Identification ofnolR-regulated proteins inSinorhizobium meliloti using proteome analysis

Abstract

Extractable proteins from Sinorhizobium meliloti strains AK631 and EK698 (a Tn5-induced noIR-deficient mutant of AK631), grown in tryptone agar (TA) medium with or without the addition of the plant signal luteolin, were separated by two-dimensional gel electrophoresis and compared. Analysis of silver-stained gels showed that the noIR mutant had 189 proteins that were significantly altered in their levels (101 protein spots up- and 88 downregulated). Coomassie-stained preparative two-dimensional (2-D) gels or polyvinylidene difluoride (PVDF) membranes blotted from preparative gels showed that at least 52 of the altered proteins could be reproducibly detected and isolated from the noIR mutant. These 52 altered protein spots were classified into five groups based on an assessment of protein abundance by computer analysis and the effect of the presence or absence of luteolin addition to the growth medium. N-terminal microsequencing of 38 proteins revealed that the most striking feature of the consequence of the noIR mutation is the number and broad spectrum of cellular functions that are affected by the loss of the NoIR function. These include proteins involved in the tricarboxylic acid (TCA) cycle, heat shock and cold shock proteins, protein synthesis, a translation elongation factor, oxidative stress and cell growth and maintenance functions. We propose that the NoIR repressor is a global regulatory protein which responds to environmental factors to fine-tune intracellular metabolism.

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

“…Several other proteome studies have been applied to map and analyse mainly Rhizobium proteins important for the establishment and maintenance of the symbiosis (e.g. [13][14][15][16][17]). Two of these studies [16,17] were devoted to the analysis of changes in protein expression in bacteroids compared to free bacteria, and in nodules compared to uninfected roots.…”

“…1, spots 2, 14) and succinate CoA synthase (Fig. 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].…”

A high-resolution reference map for cytoplasmic and membrane-associated proteins ofCorynebacterium glutamicum

“…In E. coli, proteins with N-terminal Arg, Lys, Phe, Tyr, Trp and Leu residues are highly unstable [17] and none of the N-termini of 223 cytoplasmic E. coli proteins starts with one of these amino acids [15]. In contrast, mature proteins starting with Arg, Lys, Tyr and Leu residues were detected in C. glutamicum, M. tuberculosis [14], Streptococcus pneumoniae [18], B. subtilis [19] and Sinorhizobium meliloti [20]. …”

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

“…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). Gels were stained with CBB G-250 (BioRad, Hercules, CA, USA) according to [11] with minor modifications. Silver staining was done as described in [15].…”

“…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).…”