Table 1.

The effect of altered spacers in O_R or O_L on PvuII R-M system establishment and maintenance

C box intraoperator spacers OL/OR	Relative EOT (%)a (pvuIIM+ pvuIIM−host)	Ability to replace co-existing pPvuM1.9-ACYC (M+) with pACYC177- kan (M−)b
No PvuII R-M (pBR322)	100 ± 7	+
CAT/CAA, WT (pPvuRM3.4)	98 ± 13	+
CAT/CAA, Sym (pIM20)	30 ± 3	+
CAT/CAA, Sym (pIM6)	34 ± 3	NDc
CAT/CAT, Sym (pIM17)	26 ± 7	NDc
CAA/CAA, Sym (pIM18)	0.2 ± 0.1d	−
CAA/CAT, Sym (pIM19)	0.7 ± 0.4d	−

^aEqual amounts of plasmid DNAs with PvuII R-M system and its C-box spacer variants were used to determine the efficiency of transformation (EOT) in each of two host strains. These plasmids were introduced into competent E. coli TOP10 cells that already carried either the gene for the PvuII MTase (pvuIIM; plasmid pPvuM1.9-ACYC) or a vector control (pACYC177). Relative EOT was determined as the fraction of M.PvuII^– transformants obtained relative to the number of transformants for the M.PvuIIM⁺ strain, and then normalized to the pBR322 EOT ratio. The standard deviation is indicated.

^bThe same competent cells, having indicated plasmid and pPvuM1.9-ACYC (M⁺), were transformed with pACYC-kan (M^–) and grown without antibiotics for 2 h to allow plasmid segregation. As they belong to the same incompatibility group, either pPvuM1.9-ACYC or pACYC-kan should be lost unless the plasmid is essential. Potential segregants were plated with selection for R-M system plasmid (Amp^R) and pACYC-kan (M^–) (Kan^R). Lack of transformants after overnight incubation indicates a requirement of additional MTase expression for R-M system maintenance.

^cND, not determined.

^dSmaller colonies, viability problems.