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Monomer plasmid DNA transforms Streptococcus pneumoniae
Abstract:
The covalently closed (CC) monomer form of plasmid pMV158 was found to transform pneumococcus (Streptococcus pneumoniae) and to do so with two-hit kinetics. The evidence came from analysis of the behavior of the transforming activity in fractions from preparative gel electrophoresis. Activity in the first major peak to elute (i) co-eluted with monomer CC as detected on analytical gels, (ii) banded as CC in dye-buoyancy gradients, (iii) sedimented with the velocity expected for monomer CC, and (iv) gave two-hit kinetics as functions of both concentration and time of exposure of the cells to DNA. A second major peak of activity behaved physically as though mostly due to dimer CC forms and gave single-hit response curves. Because almost no dimer was detectable optically on analytical gels of starting preparations, its specific activity was high relative to that of the monomers.
Insights
The covalently closed monomer of plasmid pMV158 transforms Streptococcus pneumoniae via two-hit kinetics. Dimer forms showed higher specific activity but were present in lower quantities.
Area of Science:
- Molecular Biology
- Microbial Genetics
- Plasmid Biology
Background:
- Plasmid DNA is crucial for bacterial transformation and genetic exchange.
- Understanding the physical forms of plasmids and their transforming efficiency is key to microbial genetics.
- Plasmid pMV158 is a well-characterized molecule used in studies of bacterial transformation.
Purpose of the Study:
- To investigate the transforming activity of different forms of plasmid pMV158 in Streptococcus pneumoniae.
- To determine the kinetics of transformation mediated by monomeric and dimeric forms of covalently closed (CC) plasmid DNA.
- To correlate the physical properties of plasmid DNA with its biological transforming efficiency.
Main Methods:
- Preparative gel electrophoresis to separate plasmid DNA forms.
- Analytical gel electrophoresis and dye-buoyancy gradients to characterize DNA species.
- Sedimentation velocity analysis to assess DNA molecular weight and conformation.
- Transformation assays with Streptococcus pneumoniae to measure biological activity.
- Kinetic analysis (concentration and time dependence) of transformation efficiency.
Main Results:
- The covalently closed (CC) monomer form of plasmid pMV158 demonstrated two-hit kinetics for transforming pneumococcus.
- A second peak of transforming activity, primarily composed of CC dimer forms, exhibited single-hit kinetics.
- Monomer CC was identified as the predominant species responsible for two-hit transformation kinetics.
- Dimeric CC forms possessed higher specific transforming activity compared to monomers, despite being less abundant.
Conclusions:
- Plasmid pMV158 transformation in Streptococcus pneumoniae is dependent on the DNA's physical form (monomer vs. dimer).
- Two-hit kinetics are characteristic of transformation mediated by monomeric CC plasmid DNA.
- The study elucidates the relationship between plasmid conformation, quantity, and transforming efficiency in bacteria.