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Expression of the mobM gene of the streptococcal plasmid pMV158 in Lactococcus lactis subsp. lactis
M E Farías1, E Grohmann, M Espinosa
1Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Abstract:
The streptococcal plasmid pMV158 is not auto-transferable, but it can be mobilised between bacteria by the use of functions supplied by plasmids of the pIP501/pAM beta 1 family. Plasmid pMV158 encodes a protein, MobM, which is involved in its mobilisation. This process initiates when MobM specifically cleaves supercoiled pMV158 plasmid DNA at the origin of transfer, oriT. Plasmid pMV158 has been transferred to Lactococcus lactis by conjugation aided by plasmid pAM beta 1. In the lactococcal host, MobM-mediated specific pMV158-relaxed molecules were detected. The intracellular amount of MobM has been quantified by immunoblot analyses and shown to be about 3500 molecules per cell. In the same host, we have mapped the initiation point of transcription of mobM. Transcription of this gene is directed from a promoter with an extended--10 region which overlaps with the pMV158-oriT.
Insights
The streptococcal plasmid pMV158 requires helper functions for bacterial transfer. Its mobilization protein, MobM, cleaves plasmid DNA at the origin of transfer, facilitating movement into new hosts like Lactococcus lactis.
Area of Science:
- Bacteriology
- Molecular Biology
- Genetics
Background:
- The streptococcal plasmid pMV158 is not self-transmissible.
- Mobilization requires functions from helper plasmids like pAM beta 1.
- The MobM protein is crucial for pMV158 mobilization.
Purpose of the Study:
- To investigate the mechanism of pMV158 mobilization by MobM.
- To characterize MobM function in a new host, Lactococcus lactis.
- To analyze the regulation of mobM gene expression in L. lactis.
Main Methods:
- Conjugation experiments to transfer pMV158 into Lactococcus lactis using pAM beta 1.
- Detection of MobM-mediated DNA cleavage products (relaxed molecules).
- Immunoblot analysis to quantify MobM intracellular levels.
- Mapping the transcription initiation site of the mobM gene.
Main Results:
- Plasmid pMV158 was successfully transferred to Lactococcus lactis.
- MobM-mediated cleavage of pMV158 DNA into relaxed molecules was observed in L. lactis.
- Approximately 3500 MobM molecules per cell were quantified.
- The mobM gene promoter was mapped, showing an extended -10 region overlapping with pMV158 origin of transfer (oriT).
Conclusions:
- MobM is essential for pMV158 mobilization, acting by cleaving the plasmid DNA at oriT.
- The mobM gene is expressed in L. lactis, and its promoter overlaps the oriT region.
- This overlap suggests a potential regulatory mechanism linking gene expression to plasmid transfer initiation.