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 subunits of the electron transfer flavoprotein were identified by N-terminal microsequencing 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.
Guerreiro
N
N
Plant-Microbe Interaction Group, Research School of Biological Sciences, Australian National University, Canberra City.
Stepkowski
T
T
Rolfe
B G
BG
Djordjevic
M A
MA
D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D010449:Peptide Mapping; D010957:Plasmids; D016961:Rhizobium leguminosarum / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins