Physical characterization of Rhizobium meliloti megaplasmids

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Rhizobium meliloti harbors large extrachromosomal DNA elements called megaplasmids.
• These megaplasmids play crucial roles in symbiosis and bacterial adaptation.
• Understanding their structure and function is key to optimizing nitrogen fixation in legumes.

Purpose of the Study:

• To isolate and characterize the intact megaplasmids of Rhizobium meliloti 2011.
• To determine the number, size, and relatedness of these megaplasmids.
• To identify the Sym plasmid responsible for nitrogen fixation.

Main Methods:

• Electron microscopy for visualizing intact megaplasmids.
• Determination of megaplasmid contour lengths.
• Plasmid isolation and transfer into Agrobacterium strains.
• Restriction pattern analysis.
• Hybridization with nifHD probe.
• Buoyant density determination.
• Reassociation kinetics for relatedness analysis.

Main Results:

• Two distinct megaplasmid classes were observed: 400 µm and up to 560 µm.
• Plasmids pRme2011a (400 µm) and pRme2011b (560 µm) were identified and characterized.
• pRme2011a hybridized with the nifHD probe, identifying it as the Sym plasmid.
• Megaplasmids pRme2011a and b showed different restriction patterns and were unrelated.
• Buoyant densities indicated pSym is similar to Agrobacterium DNA, while the second megaplasmid resembles the R. meliloti chromosome.

Conclusions:

• Rhizobium meliloti 2011 likely possesses only two distinct megaplasmids.
• Megaplasmid lengths exhibit in vivo variability.
• The Sym plasmid (pSym) is identified, crucial for nitrogen fixation.
• The characterized megaplasmids are genetically distinct and vary in size.