scholarly journals Regulatory Revolution: Evolving the “Anti-LacI” Repressor

Cell ◽  
2011 ◽  
Vol 146 (3) ◽  
pp. 350-352 ◽  
Author(s):  
Christopher J. Marx
Keyword(s):  
Laci Repressor

scholarly journals Artificial Control of Nitrate Respiration through the lac Promoter Permits the Assessment of Oxygen-Mediated Posttranslational Regulation of the nar Operon in Pseudomonas aeruginosa

2007 ◽  
Vol 189 (17) ◽  
pp. 6501-6505 ◽  
Author(s):  
Chris E. Noriega ◽  
Vandana Sharma ◽  
John J. Rowe
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrate Reduction ◽  
Methyl Viologen ◽  
Reductase Activity ◽  
Nitrate Respiration ◽  
Regulation Of Transcription ◽  
Posttranslational Regulation ◽  
Laci Repressor ◽  
Lac Promoter ◽  
Artificial Control

ABSTRACT In this study, oxygen and nitrate regulation of transcription and subsequent protein expression of the unique narK1K2GHJI respiratory operon of Pseudomonas aeruginosa were investigated. Under the control of PLAC, P. aeruginosa was able to transcribe nar and subsequently express methyl viologen-linked nitrate reductase activity under aerobic conditions without nitrate. Modulation of PLAC through the LacI repressor enabled us to assess both transcriptional and posttranslational regulation by oxygen during physiological whole-cell nitrate reduction.

scholarly journals Regulation of Endothelial-Specific Transgene Expression by the LacI Repressor Protein In Vivo

PLoS ONE ◽  
2014 ◽  
Vol 9 (4) ◽  
pp. e95980 ◽  
Author(s):  
Susan K. Morton ◽  
Daniel J. Chaston ◽  
Brett K. Baillie ◽  
Caryl E. Hill ◽  
Klaus I. Matthaei
Keyword(s):  
Transgene Expression ◽  
Repressor Protein ◽  
Laci Repressor

scholarly journals The Energetics of Molecular Adaptation in Transcriptional Regulation

2019 ◽  
Author(s):  
Griffin Chure ◽  
Manuel Razo-Mejia ◽  
Nathan M. Belliveau ◽  
Tal Einav ◽  
Zofii A. Kaczmarek ◽  
...  
Keyword(s):  
Free Energy ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Coarse Graining ◽  
Binding Domain ◽  
Biophysical Model ◽  
Model Parameters ◽  
Biophysical Parameters ◽  
Binding Domains ◽  
Laci Repressor

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.SummaryWe present a biophysical model of allosteric transcriptional regulation that directly links the location of a mutation within a repressor to the biophysical parameters that describe its behavior. We explore the phenotypic space of a repressor with mutations in either the inducer binding or DNA binding domains. Using the LacI repressor in E. coli, we make sharp, falsifiable predictions and use this framework to generate a null hypothesis for how double mutants behave given knowledge of the single mutants. Linking mutations to the parameters which govern the system allows for quantitative predictions of how the free energy of the system changes as a result, permitting coarse graining of high-dimensional data into a single-parameter description of the mutational consequences.

scholarly journals Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon

2021 ◽  
Author(s):  
Kedar Karkare ◽  
Huei-Yi Lai ◽  
Ricardo B R Azevedo ◽  
Tim F Cooper
Keyword(s):  
Promoter Region ◽  
Potential Benefit ◽  
Constitutive Expression ◽  
High Rate ◽  
Mutation Rates ◽  
Lac Operon ◽  
Historical Contingency ◽  
Fitness Effect ◽  
Fluctuating Environments ◽  
Laci Repressor

Abstract Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.

scholarly journals Predictive shifts in free energy couple mutations to their phenotypic consequences

2019 ◽  
Vol 116 (37) ◽  
pp. 18275-18284 ◽  
Author(s):  
Griffin Chure ◽  
Manuel Razo-Mejia ◽  
Nathan M. Belliveau ◽  
Tal Einav ◽  
Zofii A. Kaczmarek ◽  
...  
Keyword(s):  
Free Energy ◽  
Dna Binding ◽  
Binding Domain ◽  
Model Parameters ◽  
Free Energies ◽  
Epistatic Interactions ◽  
Binding Domains ◽  
Quantitative Accuracy ◽  
Laci Repressor ◽  
At Will

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find that the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor ofEscherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.

scholarly journals CcpA coordinates central metabolism and biofilm formation in Staphylococcus epidermidis

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3458-3468 ◽  
Author(s):  
Marat R. Sadykov ◽  
Torsten Hartmann ◽  
Theodoric A. Mattes ◽  
Megan Hiatt ◽  
Naja J. Jann ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Protein A ◽  
Tca Cycle ◽  
Gram Positive Bacteria ◽  
Catabolite Control Protein A ◽  
The Tca Cycle ◽  
Laci Repressor ◽  
Modest Effect ◽  
Catabolite Control

Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR–LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.

scholarly journals The secondary messenger ppGpp interferes with cAMP-CRP regulon by promoting CRP acetylation in Escherichia coli

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0259067
Author(s):  
Chunghwan Ro ◽  
Michael Cashel ◽  
Llorenç Fernández-Coll
Keyword(s):  
Escherichia Coli ◽  
Promoter Region ◽  
Promoter Sequence ◽  
Growth Conditions ◽  
Lysine Acetylation ◽  
Acetyl Phosphate ◽  
Secondary Messenger ◽  
Minimal Media ◽  
Operon Expression ◽  
Laci Repressor

The cAMP-CRP regulon coordinates transcription regulation of several energy-related genes, the lac operon among them. Lactose, or IPTG, induces the lac operon expression by binding to the LacI repressor, and releasing it from the promoter sequence. At the same time, the expression of the lac operon requires the presence of the CRP-cAMP complex, which promotes the binding of the RNA polymerase to the promoter region. The modified nucleotide cAMP accumulates in the absence of glucose and binds to the CRP protein, but its ability to bind to DNA can be impaired by lysine-acetylation of CRP. Here we add another layer of control, as acetylation of CRP seems to be modified by ppGpp. In cells grown in glycerol minimal media, ppGpp seems to repress the expression of lacZ, where ΔrelA mutants show higher expression of lacZ than in WT. These differences between the WT and ΔrelA strains seem to depend on the levels of acetylated CRP. During the growth in minimal media supplemented with glycerol, ppGpp promotes the acetylation of CRP by the Nε-lysine acetyltransferases YfiQ. Moreover, the expression of the different genes involved in the production and degradation of Acetyl-phosphate (ackA-pta) and the enzymatic acetylation of proteins (yfiQ) are stimulated by the presence of ppGpp, depending on the growth conditions.

scholarly journals Target Immunity of the Tn3-Family Transposon Tn4430 Requires Specific Interactions between the Transposase and the Terminal Inverted Repeats of the Transposon

2010 ◽  
Vol 192 (16) ◽  
pp. 4233-4238 ◽  
Author(s):  
Emilien Nicolas ◽  
Michaël Lambin ◽  
Bernard Hallet
Keyword(s):  
Site Selection ◽  
Inverted Repeats ◽  
Chimeric Proteins ◽  
Specific Interactions ◽  
Wild Type ◽  
Independent Manner ◽  
Terminal Inverted Repeats ◽  
Target Site Selection ◽  
Laci Repressor

ABSTRACT Specificity of the Tn4430 target immunity signal was examined by fusing the transposase TnpA to the LacI repressor of Escherichia coli. The resulting chimeric proteins failed to impose immunity to DNA targets carrying copies of the lacO operator, though they were proficient in lacO binding in vivo and remained responsive to wild-type immunity conferred by the Tn4430 inverted repeat end. Intriguingly, the presence of lacO repeats within the target was found to strongly influence target site selection by Tn4430, but in a LacI-independent manner.

scholarly journals Engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of Escherichia coli

1994 ◽  
Vol 298 (1) ◽  
pp. 189-195 ◽  
Author(s):  
C Fraipont ◽  
M Adam ◽  
M Nguyen-Distèche ◽  
W Keck ◽  
J Van Beeumen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Protein ◽  
Molar Ratio ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
Operator Expression ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Laci Repressor

Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3.

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