Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli
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Abstract:
The alpha-hemolytic Escherichia coli strain PM152 harbors three transmissible plasmids, which have molecular weights of 65 X 10(6) (pA152), 41 X 10(6) pHly152), and 32 X 10(6) (pC152). Plasmids pHly152 and pC152 belong to incompatibility groups J2 and N, respectively. By transforming E. coli K-12 with isolated plasmids, we showed that the genetic determinant required for hemolysis was located entirely on plasmid pHly152, and a physical map of this plasmid was constructed. By transposon mutagenesis, a deoxyribonucleic acid segment of about 3.5 X 10(6) daltons was identified as being essential for hemolysis. Most of the EcoRI and HindIII fragments of the hemolytic plasmid pHly152 were cloned by using pACYC184 and RSF2124 as vectors. Two classes of Tn3-induced hemolysis-negative mutants could be complemented by recombinant plasmids carrying fragments from the hemolysis region of pHly152, whereas a third class could be restored to hemolytic activity only by recombination between the mutant plasmids and a suitable recombinant deoxyribonucleic acid. These data suggest that there are at least three clustered cistrons which are required for hemolysis. Other EcoRI and HindIII fragments of pHly152 were identified as being essential for replication, incompatibility, transfer, and restriction.
Insights
The alpha-hemolytic Escherichia coli strain PM152 has three plasmids, with the hemolysis gene located on pHly152. This plasmid contains at least three clustered cistrons essential for hemolytic activity.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Escherichia coli strain PM152 exhibits alpha-hemolytic activity.
- The strain contains three transmissible plasmids: pA152 (65x10^6 Da), pHly152 (41x10^6 Da), and pC152 (32x10^6 Da).
- Plasmids pHly152 and pC152 belong to incompatibility groups J2 and N, respectively.
Purpose of the Study:
- To identify the genetic determinant responsible for hemolysis in Escherichia coli PM152.
- To construct a physical map of the hemolytic plasmid pHly152.
- To elucidate the genetic organization of the hemolytic determinant.
Main Methods:
- Plasmid isolation and transformation into E. coli K-12.
- Transposon mutagenesis using Tn3.
- Cloning of EcoRI and HindIII fragments of pHly152 into vectors pACYC184 and RSF2124.
- Complementation analysis of hemolysis-negative mutants.
Main Results:
- The genetic determinant for hemolysis is located on plasmid pHly152.
- A DNA segment of approximately 3.5x10^6 daltons on pHly152 is essential for hemolysis.
- Complementation studies suggest at least three clustered cistrons are required for hemolytic activity.
- Other fragments of pHly152 are involved in replication, incompatibility, transfer, and restriction.
Conclusions:
- The hemolytic activity of Escherichia coli PM152 is encoded by genes located on the pHly152 plasmid.
- At least three clustered cistrons within a 3.5x10^6 dalton DNA region are essential for hemolysis.
- The pHly152 plasmid also carries determinants for replication, incompatibility, transfer, and restriction.