scholarly journals Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

2019 ◽  
Author(s):  
Tomas Aparicio ◽  
Akos Nyerges ◽  
István Nagy ◽  
Csaba Pal ◽  
Esteban Martínez-García ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Mismatch Repair ◽  
Dominant Negative ◽  
Wild Type ◽  
Single Nucleotide ◽  
Important Species ◽  
E Coli ◽  
Different Types ◽  
Nucleotide Mismatch ◽  
The One

SUMMARYThe mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. On this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast’s URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G< C:C< G:A< C:A, A:A, G:G, T:T, T:G, A:C, C:T< G:T, T:C as the one prevalent in Pseudomonas putida. This information was vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.Originality-Significance StatementSingle-stranded DNA (ssDNA) recombineering has emerged in recent years as one of the most powerful technologies of genome editing in E. coli and other Enterobacteria. However, the efforts to expand the concept and the methods towards environmental microorganisms such as Pseudomonas putida have been limited thus far by several gaps in our fundamental knowledge of how nucleotide mismatch repair (MMR) operates in such non-model species. One critical bottleneck is the hierarchy of recognition of different types of base mispairings as well as the need of setting up strategies for counteracting MMR and thus enabling tolerance to all types of changes. The work presented here tackles both issues and makes P. putida amenable to sophisticated genetic manipulations that were impossible before.

scholarly journals Moraxella catarrhalis Expresses an Unusual Hfq Protein

2008 ◽  
Vol 76 (6) ◽  
pp. 2520-2530 ◽  
Author(s):  
Ahmed S. Attia ◽  
Jennifer L. Sedillo ◽  
Wei Wang ◽  
Wei Liu ◽  
Chad A. Brautigam ◽  
...  
Keyword(s):  
Moraxella Catarrhalis ◽  
Cell Envelope ◽  
Stress Sensitivity ◽  
Wild Type ◽  
Mobility Shift ◽  
Altered Expression ◽  
Reading Frame ◽  
E Coli ◽  
Different Types

ABSTRACT The Hfq protein is recognized as a global regulatory molecule that facilitates certain RNA-RNA interactions in bacteria. BLAST analysis identified a 630-nucleotide open reading frame in the genome of Moraxella catarrhalis ATCC 43617 that was highly conserved among M. catarrhalis strains and which encoded a predicted protein with significant homology to the Hfq protein of Escherichia coli. This protein, containing 210 amino acids, was more than twice as large as the Hfq proteins previously described for other bacteria. The C-terminal half of the M. catarrhalis Hfq protein was very hydrophilic and contained two different types of amino acid repeats. A mutation in the M. catarrhalis hfq gene affected both the growth rate of this organism and its sensitivity to at least two different types of stress in vitro. Provision of the wild-type M. catarrhalis hfq gene in trans eliminated these phenotypic differences in the hfq mutant. This M. catarrhalis hfq mutant exhibited altered expression of some cell envelope proteins relative to the wild-type parent strain and also had a growth advantage in a continuous flow biofilm system. The presence of the wild-type M. catarrhalis hfq gene in trans in an E. coli hfq mutant fully reversed the modest growth deficiency of this E. coli mutant and partially reversed the stress sensitivity of this E. coli mutant to methyl viologen. The use of an electrophoretic mobility shift assay showed that this M. catarrhalis Hfq protein could bind RNA derived from a gene whose expression was altered in the M. catarrhalis hfq mutant.

scholarly journals Supplementation of Intracellular XylR Leads to Coutilization of Hemicellulose Sugars

2012 ◽  
Vol 78 (7) ◽  
pp. 2221-2229 ◽  
Author(s):  
Dan Groff ◽  
Peter I. Benke ◽  
Tanveer S. Batth ◽  
Gregory Bokinsky ◽  
Christopher J. Petzold ◽  
...  
Keyword(s):  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Gene Insertion ◽  
Starting Point ◽  
Different Types ◽  
Important Challenge ◽  
Biomass Sugars ◽  
Hemicellulosic Sugars ◽  
Type Strains

ABSTRACTEscherichia colihas the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in usingE. colifor the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars. We demonstrate that, by increasing the intracellular levels of the transcription factor XylR, the preferential consumption of arabinose before xylose can be eliminated. In addition, XylR augmentation must be finely tuned for robust coutilization of these two hemicellulosic sugars. Using a novel technique for scarless gene insertion, an additional copy ofxylRwas inserted into thearaBADoperon. The resulting strain was superior at cometabolizing mixtures of arabinose and xylose and was able to produce at least 36% more ethanol than wild-type strains. This strain is a useful starting point for the development of anE. colibiocatalyst that can simultaneously convert all biomass sugars.

scholarly journals Dehalogenation of Dichlorobenzoates by Acidovorax sp. KKS102’s beta class Glutathione S-transferase and its Mutants

2021 ◽  
Vol 6 (1) ◽  
pp. 70-76
Author(s):  
D. Shehu ◽  
◽  
Zazali Alias
Keyword(s):  
Molecular Docking ◽  
Chloride Ion ◽  
Docking Study ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Molecular Docking Study ◽  
E Coli ◽  
Detection Assay ◽  
Glutathione S Transferases ◽  
The One

Glutathione s-transferases (GSTs) are ubiquitous family of enzymes well known for their detoxification function. Several different classes of the enzyme exist with beta class being the one specific to bacteria. Recently, the enzymes were found to exhibit other functions, in particular dehalogenation of some organic compounds. This property could be extremely useful especially in the bioremediation of some organochlorine pollutants. A beta class GST from Acidovorax sp. KKS102 designated as KKS-BphK was previously cloned and characterized. In this research, molecular docking study was first employed to investigate the possibility of binding of the protein to dichlorobenzoates; byproducts of polychlorobiphenyl degradation. The wild type enzyme together with other mutants were expressed using E. coli BL21 (DE3) cells and purified. The dehalogenation function of the enzymes against dichlorobenzoate derivatives was also investigated through chloride ion detection assay. The results of the molecular docking study indicated the possibility of binding of KKS-BphK to these substrates. Both the wild type and the mutants showed dehalogenation function against the model substrate 1-chloro-2,4- dinitrobenzene (CDNB). Furthermore, the enzymes also showed dehalogenation function against 2,4-dichlorobenzoate derivatives. However, in testing the activity of the enzymes toward 2,5- dichlorobenoate and 2,6-dichlorobenzoate, only K107T and A180P mutants showed some activity while the wild type and C10F mutant showed zero activity. The research indicates the usefulness of beta class GST in the dehalogenation of dichlorobenzoates in addition to their known function of dehalogenating monochlorobenzoates. Keywords: Glutathione s-transferase, Mutants, Beta class, dehalogenation, dichlorobenzoates.

scholarly journals Mouse Dspp frameshift model of human dentinogenesis imperfecta

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tian Liang ◽  
Yuanyuan Hu ◽  
Hong Zhang ◽  
Qian Xu ◽  
Charles E. Smith ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Dentinogenesis Imperfecta ◽  
Wild Type ◽  
Single Nucleotide ◽  
Dentinal Tubules ◽  
Reading Frame ◽  
Cellular Debris ◽  
Reparative Dentin ◽  
Dentin Formation

AbstractNon-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5′ mutations affecting an N-terminal targeting sequence and 3′ mutations that shift translation into the − 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp−1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp−1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp−1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5′ and 3′ Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

scholarly journals Identification of valine/leucine/isoleucine and threonine/alanine/glycine proton-spin systems of Escherichia coli adenylate kinase by selective deuteration and selective protonation

1991 ◽  
Vol 273 (2) ◽  
pp. 311-316 ◽  
Author(s):  
I Bock-Möbius ◽  
M Brune ◽  
A Wittinghofer ◽  
H Zimmermann ◽  
R Leberman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Adenylate Kinase ◽  
Spin Systems ◽  
Proton Spin ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Auxotrophic Strain ◽  
E Coli ◽  
Selective Deuteration ◽  
The One

Adenylate kinase from two types of Escherichia coli strains, a wild-type and a leucine-auxotrophic strain, was purified. On the one hand, growing the leucine-auxotrophic bacteria on a medium containing deuterated leucine yielded E. coli adenylate kinase with all leucine residues deuterated. On the other hand, by growing the wild-type bacteria on deuterated medium with phenylalanine, threonine and isoleucine present as protonated specimens, 80% randomly deuterated enzyme with protonated phenylalanine, threonine and isoleucine residues could be prepared. Use of these proteins enabled identification of the spin systems of these amino acid residues in the n.m.r. spectra of the protein.

scholarly journals Separation-of-Function Mutations inSaccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

1999 ◽  
Vol 19 (11) ◽  
pp. 7558-7567 ◽  
Author(s):  
Barbara Studamire ◽  
Gavrielle Price ◽  
Neal Sugawara ◽  
James E. Haber ◽  
Eric Alani
Keyword(s):  
Mismatch Repair ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Dominant Negative ◽  
Effector Protein ◽  
Mismatch Repair Genes ◽  
Gene Products ◽  
Wild Type ◽  
Repair Genes ◽  
Negative Phenotype

ABSTRACT Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3gene products are required to remove 3′ nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6,MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.

scholarly journals Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation.

1988 ◽  
Vol 8 (12) ◽  
pp. 5417-5424 ◽  
Author(s):  
G R Banks ◽  
S Y Taylor
Keyword(s):  
Dna Sequences ◽  
Gene Copy Number ◽  
Mutant Gene ◽  
Ustilago Maydis ◽  
Gene Copy ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Multiple Transcripts ◽  
Different Types

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

scholarly journals Spatiotemporal manipulation of the mismatch repair system of Pseudomonas putida accelerates phenotype emergence

2021 ◽  
Author(s):  
Lorena Fernández-Cabezón ◽  
Antonin Cros ◽  
Pablo I. Nikel
Keyword(s):  
Pseudomonas Putida ◽  
Mismatch Repair ◽  
Dominant Negative ◽  
Broad Host Range ◽  
General Strategy ◽  
Repair System ◽  
Dna Mismatch ◽  
Evolution Of Metabolic Pathways ◽  
Mismatch Repair System ◽  
Accelerate Evolution

ABSTRACTDeveloping complex phenotypes in industrially-relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed towards increasing mutation frequencies—yet the precise spatiotemporal control of mutagenesis processes continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in bacteria. Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutLE36K allele. Regulatable overexpression of mutLE36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in mutation frequencies and a heritable fixation of genome mutations. Following such accelerated mutagenesis-followed-by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes.

Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation

1988 ◽  
Vol 8 (12) ◽  
pp. 5417-5424
Author(s):  
G R Banks ◽  
S Y Taylor
Keyword(s):  
Dna Sequences ◽  
Gene Copy Number ◽  
Mutant Gene ◽  
Ustilago Maydis ◽  
Gene Copy ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Multiple Transcripts ◽  
Different Types

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

scholarly journals Natural IgA and TNFRSF13B polymorphism: a double edged sword fueling balancing selection

2021 ◽  
Author(s):  
Jeffrey L. Platt ◽  
Mayara Garcia de Mattos Barbosa ◽  
Daniel Huynh ◽  
Adam R. Lefferts ◽  
Juhi Katta ◽  
...  
Keyword(s):  
High Frequency ◽  
Virulence Genes ◽  
Balancing Selection ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Wild Type ◽  
E Coli ◽  
High Prevalence ◽  
Protective Antibodies ◽  
Dominant Negative Variant

AbstractTNFRSF13B encodes the “transmembrane-activator and CAML-interactor” (TACI) receptor, which drives plasma cell differentiation. Although TNFRSF13B supports host defense, dominant-negative TNFRSF13B alleles are common in humans and other species and only rarely associate with disease. We reasoned the high frequency of disruptive TNFRSF13B alleles reflects balancing selection, the loss of function conferring advantage in some settings. Testing that concept, we asked whether and how a common human dominant negative variant, TNFRSF13B A181E, imparts resistance to enteric pathogens. Mice engineered to express mono-allelic or bi-allelic A144E variants of tnrsf13B, corresponding to A181E exhibited striking resistance to pathogenicity and transmission of C. rodentium, a murine pathogen that models enterohemorrhagic E. coli, and resistance was principally owed to deficiency of natural IgA in the intestine. In wild type mice with gut IgA and in mutant mice fed IgA, binding of Ig induces expression of LEE encoded virulence genes, which confer pathogenicity and transmission. C. rodentium and probably some other enteric organisms thus appropriated binding of otherwise protective antibodies to signal induction of the virulence program and the high prevalence of TNFRSF13B dominant negative variants thus reflects balancing selection.

Sign in / Sign up

Export Citation Format

Share Document