Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotype in Escherichia coli

Abstract

A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.

Expression, Purification, Mass Spectrometry, Crystallization and Multiwavelength Anomalous Diffraction of Selenomethionyl PvuII DNA Methyltransferase (cytosine‐N4‐specific)

“…Overexpression of the short form of M.PvuII. Early characterization of M.PvuI1 revealed that it is produced in two forms, which differ due to alternative translation initiators 13 codons apart (Blumenthal et al, 1985). While approximately 95% of the methyltransferase produced is the short form, whether from native clones or overexpression vectors (Tao et al, 1989), the two forms could not be purified apart and the mixture could not be crystallized (L. Dorner and I. Schildkraut, New England Biolabs, Inc., personal communication).…”

“…Expression of a SeMet derivative protein in such a strain generally increases the chance for complete incorporation of SeMet. However, the M.PvuI1-coding plasmid was not moved into a strain auxotrophic for methionine because we did not have an auxotrophic strain that was McrBC-; expression of M.PvuII is lethal unless the host strain is McrBC- (Blumenthal et al, 1985;Sutherland et al, 1992). Instead, we tested whether or not SeMet would be efficiently incorporated into an overexpressed protein by a methionine prototroph, strain DHlOB.…”

“…The PvuII restriction-modification system is produced by the gram-negative bacterium Proteus vulgaris, and recognizes the sequence 5'-CAGCTG-3' (Gingeras et al, 1981). The genes for this system were cloned (Blumenthal et al, 1985), and specify a restriction endonuclease, a niethyltransferase, and two regulatory proteins (Blumenthal et al, 1985;Tao et al, 1992;Adams and Blumenthal, 1995). M.PvuTI was shown to methylate the internal cytosine of CAGCTG sequences (Blumenthal et al, 1985;Butkus et al, 1987), generating m4C (Butkus et al, 1987).…”

CRISPR-interference based modulation of mobile genetic elements in bacteria

“…Their results showed a striking dominance of insertions with IS transposition being responsible for 69.5 to 99.9 % of mutations in E. coli DH10B. Moreover, a significant number of case studies from basic-and applied-research have also reported the interruption of cloned DNA segments by IS elements (47)(48)(49)(50)(51)(52)(53).…”

Complementation by detached parts of GGCC-specific DNA methyltransferases

“…Strains, plasmids and media All experiments were done in E.coli strains JM107MA2 (mcrB-) (13) or HBIO (mcrB-) (14). Bacteria were grown in LB medium at 370C.…”