Determination of plasmid‐encoded functions in Rhizobium leguminosarum biovar trifolii using proteome analysis of plasmid‐cured derivatives

We have used proteome analysis of derivatives of R. leguminosarum biovar trifolii strain ANU843, cured of indigenous plasmids by a direct selection system, to investigate plasmid‐encoded functions. Under the conditions used, the plasmid‐encoded gene products contributed to only a small proportion of the 2000 proteins visualised in the two‐dimensional (2‐D) protein map of strain ANU843. The level of synthesis of thirty‐nine proteins was affected after curing of either plasmid a, c or e. The differences observed upon plasmid curing included: protein loss, up/down‐regulation of specific proteins and novel synthesis of some proteins. This suggests that a complex interplay between the cured plasmid and the remaining replicons is occurring. Twenty‐two proteins appeared to be absent in the cured strains and these presumably are encoded by plasmid genes. Of these, a small heat shock protein, a cold shock protein, a hypothetical YTFG‐29.7 kDa protein, and the alpha and beta sub‐units of the electron transfer flavoprotein were identified by N ‐terminal micro‐sequencing and predicted to be encoded by plasmid e. Four of the sequenced proteins putatively encoded on plasmid e and two encoded on plasmid c were novel. In addition, curing of plasmid e and c consistently decreased the levels of 3‐isopropylmalate dehydratase and malate dehydrogenase, respectively, suggesting that levels of these proteins may be influenced by plasmid‐encoded functions. A protein with homology to 4‐oxalocrotonate tautomerase, which is involved in the biodegradation of phenolic compounds, was found to be newly synthesised in the strain cured of plasmid e. Proteome analysis provides a sensitive tool to examine the functional organisation of the Rhizobium genome and the global gene interactions which occur between the different replicons.