scholarly journals Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Samuel Thompson ◽  
Yang Zhang ◽  
Christine Ingle ◽  
Kimberly A Reynolds ◽  
Tanja Kortemme
Keyword(s):  
Quality Control ◽  
Single Point ◽  
Point Mutations ◽  
Mechanistic Explanation ◽  
Metabolic Enzyme ◽  
Altered Expression ◽  
E Coli ◽  
Mutational Landscape ◽  
Cellular Environment

Protein mutational landscapes are shaped by the cellular environment, but key factors and their quantitative effects are often unknown. Here we show that Lon, a quality control protease naturally absent in common E. coli expression strains, drastically reshapes the mutational landscape of the metabolic enzyme dihydrofolate reductase (DHFR). Selection under conditions that resolve highly active mutants reveals that 23.3% of all single point mutations in DHFR are advantageous in the absence of Lon, but advantageous mutations are largely suppressed when Lon is reintroduced. Protein stability measurements demonstrate extensive activity-stability tradeoffs for the advantageous mutants and provide a mechanistic explanation for Lon’s widespread impact. Our findings suggest possibilities for tuning mutational landscapes by modulating the cellular environment, with implications for protein design and combatting antibiotic resistance.

scholarly journals Modulating the cellular context broadly reshapes the mutational landscape of a model enzyme

2019 ◽  
Author(s):  
Samuel Thompson ◽  
Yang Zhang ◽  
Christine Ingle ◽  
Kimberly A. Reynolds ◽  
Tanja Kortemme
Keyword(s):  
Protein Design ◽  
Single Point ◽  
Point Mutations ◽  
Mechanistic Explanation ◽  
Metabolic Enzyme ◽  
E Coli ◽  
Mutational Landscape ◽  
Highly Active ◽  
Cellular Environment ◽  
Cellular Context

AbstractProtein mutational landscapes are shaped by the cellular environment, but key factors and their quantitative effects are often unknown. Here we show that Lon, a quality control protease naturally absent in common E. coli expression strains, drastically reshapes the mutational landscape of the metabolic enzyme dihydrofolate reductase (DHFR). Selection under conditions that resolve highly active mutants reveals that 23.3% of all single point mutations in DHFR are advantageous in the absence of Lon, but advantageous mutations are largely suppressed when Lon is reintroduced. Protein stability measurements demonstrate extensive activity-stability tradeoffs for the advantageous mutants and provide a mechanistic explanation for Lon’s widespread impact. Our findings suggest possibilities for tuning mutational landscapes by modulating the cellular environment, with implications for protein design and combatting antibiotic resistance.

scholarly journals In Vivo Evidence for TonB Dimerization

2003 ◽  
Vol 185 (19) ◽  
pp. 5747-5754 ◽  
Author(s):  
Annette Sauter ◽  
S. Peter Howard ◽  
Volkmar Braun
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Ferric Citrate ◽  
Site Directed Mutagenesis ◽  
Toxin Gene ◽  
Transmembrane Region ◽  
Dimer Formation ◽  
Citrate Transport ◽  
Hybrid Proteins

ABSTRACT TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and φ80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P ctx ::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P ctx ::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

scholarly journals The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

1993 ◽  
Vol 296 (3) ◽  
pp. 851-857 ◽  
Author(s):  
T Belyaeva ◽  
L Griffiths ◽  
S Minchin ◽  
J Cole ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Growth Conditions ◽  
Upstream Region ◽  
E Coli ◽  
Run Off ◽  
A Site ◽  
Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

scholarly journals Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

2021 ◽  
Vol 20 (3) ◽  
Author(s):  
Hairul Aini Hamzah ◽  
Rahmatullah Sirat ◽  
Mohammed Imad A. Mustafa Mahmud ◽  
Roesnita Baharudin
Keyword(s):  
Mutational Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Resistant Bacteria ◽  
Single Point Mutation ◽  
Phenotypic Resistance ◽  
Drug Effectiveness ◽  
E Coli

 Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

scholarly journals Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Courtney L Klaips ◽  
Megan L Hochstrasser ◽  
Christine R Langlois ◽  
Tricia R Serio
Keyword(s):  
Quality Control ◽  
Molecular Chaperone ◽  
Protein Misfolding ◽  
Elevated Temperatures ◽  
Control Factor ◽  
Control Mechanisms ◽  
Cellular Environment ◽  
Effective Activity ◽  
Spatial Quality

The proteostasis network has evolved to support protein folding under normal conditions and to expand this capacity in response to proteotoxic stresses. Nevertheless, many pathogenic states are associated with protein misfolding, revealing in vivo limitations on quality control mechanisms. One contributor to these limitations is the physical characteristics of misfolded proteins, as exemplified by amyloids, which are largely resistant to clearance. However, other limitations imposed by the cellular environment are poorly understood. To identify cell-based restrictions on proteostasis capacity, we determined the mechanism by which thermal stress cures the [PSI+]/Sup35 prion. Remarkably, Sup35 amyloid is disassembled at elevated temperatures by the molecular chaperone Hsp104. This process requires Hsp104 engagement with heat-induced non-prion aggregates in late cell-cycle stage cells, which promotes its asymmetric retention and thereby effective activity. Thus, cell division imposes a potent limitation on proteostasis capacity that can be bypassed by the spatial engagement of a quality control factor.

PU.1 - c-Jun Interactions Are Crucial for PU.1 Function in Myeloid Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3651-3651
Author(s):  
Boris Bartholdy ◽  
Yukiya Yamamoto ◽  
Erica Evans ◽  
John Crispino ◽  
Daniel G. Tenen
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Single Point ◽  
Point Mutations ◽  
Hematopoietic Stem ◽  
Myeloid Development ◽  
Factor C

Abstract Abstract 3651 Poster Board III-587 The Ets transcription factor PU.1 is a master regulator absolutely required for the differentiation of monocytes, macrophages, and B cells in the fetal liver and in the adult bone marrow. PU.1 drives hematopoietic differentiation partly through direct protein-protein interactions with other transcription factors, such as the AP-1 transcription factor c-Jun. We have shown that c-Jun can be recruited to promoters which do not include AP-1 binding sites, such as the MCSFR promoter, and act as a PU.1-dependent co-activator. To address the functional importance of this interaction, we identified and studied PU.1 point mutants that lost the capability to physically interact with c-Jun while retaining normal DNA binding affinity. These mutants failed to efficiently transactivate a PU.1 target reporter, and, more importantly, were unable to induce monocyte/macrophage differentiation of the PU.1-deficient immature myeloid 503 cell line. Subsequently, we have generated knock-in mouse models harboring these single point mutations by means of homologous recombination. The mutant mice phenotypically resemble PU.1-deficient mice, have an early block in hematopoiesis, and die perinatally. We show that the mutant PU.1 mRNA and protein is expressed in long-term and short-term hematopoietic stem cells, but that the maturation into lymphoid primed multipotent progenitor (LMPP) and later progenitor populations is severely blocked, leading to an almost complete loss of mature B, T and myeloid cells. Collectively, our data strongly suggest that the PU.1-c-Jun interaction is crucial for normal PU.1 function in vivo during murine fetal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

2017 ◽  
Vol 71 (4) ◽  
pp. 266-274
Author(s):  
Dagnija Ārgule ◽  
Indulis Cielēns ◽  
Regīna Renhofa ◽  
Arnis Strods
Keyword(s):  
Coat Protein ◽  
Self Assembly ◽  
Rna Binding ◽  
Single Point ◽  
Point Mutations ◽  
Coat Protein Sequence ◽  
Yeast Cells ◽  
Virus Like Particles ◽  
Binding Characteristics

Abstract Bacteriophage GA coat protein formed self-assembly competent virus-like particles (VLPs) have been expressed previously in bacterial and yeast cells. On the basis of our previous experiments on the yeast vector pESC-URA / S. cerevisiae system containing two oppositely oriented promoters, new constructions were created with point-mutations in coat protein to mimic phage MS2-like RNA binding characteristics. Simultaneously, the MS2 operator sequence was added to mRNA desired for packaging. After the introduction of single-point mutations (S87N, K55N, R43K) and double-point mutations (S87N + K55N and S87N + R43K), the coat protein’s ability to form VLPs was retained, but yield from cells was decreased. Exchange of the 87th Ser to Asn in coat protein sequence in combination with bacteriophage MS2 translational operator provided specific packaging of the gene of interest (GFP). Although non-specific nucleic acid sequences were packaged, the remarkable specificity for packaging of the gene of interest can be achieved using the described approach.

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