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Molecular characterization of a plasmid-borne (pGT633) erythromycin resistance determinant (ermGT) from Lactobacillus
G W Tannock1, J B Luchansky, L Miller
1Department of Microbiology, University of Otago, Dunedin, New Zealand.
Abstract:
Lactobacillus reuteri 100-63 contains a 9.8 kb plasmid (pGT633) encoding resistance to the macrolide-lincosamide-streptogramin (MLS) type B antibiotics. The restriction endonucleases AccI, BamHI, BclI, EcoRI, EcoRV, HhaI, HindII, HpaI, KpnI, SalI, and SspI were employed to establish a physical map of pGT633. Next, genetic transfer experiments were conducted to establish the host range of pGT633. The results revealed that pGT633 replicated autonomously and encoded constitutive resistance to erythromycin in a variety of bacterial hosts, including Bacillus subtilis BD170, Streptococcus sanguis DL1, Staphylococcus aureus RN4220, and Enterococcus faecalis 19433, as well as several Lactobacillus spp. A 2.2 kb BamHI fragment of pGT633 containing the genetic determinant (ermGT) encoding resistance to erythromycin was cloned into Escherichia coli. Determination of the nucleotide sequence of ermGT revealed a methylase gene 731 bp in length that was highly related (ca. 81% nucleotide and ca. 78% amino acid identity) to the ermC gene from S. aureus plasmid pE194. The leader sequences of ermGT and ermC were identical except for a single base change at nt 51.
Insights
Lactobacillus reuteri plasmid pGT633 confers resistance to macrolide-lincosamide-streptogramin B antibiotics. The ermGT gene on this plasmid provides constitutive erythromycin resistance across various bacterial species.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Lactobacillus reuteri 100-63 harbors the pGT633 plasmid.
- This plasmid confers resistance to macrolide-lincosamide-streptogramin (MLS) type B antibiotics.
Purpose of the Study:
- To characterize the pGT633 plasmid from Lactobacillus reuteri.
- To determine the genetic basis of MLS B resistance encoded by pGT633.
- To investigate the host range and replication capabilities of pGT633.
Main Methods:
- Physical mapping of pGT633 using restriction endonucleases.
- Genetic transfer experiments to assess host range.
- Cloning and nucleotide sequencing of the erythromycin resistance determinant (ermGT).
Main Results:
- A physical map of the 9.8 kb pGT633 plasmid was established.
- pGT633 demonstrated autonomous replication and constitutive erythromycin resistance in diverse hosts including Bacillus subtilis, Streptococcus sanguis, Staphylococcus aureus, Enterococcus faecalis, and Lactobacillus spp.
- The ermGT gene, responsible for erythromycin resistance, was identified and sequenced, showing high similarity to ermC from Staphylococcus aureus plasmid pE194.
Conclusions:
- The pGT633 plasmid from Lactobacillus reuteri is a stable genetic element conferring MLS B resistance.
- The ermGT gene represents a novel determinant of erythromycin resistance with significant homology to known erm genes.
- pGT633 has a broad host range, suggesting potential for horizontal gene transfer.