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.

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid

“…Similarity searches failed to find GdhA encoded by M. loti. GdhA is probably not required for glutamate catabolism in free-living S. meliloti because a pSymA-cured strain can still use glutamate for growth (18). Interestingly, the pSymA-cured strain will not grow on minimal medium with ammonium as the sole nitrogen source, suggesting that pSymA plays a role in ammonium assimilation (M.J.B., unpublished work).…”

“…pSymA of the closely related strain Rm2011 can be cured without affecting growth in either rich or minimal-succinate media, but this strain is defective in the utilization of certain carbon sources (18). Our analysis shows that many genes on pSymA provide versatility to S. meliloti and may be adaptive in both the free-living and symbiotic states.…”

Predicting gene expression levels from codon biases in α-proteobacterial genomes

“…pSymA includes as PA genes most of the symbiotic genes that function in nodulation and nitrogen fixation. When pSymA is knocked out, SINME remains viable but no longer infects its plant host or engages in nitrogen fixation (10). The foregoing discussion may bear on the course of the evolution and development of multiple chromosomes.…”

“…pSymA carries the nod genes and most of the nitrogen-fixation genes. Although pSymA is required for nodulation, it can be cured without affecting viability, at least under laboratory conditions (10). pSymB carries genes required for successful infection and several essential genes (Arg tRNA and minCDE), making the classification of pSymB as a plasmid rather than a chromosome somewhat controversial (6).…”

A dual-genome Symbiosis Chip for coordinate study of signal exchange and development in a prokaryote–host interaction

“…S. meliloti has three circular replicons: a chromosome (3.65 Mb) that encodes most of the housekeeping and essential genes (10) and two megaplasmids, pSymA (1.4 Mb) and pSymB (1.7 Mb) (11). pSymA is not essential for viability but contains many of the genes required for symbiosis [nod, nif, and most fix genes (12,13)]. pSymB contains exo and dct genes required for symbiosis, as well as several genes essential for viability (14).…”