scholarly journals Proteome dynamics in persisting and resuscitating bacterial cells

2020 ◽  
Author(s):  
Maja Semanjski ◽  
Fabio Gratani ◽  
Till Englert ◽  
Viktor Beke ◽  
Nicolas Nalpas ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Treatment Time ◽  
Bacterial Cells ◽  
Persister Cells ◽  
Time Resolved ◽  
E Coli ◽  
Associated Proteins ◽  
Label Incorporation ◽  
Insight Into

AbstractBacterial persister cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of bacterial persistence is hindered by low count of persisting cells and the need for their isolation. Here we used sustained addition of stable isotope-labeled lysine to selectively label the proteome of hipA-induced persisting and hipB-induced resuscitating E. coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-hour measurement of label incorporation allowed detection of over 500 newly synthetized proteins in persister cells, demonstrating low but widespread protein synthesis. Many essential proteins were newly synthesized and several ribosome-associated proteins showed unusually high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized ABC transporters, restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. This dataset provides an unprecedented insight into the processes governing persistence and resuscitation of bacterial cells.

scholarly journals Bacterial Cell Inactivation Using a Single-Frequency Batch-Type Ultrasound Device

2021 ◽  
Vol 6 (1) ◽  
pp. 65-80
Author(s):  
Poetro Sambegoro ◽  
Maya Fitriyanti ◽  
Bentang Arief Budiman ◽  
Kamarisima Kamarisima ◽  
Sekar Wangi Arraudah Baliwangi ◽  
...  
Keyword(s):  
Bacterial Cell ◽  
Treatment Time ◽  
Low Cost ◽  
Single Frequency ◽  
Bacterial Cells ◽  
Cell Inactivation ◽  
E Coli ◽  
Commercial System ◽  
Ultrasound Device ◽  
Frequency Ultrasound

Ultrasound technology employs cavitation to generate high-pressure soundwaves to disrupt bacterial cells. This study reveals the effectiveness of a single frequency ultrasound device for bacterial cell inactivation. A low-cost ultrasound device having a single frequency, i.e. 22 kHz for lab-scale application, was developed first, and the prototype was mechanically designed and analyzed using the finite-element method to assure the targeted natural frequency could be achieved. The prototype was then tested inactivating bacterial cells, Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis), in a simple medium and a food system, and the results were then compared to a commercial system. A treatment time of up to 15 minutes was able to reduce E. coli and B. subtilis cells by 3.3 log and 2.8 log, respectively, and these results were similar to those of the commercial system. The effectiveness of bacterial cell inactivation using the developed single-frequency ultrasound device is then discussed. The findings are useful for designing low-cost ultrasound devices for application in the food industry.

scholarly journals Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

2016 ◽  
Author(s):  
Aaron Smargon ◽  
David B.T. Cox ◽  
Neena Pyzocha ◽  
Kaijie Zheng ◽  
Ian M. Slaymaker ◽  
...  
Keyword(s):  
Rna Interference ◽  
Essential Gene ◽  
Sequence Database ◽  
Flanking Sequence ◽  
E Coli ◽  
Adaptive Immune ◽  
Associated Proteins ◽  
Adaptive Immune Systems ◽  
Target Rna

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two Class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 or Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

scholarly journals Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Xing Jin ◽  
Weston Kightlinger ◽  
Yong-Chan Kwon ◽  
Seok Hoon Hong
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Host Cells ◽  
Persister Cells ◽  
Free Protein ◽  
E Coli ◽  
Dna And Rna ◽  
Rapid Production ◽  
Cell Free Protein Synthesis

Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

Chromatin remodeling complexes: ATP-dependent machines in action

2005 ◽  
Vol 83 (4) ◽  
pp. 405-417 ◽  
Author(s):  
Cotteka N Johnson ◽  
Nicholas L Adkins ◽  
Philippe Georgel
Keyword(s):  
Chromatin Remodeling ◽  
Atp Hydrolysis ◽  
Nucleosome Remodeling ◽  
Core Histones ◽  
Associated Proteins ◽  
Chromatin Template ◽  
Chromatin Remodeling Complexes ◽  
The Individual ◽  
Insight Into

Since the initial characterization of chromatin remodeling as an ATP-dependent process, many studies have given us insight into how nucleosome-remodeling complexes can affect various nuclear functions. However, the multistep DNA-histone remodeling process has not been completely elucidated. Although new studies are published on a nearly weekly basis, the nature and roles of interactions of the individual SWI/SNF- and ISWI-based remodeling complexes and DNA, core histones, and other chromatin-associated proteins are not fully understood. In addition, the potential changes associated with ATP recruitment and its subsequent hydrolysis have not been fully characterized. This review explores possible mechanisms by which chromatin-remodeling complexes are recruited to specific loci, use ATP hydrolysis to achieve actual remodeling through disruption of DNA-histone interactions, and are released from their chromatin template. We propose possible roles for ATP hydrolysis in a chromatin-release/target-scanning process that offer an alternative to or complement the often overlooked function of delivering the energy required for sliding or dislodging specific subsets of core histones.Key words: chromatin remodeling, SWI/SNF, ISWI, APT hydrolysis.

scholarly journals PRODUCTION AND CHARACTERIZATION OF RECOMBINANT PROTEINS CONTAINING BET V 2 ANTIGENIC EPITOPES OF BETULA PENDULA

2012 ◽  
Vol 9 (6) ◽  
pp. 28-31
Author(s):  
A V Chernysheva ◽  
V V Tyutyaeva ◽  
A V Pivovarova ◽  
I V Andreev ◽  
M N Sankov ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Inclusion Bodies ◽  
Birch Pollen ◽  
Elisa Test ◽  
Recombinant Allergen ◽  
Bacterial Cells ◽  
E Coli ◽  
Immunological Tests ◽  
Linear Epitopes

Aim of investigation. Production of immunologically active recombinant protein of Bet v 2 allergen ofbirch pollen. Materials and methods. mRNA was isolated from a sample ofbirch pollen. cDNA library was derived using SMART technology. Gene Bet v 2 was amplified by means of PCR with primers from the cDNA. The resulting PCR fragment of the gene was cloned into the vector pET29b(+). The recombinant protein Bet v 2 was expressed in cells E. coli, transformed with a plasmid. The recombinant protein was purified using NiNTA agarose. Immunological activity of the recombinant protein Bet v 2 was measured by ELISA and immunoblot methods. Results. The production system of the recombinant allergen Bet v 2 preparation suitable for immunological tests was developed during the research project. In the first phase allergen Bet v 2 gene was cloned from birch pollen collected in Russia. The gene was inserted into the vector pET29(+) for expression in bacterial cells. The expression cell strain E. coli was obtained with this plasmid. The synthesis of the recombinant protein that accumulates in inclusion bodies was activated in bacterial cells. The procedure of recombinant protein Bet v 2 isolation from inclusion bodies was developed by one round of chromatography purification. The recombinant protein isolation was carried out by chromatography on Niagarose. The highly purified preparation of the recombinant allergen was obtained as a result. The recognition of the recombinant protein Bet v 2 by sera varied in ELISA, indicating a different degree of patients sensitization to this allergen. In the immunoblot test the preparation was active only in 15% of cases. Apparently, reactive epitopes of the allergen are mainly conformational ones and are active in ELISA test, whereas linear epitopes, that are active in immunoblot, are in the minority. Conclusion.The system for production of recombinant allergen Bet v 2 preparation suitable for immunological tests has been developed.

scholarly journals Characterization of Primed Adaptation in the Escherichia coli type I-E CRISPR-Cas System

2020 ◽  
Author(s):  
Anne M. Stringer ◽  
Lauren A. Cooper ◽  
Sujatha Kadaba ◽  
Shailab Shrestha ◽  
Joseph T. Wade
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Genetic Locus ◽  
Type I ◽  
Cornell University ◽  
E Coli ◽  
Nucleic Acid Molecule ◽  
Bacterial Immune Systems ◽  
Insight Into

ABSTRACTCRISPR-Cas systems are bacterial immune systems that target invading nucleic acid. The hallmark of CRISPR-Cas systems is the CRISPR array, a genetic locus that includes short sequences known as “spacers”, that are derived from invading nucleic acid. Upon exposure to an invading nucleic acid molecule, bacteria/archaea with functional CRISPR-Cas systems can add new spacers to their CRISPR arrays in a process known as “adaptation”. In type I CRISPR-Cas systems, which represent the majority of CRISPR-Cas systems found in nature, adaptation can occur by two mechanisms: naïve and primed. Here, we show that, for the archetypal type I-E CRISPR-Cas system from Escherichia coli, primed adaptation occurs at least 1,000 times more efficiently than naïve adaptation. By initiating primed adaptation on the E. coli chromosome, we show that spacers can be acquired across distances of >100 kb from the initially targeted site, and we identify multiple factors that influence the efficiency with which sequences are acquired as new spacers. Thus, our data provide insight into the mechanism of primed adaptation.[This paper has been peer reviewed, with Ailong Ke (Cornell University) serving as the editor. Reviews and point-by-point response, and a marked-up version of the edited manuscript are provided as supplementary files.]

scholarly journals Persister cells mediate tolerance to metal oxyanions in Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3181-3195 ◽  
Author(s):  
Joe J. Harrison ◽  
Howard Ceri ◽  
Nicole J. Roper ◽  
Erin A. Badry ◽  
Kimberley M. Sproule ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Viable Cell ◽  
Small Population ◽  
Water Soluble ◽  
Bacterial Cells ◽  
Persister Cells ◽  
Term Survival ◽  
E Coli ◽  
Cell Counts ◽  
High Concentrations

Bacterial cultures produce subpopulations of cells termed ‘persisters’, reputedly known for high tolerance to killing by antibiotics. Ecologically, antibiotics produced by competing microflora are only one potential stress encountered by bacteria. Another pressure in the environment is toxic metals that are distributed ubiquitously by human pollution, volcanic activity and the weathering of minerals. This study evaluated the time- and concentration-dependent killing of Escherichia coli planktonic and biofilm cultures by the water-soluble metal(loid) oxyanions chromate (), arsenate (), arsenite (), selenite (), tellurate () and tellurite (). Correlative to previous reports in the literature, control antibiotic assays indicated that a small proportion of E. coli biofilm populations remained recalcitrant to killing by antibiotics (even with 24 h exposure). In contrast, metal oxyanions presented a slow, bactericidal action that eradicated biofilms. When exposed for 2 h, biofilms were up to 310 times more tolerant to killing by metal oxyanions than corresponding planktonic cultures. However, by 24 h, planktonic cells and biofilms were eradicated at approximately the same concentration in all instances. Coloured complexes of metals and chelators could not be generated in biofilms exposed to or , suggesting that the extracellular polymeric matrix of E. coli may have a low binding affinity for metal oxyanions. Viable cell counts at 2 and 24 h exposure revealed that, at high concentrations, all of the metal oxyanions had killed 99 % (or a greater proportion) of the bacterial cells in biofilm populations. It is suggested here that the short-term survival of <1 % of the bacterial population corresponds well with the hypothesis that a small population of persister cells may be responsible for the time-dependent tolerance of E. coli biofilms to high concentrations of metal oxyanions.

scholarly journals Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

2019 ◽  
Author(s):  
Sooyeon Song ◽  
Thomas K. Wood
Keyword(s):  
Escherichia Coli ◽  
Small Rna ◽  
23S Rrna ◽  
Bacterial Cells ◽  
Persister Cells ◽  
E Coli ◽  
Pseudouridine Synthase ◽  
Dormant Cells ◽  
Wide Range ◽  
Persister Cell

ABSTRACTUpon a wide range of stress conditions (e.g., nutrient, antibiotic, oxidative), a subpopulation of bacterial cells known as persisters survive by halting metabolism. These cells resuscitate rapidly to reconstitute infections once the stress is removed and nutrients are provided. However, how these dormant cells resuscitate is not understood well but involves reactivating ribosomes. By screening 10,000 compounds directly for stimulating Escherichia coli persister cell resuscitation, we identified that 2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (BPOET) stimulates resuscitation. Critically, by screening 4,267 E. coli proteins, we determined that BPOET activates hibernating ribosomes via 23S rRNA pseudouridine synthase RluD, which increases ribosome activity. Corroborating the increased waking with RluD, production of RluD increased the number of active ribosomes in persister cells. Also, inactivating the small RNA RybB which represses rluD led to faster persister resuscitation. Hence, persister cells resuscitate via activation of RluD.

scholarly journals Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 934
Author(s):  
Dov Borovsky ◽  
Kato Deckers ◽  
Anne Catherine Vanhove ◽  
Maud Verstraete ◽  
Pierre Rougé ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Sequence ◽  
Bacterial Cells ◽  
E Coli ◽  
Trypsin Modulating Oostatic Factor ◽  
High Affinity ◽  
Sds Page ◽  
Atpase Inhibitors ◽  
Kinetics Of

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA− was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.

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