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

Abstract

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'-dihydroxyflavone. 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-terminal sequencing was successful with 100 to 200 fmol of protein. Proteome analysis provides a sensitive new tool to examine plant-microbe interactions.

Development of a 3-hydroxypropionate resistantEscherichia colistrain

“…The cell pellet was washed twice with 30 mM phosphate potassium buffer pH7, and cellular proteins from the pellet were solubilized as described previously. 32 …”

Effect of the plant flavonoid luteolin on Ensifer meliloti 3001 phenotypic responses

“…Notably, the NodD regulon, comprising luteolin-inducible and NodDdependent genes (Barnett et al 2004;Capela et al 2005), showed that the biosynthesis of NFs is one of the major pathways induced by the luteolin, but not the only one. Indeed, other additional genes are induced by the perception of plant flavonoid signals or show in their promoter region putative binding sites for NodD besides those related to NF biosynthesis (Batista and Hungria 2012;Galardini et al 2011;Lang et al 2008;Guerreiro et al 1997;Tolin et al 2013). Similarly to other plant flavonoids, luteolin was found to induce in E. meliloti, in addition to nod genes, the expression of genes encoding the EmrAB efflux pump, the GroES and GroEL chaperonins, as well as the SyrM transcriptional regulator and three hypothetical proteins (Capela et al 2005).…”

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

“…Rhizobium strains were grown at 28°C, on a shaker at 200 rpm, in 1 liter of BIII medium (9) . During mid-exponential-phase growth (optical density at 600 nm of 0.5 to 0.6) the cells were harvested, washed and lysed as previously described (17).…”

“…2-DE and electroblotting to polyvinylidene difluoride (PVDF) membranes were done by previously described methods (17). Isoelectric focusing in the first dimension was carried out on linear pH 4 to 7 and nonlinear pH 3 to 10 18-cm immobilized pH gradient (IPG) strips (PharmaciaBiotechnology, Uppsala, Sweden) loaded with 100 g (0.5 to 1 mg for preparative 2-D gels) of total cellular protein and run for 200 kV ⅐ h. N-terminal protein sequencing was done on a PROCISE-HT sequencer system or on a PROCISE-CLC (both from Perkin-Elmer Applied Biosystems, Foster City, Calif.) for verylow-abundance proteins.…”