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

Abstract

The symbiosis between rhizobial microbes and host plants involves the coordinated expression of multiple genes, which leads to nodule formation and nitrogen fixation. As part of the transcriptional machinery for nodulation and symbiosis across a range of Rhizobium, NolR serves as a global regulatory protein. Here, we present the X-ray crystal structures of NolR in the unliganded form and complexed with two different 22-base pair (bp) double-stranded operator sequences (oligos AT and AA). Structural and biochemical analysis of NolR reveals protein-DNA interactions with an asymmetric operator site and defines a mechanism for conformational switching of a key residue (Gln56) to accommodate variation in target DNA sequences from diverse rhizobial genes for nodulation and symbiosis. This conformational switching alters the energetic contributions to DNA binding without changes in affinity for the target sequence. Two possible models for the role of NolR in the regulation of different nodulation and symbiosis genes are proposed. To our knowledge, these studies provide the first structural insight on the regulation of genes involved in the agriculturally and ecologically important symbiosis of microbes and plants that leads to nodule formation and nitrogen fixation.transcription factor | protein structure T he symbiosis between rhizobial bacteria from the Rhizobium, Sinorhizobium, Mesorhizobium, Azorhizobium, and Bradyrhizobium genera and leguminous plants leads to the formation of root nodules (1, 2). These plant organs are specialized for nitrogen fixation and assimilation and are of major ecological and agricultural importance. For example, nitrogen-fixing nodules account for one quarter of total nitrogen fixed globally each year (3). The development of nitrogen-fixing nodules by rhizobia involves a variety of interactions between the plant and microbe; however, at the center of this process are a set of nod (nodulation) genes required for the synthesis of oligosaccharide-nodulation factors, for determining host-plant specificity, and for optimizing the efficiency of symbiosis (4-6). Successful interaction between the rhizobium and host plant requires expression of both positive and negative transcriptional control of genes related to nodulation and symbiosis (7,8

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

“…C). The closest structural homolog is an HTH domain in NolR (PDB ID 4ON0, Z‐score of 8.3 and RMSD of 2.4 Å for 66 residues), a small protein of Rhizobium that controls nodulation and symbiosis genes (Lee et al ., ). The AtxA HTH2 domain structure closely resembles the NolR protein, with the exception of the positioning of terminal helices.…”

“…The superposition of AtxA‐HTH1 and KdpE (http://onlinelibrary.wiley.com/doi/10.1111/mmi.12867/suppinfo) indicates binding of helix H4 inside the DNA major groove and wings interacting with the DNA minor groove. The NolR‐DNA complex (PBD ID 4ON0) (Lee et al ., ) offers a model for DNA binding and the AtxA HTH2 domain. NolR is a global regulator of nodulation and symbiosis gene expression in rhizobia, and recognizes variable asymmetric operator sites in the control regions of these genes (Lee et al ., ).…”

“…The NolR‐DNA complex (PBD ID 4ON0) (Lee et al ., ) offers a model for DNA binding and the AtxA HTH2 domain. NolR is a global regulator of nodulation and symbiosis gene expression in rhizobia, and recognizes variable asymmetric operator sites in the control regions of these genes (Lee et al ., ). Using NolR as a model, AtxA helix H8 is inside the DNA major groove (http://onlinelibrary.wiley.com/doi/10.1111/mmi.12867/suppinfo).…”

Structural basis of hypoxic gene regulation by the Rv0081 transcription factor of Mycobacterium tuberculosis

“…From the structural superposition of NolR‐DNA complex of Sinorhizobium fredii (PDB: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4OMY), there are two kinds of residues that are seen to interact with the DNA, the residues involved in contacting the DNA bases and the residues interacting with phosphate backbone. In the structure of NolR‐DNA complex, the base recognizing residues are Gln 56, Ser 57, Ser 60 and Gln 61 . Superposition of the two structures suggests that corresponding residues in Rv0081 would be Ser 48, Ser 49, Ser 52 and Gln 53.…”

Nodulation outer proteins: double-edged swords of symbiotic rhizobia

“…Negative regulation of ttsI gene expression by the regulatory protein NolR has been also reported. NolR appears to bind to a specific operator sequence in the ttsI promoter and thereby interferes with the action of NodD transcriptional activators [49,50]. As expression of ttsI usually depends on NodD proteins and specific host flavonoids, T3 pilus formation ( Figures 1A and 1B) and subsequent secretion of T3 effectors through the T3SS are strongly induced during the rhizobial infection process.…”