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Identification of a new gene in the streptococcal plasmid pLS1: the rnaI gene
P Acebo1, A M Hernández-Arriaga, M G Kramer
1Centro de Investigaciones Biológicas, CSIC, Velázquez, 144, Madrid, E-28006, Spain.
Abstract:
The streptococcal plasmid pMV158 has been reported to harbor five genes: three involved in initiation of rolling circle replication and its control (copG, repB, and maII), one involved in conjugative mobilization (mobM), and the fifth one specifying constitutive resistance to tetracycline (tet). The mobM gene was removed in the construction of the pMV158-derivative plasmid pLS1, which was used in this study. By in vitro transcription assays, primer extension experiments, and construction of mutations, here we demonstrate the presence of another gene (the sixth of pMV158), termed maI, which is transcribed in opposite orientation with respect to the plasmid mRNAs, to render RNA I. The 5'-end of RNA I has an 8-nt sequence which is complementary to a region of the lagging-strand origin (ssoA) comprising a 6-nt consensus sequence involved in lagging strand synthesis. This suggested that RNA I could influence, positively or negatively, initiation of lagging strand synthesis from the pLS1-ssoA. However, plasmids defective in RNA I synthesis exhibited a phenotype similar to the wild type in terms of efficiency of replication from the ssoA and copy number. When the maI gene was cloned into a compatible plasmid, the resulting recombinants did not exhibit incompatibility toward plasmids with the pLS1 replicon. Thus, RNA I does not seem to be a true copy number control element. We postulate that transcription from the maI promoter may facilitate extrusion of the hairpin of the plasmid double-strand origin, which is the target of the initiator of replication protein.
Insights
Researchers identified a sixth gene, maI, on the streptococcal plasmid pMV158, which produces RNA I. This RNA I does not appear to regulate plasmid copy number, contrary to initial hypotheses.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The streptococcal plasmid pMV158 contains genes for replication, mobilization, and tetracycline resistance.
- Previous studies identified five genes, but the function of a potential sixth gene was unexplored.
Purpose of the Study:
- To identify and characterize a potential sixth gene on plasmid pMV158.
- To investigate the role of the transcribed RNA I in plasmid replication and copy number control.
Main Methods:
- In vitro transcription assays.
- Primer extension experiments.
- Construction and analysis of gene mutations.
- Cloning experiments into compatible plasmids.
Main Results:
- A sixth gene, maI, was identified, transcribed to produce RNA I in the opposite orientation to other plasmid mRNAs.
- RNA I's 5' end is complementary to the lagging-strand origin (ssoA), suggesting a regulatory role.
- Plasmids lacking functional maI or RNA I showed no significant difference in replication efficiency or copy number compared to wild-type.
- Cloning maI into a compatible plasmid did not induce plasmid incompatibility.
Conclusions:
- RNA I is not a primary determinant of plasmid copy number control for pMV158.
- The maI gene and its product, RNA I, may play a role in facilitating the initiation of replication by aiding the extrusion of the double-strand origin hairpin.