Plasmid pEC156, a Naturally Occurring Escherichia coli Genetic Element That Carries Genes of the EcoVIII Restriction-Modification System, Is Mobilizable among Enterobacteria

Abstract

Type II restriction-modification systems are ubiquitous in prokaryotes. Some of them are present in naturally occurring plasmids, which may facilitate the spread of these systems in bacterial populations by horizontal gene transfer. However, little is known about the routes of their dissemination. As a model to study this, we have chosen an Escherichia coli natural plasmid pEC156 that carries the EcoVIII restriction modification system. The presence of this system as well as the cis-acting cer site involved in resolution of plasmid multimers determines the stable maintenance of pEC156 not only in Escherichia coli but also in other enterobacteria. We have shown that due to the presence of oriT-type F and oriT-type R64 loci it is possible to mobilize pEC156 by conjugative plasmids (F and R64, respectively). The highest mobilization frequency was observed when pEC156-derivatives were transferred between Escherichia coli strains, Enterobacter cloacae and Citrobacter freundii representing coliform bacteria. We found that a pEC156-derivative with a functional EcoVIII restriction-modification system was mobilized in enterobacteria at a frequency lower than a plasmid lacking this system. In addition, we found that bacteria that possess the EcoVIII restriction-modification system can efficiently release plasmid content to the environment. We have shown that E. coli cells can be naturally transformed with pEC156-derivatives, however, with low efficiency. The transformation protocol employed neither involved chemical agents (e.g. CaCl2) nor temperature shift which could induce plasmid DNA uptake.

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.

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, Km^R] 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).

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)].