dnae1  (CH Instruments)

Journal: Nucleic Acids Research

Article Title: Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum

doi: 10.1093/nar/gkad602

Figure Lengend Snippet: Engineering of the DNA replication proofreading elements for increased spontaneous mutagenesis in C. glutamicum . ( A ) Effects of deleting dnaQ homologs ( cgl0247 , cgl1289 and cgl2116 ) on spontaneous mutagenesis. The wild-type strain was used as a control. ( B ) Schematic illustration of the DnaE1-mediated DNA replication proofreading and the increased mutagenesis caused by DnaE1 variant with impaired PHP domain (PHP*, highlighted in grey). ( C ) Effects of D20N and D223N mutations on spontaneous mutagenesis. ( D ) Coordination of three Zn ions (green dot) and one water molecule (red dot) in the PHP active site of the wild-type DnaE1 (D223) and the variant with D223N substitution. Yellow and green dotted lines represent coordination bond (between D223 and the Zn ion) and hydrogen bond (between D223 and the water molecule), respectively. The red box highlights the key 223 residue, the Zn ion and the water molecule. ( E ) Effects of IPTG-inducible overexpression of DnaE1 D223N on spontaneous mutagenesis. Relative expression level of dnaE1 was determined by RT-PCR. Values and error bars represent means and standard deviations ( n = 3).

Article Snippet: C. glutamicum strains ATCC 13032/pXMJ19-DnaE1 D223N and ATCC 13032/pXMJ19-NucS E111L were cultivated in TSB medium supplemented with chloramphenicol and different concentrations of IPTG with an initial OD 600 of 0.1 in 24-well plates at 30°C and with shaking at 800 rpm.

Techniques: Mutagenesis, Control, Variant Assay, Residue, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum

doi: 10.1093/nar/gkad602

Figure Lengend Snippet: Genome evolution of C. glutamicum for improving methanol tolerance by using DnaE1 D223N -based mutator. ( A ) Schematic illustration of the strain evolution and mutant isolation process. ( B ) Genome evolution of strains with (red line) or without (blue line) DnaE1 D223N -based mutator. The black line represents the elevated methanol concentration during evolution. The evolution was conducted using 250 ml shake flasks and the OD 600nm values were measured by using a standard 1 cm cuvette and spectrophotometer. ( C ) Growth of the wild-type strain and evolved mutants with 70 g/l methanol. Growth in the absence of methanol stress is shown in . ( D ) The type of mutations detected in the evolved methanol-tolerant mutants. ( E ) Effects of the 35 single-site mutations on methanol tolerance. For the first round of screening, the wild-type strain and 35 recombinant strains harboring single-site mutations were cultivated with 45 g/l methanol in 96-well plates. Three mutations leading to significant improvement (log 2 Fold change ≥ 0.5, P value < 0.05) in methanol tolerance are highlight in red dots. ( F ) Growth of the three recombinant strains harboring single-site mutations with 45 g/l methanol. ( G ) Growth of the three recombinant strains harboring single-site mutations with 70 g/l methanol. Growth in the absence of methanol stress is shown in . ( H ) Emergence and enrichment of the three key mutations responsible for methanol tolerance during evolution. The target gene fragments were amplified by PCR using genomic DNA of the cells collected from each passage and analyzed by NGS. Except for the evolution experiment, all the growth tests were conducted using an automatic microbial growth curve analyzer. Values and error bars represent means and standard deviations ( n = 3).

Article Snippet: C. glutamicum strains ATCC 13032/pXMJ19-DnaE1 D223N and ATCC 13032/pXMJ19-NucS E111L were cultivated in TSB medium supplemented with chloramphenicol and different concentrations of IPTG with an initial OD 600 of 0.1 in 24-well plates at 30°C and with shaking at 800 rpm.

Techniques: Mutagenesis, Isolation, Concentration Assay, Spectrophotometry, Recombinant, Amplification

Journal: Nucleic Acids Research

Article Title: Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum

doi: 10.1093/nar/gkad602

Figure Lengend Snippet: Engineering of the DNA replication proofreading elements for increased spontaneous mutagenesis in C. glutamicum . ( A ) Effects of deleting dnaQ homologs ( cgl0247 , cgl1289 and cgl2116 ) on spontaneous mutagenesis. The wild-type strain was used as a control. ( B ) Schematic illustration of the DnaE1-mediated DNA replication proofreading and the increased mutagenesis caused by DnaE1 variant with impaired PHP domain (PHP*, highlighted in grey). ( C ) Effects of D20N and D223N mutations on spontaneous mutagenesis. ( D ) Coordination of three Zn ions (green dot) and one water molecule (red dot) in the PHP active site of the wild-type DnaE1 (D223) and the variant with D223N substitution. Yellow and green dotted lines represent coordination bond (between D223 and the Zn ion) and hydrogen bond (between D223 and the water molecule), respectively. The red box highlights the key 223 residue, the Zn ion and the water molecule. ( E ) Effects of IPTG-inducible overexpression of DnaE1 D223N on spontaneous mutagenesis. Relative expression level of dnaE1 was determined by RT-PCR. Values and error bars represent means and standard deviations ( n = 3).

Article Snippet: To start the mutator-assisted evolution, the DnaE1 D223N overexpressing plasmid was transformed into C. glutamicum ATCC 13032 and three colonies were verified by PCR and subjected to continuous cultivation under gradually increased methanol concentration.

Techniques: Mutagenesis, Control, Variant Assay, Residue, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum

doi: 10.1093/nar/gkad602

Figure Lengend Snippet: Genome evolution of C. glutamicum for improving methanol tolerance by using DnaE1 D223N -based mutator. ( A ) Schematic illustration of the strain evolution and mutant isolation process. ( B ) Genome evolution of strains with (red line) or without (blue line) DnaE1 D223N -based mutator. The black line represents the elevated methanol concentration during evolution. The evolution was conducted using 250 ml shake flasks and the OD 600nm values were measured by using a standard 1 cm cuvette and spectrophotometer. ( C ) Growth of the wild-type strain and evolved mutants with 70 g/l methanol. Growth in the absence of methanol stress is shown in . ( D ) The type of mutations detected in the evolved methanol-tolerant mutants. ( E ) Effects of the 35 single-site mutations on methanol tolerance. For the first round of screening, the wild-type strain and 35 recombinant strains harboring single-site mutations were cultivated with 45 g/l methanol in 96-well plates. Three mutations leading to significant improvement (log 2 Fold change ≥ 0.5, P value < 0.05) in methanol tolerance are highlight in red dots. ( F ) Growth of the three recombinant strains harboring single-site mutations with 45 g/l methanol. ( G ) Growth of the three recombinant strains harboring single-site mutations with 70 g/l methanol. Growth in the absence of methanol stress is shown in . ( H ) Emergence and enrichment of the three key mutations responsible for methanol tolerance during evolution. The target gene fragments were amplified by PCR using genomic DNA of the cells collected from each passage and analyzed by NGS. Except for the evolution experiment, all the growth tests were conducted using an automatic microbial growth curve analyzer. Values and error bars represent means and standard deviations ( n = 3).

Article Snippet: To start the mutator-assisted evolution, the DnaE1 D223N overexpressing plasmid was transformed into C. glutamicum ATCC 13032 and three colonies were verified by PCR and subjected to continuous cultivation under gradually increased methanol concentration.

Techniques: Mutagenesis, Isolation, Concentration Assay, Spectrophotometry, Recombinant, Amplification

Journal: Nucleic Acids Research

Article Title: Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum

doi: 10.1093/nar/gkad602

Figure Lengend Snippet: Engineering of new DnaE1 variants by site-saturation mutation of D20 and D223. ( A ) Schematic illustration of CRISPR-mediated ssDNA recombineering for site-saturation mutation and screening of variants capable of increasing spontaneous mutagenesis. ( B ) Effects of amino acid substitution at D20 of the chromosomal dnaE1 copy on spontaneous mutagenesis. ( C ) Effects of amino acid substitution at D223 of the chromosomal dnaE1 copy on spontaneous mutagenesis. ( D ) Effects of episomal overexpression of DnaE1 with D20 mutations on spontaneous mutagenesis. ( E ) Effects of episomal overexpression of DnaE1 with D223 mutations on spontaneous mutagenesis. Values and error bars represent means and standard deviations ( n = 3).

Article Snippet: To start the mutator-assisted evolution, the DnaE1 D223N overexpressing plasmid was transformed into C. glutamicum ATCC 13032 and three colonies were verified by PCR and subjected to continuous cultivation under gradually increased methanol concentration.

Techniques: Mutagenesis, CRISPR, Over Expression

Journal: Nucleic Acids Research

Article Title: Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum

doi: 10.1093/nar/gkad602

Figure Lengend Snippet: Development and characterization of the unary and binary mutators. ( A ) Effects of episomal overexpression of the unary mutators based on DnaE1 or NucS variants and the binary mutator combining DnaE1 D20R and NucS E111L on spontaneous mutagenesis. The amino acid substitutions of these DnaE1 and NucS variants are shown in . Values and error bars represent means and standard deviations ( n = 3). ( B ) The mutation type of representative unary and binary mutators. For each genetic mutator and the wild-type strain control, the rpoB sequences of 70–100 rifampicin-resistant colonies were analyzed.

Article Snippet: To start the mutator-assisted evolution, the DnaE1 D223N overexpressing plasmid was transformed into C. glutamicum ATCC 13032 and three colonies were verified by PCR and subjected to continuous cultivation under gradually increased methanol concentration.

Techniques: Over Expression, Mutagenesis, Control

Journal: Frontiers in Microbiology

Article Title: Error-prone DnaE2 Balances the Genome Mutation Rates in Myxococcus xanthus DK1622

doi: 10.3389/fmicb.2017.00122

Figure Lengend Snippet: Expression levels of the dnaE1 and dnaE2 genes at different time points under the nutritional and developmental conditions. (A) M. xanthus DK1622 cells grown in the CTT growth medium. (B) The DK1622 strain grown on the TPM development plate. (C) . The YL1601 mutant cells grown on TPM plate. The expression of the dnaE1 gene in CTT at 12 h of incubation was set as 100, and the others were the relative expressions. The error bars represent the standard deviation of three independent experiments.

Article Snippet: The single DnaE gene of E. coli was in the DnaE1 group, clearly distant from the DnaE2 group; and the two DnaE-group trees had highly similar topologies.

Techniques: Expressing, Mutagenesis, Incubation, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Error-prone DnaE2 Balances the Genome Mutation Rates in Myxococcus xanthus DK1622

doi: 10.3389/fmicb.2017.00122

Figure Lengend Snippet: Overexpression of the dnaE2 gene in M. xanthus cells and cellular growth abilities. (A) Expressions of dnaE1 and dnaE2 in YL1605 (DK1622 att::dnaE2 ) and YL1604 (DK1622 att::Kan ) in CTT growth medium; (B) Expressions of dnaE1 and dnaE2 in the two mutants on TPM plate; (C) Growth curves of YL1605 and YL1604 in CTT liquid medium supplemented with kanamycin (40 μg/ml). OD600 values were measured every 4 h until 60 h of incubation. The data were analyzed using two-way ANOVA. The error bars represent the standard deviation of three independent experiments. The expressions of dnaE1 and dnaE2 in (A) and (B) of this Figure were the relative expressions, compared with the expression of the dnaE1 gene in CTT at 12 h of nutritional condition of Figure .

Article Snippet: The single DnaE gene of E. coli was in the DnaE1 group, clearly distant from the DnaE2 group; and the two DnaE-group trees had highly similar topologies.

Techniques: Over Expression, Incubation, Standard Deviation, Expressing

Journal: Frontiers in Microbiology

Article Title: Error-prone DnaE2 Balances the Genome Mutation Rates in Myxococcus xanthus DK1622

doi: 10.3389/fmicb.2017.00122

Figure Lengend Snippet: Structure modeling of DK1622 DnaE1 and DnaE2 (A) and comparison of the domain conservation of myxobacterial dnaE1 and dnaE2 (B) . (A) Three-dimensional structures showing different domains in different colors (left panels) and evolutionary conservation of surface amino acids (right panels); (B) The dN/dS values of dnaE1 and dnaE2 and the different domains of those sequenced myxobacteria.

Article Snippet: The single DnaE gene of E. coli was in the DnaE1 group, clearly distant from the DnaE2 group; and the two DnaE-group trees had highly similar topologies.

Techniques: Comparison