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ppGpp concentration, growth without PBP2 activity, and growth-rate control in Escherichia coli
D Joseleau-Petit1, D Thévenet, R D'Ari
1Institut Jacques Monod Centre National de la Recherche Scientifique, Université Paris 7, France.
Abstract:
Escherichia coli strains partially induced for the stringent response are resistant to mecillinam, a beta-lactam antibiotic which specifically inactivates penicillin-binding protein 2, the key enzyme determining cell shape. We present evidence that mecillinam resistance occurs whenever the intracellular concentration of the nucleotide ppGpp (guanosine 3'-diphosphate 5'-diphosphate), the effector of the stringent response, exceeds a threshold level. First, the ppGpp concentration was higher in a mecillinam-resistant mutant than in closely related sensitive strains. Second, the ppGpp pool was controlled by means of a plasmid carrying a ptac-relA' gene coding for a hyperactive (p)ppGpp synthetase, RelA'; increasing the ppGpp pool by varying the concentration of lac operon inducer IPTG resulted in a sharp threshold ppGpp concentration, above which cells were mecillinam resistant. Third, the ppGpp pool was increased by using poor media; again, at the lowest growth rate studied, the cells were mecillinam resistant. In all experiments, cells with a ppGpp concentration above 140 pmoles/A600 were mecillinam resistant whereas those with lower concentrations were sensitive. We discuss a possible role for ppGpp as transcriptional activator of cell division genes whose products seem to become limiting in the presence of mecillinam, when cells form large spheres. We confirmed the well-known inverse correlation between growth rate and ppGpp concentration but, surprisingly, for a given growth rate, the ppGpp concentration was lower in poor medium than in richer medium in which RelA' is induced. We conclude that, for E. coli growing in poor media, the concentration of the nucleotide ppGpp is not the major growth rate determinant.
Insights
The stringent response nucleotide ppGpp (guanosine 3'-diphosphate 5'-diphosphate) confers mecillinam antibiotic resistance in Escherichia coli when its concentration exceeds a threshold. This finding impacts understanding of antibiotic resistance mechanisms and bacterial cell division.
Area of Science:
- Microbiology
- Molecular Biology
- Antibiotic Resistance
Background:
- Mecillinam, a beta-lactam antibiotic, targets penicillin-binding protein 2, crucial for Escherichia coli cell shape.
- The stringent response, regulated by ppGpp (guanosine 3 -diphosphate 5 -diphosphate), influences bacterial adaptation to stress.
- Previous studies indicate a link between stringent response and antibiotic resistance, but the precise role of ppGpp in mecillinam resistance is unclear.
Purpose of the Study:
- To investigate the role of intracellular ppGpp concentration in mecillinam resistance in Escherichia coli.
- To determine the threshold ppGpp level associated with mecillinam resistance.
- To explore the potential regulatory mechanisms linking ppGpp to cell division and antibiotic resistance.
Main Methods:
- Comparison of ppGpp levels in mecillinam-resistant and sensitive E. coli strains.
- Manipulation of intracellular ppGpp pools using a plasmid expressing a hyperactive RelA' enzyme and varying IPTG concentrations.
- Induction of ppGpp by growing cells in nutrient-poor media.
- Quantification of ppGpp concentration and assessment of mecillinam susceptibility.
Main Results:
- Mecillinam resistance in E. coli is directly correlated with intracellular ppGpp concentration, with a resistance threshold observed above 140 pmoles/A600.
- Elevated ppGpp levels, achieved through genetic manipulation or nutrient limitation, conferred mecillinam resistance.
- A surprising inverse correlation between growth rate and ppGpp concentration was observed, with lower ppGpp in poor media at similar growth rates.
Conclusions:
- Intracellular ppGpp concentration is a critical determinant of mecillinam resistance in Escherichia coli.
- The study proposes ppGpp may act as a transcriptional activator for cell division genes, becoming limiting in the presence of mecillinam.
- ppGpp concentration is not the sole determinant of growth rate in E. coli, particularly in nutrient-poor environments.