Fig 1.
A map of plasmid pEC156 (A). The genes coding for the EcoVIII RM system, cer locus, rom gene as well as regions with oriT F-like, oriT R64-like sequences and genes that are engaged in the priming (RNA II) and controlling the initiation of plasmid DNA replication (RNA I) are indicated. Alignment of the pEC156 nucleotide sequence with oriT of F plasmid (B) and oriT of R64 plasmid (C). The minimal region that allows oriT F-dependent plasmid mobilization is in boldface. The position of nick sites (nic) are indicated. Binding sites for plasmid F TraM (sbmA, sbmB and sbmC), TraY (sbyA and sbyC) and IHF protein as well as NikA binding site on plasmid R64 are underlined. Imperfect inverted repeats are indicated by arrows. Asterisks indicate identical nucleotides. The accession numbers of nucleotide sequences of plasmids pEC156, F and R64 that have been deposited in the Genbank database are AF158026, AP001918 and AB027308, respectively.
Table 1.
Mobilization of pEC156 derivatives by IncFI and IncI1 conjugative plasmids.
Fig 2.
The effect of the presence of EcoVIII RM system in recipient cells on efficiency of conjugal transfer of plasmid F. E. coli DH5α Rif [F’ts114lac::Tn5, KmR] was used as donor.
The following recipient bacteria were assayed: Escherichia coli HB101, Enterobacter cloacae, Klebsiella oxytoca, Citrobacter freundii, and Salmonella enteritidis that carried either pEC156-derivative pIB8 (EcoVIII R+M+, black bars) or pIB9 (EcoVIII R−M−, grey bars). Each column represents the mean value (± standard deviation) from three repeats. Statistical analysis revealed correlation between presence of a plasmid with RM system in the recipient bacteria and frequency of F plasmid conjugal transfer (P<0.0007 for E. coli, E. cloacae, K. oxytoca, C. freundii; and P = 0.02 for S. enteritidis; Student t test).
Table 2.
Mobilization frequency (italics) of pEC156-derivative pIB8 (EcoVIII R+M+).
Table 3.
Mobilization frequency (italics) of pEC156-derivative pIB9 (EcoVIII R¯M¯).
Fig 3.
Effect of the EcoVIII RM system on plasmid release.
Growth curves of E. coli MG1655 [pIB8, pACYC177] and MG1655 [pIB9, pACYC177] (A). Plasmid release indicated as number of pACYC177-positive transformants (B). The efficiency of plasmid release was determined as a ratio of transformants against the titer of the host (C). Each column represents the mean value (± standard deviation) from three repeats. Statistically significant differences in plasmid release as analyzed by Student’s t test, indicated by asterisk, were observed between bacteria carrying pIB8 (EcoVIII R+M+) and those that carried pIB9 (EcoVIII R−M−) (P<0.05; Student t test).
Fig 4.
Efficiency of natural transformation of E. coli HB101 with pEC156-derivatives pIB8A (EcoVIII R+M+) and pIB9A (EcoVIII R−M−).
Plasmid pUC19 was used as a control. Each column represents the mean value (± standard deviation) from three repeats. Statistical significance analyzed by Student’s t test revealed lack of correlation between presence of EcoVIII RM system and efficiency of natural transformation (P = 0.12; Student t test).
Fig 5.
Mobility of pEC156-derivatives pIB8 EcoVIII R+M+) (A) and pIB9 (EcoVIII R−M−) (B) among enterobacteria.
Arrows indicate direction of plasmid dissemination and rate of mobilization frequency based on experimental data presented in Tables 2 and 3 [solid line, high frequency (10−3–10−5); dashed line, medium frequency (10−6–10−7); and dotted line, low frequency (10−8–10−9)].