Megaplasmid pRme2011a of Sinorhizobium meliloti is not required for viability

Abstract

We report the curing of the 1,360-kb megaplasmid pRme2011a from Sinorhizobium meliloti strain Rm2011. With a positive selection strategy that utilized Tn5B12-S containing the sacB gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.

FIG. 1

Eckhardt gel showing plasmid profiles of S. meliloti strains with deletions in pRme2011a. Lanes: A, R. leguminosarum LRS39501 (24) (size standard); B, Rm2011; C, SmA818; D, SmA146; E, Rm2011. All lanes are from the same gel, but some intervening lanes showing derivatives with smaller deletions have been removed for clarity.

FIG. 2

Schematic representation of pRme2011a showing relative positions of known genetic markers. The approximate position of syrB::Tn5 in strain MB101 (5) was determined by conjugal-transfer experiments similar to those which were described for pRmeSU47b (11). The oriT used for these experiments was that of Ω30::Tn5-11 from Rm5420 (which is linked to nifH) (18). The direction of transfer was determined to be clockwise, using fixJ2.3::Tn5 from strain GMI 5704 as a marker for conjugal transfer (15). The arc shown represents the approximate position of Δ14-6. The end points of this deletion are undefined. The large arrows indicate the positions of PmeI restriction sites (23). PmeI fragment 5 extends between sites 1 and 2, fragment 6 extends between sites 1 and 3, and fragment 7 extends between sites 2 and 3. All three fragments are missing in strain SmA818.

FIG. 3

Southern blot analysis of SmA818. Equal amounts of EcoRI-restricted DNA from Rm2011 and SmA818 were electrophoresed, blotted to a nylon membrane, and probed with pRme2011Δ14-6 DNA which was isolated from a preparative Eckhardt gel. Lanes: A, Rm2011; B, SmA818.

FIG. 4

Pulsed-field gel profiles of PacI-digested S. meliloti strains. Lanes: A, SmA818; B, SmA146; C, Rm2011; D, concatemers, molecular size markers.

FIG. 5

Unidentified dehydrogenase (Udh) and SOD activities in S. meliloti. Cell extracts of Rm2011 and SmA818 were run on nondenaturing polyacrylamide gels. The gel was stained as previously described (8). Lanes: A, Rm2011; B, SmA818.