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For E. coli, C. thermocellum, Z. mobilis: shotgun proteomics analyses of tryptic digests of cell lysates; direct infusion of cell lysates spiked with isotopically labelled peptides to perform absolute quantification of specific proteins of interest, primarily related to glycolysis
The E. coli AntiMicrobial Satellite(EcAMSat) mission will investigate space microgravity affects on the antibiotic resistance of E. coli, a bacterial pathogen responsible for urinary tract infection in humans and animals. EcAMSat is being developed through a partnership between NASA Ames Research Center and the Stanford University School of Medicine. Scientists believe that the results of this experiment could help design effective countermeasures to protect astronauts health during long duration human space missions.
The E. coli AntiMicrobial Satellite(EcAMSat) mission will investigate space microgravity affects on the antibiotic resistance of E. coli, a bacterial pathogen responsible for urinary tract infection in humans and animals. EcAMSat is being developed through a partnership between NASAs Ames Research Center and the Stanford University School of Medicine. Dr. A.C. Matin is the Stanford University Principal Investigator. EcAMSat will investigate spaceflight effects on bacterial antibiotic resistance and its genetic basis. Bacterial antibiotic resistance may pose a danger to astronauts in microgravity, where the immune response is weakened. Scientists believe that the results of this experiment could help design effective countermeasures to protect astronauts health during long duration human space missions.
In the wake of the COVID-19 pandemics, the demand for innovative and effective methods of bacterial inactivation has become a critical area of research, providing the impetus for this study. The purpose of this research is to analyze the AuNPs-mediated photothermal inactivation of E. coli. Gold nanoparticles irradiated by laser represent a promising technique for combating bacterial infection that combines high-tech and scientific progress. The intermediate aim of the work was to present the calibration of the model with respect to the gold nanorods experiment. The purpose of this work is to study the effect of initial concentration of E. coli bacteria, the design of the chamber and the laser power on heat transfer and inactivation of E. coli bacteria. Using the CFD simulation, the work combines three main concepts. 1. The conversion of laser light to heat has been described by a combination of three distinctive approximations: a- Discrete particle integration to take into account every nanoparticle within the system, b- Rayleigh-Drude approximation to determine the scattering and extinction coefficients and c- Lambert–Beer–Bourger law to describe the decrease in laser intensity across the AuNPs. 2. The contribution of the presence of E. coli bacteria to the thermal and fluid-dynamic fields in the microdevice was modeled by single-phase approach by determining the effective thermophysical properties of the water-bacteria mixture. 3. An approach based on a temperature threshold attained at which bacteria will be inactivated, has been used to predict bacterial response to temperature increases. The comparison of the thermal fields and temporal temperature changes obtained by the CFD simulation with those obtained experimentally confirms the accuracy of the light-heat conversion model derived from the aforementioned approximations. The results show a linear relationship between maximum temperature and variation in laser power over the range studied, which is in line with previous experimental results. It was also found that the temperature inside the microchamber can exceed 55 °C only when a laser power higher than 0.8 W is used, so bacterial inactivation begins. The experimental data allows to determinate the concentration of nanoparticles. This parameter is introduced into the mathematical model obtaining the same number of AuNPs. However, this assumption introduces a certain simplification, as in the mathematical model the distribution of nanoparticles is uniform. This work is directly connected to the use of gold nanoparticles for energy conversion, as well as the field of bacterial inactivation in microfluidic systems such as lab-on-a-chip. Presented mathematical and numerical models can be extended to the entire spectrum of wavelengths with particular use of white light in the inactivation of bacteria. This work represents a significant advancement in the field, as to the best of the authors’ knowledge, it is the first to employ a single-phase computational fluid dynamics (CFD) approach specifically combined with the thermal inactivation of bacteria. Moreover, this research pioneers the use of a numerical simulation to analyze the temperature threshold of photothermal inactivation of E. coli mediated by gold nanorods (AuNRs). The integration of these methodologies offers a new perspective on optimizing bacterial inactivation techniques, making this study a valuable contribution to both computational modeling and biomedical applications.
Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C–C bond formation. Here, in this study, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as expected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for reduced aromatic aldehyde reduction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supplemented to resting cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits reduced oxidation and reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions – the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard β-hydroxy-α-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.
Cultivation of crops in controlled environmental agricultural systems may limit microbial colonization and reduce diversity of the microbial communities. Practices like seed and growth medium sanitization may further impact microbial communities in the mature plant and the plant’s capacity to limit the growth of pathogens through competition. As humans expand their travels to space, understanding plant growth, health, and development in closed environments will be critical to the success of producing a safe, supplemental food source for astronauts. To determine the persistence of a potential human pathogen in plant growth and development, sanitized and unsanitized seeds from, mizuna (Brassica rapa var japonica) and red romaine lettuce (Lactuca sativa cultivar ‘Outredgeous’), were inoculated with Escherichia coli, ATCC 21445, germinated under simulated International Space Station (ISS) environmental conditions and harvested every 7 days until maturity. The persistence of E. coli in the rhizosphere was determined by plating on selective media, real time PCR (Polymerase Chain Reaction) and community sequencing of the rhizosphere communities. E. coli was detected in the crops’ roots and leaves for several weeks post germination. At day 28, plants from sanitized seeds had significantly higher counts of E. coli on the roots than those from unsanitized seeds. E. coli was also detected on a few uninoculated plants indicating airborne cross contamination among plants in the same growth chamber and suggesting an influence of the natural microbiome on human pathogen survival and persistence in leafy greens. Sequencing analysis revealed variations in composition and diversity between the communities. Understanding the microbial community of the rhizospheric microbiome is only the first step in determining the relationships between plants. Additional studies to include genotypic and phenotypic variations in the plants should be considered to determine if the natural microbes in the rhizosphere may contribute to the health and therefore, safety of the edible plants.
The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease with which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption persists within the E. coli community that inactive gRNAs are rare. To resolve these contradictions, we evaluated the activity of 463 000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional. These nonfunctional gRNAs exhibit strong spacer self-interaction, which can either be excluded using a simple design rule or “repaired” during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million empirically evaluated E. coli gRNAs as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. Lastly, we have produced a webapp to allow users to easily browse and extract gRNA sequences from the E. coli genome, which can be accessed at https://grna.ornl.gov.
It has been shown by Lacey et al. (1985) that, in general, the hydrophobicity ranking of an amino acid correlates with that of its anticodonic nucleotide, with tryptophan being one of the four amino acids for which this rule does not apply. It was proposed that this failure to correlate was due to the fact that the anticodon assignments for the four amino acids were made late, after the mutation of existing tRNAs. In this paper, the evolution of E. coli tRNA(Trp) is examined by comparing its homology with other E. coli tRNAs. The results demonstrate the presence of an evolutionary relationship between E. coli tRNA(Trp) and tRNA(Gly) or tRNA(Arg) molecules, and support the idea of the late assignment of anticodon to Trp.
Previous investigations have shown that liquid bacterial cultures grown in space flight were not killed as effectively by antibiotic treatments as were cultures grown on Earth. However, the cause for the decreased antibiotic effectiveness remains unknown. Possible explanations include modified cell proliferation and modified antibiotic transport in the culture medium. Escherichia coli cultures were grown in space flight (STS-69 and STS-73), with and without gentamicin, on a solid agar substrate thus eliminating fluid effects and reducing the unknowns associated with space-flight bacterial cultures in suspension. This research showed that E. coli cultures grown in flight on agar for 24 to 27 hours experienced a heightened growth compared to simultaneous controls. However, addition of gentamicin to the agar killed the bacteria such that both flight and ground control E. coli samples had similar final cell concentrations. Therefore, while the reported existence of a decrease in antibiotic effectiveness in liquid cultures remains unexplained, these data suggest that gentamicin in space flight was at least as effective as, if not more effective than, on Earth, when E. coli cells were grown on agar.
Gravity-induced ultrastructural alterations in e. coli b
The Arabidopsis thaliana glycosyl transferases SPINDLY (SPY) and SECRET AGENT (SEC) modify nuclear and cytosolic proteins with O-linked fucose or O-linked N-acetylglucosamine (O-GlcNAc), respectively. O-fucose and O-GlcNAc modifications can occur at the same sites. SPY interacts physically and genetically with GIGANTEA (GI), suggesting that it could be modified by both enzymes. Previously, we found that, when co-expressed in Escherichia coli, SEC modifies GI; however, the modification site was not determined. By analyzing the overlapping sub-fragments of GI, we identified a region that was modified by SEC in E. coli. Modification was undetectable when threonine 829 (T829) was mutated to alanine, while the T834A and T837A mutations reduced the modification, suggesting that T829 was the primary or the only modification site. Mapping using mass spectrometry detected only the modification of T829. Previous studies have shown that the positions modified by SEC in E. coli are modified in planta, suggesting that T829 is O-GlcNAc modified in planta.
Abstract The Escherichia coli genome‐scale metabolic model (GEM) is an exemplar systems biology model for the simulation of cellular metabolism. Experimental validation of model predictions is essential to pinpoint uncertainty and ensure continued development of accurate models. Here, we quantified the accuracy of four subsequent E. coli GEMs using published mutant fitness data across thousands of genes and 25 different carbon sources. This evaluation demonstrated the utility of the area under a precision–recall curve relative to alternative accuracy metrics. An analysis of errors in the latest (iML1515) model identified several vitamins/cofactors that are likely available to mutants despite being absent from the experimental growth medium and highlighted isoenzyme gene‐protein‐reaction mapping as a key source of inaccurate predictions. A machine learning approach further identified metabolic fluxes through hydrogen ion exchange and specific central metabolism branch points as important determinants of model accuracy. This work outlines improved practices for the assessment of GEM accuracy with high‐throughput mutant fitness data and highlights promising areas for future model refinement in E. coli and beyond.
E. coli ribosomes structure, molecular components, function and protein synthesis with artificial ribosome derivatives deficient in one component
We have successfully flown the EcAMSat (Escherichia coli Antimicrobial Satellite) free-flyer mission. This was a 6U small satellite that autonomously conducted an experiment in low Earth orbit to explore the impact of the space environment on antibiotic resistance in uropathogenic E. coli (UPEC) and the role a particular sigma factor plays in the response. After being held in stasis during transport to orbit, two strains†a wildtype UPEC and an isogenic mutant with a deleted gene that encodes a sigma factor†were grown to stationary phase in a fluidic card inside EcAMSat's payload, then incubated with three concentrations of the antibiotic gentamicin. The payload then administered alamarBlue®, a redox indicator, into all wells of the fluidic card. The cells were then incubated for 144 hours and metabolic activity was measured optically using the payloads' LED and detector system. Data were then telemetered to the ground and compared to a control experiment conducted in an identical satellite in a lab. The results of this experiment will help us better understand important therapeutic targets for treating bacterial infections on Earth and in space. Such targets are particularly relevant to deep-space and long-duration missions where crew may be more susceptible to infection and treatments for them may work differently.
In this report, we present structural studies on the large conductance mechanosensitive ion channel (MscL) from E. coli in detergent micelles and lipid vesicles. Both transmission Fourier transform infrared spectroscopy and circular dichroism (CD) spectra indicate that the protein is highly helical in detergents as well as liposomes. The secondary structure of the proteins was shown to be highly resistant towards denaturation (25-95 degrees C) based on an ellipticity thermal profile. Amide H+/D+ exchange was shown to be extensive (ca. 66%), implying that two thirds of the protein are water accessible. MscL, reconstituted in oriented lipid bilayers, was shown to possess a net bilayer orientation using dichroic ratios measured by attenuated total-reflection Fourier transform infrared spectroscopy. Here, we present and discuss this initial set of structural data on this new family of ion-channel proteins.
We have successfully flown the EcAMSat (Escherichia coli Antimicrobial Satellite) free-flyer mission. This was a 6U (six unit - CubeSat) small satellite that autonomously conducted an experiment in low Earth orbit to explore the impact of the space environment on antibiotic resistance in uropathogenic E. coli (UPEC) and the role a particular sigma factor plays in the response. After being held in stasis during transport to orbit, two strains - a wildtype UPEC and an isogenic mutant with a deleted gene that encodes a sigma factor - were grown to stationary phase in a fluidic card inside EcAMSat's payload, then incubated with three concentrations of the antibiotic gentamicin. The payload then administered alamarBlue (registered trademark), a redox indicator, into all wells of the fluidic card. The cells were then incubated for 144 hours and metabolic activity was measured optically using the payloads' LED (Light-Emitting Diode) and detector system. Data were then telemetered to the ground and compared to a control experiment conducted in an identical satellite in a lab. The results of this experiment will help us better understand important therapeutic targets for treating bacterial infections on Earth and in space. Such targets are particularly relevant to deep-space and long-duration missions where crew may be more susceptible to infection and treatments for them may work differently.
The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA–RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.
Lipopolysaccharide (LPS) is the unique feature that composes the outer leaflet of the Gram-negative bacterial cell envelope. Variations in LPS structures affect a number of physiological processes, including outer membrane permeability, antimicrobial resistance, recognition by the host immune system, biofilm formation, and interbacterial competition. Rapid characterization of LPS properties is crucial for studying the relationship between these LPS structural changes and bacterial physiology. However, current assessments of LPS structures require LPS extraction and purification followed by cumbersome proteomic analysis. This paper demonstrates one of the first high-throughput and non-invasive strategies to directly distinguish Escherichia coli with different LPS structures. Using a combination of three-dimensional insulator-based dielectrophoresis (3DiDEP) and cell tracking in a linear electrokinetics assay, we elucidate the effect of structural changes in E. coli LPS oligosaccharides on electrokinetic mobility and polarizability. We show that our platform is sufficiently sensitive to detect LPS structural variations at the molecular level. To correlate electrokinetic properties of LPS with the outer membrane permeability, we further examined effects of LPS structural variations on bacterial susceptibility to colistin, an antibiotic known to disrupt the outer membrane by targeting LPS. Our results suggest that microfluidic electrokinetic platforms employing 3DiDEP can be a useful tool for isolating and selecting bacteria based on their LPS glycoforms. Future iterations of these platforms could be leveraged for rapid profiling of pathogens based on their surface LPS structural identity.