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Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33
1Department of Microbiology, University of Tennessee, Knoxville 37996.
Abstract:
Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.
Insights
Lactobacillus sp. strain 100-33
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Lactobacillus sp. strain 100-33 exhibits resistance to macrolides, lincosamides, and streptogramin B (MLSR).
- This strain possesses major and minor plasmids, including the 18 kbp plasmid pLAR33.
Purpose of the Study:
- To investigate the genetic basis of MLSR in Lactobacillus sp. strain 100-33.
- To identify and characterize the specific plasmid(s) and genetic determinant(s) responsible for antibiotic resistance.
Main Methods:
- Protoplast regeneration to derive antibiotic-sensitive strains.
- Plasmid DNA isolation, cloning using EcoRV restriction enzyme into pBR322.
- Transformation of Escherichia coli HB101 with cloned DNA.
- Electroporation of resistant plasmid DNA into sensitive strains.
- Endonuclease digestion and Southern blot analysis of plasmid DNA.
Main Results:
- A sensitive derivative, strain ES1, was obtained by losing major plasmids including pLAR33.
- The MLSR determinant was cloned from pLAR33 on a 3-kbp fragment and expressed resistance in E. coli.
- The resistance determinant was localized to approximately 1 kbp and appears to have its own promoter.
- Electroporation of strain ES1 with parental plasmid DNA restored MLSR phenotype and plasmid complement.
Conclusions:
- The MLSR determinant is located on the plasmid pLAR33.
- Major plasmids in strain 100-33 are multimeric, deleted forms of a single archetypal extrachromosomal element.
- Understanding plasmid-mediated antibiotic resistance in Lactobacillus is crucial for food and probiotic applications.