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The femC locus of Staphylococcus aureus required for methicillin resistance includes the glutamine synthetase operon
J Gustafson1, A Strässle, H Hächler
1Institute for Medical Microbiology, University of Zürich, Switzerland.
Abstract:
Tn551 insertional inactivation of femC is known to reduce methicillin resistance levels in methicillin-resistant and -susceptible Staphylococcus aureus. By use of cotransductional crosses, femC was mapped close to thrB on the SmaI-A fragment of the S. aureus NCTC 8325 chromosome. The Tn551 insertion femC::omega 2005 was found to interrupt an open reading frame coding for a putative protein of 121 amino acids which is highly similar to the glutamine synthetase repressors (GlnR) of Bacillus spp. Downstream of femC, an open reading frame highly similar to Bacillus sp. glutamine synthetases (GlnA) was found. Northern (RNA) blots probed with putative glnR or glnA fragments revealed that 1.7- and 1.9-kb transcripts characteristic of wild-type cells were replaced by less abundant 7.0- and 7.2-kb transcripts in the femC::omega 2005 mutant. Total glutamine synthetase activity was also decreased in the mutant strain; the addition of glutamine to defined media restored the wild-type methicillin resistance phenotype of the femC mutant. This result suggests that the omega 2005 insertion in glnR has a polar effect on glnA and that glnR and glnA are transcribed together as an operon. These results suggest that the loss of wild-type levels of glutamine synthetase and the consequent decrease in glutamine availability cause a decreased level of methicillin resistance.
Insights
Tn551 insertion in the femC gene reduces methicillin resistance in Staphylococcus aureus by affecting glutamine synthetase. Restoring glutamine levels reversed this effect, indicating a link between glutamine synthetase activity and antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Methicillin resistance in Staphylococcus aureus (MRSA) is a significant public health concern.
- The femC gene has been implicated in modulating methicillin resistance levels.
- Understanding the genetic basis of antibiotic resistance is crucial for developing new therapeutic strategies.
Purpose of the Study:
- To elucidate the function of the femC gene and its role in methicillin resistance.
- To investigate the genetic and molecular mechanisms underlying the effect of femC inactivation on antibiotic resistance.
- To identify potential therapeutic targets for combating MRSA.
Main Methods:
- Tn551 insertional mutagenesis to create femC mutants in Staphylococcus aureus.
- Cotransductional crosses for genetic mapping of the femC gene.
- Northern blotting to analyze gene transcript levels.
- Enzyme activity assays to measure glutamine synthetase activity.
- Phenotypic analysis of methicillin resistance in wild-type and mutant strains.
Main Results:
- Tn551 insertion in femC disrupted an open reading frame homologous to Bacillus glutamine synthetase repressor (GlnR).
- The mutation led to decreased expression of both glnR and a downstream gene homologous to Bacillus glutamine synthetase (GlnA), suggesting an operon structure.
- Total glutamine synthetase activity was reduced in the femC mutant.
- Supplementation with glutamine restored the wild-type methicillin resistance phenotype in the femC mutant.
Conclusions:
- The femC gene in Staphylococcus aureus encodes a glutamine synthetase repressor (GlnR) that is part of an operon with glnA.
- Reduced glutamine synthetase activity due to femC inactivation leads to decreased methicillin resistance.
- Glutamine availability is a key factor influencing methicillin resistance levels in Staphylococcus aureus, offering a potential target for therapeutic intervention.