scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. IV. DIRECTED EVOLUTION OF THE EBG REPRESSOR

Genetics ◽  
1978 ◽  
Vol 90 (4) ◽  
pp. 673-681
Author(s):  
Barry G Hall
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzyme Synthesis ◽  
Regulatory Function ◽  
Wild Type ◽  
Selection For ◽  
Selection Of

ABSTRACT In Escherichia coli, the wild-type repressor of ebg (evolved β-galactosidase) enzyme synthesis, specified by the ebgR  + gene, responds very weakly to lactulose (fructose-β-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 x 10-10. ebgR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Relationship between Ceftolozane-Tazobactam Exposure and Selection for Pseudomonas aeruginosa Resistance in a Hollow-Fiber Infection Model

2014 ◽  
Vol 58 (10) ◽  
pp. 6024-6031 ◽  
Author(s):  
Brian D. VanScoy ◽  
Rodrigo E. Mendes ◽  
Mariana Castanheira ◽  
Jennifer McCauley ◽  
Sujata M. Bhavnani ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Infection Model ◽  
Drug Resistant ◽  
Wild Type ◽  
Resistance Selection ◽  
Atcc Strain ◽  
Dosing Regimens ◽  
Selection For ◽  
The Relationship ◽  
Selection Of

ABSTRACTIt is important to understand the relationship between antibiotic exposure and the selection of drug resistance in the context of therapy exposure. We sought to identify the ceftolozane-tazobactam exposure necessary to prevent the amplification of drug-resistant bacterial subpopulations in a hollow-fiber infection model. TwoPseudomonas aeruginosachallenge isolates were selected for study, a wild-type ATCC strain (ceftolozane-tazobactam MIC, 0.5 mg/liter) and a clinical isolate (ceftolozane-tazobactam MIC, 4 mg/liter). The experiment duration was 10 days, and the ceftolozane-tazobactam dose ratio (2:1) and dosing interval (every 8 h) were selected to approximate those expected to be used clinically. The studied ceftolozane-tazobactam dosing regimens ranged from 62.5/31.25 to 2,000/1,000 mg per dose in step fold dilutions. Negative-control arms included no treatment and tazobactam at 500 mg every 8 h. Positive-control arms included ceftolozane at 1 g every 8 h and piperacillin-tazobactam dosed at 4.5 g every 6 h. For the wild-type ATCC strain, resistance was not selected by any ceftolozane-tazobactam regimen evaluated. For the clinical isolate, an inverted-U-shaped function best described the relationship between the amplification of a drug-resistant subpopulation and drug exposure. The least (62.5/31.25 mg) and most (2,000/1,000 mg) intensive ceftolozane-tazobactam dosing regimens did not select for drug resistance. Drug resistance selection was observed with intermediately intensive dosing regimens (125/62.5 through 1,000/500 mg). For the intermediately intensive ceftolozane-tazobactam dosing regimens, the duration until the selection for drug resistance increased with dose regimen intensity. These data support the selection of ceftolozane-tazobactam dosing regimens that minimize the potential for on-therapy drug resistance selection.

Growth of Escherichia coli on glucosamine 6-phosphate: selection of a constitutive hexose phosphate transport system mutant

1978 ◽  
Vol 24 (3) ◽  
pp. 203-208
Author(s):  
George W. Dietz Jr.
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Phosphate Transport ◽  
Wild Type ◽  
Hexose Phosphate ◽  
Selection Of

Glucosamine 6-phosphate was found to be a substrate but not an inducer for the hexose phosphate transport system of Escherichia coli. Wild-type cells grow very poorly on glucosamine 6-phosphate. A mutant was selected that will grow rapidly on glucosamine 6-phosphate because it contains a constitutive hexose phosphate transport system.

scholarly journals Emergence of Biofilm-Forming Subpopulations upon Exposure of Escherichia coli to Environmental Bacteriophages

2006 ◽  
Vol 72 (1) ◽  
pp. 956-959 ◽  
Author(s):  
Andrea Lacqua ◽  
Oskar Wanner ◽  
Teresa Colangelo ◽  
Maria Giovanna Martinotti ◽  
Paolo Landini
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Biofilm Formation ◽  
Type I ◽  
Selection For ◽  
Dps Protein ◽  
Derivatives Of ◽  
Selection Of

ABSTRACT Exposure of Escherichia coli MG1655 to environmental bacteriophages results in rapid selection for phage-tolerant subpopulations displaying increased biofilm formation. Analysis of one phage-tolerant strain revealed large amounts of the DNA-binding Dps protein in the outer membrane protein and production of fimbria-like structures. In dps and fimA mutant derivatives of MG1655, no selection of phage-tolerant bacteria upon exposure to bacteriophages occurred, suggesting a role for Dps and type I pili in bacteriophage tolerance.

scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. III EVOLUTION OF THE ebg REPRESSOR DURING SELECTION FOR ENHANCED LACTASE ACTIVITY

Genetics ◽  
1977 ◽  
Vol 85 (2) ◽  
pp. 193-201
Author(s):  
Barry G Hall ◽  
Norma D Clarke
Keyword(s):  
Regulatory Gene ◽  
Enzyme Synthesis ◽  
Lactase Activity ◽  
Wild Type ◽  
Regulatory Mutation ◽  
E Coli ◽  
Lactose Induction ◽  
A Cell ◽  
Lactose Utilization ◽  
Selection For

ABSTRACT The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg  + strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.

scholarly journals RstA, a two-component response regulator, plays important roles in multiple virulence-associated processes in enterohemorrhagic Escherichia coli O157:H7

Gut Pathogens ◽  
2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Yutao Liu ◽  
Shujie Li ◽  
Wendi Li ◽  
Peisheng Wang ◽  
Peng Ding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Acid Tolerance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Regulatory Function ◽  
Wild Type ◽  
Two Component

Abstract Background Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC O157 encounters varied microenvironments during infection, and can efficiently adapt to these using the two-component system (TCS). Recently, a functional TCS, RstAB, has been implicated in the regulation of virulence of several bacterial pathogens. However, the regulatory function of RstAB in EHEC O157 is poorly understood. This study aimed at providing insights into the global effects of RstA on gene expression in EHEC O157. Results In the present study, we analyzed gene expression differences between the EHEC O157 wild-type strain and a ΔrstA mutant using RNA-seq technology. Genes with differential expression in the ΔrstA mutant compared to that in the wild-type strain were identified and grouped into clusters of orthologous categories. RstA promoted EHEC O157 LEE gene expression, adhesion in vitro, and colonization in vivo by indirect regulation. We also found that RstA could bind directly to the promoter region of hdeA and yeaI to enhance acid tolerance and decrease biofilm formation by modulating the concentration of c-di-GMP. Conclusions In summary, the RstAB TCS in EHEC O157 plays a major role in the regulation of virulence, acid tolerance, and biofilm formation. We clarified the regulatory function of RstA, providing an insight into mechanisms that may be potential drug targets for treatment of EHEC O157-related infections.

scholarly journals Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

2007 ◽  
Vol 189 (19) ◽  
pp. 6839-6848 ◽  
Author(s):  
Joël Caillet ◽  
Monique Graffe ◽  
Flore Eyermann ◽  
Pascale Romby ◽  
Mathias Springer
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Dominant Negative ◽  
Zinc Binding ◽  
Regulatory Function ◽  
Dominant Negative Effect ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Zinc Binding Site ◽  
Negative Effect

ABSTRACT Escherichia coli threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNAThr isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNAThr by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.

scholarly journals An Improved Cloning Vector for Construction of Gene Replacements in Listeria monocytogenes

2003 ◽  
Vol 69 (5) ◽  
pp. 3020-3023 ◽  
Author(s):  
Guojie Li ◽  
S. Kathariou
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Plasmid Vector ◽  
Gene Replacement ◽  
Direct Selection ◽  
Genetic Studies ◽  
Food Borne ◽  
Food Borne Illness ◽  
Selection For ◽  
Selection Of

ABSTRACT Listeria monocytogenes is a gram-positive, facultative intracellular bacterium implicated in severe food-borne illness (listeriosis) in humans. The construction of well-defined gene replacements in the genome of L. monocytogenes has been instrumental to several genetic studies of the virulence and other attributes of the organism. Construction of such mutations by currently available procedures, however, tends to be labor intensive, and gene replacement mutants are sometimes difficult to recover due to lack of direct selection for the construct. In this study we describe the construction and use of plasmid vector pGF-EM, which can be conjugatively transferred from Escherichia coli S17-1 to L. monocytogenes and which provides the genetic means for direct selection of gene replacements.

ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

2015 ◽  
Vol 25 (6) ◽  
pp. 394-402 ◽  
Author(s):  
Taylor L. Fischer ◽  
Robert J. White ◽  
Katherine F.K. Mares ◽  
Devin E. Molnau ◽  
Justin J. Donato
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
E Coli ◽  
Enoyl Acp Reductase ◽  
Selection For

<b><i>Background/Aims:</i></b> We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in <i>Escherichia coli</i>. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. <b><i>Methods:</i></b> ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into <i>E. coli</i>, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. <b><i>Results:</i></b> Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into <i>E. coli</i>, whereas the mutant remained susceptible to triclosan<i>. </i>Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. <b><i>Conclusion:</i></b> ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

scholarly journals Development of Improved Cellulase Variants for the Conversion of Spent Mushroom Substrate Supplemented with Wheat Straw by Directed Evolution

2021 ◽  
Author(s):  
Valentina Mauriello ◽  
Anna Pennacchio ◽  
Irantzu Alegria Dallo ◽  
Laura Garcia Saez ◽  
Petri Ihalainen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Wheat Straw ◽  
Recombinant Expression ◽  
Scale Up ◽  
Wild Type ◽  
Spent Mushroom Substrate ◽  
Glucose Yield ◽  
Hydrolysis Of ◽  
Selection Of

Abstract To improve the Spent mushroom substrate (SMS) saccharification, cloning, recombinant expression in Escherichia coli and characterization of two new GH5 family cellulases (Cel1 and Cel2) were performed. Based on enzymes properties, Cel2 was selected for the generation of 30,000 random mutants by directed evolution in order to develop improved biocatalysts. Error-prone Polymerase Chain Reaction was used for diversity generation in cel2 gene and the screening for activity of mutants allowed selection of 63 improved variants that were subjected to a scale up production. Among these, 13 clones exhibited two-fold higher activity than Cel2 and a higher thermoresistance after 72h. The performances of these mutants in the hydrolysis of pretreated SMS/ wheat straw (40/60) were compared to the wild type Cel2 in conjunction with a commercial enzymatic mixture (MetZyme® SUNO™ BOOSTER 144). All the mutants exhibited a glucose yield two-fold or four fold higher than wild-type Cel2 after 72h of incubation.

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