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Published on: August 21, 2016
Cellular localization and dynamics of the Mrr type IV restriction endonuclease of Escherichia coli
Anirban Ghosh1, Ioannis Passaris, Mehari Tesfazgi Mebrhatu
1Department of Microbial and Molecular Systems (M2S), Laboratory of Food Microbiology, KU Leuven, B-3001 Leuven, Belgium and Department of Chemistry, KU Leuven, B-3001 Leuven, Belgium.
Abstract:
In this study, we examined the intracellular whereabouts of Mrr, a cryptic type IV restriction endonuclease of Escherichia coli K12, in response to different conditions. In absence of stimuli triggering its activity, Mrr was found to be strongly associated with the nucleoid as a number of discrete foci, suggesting the presence of Mrr hotspots on the chromosome. Previously established elicitors of Mrr activity, such as exposure to high (hydrostatic) pressure (HP) or expression of the HhaII methyltransferase, both caused nucleoid condensation and an unexpected coalescence of Mrr foci. However, although the resulting Mrr/nucleoid complex was stable when triggered with HhaII, it tended to be only short-lived when elicited with HP. Moreover, HP-mediated activation of Mrr typically led to cellular blebbing, suggesting a link between chromosome and cellular integrity. Interestingly, Mrr variants could be isolated that were specifically compromised in either HhaII- or HP-dependent activation, underscoring a mechanistic difference in the way both triggers activate Mrr. In general, our results reveal that Mrr can take part in complex spatial distributions on the nucleoid and can be engaged in distinct modes of activity.
Insights
The Mrr restriction enzyme in Escherichia coli K12 localizes to nucleoid hotspots. Its distribution changes with high pressure or HhaII methyltransferase, revealing distinct activation mechanisms and impacts on cellular integrity.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Mrr is a cryptic type IV restriction endonuclease in Escherichia coli K12.
- Its intracellular localization and activity regulation are not fully understood.
Purpose of the Study:
- To investigate the intracellular localization of Mrr in Escherichia coli K12 under various conditions.
- To elucidate the distinct activation mechanisms of Mrr by high pressure and HhaII methyltransferase.
Main Methods:
- Microscopy to observe Mrr localization and nucleoid structure.
- Genetic manipulation to isolate Mrr variants with altered activation properties.
Main Results:
- Mrr localizes to discrete foci on the nucleoid in unstressed cells.
- High pressure and HhaII expression induce nucleoid condensation and Mrr foci coalescence.
- HP-induced Mrr activation leads to transient complexes and cellular blebbing, while HhaII-induced activation results in stable complexes.
- Specific Mrr variants show differential responses to HP and HhaII, indicating distinct activation pathways.
Conclusions:
- Mrr exhibits complex spatial organization on the Escherichia coli nucleoid.
- Distinct triggers like high pressure and HhaII methyltransferase engage Mrr through mechanistically different pathways.
- Mrr activation can impact chromosome and cellular integrity, suggesting a role beyond simple DNA restriction.
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