Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA703949 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13762607_1.fastq.gz | Fastqsanger.gz | |||
| SRR13762607_2.fastq.gz | Fastqsanger.gz | |||
| SRR13762608_1.fastq.gz | Fastqsanger.gz | |||
| SRR13762608_2.fastq.gz | Fastqsanger.gz | |||
| SRR13762609_1.fastq.gz | Fastqsanger.gz |
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Project description:Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854hsdM exhibited a 5-fold increase in transformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.
2021-04-14 | GSE167217 | GEO
Project description:Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.
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Project description:In order to study the mechanism of co-inactivation of RB1 and TP53 in the transformation of lung adenocarcinoma to small cell lung cancer, we established the NCI-H1975 cell line with RB1 knockdown.NCI-H1975 cells were cultured and infected with lentivirus expressing RB1-shRNA (n=3) or pLKO.1-shRNA (n=3), We then performed transcriptome sequencing (RNA-seq) on the above cells.
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