbiology↗

bioRxiv2018

Genetic, biochemical, and molecular characterization of Methanosarcina barkeri mutants lacking three distinct classes of hydrogenase

The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H2/CO2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to wild-type, produced methane and CO2 in the expected 3:1 ratio, but had a ca. 33% slower growth rate. Thus, hydrogenases play a significant, but non-essential, role during growth on this substrate. As previously observed, mutants lacking Ech fail to grow on methanol/H2 unless supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the {Delta}ech/{Delta}frh and {Delta}ech/{Delta}frh/{Delta}vht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of reducing equivalents needed for biosynthesis. Quantification of methane and CO2 produced from methanol by resting cell suspensions of various mutants supports this conclusion. Based on global transcriptional profiles, none of the hydrogenases are upregulated to compensate for loss of the others. However, transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F420. The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.

microbiology↗

bioRxiv2017

Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

The enzyme methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The subunit of this enzyme (McrA) contains several unusual post-translational modifications, including an exceptionally rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioamide-containing natural products, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA in a {Delta}ycaO-tfuA mutant of the methanogenic archaeon Methanosarcina acetivorans revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Physiological characterization of this mutant suggested a new role for the thioglycine modification in enhancing protein stability, as opposed to playing a direct catalytic role. The universal conservation of this modification suggests that MCR arose in a thermophilic ancestor.

biochemistry↗