Localization of the Type I Restriction–Modification Enzyme EcoKI in the Bacterial Cell

Holubová, I.; Vejsadová, Štěpánka; Weiserová, Marie; Firman, Keith

doi:10.1006/bbrc.2000.2375

Cited by 11 publications

(14 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, it has been shown that HsdR of EcoKI is proteolysed by ClpXP protease only when it is a part of an assembled translocation‐proficient EcoKI complex, preferentially in cytoplasmic fraction, while in the membrane fraction HsdR is not degraded (Doronina & Murray, 2001). This is in keeping with the present results showing that HsdR of EcoKI is phosphorylated at a threonine residue by an as yet unidentified cytoplasmic kinase only as part of a complex REase and with the previous finding that the membrane‐associated fraction of EcoKI is responsible for the residual restriction activity of the enzyme under a condition of restriction alleviation (Holubova et al , 2000; Doronina & Murray, 2001). It may be speculated that HsdR of EcoKI requires phosphorylation/dephosphorylation for better susceptibility to ClpXP proteolysis.…”

Section: Resultssupporting

confidence: 93%

“…The cytoplasm-localized HsdR subunits might be phosphorylated in the domain, which in the case of the membrane-associated EcoKI complex, is embedded in the cytoplasmic membrane. Trypsinization of spheroplasts revealed that solely the HsdR subunit of the complex enzyme was digested by the protease, as was demonstrated by the disappearance of the band corresponding to the HsdR polypeptide that was replaced by two smaller polypeptides T1 and T2 (Holubova et al, 2000). Using NETPHOS 2.0 Server (Blom et al, 1999), 16 threonine residues were predicted as possible targets for phosphorylation: 11 present on fragment T1 and five on fragment T2.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit

Cajthamlová

Šišáková

Weiser

et al. 2007

FEMS Microbiology Letters

View full text Add to dashboard Cite

Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I - representatives of IA, IB, and IC families, respectively - was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated. Further, evidence is presented that HsdR is phosphorylated in vivo only when coproduced with HsdM and HsdS subunits - as part of assembled EcoKI restriction endonuclease, while the individually produced HsdR subunit is not phosphorylated. In vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of a Type I R-M enzyme reported.

show abstract

Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit

Cajthamlová

Šišáková

Weiser

et al. 2007

FEMS Microbiology Letters

View full text Add to dashboard Cite

show abstract

“…As it was reported previously, using an immunocytochemical microscopy technique EcoRI endonuclease molecules were localized predominantly within the envelope part of E. coli cells, including the periplasmic space (Kohring & Mayer, 1987). Similarly, HsdR subunit of the heteromultimeric EcoKI type I R-M enzyme was found to be associated with the cytoplasmic membrane, having access to the periplasmic space (Holubova et al, 2000). Thus, it could be assumed that periplasmic localization is well-adapted for the restriction of foreign DNA.…”

Section: The Rationale Of the Projectsupporting

confidence: 58%

Periplasmic expression of a restriction endonuclease in Escherichia coli and its effect on the antiviral activity of the host

et al. 2019

View full text Add to dashboard Cite

One possible mechanism preventing phage infection of the bacterial cells is related to the presence of an effective restriction-modification system (R-M) which allows restriction of the invading DNA. However, there are some limitations to the absolute restriction of foreign DNA. Since there is a serious conflict between increase in the restriction-modification genes expression level and cell viability, we examined the antiviral effect of EcoRI restriction endonuclease after its translocation to the periplasmic space of the cell. We assumed that such reconstructed R-M system could be able to degrade foreign DNA at the stage of its passage through the cell envelope of Gram-negative bacteria, before its penetration into the bacterial cytoplasm. The Tat secretion pathway of Escherichia coli was used to export R.EcoRI fused to the TorA leader peptide across the cytoplasmic membrane. However, although we observed a huge accumulation of the TorAss-R.EcoRI pre-protein in the cytoplasm the Tat system did not provide an efficient transport across the cytoplasmic membrane. Moreover, our data strongly suggest that endonuclease cannot function under the conditions prevailing in periplasmic space, therefore, the transported endonuclease could not contribute to an increase in restriction properties of the host.

show abstract

“…The mechanism by which RA occurs appears to be family dependent, and the proteolysis of HsdR observed for EcoKI was not observed with EcoR124I. The explanation for this was unclear for a number of years until a series of elegant experiments by Holubova et al (35,36) showed first that the EcoKI holoenzyme was localized within the cell membrane of the host and later that the precise localization within the cell was also dependent on the type I family to which the enzyme belongs, providing a possible explanation for the different modes of RA activity. The EcoR124I holoenzyme was found to be particularly exposed on the periplasmic side of the cytoplasmic membrane, with EcoKI partially exposed in the periplasm, but EcoAI is not exposed to the periplasm at all.…”

Section: Cellular Localization Of Ecor124imentioning

confidence: 99%

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

Youell

Firman

2008

Microbiol Mol Biol Rev

Self Cite

View full text Add to dashboard Cite

SUMMARY Plasmid R124 was first described in 1972 as being a new member of incompatibility group IncFIV, yet early physical investigations of plasmid DNA showed that this type of classification was more complex than first imagined. Throughout the history of the study of this plasmid, there have been many unexpected observations. Therefore, in this review, we describe the history of our understanding of this plasmid and the type I restriction-modification (R-M) system that it encodes, which will allow an opportunity to correct errors, or misunderstandings, that have arisen in the literature. We also describe the characterization of the R-M enzyme EcoR124I and describe the unusual properties of both type I R-M enzymes and EcoR124I in particular. As we approached the 21st century, we began to see the potential of the EcoR124I R-M enzyme as a useful molecular motor, and this leads to a description of recent work that has shown that the R-M enzyme can be used as a nanoactuator. Therefore, this is a history that takes us from a plasmid isolated from (presumably) an infected source to the potential use of the plasmid-encoded R-M enzyme in bionanotechnology.

show abstract

Localization of the Type I Restriction–Modification Enzyme EcoKI in the Bacterial Cell

Cited by 11 publications

References 42 publications

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit

Periplasmic expression of a restriction endonuclease in Escherichia coli and its effect on the antiviral activity of the host

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

Contact Info

Product

Resources

About