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At least 1,027 records · Page 57
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

How Does Escherichia coli Allocate Proteome?

Microorganisms are shown to actively partition their intracellular resources, such as pro- teins, for growth optimization. Recent experiments have begun to reveal molecular com- ponents unpinning the partition; however, it remains unclear quantitatively how individual parts orchestrate to yield precise resource allocation that is both robust and dynamic. Here we developed a coarse-grained mathematical framework that centers on guanosine pentaphosphate (ppGpp)-mediated regulation, and used it to systematically uncover the design principles of proteome allocation in Escherichia coli. Our results showed that cellular ability of resource partition lies in an ultrasensitive, negative feedback control- ling topology with the ultrasensitivity arising from zero-order amino acid kinetics and the negative feedback from ppGpp-controlled ribosome synthesis. In addition, together with the time-scale separation between slow ribosome kinetics and fast turnovers of ppGpp and amino acids, the network topology confers the organism an optimization mechanism which mimics sliding mode control, a nonlinear optimization strategy that is widely used in man-made systems. We further showed that such a controlling mechanism is robust against parameter variations and molecular fluctuations, and is also efficient for biomass production over time. Furthermore, this work elucidates the fundamental controlling mechanism of E. coli proteome allocation, thereby providing insights into quantitative microbial physiology as well as the design of synthetic gene networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Tandem repeats in giant archaeal Borg elements undergo rapid evolution and create new intrinsically disordered regions in proteins

Borgs are huge, linear extrachromosomal elements associated with anaerobic methane-oxidizing archaea. Striking features of Borg genomes are pervasive tandem direct repeat (TR) regions. Here, we present six new Borg genomes and investigate the characteristics of TRs in all ten complete Borg genomes. We find that TR regions are rapidly evolving, recently formed, arise independently, and are virtually absent in host Methanoperedens genomes. Flanking partial repeats and A-enriched character constrain the TR formation mechanism. TRs can be in intergenic regions, where they might serve as regulatory RNAs, or in open reading frames (ORFs). TRs in ORFs are under very strong selective pressure, leading to perfect amino acid TRs (aaTRs) that are commonly intrinsically disordered regions. Proteins with aaTRs are often extracellular or membrane proteins, and functionally similar or homologous proteins often have aaTRs composed of the same amino acids. We propose that Borg aaTR-proteins functionally diversify Methanoperedens and all TRs are crucial for specific Borg–host associations and possibly cospeciation.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1996

Insect Development in Altered Gravitational Environment

When tobacco hornworm (Manduca sexta) larvae burrow underground (25-30 cm) to pupate, they reorient themselves to a relatively horizontal position indicating an ability to sense gravity. To evaluate their sensitivity to gravitational environment during metamorphosis, Manduca (pharate adults) were placed in a vertical (head-up) position. Distinct morphological changes, each one reflecting ensuing phases, were used to follow adult development. Five days after pupation, the vertical group showed accelerated (P less than 0.05) development and were nearly 4 phases ahead (P less than 0.0001) after 10 days. Differences in development in the vertical group were characterized further by increased (7-48%) hemolymph concentrations of 13 amino acids, but a decrease in cys and pro and no change in arg, his, met and val (trp, undetectable). Decreased (36%) turnover of injected H-3 - phenylalanine suggested slower utilization of amino acids contributed, at least partly, to the increased concentrations. Vertically-oriented Manduca also exhibited a greater (20 %, P less than 0.001) protein content in their flight muscles near the end of development. Analysis of hemolymph sugar levels showed a redistribution of sugars from the monosaccharide glucose to the disaccharide trehalose. Since injection of 20-hydroxyecdysone decreased (49%) turnover of H-3- phenylalanine in pharate adults and since ecdysteroids are known to increase flight muscle size and control adult development, these results are consistent with our measuring a greater (+80%, P less than 0.05) ecdysteroid titer in the vertically-oriented insects. These results suggest that gravity environment influences ecdysone output by the pharate adult. When we evaluated hemolymph flow in the head-up and control positions, we found that injected C-14-inulin was distributed somewhat more rapidly in the head-up group irrespective of the sight of injection (head or abdomen) likely because in the head-up position flow of the hemolymph is facilitated throughout the animal. Other experiments showed that an intact prothoracic gland is needed for the response. Hence vertical, head-up orientation affects release of ecdysone from the prothoracic gland.

Tischler, Marc E.↗
NASA NTRS1996

The Metabolic World: Sugars as an Energized Carbon Substrate for Prebiotic and Biotic Synthesis

To understand the origin of metabolism and biopolymer synthesis we investigated the energy sources that drive anabolic metabolism. We found that biosynthesis of amino acids and lipids from sugars is driven bz the free energy of redox disproportionation of carbon (see discussion or next page). The indispensable role of sugar disproportionation in the biosynthesis of amino acids and lipids suggests that the origin of life uses the same chemical engine, and was therefore based on nonenzymatic redox disproportionation reactions of sugars that occurred in the presence o ammonia and hydrogen sulfide. The chemistry of this 'metabolic' model of the origin of life is described.

Weber, Arthur L.↗
DOE OSTI2023

Direct observation of tRNA-chaperoned folding of a dynamic mRNA ensemble

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1972

Selective condensation of aminoacyl adenylates by nucleoproteinoid microparticles.

Microparticles composed of each of four enzymically synthesized homopolynucleotides and the same lysine-rich proteinoid have been found to influence the condensation of the AMP-anhydrides of each of four amino acids. The conditions of preparation of the particles and other variables of the experiments control the types of reaction. When a period set of conditions was identified empirically, the incorporation favored the amino acids whose present-day codons are related to the homopolynucleotide in the particle.

Nakashima, T.↗
NASA NTRS1979

Effects of gravity on contractile proteins

A method was established for the isolation and purification of nuclei in high yield from the microplasmodia of Physarum flavicomum. Purified nuclei were resistant to breakage by methods commonly employed for isolated plant and animal nuclei. Several methods for the extraction of nuclear protein were compared. Incubation of nuclear lysates with either 2 M NaCl, with or without 5 M urea, or 1 M CaCl2 resulted in the extraction of nuclear action together with histones. The histones were chemically fractionated into the 5 basic groups common to other eucaryotic tissue. Amino acid analyses of the total histone were also performed. Nuclear actin was found to have a molecular weight of 41,000 ? 4,000 daltons as determined by SDS polyacrylamide gel electrophoresis. The amino acid composition of the nuclear action was established.

Henney, H. R., Jr.↗
NASA NTRS1985

Rationalization of some genetic anticodonic assignments

The hydrophobicity of most amino acids correlates well with that of their anticodon nucleotides, with Trp, Tyr, Ile, and Ser being the exceptions to this rule. Using previous data on hydrophobicity and binding constants, and new data on rates of esterification of polyadenylic acid with several N-acetylaminoacyl imidazolides, several of the anticodon assignments are rationalized. Chemical reasons are shown supporting the idea of the inclusion of the Ile in the catalog of biological amino acids late in the evolution, through a mutation of the existing tRNA and its aminoacyl-tRNA-synthetase. It was found that an addition of hexane increases the incorporation of hydrophobic Ac-Phe into poly-A, in support of the Fox (1965) and Oparin (1965) emphasis on the biogenetic importance of phase-separated systems.

Lacey, J. C., Jr.↗
NASA NTRS2015

Magnetite Plaquettes Provide an Extraterrestrial Source of Asymmetric Components

Molecular selectivity is a crucial criterion for life. A possible abiotic mechanism that can produce chiral asymmetry in meteoritic amino acids is their formation with the presence of asymmetric catalysts. Magnetite (Fe3O4), a common mineral in some carbonaceous chondrites (CCs), has been shown to be an effective catalyst for the formation of amino acids that are commonly found in these meteorites. Magnetite sometimes takes the form of plaquettes that consist of barrel-shaped stacks of magnetite disks that resemble a spiral. However, a widely accepted description of the internal morphology of this particular magnetite form is still lacking, which is necessary in order to confirm or disprove the spiral configuration.

Chan, Q. H. S.↗
DOE OSTI2020

Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth

Cell growth and/or proliferation may require the reprogramming of metabolic pathways, whereby a switch from oxidative to glycolytic metabolism diverts glycolytic intermediates towards anabolic pathways. Herein, we identify a novel role for TRIM32 in the maintenance of glycolytic flux mediated by biochemical interactions with the glycolytic enzymes Aldolase and Phosphoglycerate mutase. Loss of Drosophila TRIM32, encoded by thin (tn), shows reduced levels of glycolytic intermediates and amino acids. This altered metabolic profile correlates with a reduction in the size of glycolytic larval muscle and brain tissue. Consistent with a role for metabolic intermediates in glycolysis-driven biomass production, dietary amino acid supplementation in tn mutants improves muscle mass. Remarkably, TRIM32 is also required for ectopic growth - loss of TRIM32 in a wing disc-associated tumor model reduces glycolytic metabolism and restricts growth. Overall, our results reveal a novel role for TRIM32 for controlling glycolysis in the context of both normal development and tumor growth.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1973

Prebiotic activation processes.

Questions regarding the combination of amino acids and ribonucleotides to polypeptides and polynucleotides are investigated. Each of the reactions considered occurs in the solid state in plausible prebiotic conditions. Together they provide the basis for a unified scheme of amino acid and nucleotide activation. Urea, imidazole and Mg(++) are essential catalytic components of the reaction mixtures. However, these compounds could probably be replaced by other organic molecules.

Lohrmann, R.↗
NASA NTRS1990

Could organic matter have been preserved on Mars for 3.5 billion years?

About 3.5 Gyr ago, when it is thought that Mars and earth had similar climates, biological evolution on earth had made considerable progress, such that life was abundant. It is therefore surmised that prior to this time period, the advent of chemical evolution and subsequent origin of life occurred on earth and may have occurred on Mars. Analysis for organic compounds in the soil buried beneath the Martian surface may yield useful information regarding the occurrence of chemical evolution and possibly biological evolution. Calculations based on the stability of amino acids lead to the conclusion that remnants of these compounds, if they existed on Mars 3.5 Gyr ago, might have been preserved buried beneath the surface oxidizing layer. For example, if phenylalanine, an amino acid of average stability, existed on Mars 3.5 Gyr ago, then 1.6 percent would remain buried today. Martian soil may exist from remnants of meteoritic and cometary bombardment, assuming that 1 percent of the organics survived impact.

Kanavarioti, Anastassia↗
DOE OSTI2025

Uncovering Sequence and Structural Characteristics of Fungal Expansin‐Related Proteins With Potential to Drive Substrate Targeting

Expansins loosen plant cell wall networks through disrupting non-covalent bonds between cellulose microfibrils and matrix polysaccharides. Whereas expansins were first discovered in plants, expansin-related proteins have since been identified in bacteria and fungi. The biological function of microbial expansins remains unclear; however, several studies have shown distinct binding preferences toward different structural polysaccharides. Earlier studies of bacterial expansin-related proteins uncovered sequence and structural features that correlate to substrate binding. Herein, 20 fungal expansin-related sequences were recombinantly produced in Komagataella phaffii, and the purified proteins were compared in terms of substrate binding to cellulosic and chitinous substrates. The impact of pH on the zeta potential of prioritized substrates was also measured, and Principal Component Analysis was performed to uncover correlations between protein characteristics (e.g., pI, hydrophobicity, surface charge distribution) and measured substrate binding preferences. Whereas acidic proteins with a predicted pI less than 5.0 preferentially bound to chitin, basic proteins with pI greater than 8.0 preferentially bound to xylan and xylan-containing fiber. Similar to many cellulases, binding to cellulose was correlated to relatively high aromatic amino acid content in the protein sequence and presence of a carbohydrate binding module (CBM), which in the case of expansins is a C-terminal CBM63. Whereas overall sequence characteristics could be correlated to substrate binding preference, the identity of amino acids occupying conserved positions that impact protein activity was better correlated with loosenin versus expansin classifications.

chitin↗
DOE OSTI2024

Bacterial vampirism mediated through taxis to serum

Bacteria of the family Enterobacteriaceae are associated with gastrointestinal (GI) bleeding and bacteremia and are a leading cause of death, from sepsis, for individuals with inflammatory bowel diseases. The bacterial behaviors and mechanisms underlying why these bacteria are prone to bloodstream entry remain poorly understood. Herein, we report that clinical isolates of non-typhoidal Salmonella enterica serovars, Escherichia coli , and Citrobacter koseri are rapidly attracted toward sources of human serum. To simulate GI bleeding, we utilized an injection-based microfluidics device and found that femtoliter volumes of human serum are sufficient to induce bacterial attraction to the serum source. This response is orchestrated through chemotaxis and the chemoattractant L-serine, an amino acid abundant in serum that is recognized through direct binding by the chemoreceptor Tsr. We report the first crystal structures of Salmonella Typhimurium Tsr in complex with L-serine and identify a conserved amino acid recognition motif for L-serine shared among Tsr orthologues. We find Tsr to be widely conserved among Enterobacteriaceae and numerous World Health Organization priority pathogens associated with bloodstream infections. Lastly, we find that Enterobacteriaceae use human serum as a source of nutrients for growth and that chemotaxis and the chemoreceptor Tsr provide a competitive advantage for migration into enterohemorrhagic lesions. We define this bacterial behavior of taxis toward serum, colonization of hemorrhagic lesions, and the consumption of serum nutrients as ‘bacterial vampirism’, which may relate to the proclivity of Enterobacteriaceae for bloodstream infections.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Odor exposure during imprinting periods increases odorant-specific sensitivity and receptor gene expression in coho salmon ( Oncorhynchus kisutch )

ABSTRACT Pacific salmon are well known for their homing migrations; juvenile salmon learn odors associated with their natal streams prior to seaward migration, and then use these retained odor memories to guide them back from oceanic feeding grounds to their river of origin to spawn several years later. This memory formation, termed olfactory imprinting, involves (at least in part) sensitization of the peripheral olfactory epithelium to specific odorants. We hypothesized that this change in peripheral sensitivity is due to exposure-dependent increases in the expression of odorant receptor (OR) proteins that are activated by specific odorants experienced during imprinting. To test this hypothesis, we exposed juvenile coho salmon, Oncorhynchus kisutch, to the basic amino acid odorant l-arginine during the parr–smolt transformation (PST), when imprinting occurs, and assessed sensitivity of the olfactory epithelium to this and other odorants. We then identified the coho salmon ortholog of a basic amino acid odorant receptor (BAAR) and determined the mRNA expression levels of this receptor and other transcripts representing different classes of OR families. Exposure to l-arginine during the PST resulted in increased sensitivity to that odorant and a specific increase in BAAR mRNA expression in the olfactory epithelium relative to other ORs. These results suggest that specific increases in ORs activated during imprinting may be an important component of home stream memory formation and this phenomenon may ultimately be useful as a marker of successful imprinting to assess management strategies and hatchery practices that may influence straying in salmon.

Dittman, Andrew H. (ORCID:000000016482359X)↗
NASA NTRS1992

Mixed anhydrides (phosphoric-carboxyl) are also formed in the esterification of 5'-amp with n-acetylaminoacyl imidazolides - Implications regarding the origin of protein synthesis

Procedure for the formation of aminoacyl esters of monoribonucleotides with aminoacyl imidazolides were first reported by Gottikh et al. (1970) and summarized in 1970. This reaction has been widely used by us and numbers of other workers as a convenient means of preparing aminoacyl esters of nucleotides. We have previously reported that, under conditions of excess imidazolide, large amounts of bis 2', 3' esters are formed in addition to the monoesters. However, to our knowledge, no one has reported that in addition to the esters, relatively large amounts of the mixed anhydride, with the amino acid carboxyl attached to the phosphate, are also formed at short reaction times. We report here on the relative amounts of anhydride and esters formed in this reaction of racemic mixtures of eleven N-acetyl amino acid imidazolides with 5'-AMP and discuss the relevance of the findings to the origin of protein synthesis.

Wickramasinghe, Nalinie S. M. D.↗
NASA NTRS2012

The Origins of Transmembrane Ion Channels

Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

Pohorille, Andrew↗
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↗