Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

doi: 10.1073/pnas.1703331114

Figure Lengend Snippet: Chromosome location of ctsM with methylation RAATTY and GAATTC sites. ( A ) WT chromosome with RAATTY sites (green) and EcoRI sites (red) shown for DNA surrounding ctsM. ORFs are indicated by yellow arrows; those smaller than 200 aa are not shown. ctsM is shown in blue. Numbers under chromosomes indicate bases. ( B ) RAATTY sites and EcoRI sites in a ctsM::kan mutant.

Article Snippet: Here 0.1 µg of purified plasmid DNA (with or without M.EcoRI methylation) per reaction was radiolabeled with 20 µCi [α-32 P] dCTP using a Nick Translation Kit (Roche) in accordance with the manufacturer’s instructions.

Techniques: Methylation, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

doi: 10.1073/pnas.1703331114

Figure Lengend Snippet: DNA from ctsM::kan is degraded by ApoI and EcoRI. gDNA was purified from either WT C. jejuni strain DRH153 (kan R ) or ctsM::kan mutants. The DNA was then either mock-digested (lanes 1 and 2) or digested (lanes 3 and 4) with the restriction enzymes EcoRI ( Left ) or ApoI ( Right ). The DNA was then electrophoresed on an agarose gel and visualized using ethidium bromide. ApoI (RAATTY) should cut approximately four times as frequently as EcoRI (GAATTC).

Article Snippet: Here 0.1 µg of purified plasmid DNA (with or without M.EcoRI methylation) per reaction was radiolabeled with 20 µCi [α-32 P] dCTP using a Nick Translation Kit (Roche) in accordance with the manufacturer’s instructions.

Techniques: Purification, Agarose Gel Electrophoresis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

doi: 10.1073/pnas.1703331114

Figure Lengend Snippet: Digests of plasmids treated with M. EcoRI and PCR transformation data. ( A ) Diagram of pJMB202 and PCR products generated from this plasmid. GAATTC (M.EcoRI sites) are shown in red, and other RAATTY sites are shown in green. Small arrows indicate primers. ( B ) pJMB202 was treated as indicated. DNA was then electrophoresed and visualized using Gel Red. ( C ) PCR fragments derived from pJMB202 as indicated in A were methylated in vitro using M.EcoRI. ctsM + . The dashed line indicates the limit of detection. n.d. indicates that colonies were not detected. Error bars indicate SD.

Article Snippet: Here 0.1 µg of purified plasmid DNA (with or without M.EcoRI methylation) per reaction was radiolabeled with 20 µCi [α-32 P] dCTP using a Nick Translation Kit (Roche) in accordance with the manufacturer’s instructions.

Techniques: Polymerase Chain Reaction, Transformation Assay, Generated, Plasmid Preparation, Derivative Assay, Methylation, In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

doi: 10.1073/pnas.1703331114

Figure Lengend Snippet: Sites 3.2 kb away from an antibiotic cassette are not sufficient for transformation. ( A ) Chromosome locations of GAATTC and RAATTY sites at gyrA. WT chromosome with RAATTY sites (green) and EcoRI sites (red) shown for DNA surrounding ctsM. ORFs are indicated by yellow arrows; those smaller than 200 aa are not shown. gyrA is shown in blue. Numbers under chromosomes indicate bases. SpeI and BglI sites are indicated in purple. A point mutation in gyrA confers naladixic acid (NA) resistance. ( B ) gDNA was purified from either WT NA R cells or ctsM::kan NA R cells. The DNA was then digested with either SpeI or BglI and in vitro methylated with M.EcoRI. The treated DNA was used to transform WT (blue) or ctsM::kan . Error bars indicate SD. Data are the results of three biological replicates.

Article Snippet: Here 0.1 µg of purified plasmid DNA (with or without M.EcoRI methylation) per reaction was radiolabeled with 20 µCi [α-32 P] dCTP using a Nick Translation Kit (Roche) in accordance with the manufacturer’s instructions.

Techniques: Transformation Assay, Mutagenesis, Purification, In Vitro, Methylation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

doi: 10.1073/pnas.1703331114

Figure Lengend Snippet: In vitro methylation at the CtsM site RAATTY, but not at other sites, significantly enhances gDNA substrate capability. DNA was purified from either DRH153 (kan r ) or ctsM::kan cells. ctsM::kan DNA was then either mock-methylated or in vitro methylated with EcoRI MTase, TaqI MTase, or HpaII MTase. EcoRI MTase methylates GA m6 ATTC, TaqI methylates TCG m6 A, and HpaII methylates C m6 CGG. WT cells (blue) or ctsM::cm cells (yellow) were transformed with the methylated DNA. Transformation efficiency was calculated as described previously. Data represent three biological replicates, each of which contained three technical replicates. Error bars indicate SD. * P

Article Snippet: Here 0.1 µg of purified plasmid DNA (with or without M.EcoRI methylation) per reaction was radiolabeled with 20 µCi [α-32 P] dCTP using a Nick Translation Kit (Roche) in accordance with the manufacturer’s instructions.

Techniques: In Vitro, Methylation, Purification, Transformation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

doi: 10.1073/pnas.1703331114

Figure Lengend Snippet: In vitro methylation by M.EcoRI allows for transformation of plasmid DNA. ( A ) Plasmid map of pJMB202 with pertinent features denoted. Kan R refers to a kanamycin resistance cassette. It is inserted in astA. Red lines indicate EcoRI sites (GAATTC) that are methylated by M.EcoRI. Green lines indicate CtsM sites (RAATTY) that cannot be methylated by M.EcoRI. ( B ) WT cells were transformed with various types of DNA as indicated. These DNA fragments were then used to transform WT cells. Transformation efficiency was calculated as described previously. The data represent three biological replicates, each of which contained three technical replicates. n.d. indicates that colonies were not detected. Error bars indicate SD. * P

Article Snippet: Here 0.1 µg of purified plasmid DNA (with or without M.EcoRI methylation) per reaction was radiolabeled with 20 µCi [α-32 P] dCTP using a Nick Translation Kit (Roche) in accordance with the manufacturer’s instructions.

Techniques: In Vitro, Methylation, Transformation Assay, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

doi: 10.1073/pnas.1703331114

Figure Lengend Snippet: Methylated RAATTY promotes DNA uptake. DRH212 cells were incubated with radiolabled pJMB204 that had been methylated with M.EcoRI (+MT) or mock-methylated (−MT) for 1 h. The methyltransferase experiments were initiated with 5 ng/µL of DNA. Cells were centrifuged, washed, split, and either mock-treated or treated with DNase. Mock-treated cells contain the bound DNA (blue), and DNase-treated cells contain DNA that has been transported into the cells (yellow); 100 ng of DNA was used in each experiment. After treatment, cells were washed and resuspended in scintillation fluid. Total cpm was measured using a scintillation counter. Data are representative of the experiment repeated three times. Error bars indicate SD of three technical replicates. * P

Article Snippet: Here 0.1 µg of purified plasmid DNA (with or without M.EcoRI methylation) per reaction was radiolabeled with 20 µCi [α-32 P] dCTP using a Nick Translation Kit (Roche) in accordance with the manufacturer’s instructions.

Techniques: Methylation, Incubation