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DOE OSTI2022

ROI-Finder : machine learning to guide region-of-interest scanning for X-ray fluorescence microscopy

The microscopy research at the Bionanoprobe (currently at beamline 9-ID and later 2-ID after APS-U) of Argonne National Laboratory focuses on applying synchrotron X-ray fluorescence (XRF) techniques to obtain trace elemental mappings of cryogenic biological samples to gain insights about their role in critical biological activities. The elemental mappings and the morphological aspects of the biological samples, in this instance, the bacterium Escherichia coli ( E. Coli ), also serve as label-free biological fingerprints to identify E. coli cells that have been treated differently. The key limitations of achieving good identification performance are the extraction of cells from raw XRF measurements via binary conversion, definition of features, noise floor and proportion of cells treated differently in the measurement. Automating cell extraction from raw XRF measurements across different types of chemical treatment and the implementation of machine-learning models to distinguish cells from the background and their differing treatments are described. Principal components are calculated from domain knowledge specific features and clustered to distinguish healthy and poisoned cells from the background without manual annotation. The cells are ranked via fuzzy clustering to recommend regions of interest for automated experimentation. The effects of dwell time and the amount of data required on the usability of the software are also discussed.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2023

Monomeric crystal structure of the vaccine carrier protein CRM 197 and implications for vaccine development

CRM 197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM 197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM 197 , where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM 197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM 197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Å resolution with the domain-swapping hinge loop (residues 379–387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer–dimer interconversion and for the optimization of conjugation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2

Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X -Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Crystal structure of Methanococcus jannaschii dihydroorotase with substrate bound

Here, we report the X-ray structural analysis of dihydroorotase from Methanococcus jannaschii co-crystallized with dihydroorotate at pH 6.5. The crystals are isomorphous with the crystals of the apoenzyme, with space group P3 2 21 and unit-cell dimensions a = b = 111.4, c = 101.2 Å. The structure was refined to R = 0.169 and R free = 0.186 at a resolution of 1.87 Å at room temperature. During crystallization the degradative reaction took place, and the electron density in the active site corresponds to the substrate carbamoyl aspartate. Carbamoyl aspartate interacts with the protein in the active site in a manner similar to that observed for Escherichia coli and human dihydroorotases. However, the flexible loop (residues 140–151) adopts both conformations in the crystal, loop-out and loop-in, with the loop-out conformation having higher occupancy. This contrasts with our expectations for the flexible loop to be exclusively in the loop-in conformation, which is the conformation that it adopts to stabilize the binding of the substrate in the known systems. Additional studies with substrate analogs that resemble carbamoyl aspartate and different crystallization conditions would provide further insight into the conformation of the flexible loop in this system.

Methanococcus jannaschii↗
DOE OSTI2023

Desiccation induces varied responses within a soil bacterial genus

Abstract Desiccation impacts a suite of physiological processes in microbes by elevating levels of damaging reactive oxygen species and inducing DNA strand breaks. In response to desiccation‐induced stress, microbes have evolved specialized mechanisms to help them survive. Here, we performed a 128‐day lab desiccation experiment on nine strains from three clades of an abundant soil bacterium, Curtobacterium . We sequenced RNA from each strain at three time points to investigate their response. Curtobacterium was highly resistant to desiccation, outlasting both Escherichia coli and a famously DNA damage‐resistant bacterium, Deinococcus radiodurans . However, within the genus, there were also 10‐fold differences in survival rates among strains. Transcriptomic profiling revealed responses shared within the genus including up‐regulation of genes involved in DNA damage repair, osmolyte production, and efflux pumps, but also up‐regulation of pathways and genes unique to the three clades. For example, trehalose synthesis gene otsB , the chaperone groEL , and the oxygen scavenger katA were all found in either one or two clades but not the third. Here, we provide evidence of considerable variation in closely related strains, and further elucidation of the phylogenetic conservation of desiccation tolerance remains an important goal for microbial ecologists.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genome reduction improves octanoic acid production in scale down bioreactors

Microorganisms in large-scale bioreactors are exposed to heterogeneous environmental conditions due to physical mixing constraints. Nutritional gradients can lead to transient expression of energetically wasteful stress responses and as a result, can reduce the titres, rates and yields of a bioprocess at larger scales. To what extent these process parameters are impacted is often unknown and therefore bioprocess scale-up comes with major risk. Designing platform strains to account for these intermittent stresses before introducing synthesis pathways is one strategy for de-risking bioprocess development. For example, Escherichia coli strain RM214 is a derivative of wild-type MG1655 that has had several genes and whole operons removed from its genome based on their metabolic cost. In this study, we engineered E. coli strain RM214 (referred to as WG02) to produce octanoic acid from glycerol in batch-flask and fed-batch bioreactor cultivations and compared it to an octanoic acid-producing E. coli MG1655 (WG01). In batch flask cultivations, the two strains performed similarly. However, in carbon limited fed-batch bioreactor cultivations, WG02 provided a greater than 22% boost to biomass compared to WG01 while maintaining similar titres of octanoic acid. Reducing the biomass accumulation of WG02 with nitrogen limited fed-batch cultivation resulted in a 16% improvement in octanoic acid titre over WG01. Finally, in a scale-down system consisting of a stirred tank reactor (representing a well-mixed zone) and plug flow reactor (representing an intermittent carbon starvation zone), WG02 again improved octanoic acid titre by almost 18% while maintaining similar biomass concentrations as WG01.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis

Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75 degrees C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.

09 BIOMASS FUELS↗
DOE OSTI2022

The dimeric form of bacterial l ‐asparaginase YpAI is fully active

l ‐asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity. Using multiple biophysical techniques that investigate enzymatic properties and quaternary structure at either high or low protein concentration, we found that dimeric YpAI is fully active, suggesting that the tetrameric form of this subfamily of enzymes does not bear significant enzymatic relevance. In this process, we extensively characterized YpAI, showing that it is a cooperative enzyme, although the mechanism of allostery is still not definitely established. We showed that, like most type I ASNases, the substrate affinity of YpAI is low and this enzyme is very similar in terms of both the structure and enzymatic properties to homologous type I ASNase from Escherichia coli (EcAI). We extended these studies to more medically relevant type II ASNases, used as anti‐leukaemia drugs. We confirmed that type II ASNases are not allosteric, and that they might also be functional in a dimeric form. However, the determination of the accurate tetramer⇆dimer dissociation constants of these enzymes that most likely lie in the picomolar range is not possible with currently available biophysical techniques.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus , Pseudomonas aeruginosa , and Francisella tularensis

Abstract Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli , finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate‐dependent resistance or susceptibility . Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa ; FTsmr from Francisella tularensis ; and SAsmr from Staphylococcus aureus . This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate‐specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate‐gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

Biochemistry & Molecular Biology↗
DOE OSTI2026

Elongation Factor Tu Acts as a Chaperone to Activate an Antibacterial RNase Toxin

Many Gram-negative bacterial species use contact-dependent growth inhibition (CDI) systems to deliver toxic proteins into neighboring competitors. CDI + strains deploy CdiA effector proteins, which translocate their C-terminal toxin (CT) domains into target bacteria through a receptor-mediated delivery pathway. To protect against auto-intoxication, CDI + bacteria also produce CdiI immunity proteins that neutralize CT toxin activity. Here, we present the crystal structure of the CT·CdiI O32:H37 complex from Escherichia coli O32:H37. CT O32:H37 adopts the same fold as the tRNase domain of colicin D, and the nucleases share similar catalytic centers. However, unlike colicin D, which cleaves the anticodon loops of tRNA Arg isoacceptors, CT O32:H37 exhibits nonspecific RNase activity. Notably, we find that endogenous elongation factor Tu (EF-Tu) co-purifies with the over-produced CT·CdiI O32:H37 complex. Although EF-Tu does not bind stably to CT O32:H37 in the absence of CdiI O32:H37 , the translation factor is required for toxic RNase activity in vitro. AlphaFold 3 modeling and site-directed mutagenesis indicate that CT O32:H37 interacts with the N-terminal GTPase domain of EF-Tu. EF-Tu appears to stabilize residue Trp52 within the hydrophobic core of the toxin, which in turn supports the RNase active site through an unusual hydrogen-bonding interaction with the catalytic His67 residue. Furthermore, EF-Tu is hijacked as an essential co-factor to organize the toxin's catalytic center.

Nhan, Dinh Quan [University of California, Santa B↗
DOE OSTI2024

Genome‐wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid

SUMMARY Genome‐wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD‐family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl‐CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP‐sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate‐starvation stress. GGES is a member of a well‐supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant‐specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

09 BIOMASS FUELS↗
DOE OSTI2026

Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing

CRISPR-associated transposases (CASTs) hold tremendous potential for microbial genome editing because of their ability to integrate large DNA cargos in a programmable, site-specific manner. However, their widespread application has been hindered by poorly understood host factor requirements for transposition. To address this gap, we conducted the first genome-wide screen for host factors affecting Vibrio cholerae CAST (VchCAST) activity using an Escherichia coli RB-TnSeq library and identified 15 genes affecting VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity, and two were inhibitory. Guided by the identification of homologous recombination effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors and increased editing efficiency by 55.2-fold in E. coli, 5.6-fold in Pseudomonas putida, and 10.8-fold in Klebsiella michiganensis while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting VchCAST activity and enhances its efficiency as a bacterial genome editor.

Song, Leo C T↗
DOE OSTI2020

Simultaneous cross-evaluation of heterogeneous E. coli datasets via mechanistic simulation

The extensive heterogeneity of biological data poses challenges to analysis and interpretation. Construction of a large-scale mechanistic model of Escherichia coli enabled us to integrate and cross-evaluate a massive, heterogeneous dataset based on measurements reported by various groups over decades. We identified inconsistencies with functional consequences across the data, including that the total output of the ribosomes and RNA polymerases described by data are not sufficient for a cell to reproduce measured doubling times, that measured metabolic parameters are neither fully compatible with each other nor with overall growth, and that essential proteins are absent during the cell cycle—and the cell is robust to this absence. Finally, considering these data as a whole leads to successful predictions of new experimental outcomes, in this case protein half-lives.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

An archaeal genetic code with all TAG codons as pyrrolysine

Multiple genetic codes developed during the evolution of eukaryotes and bacteria, yet no alternative genetic code is known for archaea. We used proteomics to confirm our prediction that certain archaea consistently incorporate pyrrolysine (Pyl) at TAG codons, supporting an alternative archaeal genetic code that we designate the Pyl code. This genetic code has 62 sense codons encoding 21 amino acids. In contrast to monophyletic genetic code distributions in bacteria, the archaeal Pyl code occurs sporadically, indicating that it arose independently in multiple lineages. We discovered that more than 1800 archaeal proteins contain Pyl, increasing the number of such proteins by two orders of magnitude. Additionally, five Pyl transfer RNA (tRNA) pyrrolysyl–tRNA synthetase pairs from Pyl-code archaea were used to introduce Pyl analogs into proteins in Escherichia coli.

Kivenson, Veronika [University of California, Berk↗
DOE OSTI2023

Characterization and Amelioration of Filtration Difficulties Encountered in Metabolomic Studies of Clostridium thermocellum at Elevated Sugar Concentrations

Clostridium thermocellum , a promising candidate for consolidated bioprocessing, has been subjected to numerous engineering strategies for enhanced bioethanol production. Measurements of intracellular metabolites at substrate concentrations high enough (>50 g/L) to allow the production of industrially relevant titers of ethanol would inform efforts toward this end but have been difficult due to the production of a viscous substance that interferes with the filtration and quenching steps during metabolite extraction. To determine whether this problem is unique to C. thermocellum , we performed filtration experiments with other organisms that have been engineered for high-titer ethanol production, including Escherichia coli and Thermoanaerobacterium saccharolyticum . Here, we addressed the problem through a series of improvements, including active pH control (to reduce problems with viscosity), investigation of different filter materials and pore sizes (to increase the filtration capacity), and correction for extracellular metabolite concentrations, and we developed a technique for more accurate intracellular metabolite measurements at elevated substrate concentrations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Regulation of L - and D -Aspartate Transport and Metabolism in Acinetobacter baylyi ADP1

Here, the regulated uptake and consumption of d-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as Escherichia coli, which utilizes only l-Asp, Acinetobacter baylyi ADP1 can consume both d-Asp and l-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control d- and l-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use d-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of aspT, aspY, racD, and aspA (which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N7-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed l- and d-Asn and l-Glu, but not d-Glu, as the sole carbon or nitrogen source using interrelated pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Trehalose Degradation by Cellvibrio japonicus Exhibits No Functional Redundancy and Is Solely Dependent on the Tre37A Enzyme

The α-diglucoside trehalose has historically been known as a component of the bacterial stress response, though it more recently has been studied for its relevance in human gut health and biotechnology development. The utilization of trehalose as a nutrient source by bacteria relies on carbohydrate-active enzymes, specifically those of the glycoside hydrolase family 37 (GH37), to degrade the disaccharide into substituent glucose moieties for entry into metabolism. Environmental bacteria using oligosaccharides for nutrients often possess multiple carbohydrate-active enzymes predicted to have the same biochemical activity and therefore are thought to be functionally redundant. In this study, we characterized trehalose degradation by the biotechnologically important saprophytic bacterium Cellvibrio japonicus This bacterium possesses two predicted α-α-trehalase genes, tre37A and tre37B, and our investigation using mutational analysis found that only the former is essential for trehalose utilization by C. japonicus Heterologous expression experiments found that only the expression of the C. japonicus tre37A gene in an Escherichia coli treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus GH37 activity determined that the tre37A gene product is solely responsible for cleaving trehalose and is an acidic α-α-trehalase. Bioinformatic and mutational analyses indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as C. japonicus does not possess a phosphotransferase system. This study facilitates the development of a comprehensive metabolic model for α-linked disaccharides in C. japonicus and more broadly expands our understanding of the strategies that saprophytic bacteria employ to capture diverse carbohydrates from the environment. The metabolism of trehalose is becoming increasingly important due to the inclusion of this α-diglucoside in a number of foods and its prevalence in the environment. Bacteria able to utilize trehalose in the human gut possess a competitive advantage, as do saprophytic microbes in terrestrial environments. While the biochemical mechanism of trehalose degradation is well understood, what is less clear is how bacteria acquire this metabolite from the environment. The significance of this report is that by using the model saprophyte Cellvibrio japonicus, we were able to functionally characterize the two predicted trehalase enzymes that the bacterium possesses and determined that the two enzymes are not equivalent and are not functionally redundant. Here, the results and approaches used to understand the complex physiology of α-diglucoside metabolism from this study can be applied broadly to other polysaccharide-degrading bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum . Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum , could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + P i → ADP + PP i .

59 BASIC BIOLOGICAL SCIENCES↗