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Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu
Abstract:
We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.
Insights
Researchers identified host inhibition of replication (Hir) mutants in Escherichia coli, revealing new genes involved in bacteriophage Mu replication and survival. These findings enhance understanding of bacterial genetics and phage-host interactions.
Area of Science:
- Microbiology
- Bacteriology
- Molecular Biology
Background:
- Bacteriophage Mu is a versatile genetic element.
- Understanding host factors affecting Mu replication is crucial.
- Previous studies identified host inhibition of replication (Hir) mutants.
Purpose of the Study:
- To isolate and characterize new Hir mutants of Escherichia coli.
- To identify the genes responsible for host inhibition of replication.
- To investigate the role of these genes in bacteriophage Mu survival and replication.
Main Methods:
- Isolation of 142 Hir- mutants from Escherichia coli K-12 Mu cts Kil- lysogen.
- Genetic mapping using P1 transduction and Tn5/Tn10 insertions.
- Complementation assays with plasmids and phage to confirm gene identity.
- Analysis of DNA supercoiling in mutant strains.
- Assessment of bacteriophage Mu growth in mutant hosts.
Main Results:
- Identified mutations in himA and hip genes, crucial for Mu replication.
- Discovered novel mutations in gyrA and gyrB, encoding DNA gyrase.
- Observed altered plasmid DNA supercoiling in gyrase mutants.
- Demonstrated differential effects of gyrase mutations on Mu cts and Kil- phage growth.
Conclusions:
- himA and hip are essential for host inhibition of replication.
- DNA gyrase (GyrA and GyrB) plays a significant role in bacteriophage Mu replication.
- Mutations in DNA gyrase impact Mu phage production and survival.
- This study expands the known genetic landscape of Mu-host interactions.