Search

Understanding how the structure of molecules affects their permeability across cell membranes is crucial for many topics in biomedical research, including the development of drugs. In this work, we examine the transport rates of structurally similar triphenylmethane dyes, malachite green (MG) and brilliant green (BG), across the membranes of living Escherichia coli (E. coli) cells and biomimetic liposomes. Using the time-resolved second harmonic light scattering technique, we found that BG passively diffuses across the E. coli cytoplasmic membrane (CM) 3.8 times faster than MG. In addition, BG exhibits a diffusion rate 3.1 times higher than MG across the membranes of liposomes made from E. coli polar lipid extracts. Measurements on these two molecules, alongside previously studied crystal violet (CV), another triphenylmethane molecule, are compared against the set of propensity rules developed by Lipinski and co-workers for assessing the permeability of hydrophobic ion-like drug molecules through biomembranes. It indicates that BG's increased diffusion rate is due to its higher lipophilicity, with a distribution coefficient 25 times greater than MG. In contrast, CV, despite having similar lipophilicity to MG, shows negligible permeation through the E. coli CM on the observation scale, attributed to its more hydrogen bonding sites and larger polar surface area. Importantly, cell viability tests revealed that BG's antimicrobial efficacy is ∼2.4 times greater than that of MG, which aligns well with its enhanced diffusion into the E. coli cytosol. These findings offer valuable insights for drug design and development, especially for improving the permeability of poorly permeable drug molecules. [ABSTRACT FROM AUTHOR]

The diffusion of proteins is significantly affected by macromolecular crowding. Molecular simulations accounting for protein interactions at atomic resolution are useful for characterizing the diffusion patterns in crowded environments. We present a comprehensive analysis of protein diffusion under different crowding conditions based on our recent docking-based approach simulating an intracellular crowded environment by sampling the intermolecular energy landscape using the Markov Chain Monte Carlo protocol. The procedure was extensively benchmarked, and the results are in very good agreement with the available experimental and theoretical data. The translational and rotational diffusion rates were determined for different types of proteins under crowding conditions in a broad range of concentrations. A protein system representing most abundant protein types in the E. coli cytoplasm was simulated, as well as large systems of other proteins of varying sizes in heterogeneous and self-crowding solutions. Dynamics of individual proteins was analyzed as a function of concentration and different diffusion rates in homogeneous and heterogeneous crowding. Smaller proteins diffused faster in heterogeneous crowding of larger molecules, compared to their diffusion in the self-crowded solution. Larger proteins displayed the opposite behavior, diffusing faster in the self-crowded solution. The results show the predictive power of our structure-based simulation approach for long timescales of cell-size systems at atomic resolution. [ABSTRACT FROM AUTHOR]

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. RNA structure prediction is not yet possible due to a lack of high-quality reference data associated with organismal phenotypes that could inform RNA function. We present GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences to experimental and predicted optimal growth temperatures of GTDB reference organisms. Using GARNET, we develop sequence- and structure-aware RNA generative models, with overlapping triplet tokenization providing optimal encoding for a GPT-like model. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identify mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

Lineages of rod-shaped bacteria such as Escherichia coli exhibit a temporal decline in elongation rate in a manner comparable to cellular or biological aging. The effect results from the production of asymmetrical daughters, one with a lower elongation rate, by the division of a mother cell. The slower daughter compared to the faster daughter, denoted respectively as the old and new daughters, has more aggregates of damaged proteins and fewer expressed gene products. We have examined further the degree of asymmetry by measuring the density of ribosomes between old and new daughters and between their poles. We found that ribosomes were denser in the new daughter and also in the new pole of the daughters. These ribosome patterns match the ones we previously found for expressed gene products. This outcome suggests that the asymmetry is not likely to result from properties unique to the gene expressed in our previous study, but rather from a more fundamental upstream process affecting the distribution of ribosomal abundance. Because damage aggregates and ribosomes are both more abundant at the poles of E. coli cells, we suggest that competition for space between the two could explain the reduced ribosomal density in old daughters. Using published values for aggregate sizes and the relationship between ribosomal number and elongation rates, we show that the aggregate volumes could in principle displace quantitatively the amount of ribosomes needed to reduce the elongation rate of the old daughters.

Many prokaryotic and eukaryotic cells metabolize glucose to organism-specific by-products instead of fully oxidizing it to carbon dioxide and water-a phenomenon referred to as the Warburg Effect. The benefit to a cell is not fully understood, given that partial metabolism of glucose yields an order of magnitude less adenosine triphosphate (ATP) per molecule of glucose than complete oxidation. Here, we test a previously formulated hypothesis that the benefit of the Warburg Effect is to increase ATP production rate by switching from high-yielding respiration to faster glycolysis when excess glucose is available and respiration rate becomes limited by proteome occupancy. We show that glycolysis produces ATP faster per gram of pathway protein than respiration in Escherichia coli, Saccharomyces cerevisiae, and mammalian cells. We then develop a simple mathematical model of energy metabolism that uses five experimentally estimated parameters and show that this model can accurately predict absolute rates of glycolysis and respiration in all three organisms under diverse conditions, providing strong support for the validity of the ATP production rate maximization hypothesis. In addition, our measurements show that mammalian respiration produces ATP up to 10-fold slower than respiration in E. coli or S. cerevisiae, suggesting that the ATP production rate per gram of pathway protein is a highly evolvable trait that is heavily optimized in microbes. We also find that E. coli respiration is faster than fermentation, explaining the observation that E. coli, unlike S. cerevisiae or mammalian cells, never switch to pure fermentation in the presence of oxygen.

The accurate and effective determination of antimicrobial resistance is essential to limiting the spread of infectious diseases and ensuring human health. Herein, a simple, accurate, and high-throughput phage-based colorimetric sensing strategy was developed for antimicrobial susceptibility testing (AST). Taking advantage of the CRISPR/Cas9 system, the genome of the T4 phage was modularly engineered to carry lacZ-α (lacZa), a marker gene encoding the α-fragment of β-galactosidase (β-gal). T4lacZa phages were identified by blue-white selection and then used for a biosensing application. In this strategy, the bacterial solution is exposed to the T4lacZa phage, causing target bacteria to overexpress β-gal. Upon the addition of a colorimetric substrate, the β-gal initiates an enzymatic reaction, resulting in a solution color change from yellow to red. This sensing strategy offers a visual way to monitor bacterial growth in the presence of antibiotics, enabling the determination of bacterial antimicrobial susceptibility. As a proof of concept, our developed sensing strategy was successfully applied to identify 9 different multidrug-resistant Escherichia coli (E. coli) in urine samples with 100% specificity. Compared with conventional disk diffusion susceptibility tests, the engineered phage-based sensing strategy can shorten the detection time by at least half without losing detection sensitivity, providing an alternative high-throughput method for AST in clinical diagnosis.

The transcriptional regulatory network (TRN) in bacteria is thought to rapidly evolve in response to selection pressures, modulating transcription factor (TF) activities and interactions. In order to probe the limits and mechanisms surrounding the short-term adaptability of the TRN, we generated, evolved, and characterized knockout (KO) strains in Escherichia coli for 11 regulators selected based on measured growth impact on glucose minimal media. All but one knockout strain (Δlrp) were able to recover growth and did so requiring few convergent mutations. We found that the TF knockout adaptations could be divided into four categories: (i) Strains (ΔargR, ΔbasR, Δlon, ΔzntR, and Δzur) that recovered growth without any regulator-specific adaptations, likely due to minimal activity of the regulator on the growth condition, (ii) Strains (ΔcytR, ΔmlrA, and ΔybaO) that recovered growth without TF-specific mutations but with differential expression of regulators with overlapping regulons to the KOed TF, (iii) Strains (Δcrp and Δfur) that recovered growth using convergent mutations within their regulatory networks, including regulated promoters and connected regulators, and (iv) Strains (Δlrp) that were unable to fully recover growth, seemingly due to the broad connectivity of the TF within the TRN. Analyzing growth capabilities in evolved and unevolved strains indicated that growth adaptation can restore fitness to diverse substrates often despite a lack of TF-specific mutations. This work reveals the breadth of TRN adaptive mechanisms and suggests these mechanisms can be anticipated based on the network and functional context of the perturbed TFs.

Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we used complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq's utility is demonstrated, with future applications in ecology, health, and engineering.

UNLABELLED: Microorganisms with simplified genomes represent interesting cell chassis for systems and synthetic biology. However, genome reduction can lead to undesired traits, such as decreased growth rate and metabolic imbalances. To investigate the impact of genome reduction on Escherichia coli strain DGF-298, a strain in which ~ 36% of the genome has been removed, we reconstructed a strain-specific metabolic model (iAC1061), investigated the regulation of gene expression using iModulon-based transcriptome analysis, and performed adaptive laboratory evolution to let the strain correct potential imbalances that arose during its simplification. The model notably predicted that the removal of all three key pathways for glycolaldehyde disposal in this microorganism would lead to a metabolic bottleneck through folate starvation. Glycolaldehyde is also known to cause self-generation of reactive oxygen species, as evidenced by the increased expression of oxidative stress resistance genes in the SoxS iModulon. The reintroduction of the aldA gene, responsible for one native glycolaldehyde disposal route, alleviated the constitutive oxidative stress response. Our results suggest that systems-level approaches and adaptive laboratory evolution have additive benefits when trying to repair and optimize genome-engineered strains. IMPORTANCE: Genomic streamlining can be employed in model organisms to reduce complexity and enhance strain predictability. One of the most striking examples is the bacterial strain Escherichia coli DGF-298, notable for having over one-third of its genome deleted. However, such extensive genome modifications raise the question of how similar this simplified cell remains when compared with its parent, and what are the possible unintended consequences of this simplification. In this study, we used metabolic modeling along with iModulon-based transcriptomic analysis in different growth conditions to assess the impact of genome reduction on metabolism and gene regulation. We observed little impact of genomic reduction on the regulatory network of E. coli DGF-298 and identified a potential metabolic bottleneck leading to the constitutive activity of the SoxS iModulon. We then leveraged the models predictions to successfully restore SoxS activity to the basal level.

All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. Here, we quantify enzymes and metabolic intermediates of the Escherichia coli fatty acid and phospholipid synthesis pathways, to describe how this organism measures temperature and restores optimal membrane fluidity within a single generation after a temperature shock. A first element of this regulatory system is a temperature-sensitive metabolic valve that allocates flux between the saturated and unsaturated fatty acid synthesis pathways via the branchpoint enzymes FabI and FabB. A second element is a transcription-based negative feedback loop that counteracts the temperature-sensitive valve. The combination of these elements accelerates membrane adaptation by causing a transient overshoot in the synthesis of saturated or unsaturated fatty acids following temperature shocks. This strategy is comparable to increasing the temperature of a water bath by adding water that is excessively hot rather than adding water at the desired temperature. These properties are captured in a mathematical model, which we use to show how hard-wired parameters calibrate the system to generate membrane compositions that maintain constant fluidity across temperatures. We hypothesize that core features of the E. coli system will prove to be ubiquitous features of homeoviscous adaptation systems.

Salmonella translocate to the gut epithelium via microfold cells lining the follicle-associated epithelium (FAE). How Salmonella localize to the FAE is not well characterized. Here we use live imaging and competitive assays between wild-type and chemotaxis-deficient mutants to show that Salmonella enterica serotype Typhimurium (S. Typhimurium) localize to the FAE independently of chemotaxis in an ex vivo mouse caecum infection model. Electrical recordings revealed polarized FAE with sustained outward current and small transepithelial potential, while the surrounding villus is depolarized with inward current and large transepithelial potential. The distinct electrical potentials attracted S. Typhimurium to the FAE while Escherichia coli (E. coli) localized to the villi, through a process called galvanotaxis. Chloride flux involving the cystic fibrosis transmembrane conductance regulator (CFTR) generated the ionic currents around the FAE. Pharmacological inhibition of CFTR decreased S. Typhimurium FAE localization but increased E. coli recruitment. Altogether, our findings demonstrate that bioelectric cues contribute to S. Typhimurium targeting of specific gut epithelial locations, with potential implications for other enteric bacterial infections.

Hydrogen sulfide (H2S), mainly produced from L-cysteine (Cys), renders bacteria highly resistant to oxidative stress and potentially increases antimicrobial resistance (AMR). CyuR is a Cys-dependent transcription regulator, responsible for the activation of the cyuPA operon and generation of H2S. Despite its potential importance, its regulatory network remains poorly understood. In this study, we investigate the roles of the CyuR regulon in a Cys-dependent AMR mechanism in E. coli strains. We show: (1) Generation of H2S from Cys affects the sensitivities to growth inhibitors; (2) Cys supplementation decreases stress responses; (3) CyuR negatively controls the expression of mdlAB encoding a potential transporter for antibiotics; (4) CyuR binds to a DNA sequence motif GAAwAAATTGTxGxxATTTsyCC in the absence of Cys; and (5) CyuR may regulate 25 additional genes which were not reported previously. Collectively, our findings expand the understanding of the biological roles of CyuR relevant to antibiotic resistance associated with Cys.

In swine farming, animals develop diseases that require the use of antibiotics. In-feed antibiotics as growth promoters have been banned due to the increasing concern of antimicrobial resistance. Seaweeds offer bioactive molecules with antibacterial and antioxidant properties. The aim was to estimate the in vitro properties of seaweed extracts: Ascophyllum nodosum (AN), Palmaria palmata (PP), Ulva lactuca (UL), and 1:1 mixes (ANPP, ANUL, PPUL). Escherichia coli strains were used to test for growth inhibitory activity, and chemical-based assays were performed for antioxidant properties. The treatments were 2 (with/without Escherichia coli) × 2 (F4 + and F18 +) × 5 doses (0, 1.44, 2.87, 5.75, 11.50, and 23.0 mg/mL). Bacteria were supplemented with seaweed extracts, and growth was monitored. The antioxidant activity was assessed with 6 doses (0, 1, 50, 100, 200, 500, and 600 mg/mL) × 6 compounds using two chemical assays. Data were evaluated through SAS. The results showed that AN and UL significantly inhibited (p

Epistasis is caused by genetic interactions among mutations that affect fitness. To characterize properties and potential mechanisms of epistasis, we engineered eight double mutants that combined mutations from the rho and rpoB genes of Escherichia coli. The two genes encode essential functions for transcription, and the mutations in each gene were chosen because they were beneficial for adaptation to thermal stress (42.2 °C). The double mutants exhibited patterns of fitness epistasis that included diminishing returns epistasis at 42.2 °C, stronger diminishing returns between mutations with larger beneficial effects and both negative and positive (sign) epistasis across environments (20.0 °C and 37.0 °C). By assessing gene expression between single and double mutants, we detected hundreds of genes with gene expression epistasis. Previous work postulated that highly connected hub genes in coexpression networks have low epistasis, but we found the opposite: hub genes had high epistasis values in both coexpression and protein-protein interaction networks. We hypothesized that elevated epistasis in hub genes reflected that they were enriched for targets of Rho termination but that was not the case. Altogether, gene expression and coexpression analyses revealed that thermal adaptation occurred in modules, through modulation of ribonucleotide biosynthetic processes and ribosome assembly, the attenuation of expression in genes related to heat shock and stress responses, and with an overall trend toward restoring gene expression toward the unstressed state.

Indigo is widely used in textile industries for denim garments dyeing and is mainly produced by chemical synthesis which, however, raises environmental sustainability issues. Bio-indigo may be produced by fermentation of metabolically engineering bacteria, but current methods are economically incompetent due to low titer and the need for an inducer. To address these problems, we first characterized several synthetic promoters in E. coli and demonstrated the feasibility of inducer-free indigo production from tryptophan using the inducer-free promoter. We next coupled the tryptophan-to-indigo and glucose-to-tryptophan pathways to generate a de novo glucose-to-indigo pathway. By rational design and combinatorial screening, we identified the optimal promoter-gene combinations, which underscored the importance of promoter choice and expression levels of pathway genes. We thus created a new E. coli strain that exploited an indole pathway to enhance the indigo titer to 123 mg/L. We further assessed a panel of heterologous tryptophan synthase homologs and identified a plant indole lyase (TaIGL), which along with modified pathway design, improved the indigo titer to 235 mg/L while reducing the tryptophan byproduct accumulation. The optimal E. coli strain expressed 8 genes essential for rewiring carbon flux from glucose to indole and then to indigo: mFMO, ppsA, tktA, trpD, trpC, TaIGL and feedback-resistant aroG and trpE. Fed-batch fermentation in a 3-L bioreactor with glucose feeding further increased the indigo titer (≈965 mg/L) and total quantity (≈2183 mg) at 72 h. This new synthetic glucose-to-indigo pathway enables high-titer indigo production without the need of inducer and holds promise for bio-indigo production.

This article details how to use a vortex fluidic device (VFD) to accelerate protein purification via immobilized metal affinity chromatography (IMAC). Building upon a previous report of VFD-based purification, we introduce a membrane insert to simplify the purification protocol and the resin recovery step. This new platform can be adapted to different types of IMAC resins and purification membranes. Proteins can be purified directly from clarified lysate, non-clarified lysate, and even non-lysed cultures without concerns of system clogging. Strong binding between the Ni2+ and the target protein's His6-tag effectively captures the target protein on IMAC resins or membranes placed in the VFD. Continuous flow of different solutions through the VFD allows dynamic binding, washing, and elution of the target protein. Furthermore, the system dramatically accelerates protein purification; a typical purification from cell lysate requires approximately 4 min. Herein, we demonstrate the single-step purification of two His6-tagged proteins from both clarified and non-clarified cell lysates without requiring batch binding. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of the resin-loaded membrane insert and the vortex fluidic device (VFD) setup prior to purification Basic Protocol 2: Purification of His6-tagged proteins using the VFD Alternate Protocol: VFD-mediated His6-tagged protein purification from non-clarified lysate Support Protocol: Preparation of chemically modified glass fiber membrane for VFD-mediated immobilized metal affinity chromatography purification.

UNLABELLED: Infections caused by antimicrobial-resistant Escherichia coli are the leading cause of death attributed to antimicrobial resistance (AMR) worldwide, and the known AMR mechanisms involve a range of functional proteins. Here, we employed a pan-genome wide association study (GWAS) approach on over 1,000 E. coli isolates from sick dogs collected across the US and Canada and identified a strong statistical association (empirical P < 0.01) of AMR, involving a range of antibiotics to a group 1 capsular (CPS) gene cluster. This cluster included genes under relaxed selection pressure, had several loci missing, and had pseudogenes for other key loci. Furthermore, this cluster is widespread in E. coli and Klebsiella clinical isolates across multiple host species. Earlier studies demonstrated that the octameric CPS polysaccharide export protein Wza can transmit macrolide antibiotics into the E. coli periplasm. We suggest that the CPS in question, and its highly divergent Wza, functions as an antibiotic trap, preventing antimicrobial penetration. We also highlight the high diversity of lineages circulating in dogs across all regions studied, the overlap with human lineages, and regional prevalence of resistance to multiple antimicrobial classes. IMPORTANCE: Much of the human genomic epidemiology data available for E. coli mechanism discovery studies has been heavily biased toward shiga-toxin producing strains from humans and livestock. E. coli occupies many niches and produces a wide variety of other significant pathotypes, including some implicated in chronic disease. We hypothesized that since dogs tend to share similar strains with their owners and are treated with similar antibiotics, their pathogenic isolates will harbor unexplored AMR mechanisms of importance to humans as well as animals. By comparing over 1,000 genomes with in vitro antimicrobial susceptibility data from sick dogs across the US and Canada, we identified a strong multidrug resistance association with an operon that appears to have once conferred a type 1 capsule production system.

Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix-turn-helix-turn-helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression in Escherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy.

Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the proteins central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.

In this study, poly(lactic acid), polyethylene glycol), and benzalkonium chloride with different concentrations (3, 5, 7, and 9%wt.) (PLA/PEG/RCL) composite electrospun mats were produced. PLA is a non-toxic polymer with high biocompatibility and biodegradability. However, it may be fragile due to its structure. Therefore, in this study, PEG was used as a plasticizer to improve the structural properties of PLA and it was aimed at providing antibacterial properties by adding BCL salt. Its use as an antibacterial composite nanomaterial effective against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coti (E. coli) bacterial cultures and as a dermal wound dressing material has been examined in two different areas. The addition of BCL salt reduced the bead formation in PLA/PEG nanofibers and increased the homogeneity of fiber dispersion. 9% BCL-doped composite nanofiber was obtained as the smoothest and most homogeneous surface. This mat was reported to have the highest ductility. The low [T.sub.m] of pure BCL salt enabled the [T.sub.g] temperature of PLA/PEG/BCL composite nanofibers to be observed. It was observed that as the BCL salt ratio increased, the [T.sub.5] and [T.sub.10] temperatures of the nanofibers decreased and then increased. BCL-doped mats exhibited liquid absorption behavior in the range of 497%-708%. PLA/PEG/BCL composite nanofibers showed high toxicity to the L929 fibroblast cell line. So, it has been reported that it cannot be used as a dermal wound dressing. PLA/PEG/ BCI. composite nanomaterials were reported to have 99.99% antibacterial activity against E. coli and S. aureus. It was suggested that it could be used in antibacterial coating applications by taking into account modem nanocoating technology. Highlights * Poly(lactic acid), poly(ethylene glycol), and benzalkonium chloride (PLA/PEG/BCL) composite electrospun mats were produced. * The addition of BCL salt reduced the bead formation in PLA/PEG nanofibers and increased the homogeneity of fiber dispersion. * 9% BCL-doped composite nanofiber was obtained as the smoothest and most homogeneous surface. * PLA/PEG/BCL composite nanoiibers showed high toxicity to the L929 fibroblast cell line. * PLA/PEG/BCL composite nanomaterials were reported to have 99.99% antibacterial activity against E, coli and S. aureus. KEYWORDS antibacterial effect, electrospinning, PLA nanofiber, quaternary ammonium salt benzalkonium chloride, 1 | INTRODUCTION With the nanotechnology revolution, nanofibers with tremendous properties such as a high surface/volume ratio, very good mechanical performance, and high porosity have emerged. (1,2) It has been [...]

Hydrostatic pressure increases with depth in the ocean, but little is known about the molecular bases of biological pressure tolerance. We describe a mode of pressure adaptation in comb jellies (ctenophores) that also constrains these animals depth range. Structural analysis of deep-sea ctenophore lipids shows that they form a nonbilayer phase at pressures under which the phase is not typically stable. Lipidomics and all-atom simulations identified phospholipids with strong negative spontaneous curvature, including plasmalogens, as a hallmark of deep-adapted membranes that causes this phase behavior. Synthesis of plasmalogens enhanced pressure tolerance in Escherichia coli, whereas low-curvature lipids had the opposite effect. Imaging of ctenophore tissues indicated that the disintegration of deep-sea animals when decompressed could be driven by a phase transition in their phospholipid membranes.

UNLABELLED: Cryptic conjugative plasmids lack antibiotic-resistance genes (ARGs). These plasmids can capture ARGs from the vast pool of the environmental metagenome, but the mechanism to recruit ARGs remains to be elucidated. To investigate the recruitment of ARGs by a cryptic plasmid, we sequenced and conducted mating experiments with Escherichia coli SW4848 (collected from a lake) that has a cryptic IncX (IncX4) plasmid and an IncF (IncFII/IncFIIB) plasmid with five genes that confer resistance to aminoglycosides (strA and strB), sulfonamides (sul2), tetracycline [tet(A)], and trimethoprim (dfrA5). In a conjugation experiment, a novel hybrid Tn21/Tn1721 transposon of 22,570 bp (designated Tn7714) carrying the five ARG mobilized spontaneously from the IncF plasmid to the cryptic IncX plasmid. The IncF plasmid was found to be conjugative when it was electroporated into E. coli DH10B (without the IncX plasmid). Two parallel conjugations with the IncF and the new IncX (carrying the novel Tn7714 transposon) plasmids in two separate E. coli DH10B as donors and E. coli J53 as the recipient revealed that the conjugation rate of the new IncX plasmid (with the novel Tn7714 transposon and five ARGs) is more than two orders of magnitude larger than the IncF plasmid. For the first time, this study shows experimental evidence that cryptic environmental plasmids can capture and transfer transposons with ARGs to other bacteria, creating novel multidrug-resistant conjugative plasmids with higher dispersion potential. IMPORTANCE: Cryptic conjugative plasmids are extrachromosomal DNA molecules without antibiotic-resistance genes (ARGs). Environmental bacteria carrying cryptic plasmids with a high conjugation rate threaten public health because they can capture clinically relevant ARGs and rapidly spread them to pathogenic bacteria. However, the mechanism to recruit ARG by cryptic conjugative plasmids in environmental bacteria has not been observed experimentally. Here, we document the first translocation of a transposon with multiple clinically relevant ARGs to a cryptic environmental conjugative plasmid. The new multidrug-resistant conjugative plasmid has a conjugation rate that is two orders of magnitude higher than the original plasmid that carries the ARG (i.e., the new plasmid from the environment can spread ARG more than two orders of magnitude faster). Our work illustrates the importance of studying the mobilization of ARGs in environmental bacteria. It sheds light on how cryptic conjugative plasmids recruit ARGs, a phenomenon at the root of the antibiotic crisis.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (

It has proved challenging to quantitatively relate the proteome to the transcriptome on a per-gene basis. Recent advances in data analytics have enabled a biologically meaningful modularization of the bacterial transcriptome. We thus investigate whether matched datasets of transcriptomes and proteomes from bacteria under diverse conditions can be modularized in the same way to reveal novel relationships between their compositions. We find that; (1) the modules of the proteome and the transcriptome are comprised of a similar list of gene products, (2) the modules in the proteome often represent combinations of modules from the transcriptome, (3) known transcriptional and post-translational regulation is reflected in differences between two sets of modules, allowing for knowledge-mapping when interpreting module functions, and (4) through statistical modeling, absolute proteome allocation can be inferred from the transcriptome alone. Quantitative and knowledge-based relationships can thus be found at the genome-scale between the proteome and transcriptome in bacteria.

Escherichia coli has been attributed to playing a major role in a cascade of events that affect the prevalence and severity of uterine disease in cattle. The objectives of this project were to (i) define the association between the prevalence of specific antimicrobial resistance and virulence factor genes in E. coli with the clinical status related to uterine infection, (ii) identify the genetic relationship between E. coli isolates from cows with diarrhea, with mastitis, and with and without metritis, and (iii) determine the association between the phenotypic and genotypic antimicrobial resistance identified on the E. coli isolated from postpartum cattle. Bacterial isolates (n = 148) were obtained from a larger cross-sectional study. Cows were categorized into one of three clinical groups before enrollment: metritis, cows with purulent discharge, and control cows. For genomic comparison, public genomes (n = 130) from cows with diarrhea, mastitis, and metritis were included in a genome-wide association study, to evaluate differences between the drug classes or the virulence factor category among clinical groups. A distinct E. coli genotype associated with metritis could not be identified. Instead, a high genetic diversity among the isolates from uterine sources was present. A virulence factor previously associated with metritis (fimH) using PCR was not associated with metritis. There was moderate accuracy for whole-genome sequencing to predict phenotypic resistance, which varied depending on the antimicrobial tested. Findings from this study contradict the traditional pathotype classification and the unique intrauterine E. coli genotype associated with metritis in dairy cows.IMPORTANCEMetritis is a common infectious disease in dairy cattle and the second most common reason for treating a cow with antimicrobials. The pathophysiology of the disease is complex and is not completely understood. Specific endometrial pathogenic Escherichia coli have been reported to be adapted to the endometrium and sometimes lead to uterine disease. Unfortunately, the specific genomic details of the endometrial-adapted isolates have not been investigated using enough genomes to represent the genomic diversity of this organism to identify specific virulence genes that are consistently associated with disease development and severity. Results from this study provide key microbial ecological advances by elucidating and challenging accepted concepts for the role of Intrauterine E. coli in metritis in dairy cattle, especially contradicting the existence of a unique intrauterine E. coli genotype associated with metritis in dairy cows, which was not found in our study.

We announce the sequence of the Escherichia coli MTR_GS_S1457 strain isolated from a soil sample of a vegetable gardening system for the first time in Bangladesh. With a length of 4,918,647 bp, this strain contained one plasmid, two CRISPR arrays, 54 predicted antibiotic resistance genes, and 81 predicted virulence factor genes.

Intraspecific genomic diversity brings the potential for an unreported and diverse reservoir of cryptic antibiotic resistance genes in pathogens, as cryptic resistance can occur without major mutations and horizontal transmission. Here, we predicted the differences in the types of antibiotics and genes that induce cryptic and latent resistance between micro-diverse Escherichia coli strains. For example, we hypothesize that known resistance genes will be the culprit of latent resistance within clinical strains. We used a modified functional metagenomics method to induce expression in eight E. coli strains. We found a total of 66 individual genes conferring phenotypic resistance to 11 out of 16 antibiotics. A total of 14 known antibiotic resistance genes comprised 21% of total identified genes, whereas the majority (52 genes) were unclassified cryptic resistance genes. Between the eight strains, 1.2% of core orthologous genes were positive (conferred resistance in at least one strain). Sixty-four percent of positive orthologous genes conferred resistance to only one strain, demonstrating high intraspecific variability of latent resistance genes. Cryptic resistance genes comprised most resistance genes among laboratory and clinical strains as well as natural, semisynthetic, and synthetic antibiotics. Known antibiotic resistance genes primarily conferred resistance to multiple antibiotics from varying origins and within multiple strains. Hence, it is uncommon for E. coli to develop cross-cryptic resistance to antibiotics from multiple origins or within multiple strains. We have uncovered prospective and previously unknown resistance genes as well as antibiotics that have the potential to trigger latent antibiotic resistance in E. coli strains from varying origins.IMPORTANCEIntraspecific genomic diversity may be a driving force in the emergence of adaptive antibiotic resistance. Adaptive antibiotic resistance enables sensitive bacterial cells to acquire temporary antibiotic resistance, creating an optimal window for the development of permanent mutational resistance. In this study, we investigate cryptic resistance, an adaptive resistance mechanism, and unveil novel (cryptic) antibiotic resistance genes that confer resistance when amplified within eight E. coli strains derived from clinical and laboratory origins. We identify the potential of cryptic resistance genes to confer cross-resistance to antibiotics from varying origins and within multiple strains. We discern antibiotic characteristics that promote latent resistance in multiple strains, considering intraspecific diversity. This study may help detect novel resistance genes and functional genes that could become responsible for cryptic resistance among diverse strains and antibiotics, thus also identifying potential novel antibiotic targets and mechanisms.