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RecA-Mediated gene conversion and aminoglycoside resistance in strains heterozygous for rRNA
T Prammananan1, P Sander, B Springer
1Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Abstract:
Clinical resistance to aminoglycosides in general is due to enzymatic drug modification. Mutational alterations of the small ribosomal subunit rRNA have recently been found to mediate acquired resistance in bacterial pathogens in vivo. In this study we investigated the effect of 16S rRNA heterozygosity (wild-type [wt] and mutant [mut] operons at position 1408 [1408wt/1408mut]) on aminoglycoside resistance. Using an integrative vector, we introduced a single copy of a mutated rRNA operon (1408 A-->G) into Mycobacterium smegmatis, which carries two chromosomal wild-type rRNA operons; the resultant transformants exhibited an aminoglycoside-sensitive phenotype. In contrast, introduction of the mutated rRNA operon into an M. smegmatis rrnB knockout strain carrying a single functional chromosomal wild-type rRNA operon resulted in aminoglycoside-resistant transformants. Subsequent analysis by DNA sequencing and RNase protection assays unexpectedly demonstrated a homozygous mutant genotype, rRNAmut/rRNAmut, in the resistant transformants. To investigate whether RecA-mediated gene conversion was responsible for the aminoglycoside-resistant phenotype in the rRNAwt/rRNAmut strains, recA mutant strains were generated by allelic exchange techniques. Transformation of the recA rrnB M. smegmatis mutant strains with an integrative vector expressing a mutated rRNA operon (Escherichia coli position 1408 A-->G) resulted in transformants with an aminoglycoside-sensitive phenotype. Subsequent analysis showed stable heterozygosity at 16S rRNA position 1408 with a single wild-type allele and a single resistant allele. These results demonstrate that rRNA-mediated mutational resistance to aminoglycosides is recessive.
Insights
Mutational resistance to aminoglycoside drugs in bacteria is recessive. This study found that resistance only occurs when both copies of the 16S ribosomal RNA (rRNA) gene are mutated, not when only one is.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Clinical resistance to aminoglycosides often stems from enzymatic drug modification.
- Recent findings indicate that mutations in the small ribosomal subunit rRNA can confer acquired resistance in bacterial pathogens.
- Understanding the genetic basis of this resistance is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the impact of 16S rRNA heterozygosity on aminoglycoside resistance.
- To determine the genetic mechanisms underlying acquired aminoglycoside resistance mediated by rRNA mutations.
- To elucidate the dominance or recessiveness of rRNA-mediated aminoglycoside resistance.
Main Methods:
- Introduction of mutated rRNA operons into Mycobacterium smegmatis using integrative vectors.
- Generation of recA mutant strains via allelic exchange techniques.
- Analysis of transformants using DNA sequencing and RNase protection assays to determine genotype and phenotype.
Main Results:
- Introduction of a single mutated rRNA operon into wild-type M. smegmatis resulted in an aminoglycoside-sensitive phenotype.
- Introduction of a mutated rRNA operon into a strain with a single wild-type rRNA operon unexpectedly led to a homozygous mutant genotype and aminoglycoside resistance.
- Transformation of recA mutant strains with a mutated rRNA operon resulted in stable heterozygosity and an aminoglycoside-sensitive phenotype, indicating resistance is recessive.
Conclusions:
- Aminoglycoside resistance mediated by 16S rRNA mutations is a recessive trait.
- RecA-mediated gene conversion plays a role in the development of aminoglycoside resistance in heterozygous strains.
- The genetic context (homozygous vs. heterozygous rRNA operons) is critical for determining aminoglycoside resistance phenotype.