Kamila Rachwał

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

Background and aimsThe acidic exopolysaccharide (EPS) produced by Rhizobium leguminosarum bv. trifolii is required for the establishment of effective symbiosis with compatible host plants (Trifolium spp.). In the rhizobium-legume interaction, early stages of root infection and nodule development have been well studied from a genetic standpoint. However, factors important for colonization of several surfaces by rhizobia, including soil particles and roots, have not yet been thoroughly investigated. The aim of this study was establishing of environmental factors affecting production of EPS by R. leguminosarum bv. trifolii strain 24.2 and the role of this polysaccharide in bacterial surface properties and attachment ability.MethodsBesides the wild-type strain, its derivatives differing in the level of EPS produced were used to these analyses. The ability of attachment to abiotic and biotic surfaces of these strains were established using CFU counting experiments. Three-dimensional structure and other parameters of biofilms formed were characterized in confocal laser scanning microscopy. Electrokinetic (zeta) potential of rhizobial cells were determined using Laser Doppler Velocimetry.ResultsIt was evidenced that the ability of R. leguminosarum bv. trifolii to produce EPS significantly affected bacterial attachment and biofilm formation on both abiotic and biotic surfaces. In addition, the presence of this polysaccharide influenced the zeta potential of rhizobial cells. Mutant strains having a mutation in genes involved in EPS synthesis were significantly impaired in attachment, whereas strains overproducing this polysaccharide showed higher adhesion efficiency to all of the tested materials. EPS facilitated attachment of bacterial cells to the tested surfaces most probably due to hydrophobic interactions and heterogeneity of the envelope surface.ConclusionsEPS produced by R. leguminosarum bv. trifolii plays a significant role in attachment and biofilm formation to both abiotic and biotic surfaces as well as bacterial surface properties.

Mutation in the pssA Gene Involved in Exopolysaccharide Synthesis Leads to Several Physiological and Symbiotic Defects in Rhizobium leguminosarum bv. trifolii

The symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii 24.2 secretes large amounts of acidic exopolysaccharide (EPS), which plays a crucial role in establishment of effective symbiosis with clover. The biosynthesis of this heteropolymer is conducted by a multi-enzymatic complex located in the bacterial inner membrane. PssA protein, responsible for the addition of glucose-1-phosphate to a polyprenyl phosphate carrier, is involved in the first step of EPS synthesis. In this work, we characterize R. leguminosarum bv. trifolii strain Rt270 containing a mini-Tn5 transposon insertion located in the 3′-end of the pssA gene. It has been established that a mutation in this gene causes a pleiotropic effect in rhizobial cells. This is confirmed by the phenotype of the mutant strain Rt270, which exhibits several physiological and symbiotic defects such as a deficiency in EPS synthesis, decreased motility and utilization of some nutrients, decreased sensitivity to several antibiotics, an altered extracellular protein profile, and failed host plant infection. The data of this study indicate that the protein product of the pssA gene is not only involved in EPS synthesis, but also required for proper functioning of Rhizobium leguminosarum bv. trifolii cells.

Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover

Background and aimsIn the symbiotic bacterium Rhizobium leguminosarum bv. trifolii, a majority of proteins involved in exopolysaccharide (EPS) synthesis are encoded by genes located in a large polysaccharide synthesis cluster (Pss). The aim of this study was genetic characterization of the Pss region in the Rt24.2 strain in the context of EPS production and symbiosis with red clover (Trifolium pratense).MethodsThe expression of genes located in the Pss cluster was determined using constructed pss-lacZ transcriptional fusions. The role of transcriptional regulator RosR in pss transcription was confirmed using a rosR mutant and the Rt24.2(pBR1) strain carrying multiple rosR copies. An EPS-deficient mutant, Rt770 was obtained using a random mutagenesis and mTn5SSgusA40 transposon. Symbiotic properties of the Rt770 strain in interaction with clover were characterized in inoculation experiments. Infection of host roots and nodule occupancy by this mutant were investigated using both light and electron microscopy.ResultsTranscriptional levels of particular pss genes differed significantly; the genes encoding glycosyltransferases and enzymes modifying EPS have promoters of weak activities, whereas those encoding proteins involved in EPS polymerization and export possess stronger promoters. Furthermore, RosR affected expression of some pss genes. A mutation in Rt24.2 pssS encoding glucosyltransferase totally abolished EPS synthesis, decreased motility, and increased sensitivity to some stressors. The pssS mutant Rt770 induced formation of nodules on clover roots, which were ineffective in nitrogen fixation.ConclusionEPS secreted by Rt24.2 is required for both adaptation to soil conditions and the establishment of effective symbiosis with clover plants.

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

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 Ca2+-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.

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

Background and aimsA considerable majority of the proteins involved in exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii are encoded by pss genes located in a large chromosomal region named Pss-I. The aim of this work was to characterize the phenotypic and symbiotic properties of strain Rt1933, which has a mutation in pssE. This gene encodes an enzyme responsible for the second step of EPS unit assembly.MethodsThe EPS-deficient Rt1933pssE strain was obtained via random mutagenesis using the mTn5SSgusA40 transposon. The mutation site in the Rt1933 genome was identified using hybridization, PCR amplification, and sequence analysis. Complementation of the pssE mutation was performed using biparental conjugation and a set of plasmids containing different fragments of the Pss-I region. The phenotypic properties of this mutant were established in growth kinetics experiments, as well as motility, sensitivity and hydrophobicity assays. The symbiotic proficiency of the Rt1933 strain in interaction with red clover (Trifolium pratense) was determined in plant tests, whereas occupation of root nodules by this mutant was investigated using light microscopy and bacteria harboring gusA for β-glucuronidase.ResultsAn exo99 mutation in Rt1933 was identified at 3′-end of the pssE gene located in region Pss-I, which resulted in a lack of 16 amino acids at the C-end of PssE. This mutation totally abolished EPS synthesis in R. leguminosarum bv. trifolii. Strain Rt1933 was characterized by considerably decreased growth kinetics and motility, and an increased sensitivity to some stress factors. Also, the hydrophobicity of the mutant cells differed significantly from that of the wild-type Rt24.2 and the complemented Rt1933 cells. Moreover, the pssE mutant showed strong disturbances in symbiosis with clover; it induced much fewer nodules on clover roots at a later time than normal, and the mass of the plants inoculated with the mutant was significantly lower than that of the plants inoculated with the wild-type strain.ConclusionsThe pssE gene plays a crucial role in EPS synthesis in R. leguminosarum bv. trifolii, and the presence of this polysaccharide affects the cell-surface properties of the bacterium and is required for both adaptation to stress conditions and the establishment of effective symbiosis with clover plants.

Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis

Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min(-1), respectively.

Regulatory Elements Located in the Upstream Region of the Rhizobium leguminosarum rosR Global Regulator Are Essential for Its Transcription and mRNA Stability

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a symbiotic relationship with clover (Trifolium spp.). Previously, the rosR gene, encoding a global regulatory protein involved in motility, synthesis of cell-surface components, and other cellular processes was identified and characterized in this bacterium. This gene possesses a long upstream region that contains several regulatory motifs, including inverted repeats (IRs) of different lengths. So far, the role of these motifs in the regulation of rosR transcription has not been elucidated in detail. In this study, we performed a functional analysis of these motifs using a set of transcriptional rosR-lacZ fusions that contain mutations in these regions. The levels of rosR transcription for different mutant variants were evaluated in R. leguminosarum using both quantitative real-time PCR and β-galactosidase activity assays. Moreover, the stability of wild type rosR transcripts and those with mutations in the regulatory motifs was determined using an RNA decay assay and plasmids with mutations in different IRs located in the 5′-untranslated region of the gene. The results show that transcription of rosR undergoes complex regulation, in which several regulatory elements located in the upstream region and some regulatory proteins are engaged. These include an upstream regulatory element, an extension of the -10 element containing three nucleotides TGn (TGn-extended -10 element), several IRs, and PraR repressor related to quorum sensing.