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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

1,2-propanediol utilization MCP↗
DOE OSTI2024

AlphaFold -assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

molecular replacement↗
DOE OSTI2021

Resistance, Resilience, and Recovery of Dryland Soil Bacterial Communities Across Multiple Disturbances

Dryland ecosystems are sensitive to perturbations and generally slow to recover post disturbance. The microorganisms residing in dryland soils are especially important as they contribute to soil structure and nutrient cycling. Disturbance can have particularly strong effects on dryland soil structure and function, yet the natural resistance and recovery of the microbial components of dryland soils has not been well documented. In this study, the recovery of surface soil bacterial communities from multiple physical and environmental disturbances is assessed. Samples were collected from three field sites in the vicinity of Moab, UT, United States, 6 to 7 years after physical and climate disturbance manipulations had been terminated, allowing for the assessment of community recovery. Additionally, samples were collected in a transect that included three habitat patches: the canopy zone soils under the dominant shrubs, the interspace soils that are colonized by biological soil crusts, and edge soils at the plot borders. Field site and habitat patch were significant factors structuring the bacterial communities, illustrating that sites and habitats harbored unique soil microbiomes. Across the different sites and disturbance treatments, there was evidence of significant bacterial community recovery, as bacterial biomass and diversity were not significantly different than control plots. There was, however, a small number of 16S rRNA gene amplicon sequence variants that distinguished particular treatments, suggesting that legacy effects of the disturbances still remained. Taken together, these data suggest that dryland bacterial communities may possess a previously unappreciated potential to recover within years of the original disturbance.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

The Combined Detection of Morphological and Molecular Biomarkers: Implications for Astrobiology

Experience gathered by previous researchers during their hunt for evidence of early Earth life has shown the complexity in interpreting observations of possible microfossils and to establish the evidence to be positive. Similarly, the stillsimmering controversy on the nature of the nano-structures in Martian meteorite ALH84001 described by McKay et al. (1996) emphasizes the difficulties of conclusively identifying those structures as (a) fossilized bacterial cells and (b) establish their indigeneity. A better understanding of biological signatures in rocks is needed in order to identify traces of microbial life, which include morphological, mineralogical and chemical traces. It is thus considered crucial to tackle the problems emerging in the search for evidence of early life on Earth and in exopaleontological research with a multidisciplinary approach. With this is mind we applied surface sensitive Time of Flight-Secondary Ion Mass Spectroscopy (ToF-SIMS) to a previously described 25 m.y. old fossil bacterial biofilm. This technique allows in situ analysis with high mass resolution as well as molecular imaging of micron sized structures. As no extraction or derivatisation of the sample is required for ToF-SIMS analysis, electron microscopical investigation of the same samples subsequent to analysis is possible, thus allowing the combination of molecular and morphological biomarkers. The analysed fossil bacterial biofilms were associated with macrofossils from volcanoclastic lacustrine sediments from the Upper Oligocene Enspel formation (Germany). Preliminary scanning electron microscopy (SEM) studies have shown that a fossil structure interpreted as a coprolite purely consisted of fossilized bacterial biofilm. For ToF-SIMS investigation small particles were taken from the fossil biofilm and mounted onto Au-coated In-foil and analysed in a Phi Evans T-2000 TRIFT system. The ToF-SIMS analysed samples were Au/Pd-sputter coated and imaged using a Philips XL40 Field Emission Gun SEM (FEG-SEM). ToF-SIMS analysis of the organic rich fossil biofilm (TOC 29%) in the 0-100 Dalton (Da) range showed significant amounts of inorganic species, confirming the results obtained previously by EDX analysis, clearly showing the bacterial fossils to be mineralised. ToF-SIMS furthermore revealed the presence of a variety of low- and high-mass organic molecules and fragments thereof. These include peaks indicative of alkenes and alkanes, aromatic organic species and the polycyclic aromatic hydrocarbon naphthalene. More tentatively, peaks indicative of alkyl pyrroles and pyridyl-CH2 were identified. Other peaks of interest include peaks indicative of C(n)H(2n)O2 and C(n)H(2n-2)O2, which according to their general formula would suggest the presence of both saturated and unsaturated fatty acids although further in situ derivatisation experiments and GC-MS (Gas Chromatography MS) need to be applied to verify this beyond doubt. Furthermore, peaks at m/z 370, 384, 398, 412, 426, 440, 454 and 468 were identified, which indicate the potential presence of bacterial hopanes, a class of biomarkers indicative of bacteria. The main diagnostic peak for this group of chemicals is the fragment at m/z 191.18. Our studies conducted on purified hopane standards have shown that in the high-mass resolution mode differentiation of this diagnostic hopane peak and polyethylene at m/z 191.05 is possible. However, the spectra discussed here were collected in the lower resolution mapping mode, therefore this differentiation was not possible. The centroids of the possible hopane peaks obtained on the fossil biofilms are well within the range associated with bacterial hopanes. There is a strong possibility therefore that hopanoids may be associated with the fossil bacterial cells. Due to the non-destructive nature of ToF-SIMS, analysed samples can be studied using SEM, thus allowing the combination of morphological and molecular biomarkers. Subsequent SEM analysis of the ToF-SIMS analysed samples confirmed that the analysed material purely consists of fossil bacterial cells. This is thus the first successful effort to demonstrate the combination of spectral and morphological biomarkers. The advantages of highly sensitive non-destructive in situ analysis techniques for biomarker detection are invaluable, particularly with respect to envisaged Mars sample return missions, as it may allow us to identify remains and traces of former microbial life in both ancient terrestrial and extraterrestrial materials. This technique may prove particularly useful in the quest for extraterrestrial life with respect to precious extraterrestrial materials, as minute quantities are sufficient to conduct analysis.

Toporski, J.↗
DOE OSTI2020

The effects of warming and soil chemistry on bacterial community structure in Arctic tundra soils

Soil microbial communities in the Arctic play a critical role in regulating the global carbon (C) cycle. Vast amounts of C are stored in northern latitude soils, and rising temperatures in the Arctic threaten to thaw permafrost, making relatively inaccessible C sources more available for mineralization by soil microbes. Few studies have characterized how microbial community structure responds to thawing permafrost in the context of varying soil chemistries associated with contrasting tundra landscapes. Here, we subjected active layer and permafrost soils from upland and lowland tundra sites on the North Slope of Alaska to a soil-warming incubation experiment and compared soil bacterial community profiles (obtained by 16S rRNA amplicon sequencing) before and after incubation. The influence of soil composition (characterized by mid-infrared [MIR] spectroscopy) on bacterial community structure and class abundance was analyzed using redundancy and correlation analyses. We found increased abundances of Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes [Sphingobacteriia] post incubation, particularly in permafrost soils. The categorical descriptors site and soil layer had the most explanatory power in our predictive models of bacterial community structure, highlighting the close relationship between soil bacteria and the soil environment. Specific soil chemical attributes characterizing the soil environments that were found to be the best predictors include MIR spectral bands associated with inorganic C, silicates, amide II (C=N stretch), and carboxylics (C-O stretch), and MIR peak ratios representing C substrate quality. Overall, these results further characterize soil bacterial community shifts that may occur as a frozen environment with limited access to C sources, such as is found in undisturbed permafrost, transitions to warmer and more C-available environments, such as is predicted in thawing permafrost due to climate change.

16S rRNA sequencing↗
DOE OSTI2022

The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease SgrAI

Filament formation by metabolic, biosynthetic, and other enzymes has recently come into focus as a mechanism to fine-tune enzyme activity in the cell. Filamentation is key to the function of SgrAI, a sequence-specific DNA endonuclease that has served as a model system to provide some of the deepest insights into the biophysical characteristics of filamentation and its functional consequences. Structure-function analyses reveal that, in the filamentous state, SgrAI stabilizes an activated enzyme conformation that leads to accelerated DNA cleavage activity and expanded DNA sequence specificity. The latter is thought to be mediated by sequence-specific DNA structure, protein–DNA interactions, and a disorder-to-order transition in the protein, which collectively affect the relative stabilities of the inactive, non-filamentous conformation and the active, filamentous conformation of SgrAI bound to DNA. Full global kinetic modeling of the DNA cleavage pathway reveals a slow, rate-limiting, second-order association rate constant for filament assembly, and simulations of in vivo activity predict that filamentation is superior to non-filamenting mechanisms in ensuring rapid activation and sequestration of SgrAI's DNA cleavage activity on phage DNA and away from the host chromosome. In vivo studies demonstrate the critical requirement for accelerated DNA cleavage by SgrAI in its biological role to safeguard the bacterial host. Collectively, these data have advanced our understanding of how filamentation can regulate enzyme structure and function, while the experimental strategies used for SgrAI can be applied to other enzymatic systems to identify novel functional roles for filamentation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction

Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli , Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E . coli and B . subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E . coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 μg/ml of protein on day 0 to 464 μg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E . coli transformants expressing mCherry yielded 2.7-fold increase (93 μg/mL to 254 μg/mL) after 10 mins, while 4-fold increase (380 μg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in -80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Impact of Stand Age and Fertilization on the Soil Microbiome of Miscanthus × giganteus

Yield of the perennial grass Miscanthus × giganteus has shown an inconsistent and unpredictable response to nitrogen (N) fertilizer, yet fertilization underpins the crop’s environmental and economic sustainability. The interactions among soil microbial communities, N availability, and Miscanthus × giganteus and management may explain changes in plant productivity. In this study, soil samples from different stand ages of Miscanthus × giganteus in a replicated chronosequence field trial were used to investigate the effects of stand age and N fertilizer rates on microbial community structure. We hypothesized that there is a definable Miscanthus × giganteus soil microbiome and that this community varies significantly with stand age and fertilization. Our results showed that the main phyla in soil microbial communities, regardless of plant age, are similar but microbial community structures are significantly different. The variation in observed microbial communities generally decreases with older stand ages. The amount of N fertilizer applied also affected the microbial community structure associated with different aged Miscanthus × giganteus. Specifically, the relative abundance of Proteobacteria (Alphaproteobacteria and Gammaproteobacteria) and Acidobacteria (subgroup Gp1) increased shortly after fertilization and was more associated with younger Miscanthus × giganteus. Furthermore, our results show a significant relationship between bacterial α diversity and fertilization rates and that this response is also affected by stand age. Overall, our results emphasize linkages between microbial community structure, plant age, and fertilization in Miscanthus × giganteus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors

Plant cell surface receptors perceive carbohydrate signaling molecules and hereby establish communication with surrounding microbes. Genetic studies have identified two different classes of lysin motif receptor kinases as gatekeepers that together trigger the symbiotic pathway in plants; however, no structural or functional data of the perception mechanisms switching these receptors from resting state into activation is known. In this study, we use structural biology, biochemical, and genetic approaches to demonstrate how the NFP/NFR5 class of lipochitooligosaccharide (LCO) receptors discriminate bacterial symbionts based on a kinetic proofreading mechanism that controls receptor activation and signaling specificity. We show that the LCO binding site can be engineered to support symbiotic functions, which greatly advance future opportunities for receptor engineering in legumes and nonlegumes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Assembly and Analysis of Cell-Scale Membrane Envelopes

The march toward exascale computing will enable routine molecular simulation of larger and more complex systems, for example, simulation of entire viral particles, on the scale of approximately billions of atoms-a simulation size commensurate with a small bacterial cell. Anticipating the future hardware capabilities that will enable this type of research and paralleling advances in experimental structural biology, efforts are currently underway to develop software tools, procedures, and workflows for constructing cell-scale structures. Herein, we describe our efforts in developing and implementing an efficient and robust workflow for construction of cell-scale membrane envelopes and embedding membrane proteins into them. A new approach for construction of massive membrane structures that are stable during the simulations is built on implementing a subtractive assembly technique coupled with the development of a structure concatenation tool (fastmerge), which eliminates overlapping elements based on volumetric criteria rather than adding successive molecules to the simulation system. Using this approach, we have constructed two "protocells" consisting of MARTINI coarse-grained beads to represent cellular membranes, one the size of a cellular organelle and another the size of a small bacterial cell. The membrane envelopes constructed here remain whole during the molecular dynamics simulations performed and exhibit water flux only through specific proteins, demonstrating the success of our methodology in creating tight cell-like membrane compartments. Extended simulations of these cell-scale structures highlight the propensity for nonspecific interactions between adjacent membrane proteins leading to the formation of protein microclusters on the cell surface, an insight uniquely enabled by the scale of the simulations. We anticipate that the experiences and best practices presented here will form the basis for the next generation of cell-scale models, which will begin to address the addition of soluble proteins, nucleic acids, and small molecules essential to the function of a cell.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Assessing electrogenetic activation via a network model of biological signal propagation

Introduction: Molecular communication is the transfer of information encoded by molecular structure and activity. We examine molecular communication within bacterial consortia as cells with diverse biosynthetic capabilities can be assembled for enhanced function. Their coordination, both in terms of engineered genetic circuits within individual cells as well as their population-scale functions, is needed to ensure robust performance. We have suggested that “electrogenetics,” the use of electronics to activate specific genetic circuits, is a means by which electronic devices can mediate molecular communication, ultimately enabling programmable control. Methods: Here, we have developed a graphical network model for dynamically assessing electronic and molecular signal propagation schemes wherein nodes represent individual cells, and their edges represent communication channels by which signaling molecules are transferred. We utilize graph properties such as edge dynamics and graph topology to interrogate the signaling dynamics of specific engineered bacterial consortia. Results: We were able to recapitulate previous experimental systems with our model. In addition, we found that networks with more distinct subpopulations (high network modularity) propagated signals more slowly than randomized networks, while strategic arrangement of subpopulations with respect to the inducer source (an electrode) can increase signal output and outperform otherwise homogeneous networks. Discussion: We developed this model to better understand our previous experimental results, but also to enable future designs wherein subpopulation composition, genetic circuits, and spatial configurations can be varied to tune performance. We suggest that this work may provide insight into the signaling which occurs in synthetically assembled systems as well as native microbial communities.

Chun, Kayla↗
DOE OSTI2024

Potent Inhibition of E. coli DXP Synthase by a gem -Diaryl Bisubstrate Analog

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy- D -xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate ( D -GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)-based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure–activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Structural basis of cell-surface signaling by a conserved sigma regulator in Gram-negative bacteria

Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRSDate not supplied

Microgravity Effect on Bacterial Growth: Further Clarification of the Underlying Mechanism

Gravity interacts with other physical environmental factors to impact the formation of today’s Earth and contribute to biological variations between water and land species. With their simple structures and small sizes, microbes are expected to be less gravity-sensitive than larger species. However, microgravity can significantly impact the mass transfer and interface behavior in the extracellular environment, and various effects of spaceflight on bacterial growth have been observed. Our earlier literature review summarizes the systematic efforts to understand the spaceflight effect on microbial growth through the extracellular mass transfer mechanism. It provides an in-depth literature review to address some discrepancies observed in the literature. This paper analyzes the effects of microgravity on the extracellular environment and their potential impact on bacterial growth to further clarify the underlying mechanism of the microgravity effect on bacterial growth.

Wenyan Li↗
DOE OSTI2023

Changes in soil pore structure generated by the root systems of maize , sorghum and switchgrass affect in situ N 2 O emissions and bacterial denitrification

Due to the heterogeneous nature of soil pore structure, processes such as nitrification and denitrification can occur simultaneously at microscopic levels, making prediction of small-scale nitrous oxide (N 2 O) emissions in the field notoriously difficult. We assessed N 2 O+N 2 emissions from soils under maize (Zea mays L.), switchgrass (Panicum virgatum L.), and energy sorghum (Sorghum bicolor L.), three potential bioenergy crops in order to identify the importance of different N 2 O sources to microsite production, and relate N 2 O source differences to crop-associated differences in pore structure formation. The combination of isotopic surveys of N 2 O in the field during one growing season and X-ray computed tomography (CT) enabled us to link results from isotopic mappings to soil structural properties. Further, our methodology allowed us to evaluate the potential for in situ N 2 O suppression by biological nitrification inhibition (BNI) in energy sorghum. Our results demonstrated that the fraction of N 2 O originating from bacterial denitrification and reduction of N 2 O to N 2 is largely determined by the volume of particulate organic matter occluded within the soil matrix and the anaerobic soil volume. Bacterial denitrification was greater in switchgrass than in the annual crops, related to changes in pore structure caused by the coarse root system. This led to high N-loses through N 2 emissions in the switchgrass system throughout the season a novel finding given the lack of data in the literature for total denitrification. Isotopic mapping indicated no differences in N 2 O-fluxes or their source processes between maize and energy sorghum that could be associated with the release of BNI by the investigated sorghum variety. The results of this research show how differences in soil pore structures among cropping systems can determine both N 2 O production via denitrification and total denitrification N losses in situ.

59 BASIC BIOLOGICAL SCIENCES↗