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New Rhizobium leguminosarum Flavonoid-Induced Proteins Revealed by Proteome Analysis of Differentially Displayed Proteins

Proteome analysis was used to establish the first two-dimensional protein map of Rhizobium. R. leguminosarum bv. trifolii strain ANU843 was grown in defined medium in the presence and absence of the flavonoid 7,4′-dihydroxy-flavone. Over 1,700 constitutive proteins were resolved, representing about 30% of the estimated genomic output. Proteome analysis of flavonoid-treated cells was done to reveal differentially displayed proteins. The results showed that while the global expression pattern of proteins was largely unaltered by the treatment, four inducible proteins were observed. The four inducible proteins and 20 constitutively expressed proteins were subjected to sequence analysis to provide internal standards for the construction of a two-dimensional Rhizobium protein data base. The identity of 12 proteins, including NodE and NodB, was established. NodE was present throughout the growth of the cells but was diminished in amount in stationary phase cells whereas NodB was not detected in the later stages of growth. Two of the induced proteins sequenced did not match any known nodulation gene product, with one of these being present in mid-late log and stationary phase cells and possessing four consecutive His residues at the N terminus. N-terminal sequencing was successful with 100 to 200 fmol of protein. Proteome analysis provides a sensitive new tool to examine plant-microbe interactions.