Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “E. coli”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Expanding the Landscape of Dual Action Antifolate Antibacterials through 2,4-Diamino-1,6-dihydro-1,3,5-triazines

Antibiotics that operate via multiple mechanisms of action are a promising strategy to combat growing resistance. Previous studies have shown that dual action antifolates formed from a pyrroloquinazolinediamine core can inhibit the growth of bacterial pathogens without developing resistance. Here, in this work, we expand the scope of dual action antifolates by repurposing the 2,4-diamino-1,6-dihydro-1,3,5-triazine (DADHT) cycloguanil scaffold to a variety of derivatives designed to inhibit dihydrofolate reductase (DHFR) and disrupt bacterial membranes. Dual mechanism DADHTs have activity against a variety of target pathogens, including Mycobacterium tuberculosis, Mycobacterium abscessus, and Pseudomonas aeruginosa, among other ESKAPEE organisms. Through X-ray crystallography, we confirmed engagement of the Escherichia coli DHFR target and found that some DADHTs stabilize a previously unobserved conformation of the enzyme but, broadly, bind in the occluded conformation. Using in vitro inhibition of purified E. coli and Staphylococcus aureus DHFR and disruption of E. coli membranes, we determined that alkyl substitution of dihydrotriazine at the 6-position best optimizes the DADHT's two mechanisms of action. By employing both mechanisms, the DADHT spectrum of activity was extended beyond the scope of traditional antifolates. Finally, we are optimistic that the dual mechanism approach, particularly through the action of antifolates, offers a unique means of combating hard-to-treat bacterial infections.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Granulibacter bethesdensis, a Pathogen from Patients with Chronic Granulomatous Disease, Produces a Penta-Acylated Hypostimulatory Glycero-D-talo-oct-2-ulosonic Acid–Lipid A Glycolipid (Ko-Lipid A)

Granulibacter bethesdensis can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact G. bethesdensis (Gb) is hypostimulatory compared to Escherichia coli, i.e., cytokine production in human blood requires 10–100 times more G. bethesdensis CFU/mL than E. coli. To better understand the pathogenicity of G. bethesdensis, we isolated its lipopolysaccharide (GbLPS) and characterized its lipid A. Unlike with typical Enterobacteriaceae, the release of presumptive Gb lipid A from its LPS required a strong acid. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of α-Manp-(1→4)-β-GlcpN3N-(1→6)-α-GlcpN-(1⇿1)-α-GlcpA tetra-saccharide substituted with five acyl chains: the amide-linked N-3′ 14:0(3-OH), N-2′ 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constituent of the core region of the LPS that is covalently attached to GlcpN3N of the lipid backbone may account for the acid resistance of GbLPS. In addition, the presence of Ko and only five acyl chains may explain the >10-fold lower proinflammatory potency of GbKo–lipidA compared to E. coli lipid A, as measured by cytokine induction in human blood. These unusual structural properties of the G.bethesdensis Ko–lipid A glycolipid likely contribute to immune evasion during pathogenesis and resistance to antimicrobial peptides.

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

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB

ABSTRACT Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase. IMPORTANCE Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Bioconversion of self-neutralized chemically depolymerized lignin streams into polyhydroxyalkanoates

Lignin is a chemically complex, diverse, and abundant plant polymer mainly composed of aromatic monomers. These aromatic monomers make lignin a potential source of aromatics and a viable substitute for petrochemically-derived aromatics. However, the structural recalcitrance of lignin requires harsh reagents from the chemical process and expensive catalysts for effective depolymerization. This chemical process often results in poor yields of chemical intermediate mixtures of varying bioavailability and/or toxicity. Furthermore, the cost of additional reagents required to separate or detoxify these intermediates makes processing lignin impractical. We report progress towards the use of such chemically depolymerized lignin streams by employing bacterial strains to produce polyhydroxyalkanoates (PHA). PHAs are a group of biodegradable microbial polyesters that have potential as a replacement for petroleum-based plastics. In this study we utilized two distinct lignin streams obtained after chemical depolymerization of lignin under alkaline and acidic pH in the presence of catalysts. We mixed the alkali-treated depolymerized stream with the acid-treated depolymerized stream to create a solution of neutral-pH chemically depolymerized lignin (CDL). We found moderate to substantial growth of both native and non-native PHA producers on the mixture of CDL as well as its aliphatic and aromatic components. PHA was detected by Sudan Black B staining in C. necator H16, P. putida KT2440, and E. coli LSBJ STQKAB grown on mixed CDL as the sole carbon source. For C. necator H16 and E. coli LSBJ STQKAB we found that PHA content was greater when grown on mixed CDL when compared to their preferred carbon source by GC-FID quantification. Our study provided progress towards a cost-competitive, sustainable, and industrially relevant use for lignin.

Winkler, Gordon L. W. [Univ. of Wisconsin, Madison↗
DOE OSTI2025

A large-scale screening campaign of putative carbohydrate-active enzymes reveals a novel xylanase from anaerobic gut fungi

The genomes of anaerobic gut fungi (AGF) encode a diverse array of carbohydrate-active enzymes (CAZymes), yet exceedingly few of these enzymes have been experimentally validated or expressed in heterologous systems. Here, we developed a predictive bioinformatic pipeline to annotate novel putative CAZymes from anaerobic fungi and validate their activity through large-scale heterologous expression in Escherichia coli. A total of 173 fungal proteins from Piromyces finnis associated with biomass degradation were synthesized and expressed in E. coli, and 9.8% were soluble with expression levels exceeding 5% of the total proteome using high-throughput proteomic screening. Among these 17 heterologously expressed proteins, analysis with AlphaFold and FoldSeek predicted 13 multi-functional proteins containing catalytic domains fused with repetitive fungal dockerins, and half of the substrate predictions were experimentally validated. One promising enzyme, celsome_012, exhibited robust and specific activity against beechwood xylan at 37°C and pH 6.4, with titers that were also fivefold higher than those of other recombinant proteins screened here. Both Michaelis-Menten kinetics and the linearized Lineweaver-Burk equation yielded consistent values for K m , and its activation energy was estimated at 51.9 kJ/mol based on the Arrhenius model. This work supports the industrial translation of anaerobic fungal CAZymes due to their robust lignocellulolytic activity and provides a framework for prioritizing AGF proteins for efficient E. coli heterologous expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Engineering controllable alteration of malonyl-CoA levels to enhance polyketide production

Heterologous expression of polyketide synthase (PKS) genes in Escherichia coli has enabled the production of various valuable natural and synthetic products. However, the limited availability of malonyl-CoA (M-CoA) in E. coli remains a substantial impediment to high-titer polyketide production. Here we address this limitation by disrupting the native M-CoA biosynthetic pathway and introducing an orthogonal pathway comprising a malonate transporter and M-CoA ligase, enabling efficient M-CoA biosynthesis under malonate supplementation. This approach substantially increases M-CoA levels, enhancing fatty acid and polyketide titers while reducing the promiscuous activity of PKSs toward undesired acyl-CoA substrates. Subsequent adaptive laboratory evolution of these strains provides insights into M-CoA regulation and identifies mutations that further boost M-CoA and polyketide production. This strategy improves E. coli as a host for polyketide biosynthesis and advances understanding of M-CoA metabolism in microbial systems.

Klass, Sarah H↗
DOE OSTI2022

Scalable and automated CRISPR-based strain engineering using droplet microfluidics

Abstract We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system’s capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase ( galK ) in E. coli and (2) targeted engineering of the glutamine synthetase gene ( glnA ) and the blue-pigment synthetase gene ( bpsA ) to improve indigoidine production in E. coli .

42 ENGINEERING↗
DOE OSTI2021

Potentiating antibiotic efficacy via perturbation of non-essential gene expression

Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization of E. coli without a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing a Salmonella enterica intracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies, csgD, fnr, recA and acrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates of Klebsiella pneumoniae and E. coli showed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Genome reduction improves octanoic acid production in scale down bioreactors

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

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Templated Mesoporous Silica Outer Shell for Controlled Silver Release of a Magnetically Recoverable and Reusable Nanocomposite for Water Disinfection

Here, we encapsulated Fe 3 O 4 @SiO 2 @Ag (MS-Ag), a bifunctional magnetic silver core–shell structure, with an outer mesoporous silica (mS) shell to form an Fe 3 O 4 @SiO 2 @ 2 Ag@mSiO 2 (MS-Ag-mS) nanocomposite using a cationic CTAB (cetyltrimethylammonium bromide) micelle templating strategy. The mS shell acts as protection to slow down the oxidation and detachment of the AgNPs and incorporates channels to control the release of antimicrobial Ag + ions. Results of TEM, STEM, HRSEM, EDS, BET, and FTIR showed the successful formation of the mS shells on MS-Ag aggregates 50–400 nm in size with highly uniform pores ~4 nm in diameter that were separated by silica walls ~2 nm thick. Additionally, the mS shell thickness was tuned to demonstrate controlled Ag + release; an increase in shell thickness resulted in an increased path length required for Ag + ions to travel out of the shell, reducing MS-Ag-mS’ ability to inhibit E. coli growth as illustrated by the inhibition zone results. Through a shaking test, the MS-Ag-mS nanocomposite was shown to eradicate 99.99+% of a suspension of E. coli at 1 × 10 6 CFU/mL with a silver release of less than 0.1 ppb, well under the EPA recommendation of 0.1 ppm. This high biocidal efficiency with minimal silver leach is ascribed to the nanocomposite’s mS shell surface characteristics, including having hydroxyl groups and possessing a high degree of structural periodicity at the nanoscale or “smoothness” that encourages association with bacteria and retains high Ag + concentration on its surface and in its close proximity. Furthermore, the nanocomposite demonstrated consistent antimicrobial performance and silver release levels over multiple repeated uses (after being recovered magnetically because of the oxidation-resistant silica-coated magnetic Fe 3 O 4 core). It also proved effective at killing all microbes from Long Island Sound surface water. The described MS-Ag-mS nanocomposite is highly synergistic, easy to prepare, and readily recoverable and reusable and offers structural tunability affecting the bioavailability of Ag + , making it excellent for water disinfection that will find wide applications.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2022

Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China

Escherichia coli is considered an opportunistic pathogen and an indicator for antimicrobial resistance (AMR) monitoring. Despite many reports on its AMR monitoring, studies based on genome-based analysis of AMR genes are still insufficient. Here, 181 E. coli strains were isolated from anal swab samples collected from pigs and chickens of animal farms located in Eastern China and sequenced through the Illumina platform. The results showed that 87.85% (159/181) of the E. coli isolates were multidrug-resistant (MDR). Ampicillin (AMP)- spectinomycin (SPT)- tetracycline (TET)- florfenicol (FFC)- sulfisoxazole (SF)- trimethoprim/sulfamethoxazole (SXT) was the predominant AMR pattern. By whole-genome sequencing, we found that ST10 (10.49%, 19/181) and ST48 (7.18%, 13/181) were major sequence types. IncFIB and IncX1 were the most prevalent plasmid replicons. The AMR genes bla NDM-5 (1.10%, 2/181), mcr -1 (1.10%, 2/181), tet (X4) (1.10%, 2/181), and cfr (6.08%, 11/181) were also found in these isolates. In addition, among the 169 virulence genes detected, we identified ast A (37.02%, 67/181), hly A (1.66%, 3/181), hly B (1.66%, 3/181) and hly D (1.66%, 3/181), which were closely related to heat-stable enterotoxin 1 and α-hemolysin. In addition, there were 33 virulence genes associated with the iron uptake system, and 46 were adhesion-related genes. Our study highlighted the need for routine surveillance of AMR with advanced genomic approaches, providing up-to-date data on the prevalence of AMR for the development and execution of antimicrobial stewardship policy.

Zhou, Wei↗
DOE OSTI2022

Novel reusable animal model for comparative evaluation of in vivo growth and protein-expression of Escherichia coli O157 strains in the bovine rumen

Previously, we had demonstrated that Escherichia coli O157:H7 (O157) strain 86–24 expresses proteins involved in survival rather than virulence in vitro in rumen fluid from dairy cattle limit fed a maintenance diet. Here, we verified if this observation would be true for different O157 strains grown in vitro in rumen fluid from, and in vivo in the rumen of, animals on contrasting maintenance (high fiber) and lactation (high energy-protein) diets usually limit fed to dairy cattle. For the in vivo studies, an economical, novel, reusable and non-terminal rumen-fistulated animal model permitting simultaneous evaluation of multiple bacterial strains in the bovine rumen was developed. All experiments were conducted in duplicate using different animals to account for host-related variations. The O157 strains included, 86–24, EDL933 and the super shed SS-17. E. coli Nal R (#5735), derived from a bovine intestinal commensal E. coli, was included as a control. As expected, diet influenced ruminal pH and volatile fatty acid (VFA) composition. The pH ranged from 6.2–7.0 and total VFA concentrations from 109–141 μM/ml, in animals fed the maintenance diet. In comparison, animals fed the lactation diet had a ruminal pH ranging between 5.18–6.0, and total VFA of 125–219 μM/ml. Strain dependent differences in O157 recovery from the rumen fluid of cattle fed either diet was observed, both in vitro and in vivo, with O157 strains 86–24 and EDL933 demonstrating similar survival patterns. Analysis of the O157 proteomes expressed in the rumen fluid/rumen verified previous observations of adaptive responses. Any difference in the adaptive response was mainly influenced by the animal’s diet and growth conditions (in vitro and in vivo) and not the O157 strain. These new insights into the O157 responses could help formulate modalities to control O157 across strains in cattle at all stages of husbandry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Enhancing Photosynthetic Production of Glycogen-Rich Biomass for Use as a Fermentation Feedstock

Current sources of fermentation feedstocks, i.e., corn, sugar cane, or plant biomass, fall short of demand for liquid transportation fuels and commodity chemicals in the United States. Aquatic phototrophs including cyanobacteria have the potential to supplement the supply of current fermentable feedstocks. In our strategy, cells are engineered to accumulate storage molecules including glycogen, cellulose, and/or lipid oils that can be extracted from harvested biomass and fed to heterotrophic organisms engineered to produce desired chemical products. Here in this manuscript, we examine the production of glycogen in the model cyanobacteria, Synechococcus sp. strain PCC 7002, and subsequent conversion of cyanobacterial biomass by an engineered Escherichia coli to octanoic acid as a model product. In effort to maximize glycogen production, we explored the deletion of catabolic enzymes and overexpression of GlgC, an enzyme that catalyzes the first committed step toward glycogen synthesis. We found that deletion of glgP increased final glycogen titers when cells were grown in diurnal light. Overexpression of GlgC led to a temporal increase in glycogen content but not in an overall increase in final titer or content. The best strains were grown, harvested, and used to formulate media for growth of E. coli. The cyanobacterial media was able to support the growth of an engineered E. coli and produce octanoic acid at the same titer as common laboratory media.

09 BIOMASS FUELS↗
DOE OSTI2020

Release Factor Inhibiting Antimicrobial Peptides Improve Nonstandard Amino Acid Incorporation in Wild-type Bacterial Cells

We report a tunable chemical genetics approach for enhancing genetic code expansion in different wild-type bacterial strains that employs apidaecin-like, anti-microbial peptides observed to temporarily sequester and thereby inhibit Release Factor 1 (RF1). In a concentration-dependent matter, these peptides granted a conditional lambda phage resistance to a recoded Escherichia coli strain with non-essential RF1 activity and promoted multi-site non-standard amino acid (nsAA) incorporation at in-frame amber stop codons in vivo and in vitro. When exogenously added, the peptides stimulated specific nsAA incorporation in a variety of sensitive, wild-type (RF1+) strains including Agrobacterium tumefaciens, a species in which nsAA incorporation has not been previously reported. Improvement in nsAA incorporation was typically 2–15-fold in E. coli BL21, MG1655, DH10B strains and A. tumefaciens with the >20-fold improvement observed in probiotic E. coli Nissle 1917. In-cell expression of these peptides promoted multi-site nsAA incorporation in transcripts with up to 6 amber codons, with a >35-fold increase in BL21 showing moderate toxicity. Leveraging this RF1 sensitivity allowed multiplexed partial recoding of MG1655 and DH10B that rapidly resulted in resistant strains that showed an additional ~2 fold boost to nsAA incorporation independent of the peptide. Lastly, in-cell expression of an apidaecin-like peptide library allowed the discovery of new peptide variants with reduced toxicity that still improved multi-site nsAA incorporation >25-fold. In parallel to genetic reprogramming efforts, these new approaches can facilitate genetic code expansion technologies in a variety of wild-type bacterial strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin

The siderophore rhizoferrin (N 1 ,N 4 -dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.

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

Discovery of multidrug efflux pump inhibitors with a novel chemical scaffold

Multidrug efflux is a major contributor to antibiotic resistance in Gram-negative bacterial pathogens. Inhibition of multidrug efflux pumps is a promising approach for reviving the efficacy of existing antibiotics. Previously, inhibitors targeting both the efflux transporter AcrB and the membrane fusion protein AcrA in the Escherichia coli AcrAB-TolC efflux pump were identified. In this work we use existing physicochemical property guidelines to generate a filtered library of compounds for computational docking. We then experimentally test the top candidate coumpounds using in vitro binding assays and in vivo potentiation assays in bacterial strains with controllable permeability barriers. We thus identify a new class of inhibitors of E. coli AcrAB-TolC. Six molecules with a shared scaffold were found to potentiate the antimicrobial activity of erythromycin and novobiocin in hyperporinated E. coli cells. Importantly, these six molecules were also active in wild-type strains of both Acinetobacter baumannii and Klebsiella pneumoniae, potentiating the activity of erythromycin and novobiocin up to 8-fold.

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

Enhancing transcription in Escherichia coli and Pseudomonas putida using bacteriophage lambda anti-terminator protein Q

Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli . Functional expression of heterologous genes can suffer from limitations due to the lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA between a vector-based promoter and the transcription start site. Anti-terminator proteins are a possible solution to overcome this limitation. When bacteriophage lambda infects E. coli , it relies on the host transcription machinery to transcribe and express phage DNA. Lambda anti-terminator protein Q (λQ) regulates the expression of late-genes of phage lambda. E. coli RNA polymerase recognizes the P R ' promoter on the lambda genome and forms a complex with λQ, to overcome the terminator t R '. In this study we show the use of λQ to efficiently transcribe a capsular polysaccharide cluster, cps3 , from Lactobacillus plantarum containing intrinsic terminators in Escherichia coli . In addition, we expand the use of anti-terminator λQ in Pseudomonas putida . The results show ~ fivefold higher expression of a fluorescent reporter locate ~ 12.5kbp downstream from the promoter, when the transcription is driven by P R ' promoter in presence of λQ compared to a lac promoter. These results suggest that λQ could be used in metabolic engineering to enhance expression of heterologous DNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

MassIVE MSV000095714

Proteomic analysis reveals translation-related proteins are significantly altered in response to stabilized G4s. Identification of the importance of translation factors in overcoming G4s led to the question of how E. coli cells generally respond to chemicals that stabilize such structures. As a first step in addressing this question, a proteomic analysis was carried out to measure the quantitative effects of NMM on the levels of individual proteins in E. coli. Protein levels from early log-phase cultures of delta-tolC and delta-tolC tufA::kan strains grown in the presence or absence of NMM were measured to assess how reduced EF- Tu levels and NMM impacted expression.

Bottom-up proteomics↗