Cloning and characterization of a methyl -dependent restriction endonuclease and a cell cycle regulating DNA methyltransferase from Zymomonas mobilis subspecies mobilis CP4

A Zymomonas mobilis CP4 genomic library was screened using the E. coli indicator strains API-200-9 and ER1992 to isolate clones of enzymes that cause DNA damage. Sequence analysis of positive clones identified two open reading frames encoding DNA modification enzymes: (1) a 924 base pair open reading frame with sequence similarity to mrr, a methyl-dependant restriction endonuclease, which was designated ZmCP4mrr, and (2) a 1149 by open reading frame with amino acid sequence similarity to ccrM, a cell cycle regulating DNA methyltransferase, which was designated ZmCP4ccrM .;Sequence analysis indicates that ZmCP4mrr is a solitary methyl-dependent restriction endonuclease without a cognate DNA methyltransferase. Transformation of Escherichia coli K12 strains with various DNA methyltransferase backgrounds demonstrated that a plasmid borne ZmCP4mrr gene readily transforms E. coli strains that express dcm, hsdM, and EcoKccrM DNA methyltransferases, indicating that ZmCP4Mrr does not recognize and restrict sites methylated by these DNA methyltransferases. E. coli strains that express the dam DNA methyltransferase could only be transformed if expression of plasmid borne ZmCP4mrr was repressed. Subsequent induction of ZmCP4mrr expression in these cells resulted in inhibition of growth and cell death, indicating that ZmCP4Mrr specifically restricts Dam N6-adenine methylated DNA (5'-GmATC-3'). Plasmid DNA originating from dam deficient E. coli strains did not improve transformation efficiency, indicating that Z. mobilis CP4 has a restriction system in addition to ZmCP4Mrr contributing to low frequency of gene transfer from foreign DNA.;Sequence analysis indicates that ZmCP4ccrM is a solitary DNA methyltransferase with two possible in-frame translation initiation sites. A ribosomal binding site containing a sequence, 5'-AGGA-3', conserved in Z. mobilis promoters of highly expressed genes is located adjacent to the first possible translation initiation site and not the second, suggesting that ZmCP4ccrM is being expressed from the first translational initiation site. The specificity for ZmCP4CcrM methylation was directly determined to be the N6-adenine of its recognition site 5'-GANTC-3'. Z. mobilis CP4 cells overexpressing ZmCP4ccrM exhibited a subpopulation of filamentous cells, ranging from 10-90 muM in length, with multiple chromosomes. Overexpression of ZmCP4ccrM caused disruption of normal cell division and chromosomal segregation, suggesting that ZmCP4CcrM is involved in cell cycle regulation.