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 Cys 2 -His 2 -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 Rachwał et al.RosR Affects Rhizobial Cell-Surface Properties 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.

Extracellular polysaccharide protects Rhizobium leguminosarum cells against zinc stress in vitro and during symbiosis with clover

“…Under control conditions, the hydrophobicity of the EPS‐deficient mutant was the lowest among the studied strains, suggesting important role of this polymer in cell surface properties. However, as the hydrophobicity of the rosR mutant was higher than that of the wild type, we propose that additional changes in this strain, other than diminished EPS synthesis, caused this phenomenon (i.e., alteration of membrane permeability and reduced flagellation) (Rachwał et al ., ).…”

“…To determine hydrophobicity of the rhizobial cells, a twophase method according to Neu and Poralla (1990) was used, with minor modifications (Rachwał et al, 2016). Bacterial pellets from 24 h cultures containing no, or 1 or 10 mM ZnSO 4 3H 2 O were suspended in the PUM buffer (22.2 g of K 2 HPO 4 , 7.26 g of KH 2 PO 4 , 1.8 g of urea and 0.2 g of MgSO 4 3 7H 2 O in 1 l of deionized water) to an optical density (OD 405 ) of ca.…”

Mutation in the pssZ Gene Negatively Impacts Exopolysaccharide Synthesis, Surface Properties, and Symbiosis of Rhizobium leguminosarum bv. trifolii with Clover

“…The hydrophobicity of rhizobial strains was determined using a two-phase method and dodecane (Sigma-Aldrich, St. Louis, MO, USA) according to [ 56 ]. For this assay, bacterial pellets obtained from 24 h cultures and resuspended in PUM buffer were used.…”

“…Bacterial samples for atomic force microscopy (AFM) were prepared according to a method described earlier [ 60 ] with a minor modification [ 56 ]. Briefly, 6-h cultures of rhizobial strains were diluted in a fresh portion of the medium to OD 600 = 0.1, centrifuged, and the bacterial pellets obtained were resuspended in 5 μL water, loaded onto 10-mm mica disks (Continental Trade, Warsaw, Poland), and allowed to dry in room temperature.…”

A mutation in pssE affects exopolysaccharide synthesis by Rhizobium leguminosarum bv. trifolii, its surface properties, and symbiosis with clover

“…The hydrophobicity of Rt24.2, Rt1933, and Rt1933(pM3) cells was determined using a two-phase method and dodecane (Sigma-Aldrich, St. Louis, MO, USA) according to Neu and Poralla (1990) with minor modifications (Rachwał et al 2016). Bacteria were collected from 79CA agar plates and suspended in PUM buffer (22.2 g K 2 HPO 4 × H 2 O, 7.26 g KH 2 PO 4 , 1.8 g urea, 0.2 g MgSO 4 × 7H 2 O, 1 l water) to an optical density of~0.5 (OD 1 ) at 405 nm.…”