The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

Abstract

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants.

Keywords: Rhizobium leguminosarum; clover; envelope properties; extracellular proteins; membrane proteins; rosR gene; symbiosis.

Figure 1

Extracellular (A), membrane (B), and periplasmic (C) protein fractions of R. leguminosarum bv. trifolii Rt24.2, Rt2472, and Rt2472(pRC24) separated by 1D electrophoresis. Proteins (10 μg) were loaded on each lane. Prestained protein ladder, 10–250 kDa (Thermo Scientific), was used as a molecular weight marker.

Figure 2

Western blotting of extracellular, membrane, periplasmic, and cytoplasmic protein fractions of R. leguminosarum bv. trifolii Rt24.2 and Rt2472 strains (A). The assay was performed using polyclonal rabbit antibodies against 32-kDa cytoplasmic inositol monophosphatase PssB and 43-kDa outer membrane lipoprotein PssN with N-terminal domain directed to the periplasmic space. Ten micrograms of individual protein fractions was loaded on each lane. Abbreviations: 24- Rt24.2, 2472- Rt2472. (B) Two-dimensional electrophoretic profiles of extracellular proteins of Rt24.2 and Rt2472. Hundred micrograms of individual protein fractions were analyzed (molecular weight marker—prestained protein ladder, 10–250 kDa, Thermo Scientific). Spots containing proteins in higher amounts which were excised from the gel are marked by red arrows, whereas those containing diminished protein amounts were marked by blue circles.

Figure 3

Two-dimensional electrophoretic profiles of membrane (A) and periplasmic proteins (B) of Rt24.2 and Rt2472. Individual protein fractions (100 μg) were analyzed (molecular weight marker, prestained protein ladder, 10–250 kDa, Thermo Scientific). Spots containing proteins in higher amounts which were excised from the gel are marked by red arrows, whereas those containing diminished protein amounts were marked by blue circles.

Figure 4

Outer membrane permeability of R. leguminosarum bv. trifolii Rt24.2, Rt2472 and Rt2472(pRC24) strains using the N-phenyl-1-naphthylamine uptake assay. The experiment was repeated twice with three biological repetitions for each strain analyzed.

Figure 5

AFM imaging of the wild-type R. leguminosarum bv. trifolii Rt24.2 (A), Rt2472 (B), and Rt2472(pRC24) (C) strains. The height, peak force error, elasticity (DMT modulus), adhesion, and deformation images are presented. The brighter and darker image areas correspond to the higher and lower parameter values, respectively.

Figure 6

AFM imaging of the wild-type R. leguminosarum bv. trifolii Rt24.2 (A), Rt2472 (B), and Rt2472(pRC24) (C) strains. 3D images, height images, and section profiles corresponding to lines in the height images are presented.

Figure 7

The number of plants with nodules (A) and the number of nodules induced on clover (T. pratense) roots (B) by the wild-type R. leguminosarum bv. trifolii Rt24.2, Rt2472, and Rt2472(pRC24) strains. Statistically significant differences between Rt2472 and Rt24.2, and Rt2472 and Rt2472(pRC24) are shown (^*P < 0.05, ^**P < 0.005; Student's t-test).

Figure 8

Light microscopy of clover (T. pratense) root nodules induced by the R. leguminosarum bv. trifolii strain Rt24.2, Rt2472 rosR mutant and Rt2472(pBR1) strains harboring pJBA21Tc plasmid with gusA reporter gene for β-glucuronidase. (A,B) Rt24.2 nodules at 7 and 28 days post inoculation (dpi), respectively; (C,D) Rt2472(pBR1) nodules at 7 and 28 dpi, respectively; (E–L) nodules occupied by Rt2472: (E,F) 7-dpi nodules, (G–I) 14-dpi nodules, (J) a 21-dpi nodule, (K) a 24-dpi nodule, and (L) a 30-dpi nodule, respectively. Bar = 150 μm (A,C,E–H); bar = 300 μm (B,D,I–L).

Figure 9

A semi-thin section of a 21-dpi T. pratense root nodule induced by the R. leguminosarum bv. trifolii rosR mutant Rt2472. Abbreviations: M, meristematic zone; IZ, infection zone; OC, outer cortex; R, root; arrow, inner cortex; arrow head, nodule endodermis; white asterisk, infected cells; black asterisk, uninfected cells. Bar = 100 μm.

Figure 10

Ultrastructure of wild-type (A) and rosR mutant infection threads (B,C). Abbreviations: IT, infection thread; TM, thread matrix; B, bacteria; D, dictyosome; ER, endoplasmic reticulum; short black arrow, thread wall; long black arrows, deposits of more translucent material between osmophilic layers of the thread wall; white long arrows, short ER cisterns in close contact with the thread membrane; short white arrows, transport vesicles. Bar = 5 μm. (D,E) Infection droplets containing rosR mutant bacteria. (D) A lateral infection droplet on an infection thread. (E) A large infection droplet inside the host cell. B, bacteria; ID, droplet; IT infection thread; N, nucleus; black triangle, degenerated bacteroids; black arrow heads, vesicles separated from the thread membrane surrounding the infection droplet. Bar = 2 μm for (D) and 5 μm for (E). (F) Symbiosomes containing rosR mutant bacteroids in the young infected host cell; note three types of bacteroids present: BA, bacteroids similar to those observed in the early symbiosis zone of wild-type nodules; double triangle, differentiating bacteroids; triangle, degenerated bacteroids. Bar = 5 μm. (G) A mature infected host cell: S, starch grain; rosette, abnormally differentiated rosR mutant bacteroids; black arrows, lytic compartments with degrading bacteroids. Bar = 5 μm. (H) Symbiosomes containing wild-type bacteroids in a mature infected host cell: BA, normal bacteroids. Bar = 5 μm. H₂O₂ detection in Rt24.2 and Rt2472 nodules as electron-dense precipitates formed in the presence of cerium chloride (I–L). H₂O₂ localization in the cell wall of a wild-type infection thread (I,J). H₂O₂ precipitates (white arrows) present in the cell wall of the infection threads containing rosR mutant bacteria and on the surface of bacteroids and their peribacteroidal membranes (K,L). Bar = 2.5 μm (for I,J), Bar = 5 μm (for K,L).