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Physical characterization of Rhizobium meliloti megaplasmids
Abstract:
Intact megaplasmids of Rhizobium meliloti 2011 have been isolated and visualized by electron microscopy. The contour lengths of 64 megaplasmid molecules were determined. One definite class of molecules of 400 micron length and a range of larger molecules with lengths of up to 560 micron was observed. The contour lengths of the megaplasmids pRme2011a and pRme2011b were measured after isolation from plasmid-free Agrobacterium strains into which they had been individually transferred. Plasmid pRme2011a corresponds to the 400-micron class of megaplasmids while plasmid pRme2011b belongs to the 560-micron class. Preparatively isolated megaplasmids pRme2011a and b showed completely different restriction patterns. The pattern of total megaplasmid DNA from R. meliloti 2011 is composed of those from pRme2011a and b, suggesting that no more than two different megaplasmids exist. Because the length distributions of measured molecules were broad, R. meliloti 2011 megaplasmids seem to vary in length in vivo. Because only pRme2011a hybridized with a nifHD probe, this is the Sym plasmid. For R. meliloti strain MVII-1, which carries the megaplasmids pRmeMVII-1f and pRmeMVII-1g, pRmeMVII-1f was shown to be the Sym plasmid. Buoyant density determinations of R. meliloti 2011 and MVII-1 megaplasmids gave a value of 1.717 g/cm3 for pSym, which is that of Agrobacterium DNA. The buoyant density of the second megaplasmid was 1.721 g/cm3, corresponding to the density of the R. meliloti chromosome. As determined by reassociation kinetics, pRme2011a and b are unrelated. The degree of relatedness between strains MVII-1 and 2011 was 82%.
Insights
Rhizobium meliloti megaplasmids were visualized and measured, revealing two distinct types, pRme2011a and pRme2011b. One is the Sym plasmid, essential for nitrogen fixation, with both unrelated and varying in length.
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.