A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid

Abstract

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.

FIG. 1.

Construction of the pKILLER plasmid and its use in selecting clones carrying a specific MTase gene. (A) Construction of pKILLER. An antibiotic cassette (cat gene, Cm^r) was inserted into the pEC156 plasmid (NheI site), resulting in pIB8. pKILLER was then created by deleting the NruI-EcoRV DNA fragment from the ecoVIIIM gene. Thus, pKILLER contains a functional EcoVIII ENase gene but not a functional gene coding for the cognate MTase. (B) Screening the clones obtained as a result of the selection procedure based on the use of pKILLER. Clones 1 to 3 are from the Bacillus stearothermophilus 14P genomic library; clones 4 to 6 are from the Citrobacter sp. strain RFL231 genomic library. Lanes: a, uncut plasmid DNA; b, plasmid DNA cleaved with HindIII; c, plasmid DNA cleaved with EcoRI. The arrows indicate the linear forms of vector (pBR322) and pKILLER. The appropriate inserts carrying the MTase gene (10 kbp in the case of BstZ1II and 3.1 kbp for Csp231I) are indicated with arrows labeled “insert.”

FIG. 2.

Genetic organization of the BstZ1II (A) and Csp231I (B) R-M systems. The DNA fragments that carry a specific MTase gene were isolated from the genomic libraries of Bacillus stearothermophilus 14P (A) and Citrobacter sp. strain RFL231 (B). The genes of the R-M systems BstZ1II (A) and Csp231I (B) are marked. Details concerning the reconstruction of the BstZ1II and Csp231I R-M systems are provided in the text.

FIG. 3.

Comparison of amino acid sequences of HindIII-isospecific R-M enzymes: (A) MTases, (B) ENases, and (C) putative regulatory C proteins of Csp231I and EcoO109I R-M systems. The numbers on the right margin denote the amino acid positions relative to the N terminus. The conserved motifs of m⁶N-adenine MTases are boxed and denoted by Roman numerals. The position of the putative TRD is indicated. The region of pronounced similarity between all isospecific ENases is boxed. The amino acids of the putative catalytic/magnesium binding motif PD/EX_nDXK and putative DNA binding motif RXXR are indicated. Sequences were aligned using the CLUSTAL W computer program. Asterisks indicate identical amino acids; colons and periods indicate very similar amino acids and somewhat similar amino acids, respectively; dashes indicate gaps in the aligned sequences. The accession numbers for the nucleotide sequences of the HindIII, EcoVIII, LlaCI, BstZ1II, Csp231, BbrRORF307, and EsaSS1092P R-M genes that have been deposited in the GenBank database are L15391, AF158026, AJ002064, AY789018, AY787793, BX640437, and AACY01401088, respectively.