Figure 1.
The chemical structure of the repeating unit of EPS produced by R. leguminosarum bv. trifolii [90].
Abbreviations: Glc, glucose; GlcA, glucuronic acid; Gal, galactose; Ac, acetyl.
Table 1.
Strains, plasmids and oligonucleotide primers used in this work.
Figure 2.
Organization of genes in the Pss-II region, constructs used in the pssP2 gene functional analyses and the results of PssP2 protein amino acid sequence analyses.
A) Physical and genetic map of the R. leguminosarum bv. trifolii Pss-II region; genes encoding putative proteins similar to elements of the Wzx/Wzy-dependent polysaccharide polymerization pathway are indicated above the map; Rho-independent terminator predicted downstream orf5 gene is marked with a black rectangle, promoters predicted between the pssY and pssP2 genes are marked with red rectangles; B) Constructs used for integration mutagenesis of the pssP2 gene (pKP2; green bar) and probing putative promoters identified upstream the pssP2 (pMP-P2) and pssY (pMP-Y) genes (blue bars). Small black rectangles mark positions of primers used for amplification of promoter regions. Red rectangles below the pMP-P2 and pMP-Y constructs mark positions of identified promoters and the scores obtained for each predicted promoter. C) Genomic organization of the integration mutant pssP2::pKP2. Position of the plac promoter (red rectangle) in the vector part and the direction of transcription from the promoter are shown. D) Sequence alignment of PssP2 and PssP proteins of R. leguminosarum bv. trifoli TA1. The alignment was produced in ClustalW and visualised by Alignment Viewer; amino acids were coloured according to their biochemical properties, thus the same colour means either identity or similarity, e.g. positively charged amino acids Arg and Lys are marked in red. E) Scheme of PssP2 topology and specific motifs found in silico. Blocks representing domains are aligned respective to the location in the polypeptide; TMS, transmembrane segment.
Table 2.
Putative homologues of PssP2 protein (586 aa) (ABD36550) identified through BLASTp searches.
Table 3.
Summary of the highest scoring results from the HHpred search of the PssP2 protein against the PDB database.
Figure 3.
Transcriptional activity of the predicted pssP2 and pssY promoters, as determined by measuring β-galactosidase activity in R. leguminosarum bv. trifolii TA1.
The strains carrying a plasmid with appropriate promoter fusion were cultured in TY (orange bars), 79CA (light grey) or M1 (dark grey) medium. Values are the means ± standard errors (extended bars) of at least four independent assays and are expressed in Miller units. The bars labeled with asterisks represent β-galactosidase activity values which are significantly different from the empty pMP220 vector control at p<0.05. The results were compared within groups for the three different media. The original names of the constructs for pssO and pssP genes were changed to avoid confusion only for the sake of data presentation: pMP-P stands for the original pMP2P [22] and pMP-O stands for the pMPO1 [38].
Figure 4.
Western immunoblot analysis with anti-His antibodies of subcellular localisation of His6-PssP2 protein.
Fractions analysed contained soluble proteins (SOL) and membrane proteins (TM) of the pssP2::pKP2 mutant and the complemented strain.
Figure 5.
Symbiotic capabilities of the pssP2::pKP2 mutant and its complemented derivative compared with the wild type RtTA1 strain in a standard plant test.
The mean values with standard error presented in the graph result from averaging the nodule number (pink bars), wet masses of plant shoots (mg/plant; green bars) and roots (mg/plant; brown bars) of 20 clover plants. The bars labeled with the same letters represent values which are not significantly different at p<0.05, while various letters represent values which are significantly different at p<0.05. Control plants were not inoculated with bacteria.
Figure 6.
Exopolysaccharide production and autoaggregation properties of the pssP2::pKP2 mutant and its complemented derivative.
A) Autoaggregation of RtTA1 wild type, pssP2::pKP2 mutant and the mutant carrying the pQBP2his plasmid as estimated quantitatively after growth and sedimentation in the 79CA medium. Autoaggregation is expressed in %; the higher the value the higher the autoaggregation. The bars labeled with the same letters represent values which are not significantly different at p<0.05, while various letters represent values which are significantly different at p<0.05. The extended bars represent standard error. B) Example photographs showing the autoaggregation of the studied rhizobia in 79CA liquid cultures (photographs were taken after the 24 hours of sedimentation). C) The table summarizes the EPS production by the studied strains. The amount of EPS produced is shown relative the amount produced by the wild type strain, which was 0.8 mg EPS/mg dry weight bacteria. D) Results of gel filtration chromatography of exopolysaccharides; the high-molecular-weight/low-molecular-weight (HMW/LMW) proportions were calculated as a ratio of peak areas: RtTA1 (dotted line in each graph), (i) pssP2::pKP2 mutant and (ii) pssP2::pKP2 mutant complemented with the pQBP2 plasmid. The retention times of dextran blue (2 MDa), dextran T550 and dextran T10 (10 kDa) molecular mass markers are indicated in each graph.
Figure 7.
Silver-stained Tricine SDS-PAGE profiles of LPS from the wild type, the pssP2::pKP2 and pssP2::pKP2(pQBP2his) strains. LPS I, high-molecular-weight LPS which carries the O-antigen; LPS II, low-molecular-weight LPS, representing the core oligosaccharide and lipid A.
Figure 8.
Interactions between PssP2 and other proteins involved in EPS production as analysed through the bacterial two-hybrid system.
The graphs present the results of measurements of β-galactosidase activity in E. coli DHM1 carrying respective bait and prey plasmids, the combination of which are presented at the top of panels A and B. Each analysed protein, i.e. PssT, PssP, PssL, PssP2, PssA, and PssC, was encoded as a fusion protein with an adenylate cyclase fragment in vectors pUT18 (N-terminal fusions), pUT18C (C-terminal fusions) (baits), and pKT25 (C-terminal fusions) (preys) differentiated by colour. The β-galactosidase activity was measured in at least 3 independent assays for two colonies (biological repeats) in order to exclude clone-by-clone variation, averaged and expressed in U/mg of bacterial dry weight ± standard error. Positive and negative control values are presented at the bottom of each graph. The controls were: the two interacting leucine zipper domains expressed from pUT18Czip and pKT25zip (positive control), and T18 and T25 cyclase fragments in non-recombinant pUT18(pUT18C) and pKT25 (the negative control). The bars labeled with asterisks represent β-galactosidase activity values which are significantly different from the control at p<0.05.
Figure 9.
Analysis of the interaction between PssP2 and PssP in the pssP2::pKP2 mutant carrying the pQBP2his plasmid by a co-purification strategy.
Samples of 40 µl of each fraction were separated by SDS-PAGE and visualized. (A) PssP is present in the fraction eluted from the resin, which equals to interaction between PssP and His6-PssP2, that was bound to affinity resin via a His-tag. (B) Negative control verifying that co-purification of PssP is dependent on its interaction with the His-tagged PssP2. L, material loaded to the resin; W, last wash (10 resin volumes); E, elution (1 resin volume). Western blots for each protein are shown below the corresponding gels.