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There have been several conflicting reports regarding the cellular localisation of Type I restriction-modification (R-M) enzymes. Consequently we decided to investigate the presence/absence of the EcoR124I restriction endonuclease (ENase), and its associated DNA methyltransferase (MTase), in the periplasmic and cytoplasmic fractions of Escherichia coli. This was simplified following the overproduction of the MTase and the production of polyclonal antibodies to this enzyme and, more recently, the overproduction of the endonuclease and production of polyclonal antibodies to both the HsdR subunit and the ENase. Production and localisation of MTase was monitored using the un-induced expression system and a plasmid carrying hsdM and hsdS genes expressed from their natural promoter (pCFD12). The MTase was always found in the cytoplasmic fraction (not surprisingly) but NOT in the periplasmic fraction. This experiment was repeated using a plasmid producing the endonuclease from the natural promoters (pCP1005) and the endonuclease was found in both periplasmic and cytoplasmic fractions.
Dark = cytoplasm Order of loading = control/REase; control MTase At about the same time that this work was being carried out we were also attempting to isolate temperature-sensitive mutations of the EcoR124I R-M system using EMS mutagenesis, and it was observed that cold-storage of bacteria carrying the above plasmid (pCP1005) resulted in loss of restriction activity. Furthermore, treatment of bacteria carrying pCP1005 with the non-mutagenic compound DMSO (dimethyl sulfoxide) also produced restriction-deficiency. However, a plasmid carrying the related system EcoKI was not subject to this effect:
Only the EcoR124I R-M system on pCP1005 is affected by DMSO.
This led us to speculate that the two systems may be located in different environments within the bacterial cell. It was interesting to note that the natural plasmid carrying the EcoR124I R-M system was not DMSO-sensitive, and that this plasmid has been shown to effect the level of production of the OmpF protein. it is tempting to speculate that this is related to some type of transport mechanism and that the HsdR subunit of EcoR124I is responsible for transport of the EcoR124I ENase to the periplasm and that it is this transport which is responsible for the different locations of EcoKI and EcoR124I. To localise the Type I Restriction-Modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques (Holubova et al., 2000). The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when co-produced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinisation of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. Holubova et al postulated that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.
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Pale
= periplasm
This work was extended in