DETERMINATION OF PLASMID-ENCODED FUNCTIONS IN RHIZOBIUM-LEGUMINOSARUMBIOVAR TRIFOLII USING PROTEOME ANALYSIS OF PLASMID-CURED DERIVATIVES

We have used proteome analysis of derivatives of R. leguminosarum biov ar trifolii strain ANU843, cured of indigenous plasmids by a direct se lection system, to investigate plasmid-encoded functions. Under the co nditions used, the plasmid-encoded gene products contributed to only a small proportion of the 2000 proteins visualised in the two-dimension al (2-D) protein map of strain ANU843. The level of synthesis of thirt y-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 pr oteins. This suggests that a complex interplay between the cured plasm id and the remaining replicons is occurring. Twenty-two proteins appea red 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 subunits of the electron transfer flavoprotein were identified by N-te rminal microsequencing and predicted to be encoded by plasmid e. Four of the sequenced proteins putatively encoded on plasmid e and two enco ded on plasmid c were novel. In addition, curing of plasmid e and c co nsistently decreased the levels of 3-isopropylmalate dehydratase and m alate dehydrogenase, respectively, suggesting that levels of these pro teins may be influenced by plasmid-encoded functions. A protein with h omology to 4-oxalocrotonate tautomerase, which is involved in the biod egradation 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 a nd the global gene interactions which occur between the different repl icons.