Transfer of the symbiotic plasmid of Rhizobium etli CFN42 requires cointegration with p42a, which may be mediated by site-specific recombination

Abstract

Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.

FIG. 1.

Structural analysis of RpSyms*. The pSym is represented by solid lines, and p42a is represented by cross-hatched lines. (A) Map of the wild-type pSym. The numbers represent BamHI fragments; the localization of the three nifH reiterations and the localization of the replication region are indicated. (B to G) Structure of 10 different RpSyms*, indicating BamHI bands deleted in the pSym and substituted by p42a. (B) RpSyms* lacking sequences from the end of band 78 to band 83 of the pSym. The relative localization of the rep and tra regions is indicated. The strains examined were GMI9023/p643, GMI9023/p646, GMI9023/p648-1 (this strain additionally had a duplication of the nifHa-nifHb region), GMI9023/p648-2, and GMI9023/p649. (C) RpSym* lacking sequences from the end of band 78 to the middle of band 82. The strain examined was GMI9023/p651-1. (D) RpSym* lacking sequences from the end of band 78 to band 20. The strain examined was GMI9023/p644. (E) RpSym* containing the whole pSym, with p42a integrated in band 78. The strain examined was GMI9023/p651-2. (F) RpSym* lacking bands 79 to 81. The strain examined was GMI9023/p652-1. (G) RpSym* lacking bands 26 to 1. The strain was GMI9023/p652-2. Similar endpoints for the pSym-p42a border are indicated by ellipses; the number 4 in panel B refers to EcoRI band 4 mentioned in the text. The maps are not drawn to scale.

FIG. 2.

Southern hybridization of restricted DNA from different strains with whole cosmids and individual bands from the pSym and p42a. The probes used were pSym cosmid 47 (A), pSym cosmid 7 (B), pSym BamHI band 79 (C), pSym BamHI band 80 (D), pSym BamHI band 83 (E), pSym BamHI band 84 (F), p42a EcoRI band 4 (G), and pSym BamHI band 78 (H). (A to D) BamHI-digested DNA from wild-type pSym from GMI9023/p645 (lane 1) and RpSym* from GMI9023/p643 (lane 2). (E and F) BamHI-digested DNA from RpSym* from GMI9023/p646 (lane 1), RpSym* from GMI9023/p643 (lane 2), wild-type pSym from GMI9023/p42d (lane 3), wild-type CFN42 (lane 4), and p42a from GMI9023/p42a (lane 5). (G and H) EcoRI-digested DNA from wild-type p42a from GMI9023/p42a (lane 1), RpSym* from GMI9023/p646 (lane 2), RpSym* from GMI9023/p643 (lane 3), wild-type pSym from GMI9023/p42d (lane 4), wild-type CFN42 (lane 5), and GMI9023 (lane 6), r, reiteration. The sizes of markers (in kilobases) are indicated on the right in all of the panels.

FIG. 3.

Sequence of the left pSym-p42a border in most RpSyms* and the attachment-like sequences shared by the pSym and p42a. (A) pSym; (B) p42a; (C) RpSym* from CFNX643. The sequence of p42a is indicated by cross-hatching, the homologous region between the pSym and p42a is enclosed in a box, and the inverted repeats are underlined with arrows; asterisks indicate the bases that are different in the two plasmids. The possible recombination region is indicated by two crossed lines framed by dotted lines. The locations of the attD and attA sites are indicated. The relative location of the open reading frame encoding a putative integrase (intA) is also indicated.

FIG. 4.

Alignment of the conserved regions (boxes A, B, and C) of tyrosine recombinases from R. etli (Re) (accession number AF538364), S. meliloti (Sm) (accession number AAK65874), A. tumefaciens (At) (accession number AAL45708), and M. loti (Ml) (accession number BAB54967) and a representative consensus sequence for 38 members of the phage integrase family Pfam00589 (Pf) (accession number PF00589). The invariant catalytic site residues (R-H-R-Y) are enclosed in boxes, and the length of the space between these residues is indicated by numbers. Residues that are identical in all five sequences are indicated by boldface type, and residues that are identical in >50% of the sequences are indicated by normal uppercase letters. Lowercase letters indicate residues that are identical in <50% of the sequences.

FIG. 5.

Plasmid patterns of CFN42 derivatives with an RpSym* and constructions simultaneously containing a wild-type pSym and an RpSym*. a to f indicate the positions of p42a (195 kb), p42b (185 kb), p42c (270 kb), p42d (pSym, 371 kb), p42e (500 kb), and p42f (600 kb), respectively. The position of wild-type pSym is indicated by white dots, and the position of RpSym* is indicated by asterisks. Lane 1, CFNX653; lane 2, CFNX654; lane 3, CFNX646; lane 4, CFNX657; lane 5, CFNX647; lane 6, CFNX658; lane 7, CFNX649; lane 8, CFNX648; lane 9, CFNX655; lane 10, CFNX656; lane 11, CFNX650; lane 12, CFNX659; lane 13, CFNX652; lane 14, CFNX660; lane 15, CFNX653.

FIG. 6.

Model for the mechanism of pSym conjugative transfer and generation of RpSyms*. The pSym is indicated by solid lines, and p42a is indicated by cross-hatched lines. Triangles represent the attA and attD sequences, and open rectangles represent large sequences shared by both plasmids. Solid lines and arrows labeled with the letter S indicate site-specific recombination among attachment-like sequences. Dashed lines and arrows labeled with the letter H followed by a number indicate homologous recombination among large repeated sequences. (A) When cointegration is the result of site-specific recombination, resolution through the same system regenerates the wild-type plasmids (S), but if the cointegrate is resolved through homologous recombination among other sequences (H1, H2, or H3), RpSyms* are generated. (B) Cointegrates formed and resolved through homologous recombination between two repeated sequences regenerate the wild-type plasmids (H1) but give rise to RpSyms* when they are resolved through homologous recombination among different repeats (H2) or through site-specific recombination (S).