A functional lagging strand origin does not stabilize plasmid pMV158 inheritance in Escherichia coli

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Plasmid rolling circle replication produces single-stranded DNA intermediates.
• Efficient conversion of single-stranded to double-stranded plasmid DNA is crucial for stability.
• This conversion depends on the functionality of the lagging strand origin (sso).

Purpose of the Study:

• To investigate the functionality of the streptococcal plasmid pMV158's two lagging strand origins (ssos) in different bacterial hosts.
• To assess the impact of heterologous ssos on plasmid stability across host species.
• To understand the host-specific requirements for sso function in plasmid replication.

Main Methods:

• Comparative analysis of pMV158 plasmid stability in *Streptococcus pneumoniae* and *Escherichia coli*.
• Functional characterization of pMV158's native ssos in both gram-positive and gram-negative hosts.
• Introduction and evaluation of the coliphage f1 sso in a pMV158 derivative within both bacterial species.

Main Results:

• The two native ssos of pMV158 function efficiently in *S. pneumoniae* but poorly in *E. coli*, correlating with plasmid stability.
• A pMV158 derivative lacking its native ssos is unstable in both hosts.
• The coliphage f1 sso functions effectively in *E. coli* but shows no activity in *S. pneumoniae*, and its presence did not confer stability to the pMV158 derivative in either host.

Conclusions:

• Lagging strand origin (sso) functionality is host-specific, influencing plasmid stability.
• The pMV158 plasmid's stability is dependent on its native ssos, which are optimized for *S. pneumoniae*.
• Heterologous ssos may not be functionally interchangeable between phylogenetically distant bacterial hosts, highlighting species-specific replication machinery interactions.