More Like this

1. Revisiting Replication‐Induced Transcription in Escherichia coli

   https://doi.org/10.1002/bies.201900193  

   Golding, Ido (November 2019, BioEssays)
2. Controlling and quantifying protein concentration in Escherichia coli

   https://doi.org/10.1002/pro.3637  

   Speer, Shannon L.; Guseman, Alex J.; Patteson, Jon B.; Ehrmann, Brandie M.; Pielak, Gary J. (May 2019, Protein Science)

   Abstract The cellular environment is dynamic and complex, involving thousands of different macromolecules with total concentrations of hundreds of grams per liter. However, most biochemistry is conducted in dilute buffer where the concentration of macromolecules is less than 10 g/L. High concentrations of macromolecules affect protein stability, function, and protein complex formation, but to understand these phenomena fully we need to know the concentration of the test protein in cells. Here, we quantify the concentration of an overexpressed recombinant protein, a variant of the B1 domain of protein G, in Tuner (DE3)™Escherichia colicells as a function of inducer concentration. We find that the protein expression level is controllable, and expression saturates at over 2 mMupon induction with 0.4 mMisopropyl β‐d‐thiogalactoside. We discuss the results in terms of what can and cannot be learned from in‐cell protein NMR studies inE. coli. 

   more » « less
3. In Vivo Targeting of Escherichia coli with Vancomycin-Arginine

   https://doi.org/10.1128/AAC.02416-20  

   Neville, Lewis F.; Shalit, Itamar; Warn, Peter A.; Scheetz, Marc H.; Sun, Jiuzhi; Chosy, Madeline B.; Wender, Paul A.; Cegelski, Lynette; Rendell, Jacob T. (March 2021, Antimicrobial Agents and Chemotherapy)

   null (Ed.)

   ABSTRACT The ability of vancomycin-arginine (V-r) to extend the spectrum of activity of glycopeptides to Gram-negative bacteria was investigated. Its MIC towards Escherichia coli , including β-lactamase expressing Ambler classes A, B, and D, was 8 to 16 μg/ml. Addition of 8 times the MIC of V-r to E. coli was acutely bactericidal and associated with a low frequency of resistance (<2.32 × 10 −10 ). In vivo , V-r markedly reduced E. coli burden by >7 log 10 CFU/g in a thigh muscle model. These data warrant further development of V-r in combatting E. coli , including resistant forms. 

   more » « less
4. The multidrug efflux pump regulator AcrR directly represses motility in Escherichia coli

   https://doi.org/10.1128/msphere.00430-23  

   Maldonado, Jessica; Czarnecka, Barbara; Harmon, Dana E.; Ruiz, Cristian (October 2023, mSphere)

   Bradford, Patricia A. (Ed.)

   ABSTRACT Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility inEscherichia coliand other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump inE. coliand otherEnterobacteriaceae. However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility inE. coli. By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility inE. coliby directly repressing transcription of theflhDCoperon, but not the other flagellum genes/operons tested.flhDCencodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to theflhDCpromoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed inE. colistrains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility inE. colito maintain homeostasis and escape hazards. IMPORTANCEEfflux and motility play a major role in bacterial growth, colonization, and survival. InEscherichia coli, the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824–1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of theflhDCmaster regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress inE. coli. 

   more » « less
5. Engineered citrate synthase improves citramalic acid generation in Escherichia coli

   https://doi.org/10.1002/bit.27450  

   Wu, Xianghao; Tovilla‐Coutiño, D_Brisbane; Eiteman, Mark_A (June 2020, Biotechnology and Bioengineering)

   Abstract The microbial product citramalic acid (citramalate) serves as a five‐carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized inEscherichia colidirectly by the condensation of pyruvate and acetyl‐CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl‐CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in thegltAgene coding citrate synthase prevents acetyl‐CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study theE. colicitrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl‐CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed‐batch process using MEC626/pZE12‐cimAharboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals. 

   more » « less