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

Abstract

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.

Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes

“…1, spot 15) were also affected by the nolR mutant in Sinorhizobium meliloti [14]. Luteolin induced MDH and EF-TU and EF-G in freeliving bacteria [14,15], while MDH and GroEL were lower in plasmid-cured strains [33].…”

Evaluation of proteome reference maps for cross-species identification of proteins by peptide mass fingerprinting

“…Total bacterial or plant proteins were extracted as previously described for S. meliloti [9], rice [16], M. truncatula [15], M. alba [13] and T. subterraneum [14]. Proteins were separated in the first dimension using 18 cm Immobiline DryStrips with linear pH gradients from 4-7 (Amersham Biosciences, Uppsala, Sweden) as described previously [9].…”

“…Proteins were separated in the first dimension using 18 cm Immobiline DryStrips with linear pH gradients from 4-7 (Amersham Biosciences, Uppsala, Sweden) as described previously [9]. For second dimension SDS-PAGE, a horizontal Multiphor II electrophoresis system and precast Excel gels with a 12-14% acrylamide gradient were used (Amersham Biosciences).…”

“…S. meliloti is a nitrogen fixing soil bacterium that has been extensively studied over several decades and has been chosen as a model system for rhizobia [8]. We have comprehensively studied the protein profile of S. meliloti and developed reproducible protocols for 2-DE and protein identification [9][10][11][12]. The recent completion of the genome sequence of S. meliloti strain Rm1021 [8] has enabled us to confidently identify proteins analysed by PMF by comparing tryptic digestion products of proteins to the theoretical digestion products of all possible translation products predicted for this species (unpublished data).…”

Elevated Levels of Synthesis of over 20 Proteins Results after Mutation of the Rhizobium leguminosarum Exopolysaccharide Synthesis Gene pssA

“…Silver-stained 2-D protein arrays were analyzed using the MELANIE II program (Bio-Rad, Hercules, Calif.) for the qualitative and quantitative analysis of differentially displayed protein spots. Internal protein standards were used as pI and M r reference points (17,18). Three independent experiments with each mutant strain were performed for the analysis of the differences in protein synthesis.…”