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Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range
Abstract:
The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.
Insights
Streptococcal plasmids pMV158 and pLS1 replicate in diverse bacteria, conferring tetracycline resistance. A specific deletion mutation enhanced plasmid copy number by altering regulatory RNA, impacting replication and resistance protein expression.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Streptococcal plasmids like pMV158 and pLS1 exhibit broad-host-range replication, functioning in both Gram-positive and Gram-negative bacteria.
- These plasmids confer tetracycline resistance, a crucial trait in microbial genetics and biotechnology.
- Understanding plasmid replication and copy number control is vital for genetic engineering and molecular studies.
Purpose of the Study:
- To investigate the replication mechanism and copy number control of streptococcal plasmids pMV158 and pLS1.
- To identify the genes and regulatory elements responsible for plasmid replication and tetracycline resistance.
- To analyze the DNA sequence of pLS1 and characterize its functional components.
Main Methods:
- Plasmid replication and copy number determination in various bacterial species (Streptococcus pneumoniae, Bacillus subtilis, Escherichia coli).
- Genetic analysis involving functional gene studies (polA, recA) and site-directed mutagenesis (deletion mutation).
- DNA sequencing of pLS1 to identify open reading frames, transcriptional signals, and regulatory elements. Protein identification via specific labeling.
Main Results:
- Plasmid pLS1 copy numbers varied across species: 24 in S. pneumoniae, 4 in B. subtilis, and 4 in E. coli.
- Replication in E. coli necessitated functional polA and recA genes.
- A 332 bp deletion in pLS1 (copy-control mutation) doubled its copy number in all tested species.
- Sequence analysis revealed four open reading frames, including repB (replication protein) and tet (tetracycline resistance).
- A potential inhibitory RNA was identified in the deleted region, suggesting a role in copy number regulation.
- The pLS1 tet gene showed homology to Gram-positive tet genes but differed from Gram-negative counterparts.
Conclusions:
- The repB gene encodes a essential 23,000 Mr replication protein.
- The tet gene encodes a 50,000 Mr hydrophobic protein conferring tetracycline resistance.
- A regulatory RNA likely controls pLS1 copy number, with its deletion leading to increased plasmid levels.
- The tet gene's distinct characteristics suggest specific evolutionary pathways in Gram-positive bacteria.