Identification of nolR-regulated proteins in Sinorhizobium 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.

Systematic Targeted Mutagenesis ofBrucella melitensis16M Reveals a Major Role for GntR Regulators in the Control ofVirulence

“…NolR is known to repress expression of the nodulation genes. Moreover, a proteomic study showed that nolR encodes a global regulatory protein (7,32). In response to environmental stresses, NolR regulates diverse factors such as heat shock proteins, protein synthesis, and cell growth.…”

Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR

“…The shift in position of Gln56 in each half-site of the oligo AT DNA, either to interact with A2 in the first site or away from T7' in the second site, suggested that movement of this residue may play a role in the previously observed recognition by NolR of variable operator sites (17)(18)(19)(20)(21)(22)(23)(24). To examine the possible role of Gln56 as a conformational switch in DNA binding, NolR was crystallized with oligo AA DNA (Fig.…”

“…1-3) more accurately define the interaction sequences in each asymmetric half site as ATTAG on the 5′ strand and CTTC on the 3′ strand. A remarkable feature of NolR is that the homodimer binds to an asymmetric operator site with variable sequences at two positions, one in each half site, which allows versatility in recognition of NolR operator sites of multiple target genes (19,20,(22)(23)(24).…”

“…In rhizobia, NolR modulates expression of the NodD activator protein, the core nod genes, and multiple genes involved in symbiosis (17,(19)(20)(21)(22)(23)(24). Based on amino acid sequence homology, NolR was proposed to be a helix-turn-helix family member that binds to a nonpalindromic consensus motif -(A/T)TTAG-N 9 -A(T/A) (17).…”

“…Transcript levels of nolR are high in freeliving rhizobia and in the bacteroid but are down-regulated by luteolin, a nod gene inducer (24). Subsequent studies implicated NolR as a global regulatory factor that responds to environmental factors to fine-tune a range of symbiotic signals, not just the genes required for nodulation, and that the absence or presence of NolR affects symbiotic interactions with host plants (19,20,23,24). For example, NolR represses expression of the type III secretion system ttsI gene, which is required for secretion of nodulation outer proteins (nops) that are beneficial for symbiosis of Sinorhizobium fredii with some soybean cultivars (24).…”