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Properties and transforming activities of two plasmids in Streptococcus pneumoniae
Abstract:
Two plasmids from group B streptococcus were introduced into pneumococcus (Streptococcus pneumoniae) and examined for copy number, stability, and some features of the process by which they transform pneumococcal recipients. The 3.6 Mdal pMV158 (tet) was present at a minimum of 12 to 16 copies per chromosome and was never observed to be cured. The 20 Mdal pIP501 (cat erm) had a minimum copy number of 3 to 4 per chromosome and was lost spontaneously at a frequency near 0.03 per division. The presence of novobiocin increased this frequency 2 to 3-fold. Competence for chromosomal transformation and the membrane endonuclease needed for normal DNA entry were required for plasmid transformation. Plasmid transformants segregated transformed cells one generation ahead of chromosomal transformants. Both single and multiple hit components of the transformation reaction kinetics were observed, but the latter could not be seen in the presence of competing chromosomal DNA. The major of the transforming activity behaved as covalently closed circular DNA in dye-buoyancy gradients. Although most of the activity for both plasmids sedimented in sucrose gradients more rapidly than did monomeric closed circular DNA, a significant fraction was found at a position suggesting that it may have been due to monomeric plasmids.
Insights
Group B streptococcus plasmids were introduced into pneumococcus (Streptococcus pneumoniae), revealing distinct copy numbers, stability, and transformation requirements. Plasmid transformation in pneumococcus necessitates competence and specific DNA entry mechanisms.
Area of Science:
- Molecular Biology
- Microbiology
- Bacterial Genetics
Background:
- Group B Streptococcus (GBS) plasmids are crucial for understanding streptococcal genetics.
- Pneumococcus (Streptococcus pneumoniae) is a significant human pathogen requiring genetic manipulation tools.
- Investigating interspecies plasmid transfer provides insights into bacterial transformation mechanisms.
Purpose of the Study:
- To assess the feasibility and characteristics of introducing GBS plasmids into S. pneumoniae.
- To determine plasmid copy number, stability, and segregation in a new host.
- To elucidate the requirements and kinetics of plasmid transformation in pneumococcus.
Main Methods:
- Introduction of two GBS plasmids (pMV158 and pIP501) into S. pneumoniae.
- Quantification of plasmid copy number per chromosome using quantitative methods.
- Assessment of plasmid stability and spontaneous loss rates.
- Evaluation of transformation competence and DNA entry mechanisms.
- Analysis of transformation kinetics and DNA forms using sedimentation and buoyancy gradients.
Main Results:
- The pMV158 plasmid exhibited high copy number (12-16/chromosome) and was stably maintained.
- The pIP501 plasmid showed lower copy number (3-4/chromosome) and was spontaneously lost at a frequency of ~0.03/division, increased by novobiocin.
- Plasmid transformation required pneumococcal competence and membrane endonuclease activity, with faster segregation than chromosomal transformants.
- Transformation activity was primarily associated with covalently closed circular DNA, with some evidence of monomeric forms.
Conclusions:
- GBS plasmids can be stably maintained in S. pneumoniae, with varying copy numbers and stability.
- Plasmid transformation in pneumococcus shares requirements with chromosomal transformation, indicating conserved entry pathways.
- The study provides a foundation for using GBS plasmids as tools for genetic manipulation in S. pneumoniae.