Structural elements required for replication and incompatibility of the Rhizobium etli symbiotic plasmid

Abstract

The symbiotic plasmid of Rhizobium etli CE3 belongs to the RepABC family of plasmid replicons. This family is characterized by the presence of three conserved genes, repA, repB, and repC, encoded by the same DNA strand. A long intergenic sequence (igs) between repB and repC is also conserved in all members of the plasmid family. In this paper we demonstrate that (i) the repABC genes are organized in an operon; (ii) the RepC product is essential for replication; (iii) RepA and RepB products participate in plasmid segregation and in the regulation of plasmid copy number; (iv) there are two cis-acting incompatibility regions, one located in the igs (incalpha) and the other downstream of repC (incbeta) (the former is essential for replication); and (v) RepA is a trans-acting incompatibility factor. We suggest that incalpha is a cis-acting site required for plasmid partitioning and that the origin of replication lies within incbeta.

FIG. 1

Replication capabilities and incompatibility properties of pH3. (a) Restriction map of the pH3 insert. The shaded bar indicates regions involved in partition and replication. The orientations of the three open reading frames are indicated by arrows. The solid boxes indicate the locations of the cis-acting regions, which exhibit incompatibility with the symbiotic plasmid. (b) The open boxes represent the DNA inserts contained in each plasmid derivative. The thin lines connecting the open boxes indicate the DNA regions lost in the deletion derivatives. The hatched boxes represent ΩKm cassettes. In the column labeled “replication,” R indicates that the plasmid derivative has the ability to replicate in the R. etli strain CFNX107, and NR indicates that the plasmid is unable to replicate in this strain. (c) The open boxes represent the DNA inserts contained in each plasmid derivative. The thin lines between the open boxes indicate the DNA regions lost in the deletion derivatives. In the column labeled “incompatibility,” I indicates that the plasmid derivative is incompatible with pSym of R. etli strain CFNX101, and C indicates that the construct is compatible with pSym of R. etli strain CFNX101. The solid boxes indicate the positions of the DNA regions containing cis-acting incompatibility determinants.

FIG. 2

Plasmid stability. Plasmid loss from populations of cells carrying different constructions with chloramphenicol resistance genes. ○, CFNX107 cells carrying plasmid pH3; □, CFNX107 cells carrying plasmid pRE-prepΔA-BC; ▿, CFNX107 cells carrying plasmid pRE-ΔA1; ▵, CFNX107 cells carrying plasmid pRE-prepA-ΔB-C. The graph shows the plasmid retention means (x) of three independent experiments ± the standard deviations.

FIG. 3

Plasmid copy number. Autoradiogram of a Southern blot of total DNA digested with EcoRI and HindIII and probed simultaneously with a chromosomal detector (recA) and with a repC detector. The plasmid copy number of each strain was calculated as the ratio of the integrated hybridization signal of repC (plasmid) and the integrated hybridization signal of recA (chromosome). Lane 1, CFNX107; lane 2, CFNX107(pH3); lane 3, CFNX107(pRE-ΔA1); lane 4, CFNX107(pRE-prepΔA-BC); lane 5, CFNX107(pRE-prepA-ΔB-C); and lane 6, CFNX107.