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A complex attenuator regulates inducible resistance to macrolides, lincosamides, and streptogramin type B antibiotics
Abstract:
Macrolide-lincosamide-streptogramin B resistance specified by Streptococcus sanguis plasmid pAM77 involves an adenine methylase, whose synthesis, demonstrable both phenotypically and by analysis of methionine-labeled proteins made in Bacillus subtilis minicells, is inducible by erythromycin, lincomycin, and streptogramin type B antibiotics. Localization of the methylase structural gene, including its control region in DNA fragments obtained with restriction endonucleases, has been deduced from DNA blot experiments with characterized target and probe DNAs from other streptococci, combined with DNA sequence analysis and comparison of the putative streptococcal methylase sequence with that of a cognate methylase in staphylococcal plasmid pE194. The streptococcal methylase migrates electrophoretically in polyacrylamide gels with the mobility of a 29,000-dalton protein. The sequence organization of the putative streptococcal methylase mRNA leader sequence partially resembles its staphylococcal counterpart and can support a similar mechanism of secondary structure rearrangement leading to methylase synthesis. The deduced 5' leader sequence preceding the pAM77 methylase structural gene sequence comprises approximately 155 nucleotides within which one can identify a putative control peptide 36 amino acid residues in length (in contrast to 19 in the pE194 peptide) and at least 14 possible classes of overlapping inverted complementary repeat sequences (in contrast to 3 in the pE194 control region), one of which can sequester the sequence AGGAG 7 nucleotides upstream from the putative (methionine) start codon of the streptococcal methylase. Comparison of the pAM77 and pE194 methylase amino acid sequences and their respective nucleotide sequences shows 51% conservation of amino acid residues (124 of 244) and 59% conservation of nucleotide residues (433 of 738), which suggests a common origin for the two methylase structural gene sequences. Differences in mRNA base composition associated with conserved amino acid residues occur mostly in the third nucleotide ("wobble") position of codons and may reflect adaptation of methylase genes to optimal expression in host cells with differing codon use patterns.
Insights
Macrolide-lincosamide-streptogramin B resistance in Streptococcus sanguis is mediated by an inducible adenine methylase. This methylase gene shares significant sequence similarity with a staphylococcal counterpart, suggesting a common evolutionary origin.
Area of Science:
- Molecular Microbiology
- Antimicrobial Resistance
- Bacterial Genetics
Background:
- Macrolide-lincosamide-streptogramin B (MLSB) resistance in bacteria is a significant clinical concern.
- Plasmid-mediated resistance mechanisms, such as those involving adenine methylases, contribute to the spread of antibiotic resistance.
- Streptococcus sanguis plasmid pAM77 carries a gene responsible for MLSB resistance.
Purpose of the Study:
- To characterize the adenine methylase responsible for MLSB resistance encoded by Streptococcus sanguis plasmid pAM77.
- To investigate the regulation and genetic organization of the methylase gene and its control region.
- To compare the streptococcal methylase with its staphylococcal counterpart to understand evolutionary relationships.
Main Methods:
- Phenotypic and biochemical analysis of methylase synthesis in Bacillus subtilis minicells.
- DNA blot experiments using restriction endonucleases and characterized DNA fragments for gene localization.
- DNA sequence analysis and comparison of streptococcal and staphylococcal methylase genes and their regulatory regions.
Main Results:
- The synthesis of the adenine methylase is inducible by erythromycin, lincomycin, and streptogramin B antibiotics.
- The methylase gene and its control region were localized using DNA blot experiments and sequence analysis.
- The streptococcal methylase shares 51% amino acid and 59% nucleotide sequence conservation with the staphylococcal methylase from plasmid pE194, indicating a common origin.
Conclusions:
- The adenine methylase encoded by pAM77 is a key determinant of MLSB resistance in Streptococcus sanguis.
- The regulatory region of the streptococcal methylase exhibits structural similarities to its staphylococcal counterpart, suggesting a conserved mechanism for translational control.
- The high degree of sequence conservation implies a shared evolutionary history for these MLSB resistance methylase genes.