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

Janczarek, Monika; Rachwał, Kamila; Kopcińska, Joanna

doi:10.1007/s11104-015-2567-5

Cited by 24 publications

(52 citation statements)

References 84 publications

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“…NGR234, MucR homologue regulates exopolysaccharide production . Overall the MucR orthologues characterized are mainly transcriptional repressors that control various cell envelope modifications, motility, quorum sensing and host–bacterial interaction .…”

Section: Ros\mucr Familymentioning

confidence: 99%

The prokaryotic zinc‐finger: structure, function and comparison with the eukaryotic counterpart

et al. 2015

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Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. The Ros Cys 2 His 2 ZF binds DNA in a peculiar mode and folds in a domain significantly larger than its eukaryotic counterpart consisting of 58 amino acids (the 9-66 region) arranged in a bbbaa topology, and stabilized by a conserved, extensive, 15-residue hydrophobic core. The prokaryotic ZF domain, then, shows some intriguing new features that make it interestingly different from its eukaryotic counterpart. This review will focus on the prokaryotic ZFs, summarizing and discussing differences and analogies with the eukaryotic domains and providing important insights into their structure/function relationships.

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Section: Ros\mucr Familymentioning

confidence: 99%

The prokaryotic zinc‐finger: structure, function and comparison with the eukaryotic counterpart

et al. 2015

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“…β-glucuronidase activity in the rhizobial cells occupying clover nodules was detected by staining whole roots in 50 mM sodium phosphate buffer (pH 7.2) containing 50 μg ml −1 of 5-bromo-4-chloro-3-indolyl-β-D-glucuronide for 3 h (Janczarek et al, 2015a). The nodules were imaged under an OPTIPHOT2 light microscope equipped with a DS-Fil, 5 Megapixel color camera (Nikon Instruments, Melville, NY, U.S.A).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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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.

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“…Glucuronosyl-(β1 → 4)-glucosyltransferase PssDE is engaged in the second step, whereas glucuronosyl-(β1 → 4)-glucuronosyltransferase PssC in the third step of EPS unit assembly. The fourth sugar is added to the growing subunit by glucosyl-(α1 → 4)glucuronosyltransferase PssS (Ivashina and Ksenzenko 2012;Janczarek et al 2015b). The PssD, PssE, PssC, and PssS proteins are encoded by genes located in a large chromosomal cluster named Pss-I (Król et al 2007).…”

Section: Introductionmentioning

confidence: 99%

“…The only exception is PssA encoded by a gene located at a long distance from this EPS synthesis region (Ivashina et al 1994;van Workum et al 1997). The structural and functional organization of the Pss region has been characterized in three R. leguminosarum strains (VF39, TA1, Rt24.2) (Sadykov et al 1998;Król et al 2007;Janczarek et al 2015b). Mutations in pssA, pssD, and pssS genes totally abolish EPS production, whereas a mutation located in the pssC upstream region results in a nearly twofold decrease in EPS biosynthesis in comparison to the wild-type strain (Ivashina et al 1994;van Workum et al 1997;Janczarek et al 2015b).…”

Section: Introductionmentioning

confidence: 99%

“…The structural and functional organization of the Pss region has been characterized in three R. leguminosarum strains (VF39, TA1, Rt24.2) (Sadykov et al 1998;Król et al 2007;Janczarek et al 2015b). Mutations in pssA, pssD, and pssS genes totally abolish EPS production, whereas a mutation located in the pssC upstream region results in a nearly twofold decrease in EPS biosynthesis in comparison to the wild-type strain (Ivashina et al 1994;van Workum et al 1997;Janczarek et al 2015b). pssJ encodes a protein which is probably engaged in the last step of subunit assembly, since the exo344::Tn5 strain carrying a mutation in this gene has been found to synthesize only residual amounts of EPS, whose units lack a terminal Dgalactose (Breedveld et al 1993).…”

Section: Introductionmentioning

confidence: 99%

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

2017

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Background and aims A 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. Methods The 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. Results An 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. Conclusions The 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.

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

Cited by 24 publications

References 84 publications

The prokaryotic zinc‐finger: structure, function and comparison with the eukaryotic counterpart

The prokaryotic zinc‐finger: structure, function and comparison with the eukaryotic counterpart

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

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

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