Carbohydrate binding in site III^C of LlpA_BW.

<p>(A) Stereoview of methyl-α-D-mannopyranoside bound to subdomain III^C. Methyl-α-D-mannopyranoside is shown in blue and indicated by M. Residues belonging to the QxDxNxVxY motif and hydrogen bonding to the sugar as well as Asn188 are labeled. Water molecules bridging protein and carbohydrate are shown in cyan (B) Similar view of the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. The mannose residue occupying the primary binding site is shown in blue and labeled M. The additional two mannoses (labeled +1 and −1) and two <i>N</i>-acetyl glucosamine residues (labeled +2 and −2) are shown in green. Other colors are as in panel A. (C) Binding of the disaccharide Manα(1–2)Man. The non-reducing mannose residue occupying the primary binding site is shown in blue and labeled M. The second, reducing mannose is shown in green. Other colors are as in panel A.</p

Differential inhibitory activity of wild-type LlpA_BW and LlpA/LlpA1 domain chimers.

<p>The domain structures of LlpA_BW (as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003199#ppat-1003199-g004" target="_blank">Figure 4</a>) and of LlpA1 (inferred by pairwise alignment; N-domain in orange, C-domain in purple and C-terminal extension in grey) are depicted, along with those of chimeric forms (in dashed box). The LlpA variant lacking the terminal phenylalanine residue is marked with a yellow hexagon. Inhibitory activity of the respective <i>E. coli</i> recombinants was tested with diagnostic indicators for LlpA_BW (<i>P. syringae</i> GR12-2R3) and LlpA1 (<i>P. fluorescens</i> LMG 1794). Halo sizes are semi-quantified according to size of the growth inhibition halo (+++, native halo size of LlpA_BW and LlpA1; ++, halo size reduced; C, local clearing confined to producer colony spot; −, no halo or clearing; NT, not tested). Additional chimeric and domain deletion constructs not conferring bacteriocin activity against one of the indicator strains are specified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003199#ppat.1003199.s011" target="_blank">Figure S11</a>.</p

ITC analysis of carbohydrate binding to LlpA_BW and mutants.

<p>(A) Binding of LlpA_BW to the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. (B) Binding of LlpA_BW (blue circles, wild type) and the mutants LlpA_V177Y (green circles, site III^C knockout), LlpA_V208Y (red circles, site II^C knockout) and LlpA_V177Y-V208Y (black circles, site II^C and III^C knockout) to α-methyl mannoside. There is no heat exchanged in the titration of the double mutant or the site III^C knockout LlpA_V177Y, whereas the site II^C knockout LlpA_V208Y, binds the monosaccharide in a “wildtype”-like fashion, showing that only site III^C is involved in sugar binding.</p

Inhibitory activity of wild-type LlpA_BW and selected mutants with modified (potential) mannose-binding sites.

<p>The domain structure (N-domain in red, C-domain in blue and C-terminal extension in green) and the position of the MMBL motifs (potentially active binding sites in orange, inactive ones in grey) are shown. The positions of conserved valine residues converted to tyrosine residues by site-directed mutagenesis are indicated with a black bar. Inhibitory activity of <i>E. coli</i> strains expressing mutant LlpA_BW forms was assayed against <i>P. syringae</i> GR12-2R3 and semi-quantified according to the size (inner zone radius) of the growth inhibition halo relative to LlpA_BW (+++, native LlpA_BW; ++, halo size reduced; + halo size strongly reduced; −, no halo; NT, not tested). For wild-type LlpA_BW and three purified His-tagged mutant forms (LlpA_V177Y, LlpA_V208Y and LlpA_V177Y-V208Y) the MIC values were determined with indicator <i>P. syringae</i> GR12-2R3. Molar minimal inhibitory concentrations of recombinant proteins (with standard deviations): LlpA, 2.08 nM (±0.58 nM); LlpA_V177Y, 10.9 nM (±0.66 nM); LlpA_V208Y, 1.98 nM (±0.066 nM); 65.72 nM (±2.80 nM).</p

Structural Determinants for Activity and Specificity of the Bacterial Toxin LlpA

<div><p>Lectin-like bacteriotoxic proteins, identified in several plant-associated bacteria, are able to selectively kill closely related species, including several phytopathogens, such as <i>Pseudomonas syringae</i> and <i>Xanthomonas</i> species, but so far their mode of action remains unrevealed. The crystal structure of LlpA_BW, the prototype lectin-like bacteriocin from <i>Pseudomonas putida</i>, reveals an architecture of two monocot mannose-binding lectin (MMBL) domains and a C-terminal β-hairpin extension. The C-terminal MMBL domain (C-domain) adopts a fold very similar to MMBL domains from plant lectins and contains a binding site for mannose and oligomannosides. Mutational analysis indicates that an intact sugar-binding pocket in this domain is crucial for bactericidal activity. The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site. Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity. Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity.</p> </div

Domain interactions within LlpA_BW.

<p>(A) The C-terminal hairpin extension (green cartoon) covers the interface between the N-domain (red surface representation) and the C-domain (blue surface representation). (B) Stereo view of the interactions between loop segments Val140-Asp145 (cyan) of the C-domain and Val115-Asp118 (yellow) and Ser31-Gln34 (orange) of the N-domain. Other structural elements are colored according to panel A. (C) Stereo view of the two-stranded β-sheet formed by strands β11a,b and β22a,b that links the N- and the C-domains and gives rise to domain swapping. Colors according to panel A and B.</p

Overall structure of LlpA_BW.

<p>(A) Topology diagram of LlpA_BW. The N-domain is shown in red, the C-domain in blue and the C-terminal extension in green. The different strands and subdomains are labeled. Domain swapping involves β-strand segments β11b and β22b, which together with β-strand segments β11a and β22a link both MMBL domains. (B) Cartoon representation of LlpA_BW with the different domains colored as in panel A. The bound Me-Man residue is shown as an orange stick representation. (C) Domain orientations of LlpA_BW compared with the heterodimeric MMBL ASA I (<i>Allium sativum</i> agglutinin, PDB entry 1KJ1) and tandem MMBL SCAfet (<i>Scilla campanulata</i> fetuin-binding lectin, PDB entry 1DLP). In each case, the C-domain is shown in the same orientation, highlighting the different relative orientation of the N-domain in LlpA_BW. Domain-swapped dimers in homo-oligomeric plant MMBL lectins such as snowdrop lectin have their domain orientation similar to ASA I and SCAfet.</p

Binding affinities and thermodynamic parameters obtained from ITC titrations.

<p>The reported values for K_d, ΔG°, ΔH° and −TΔS° were determined from fitting a single site interaction model (n = 1) to the experimental ITC data. The interaction of the mutants LlpA_V177Y and LlpA_V177-V208Y with the different sugars is negligible and no heat effect was observed. Therefore they are not included in this table.</p

Additional file 1: Figure S1. of Influence of rhizobacterial volatiles on the root system architecture and the production and allocation of biomass in the model grass Brachypodium distachyon (L.) P. Beauv.

Impact of individual strain volatile compounds on Total Secondary Root Length (A), Total Adventitious Root Length (B) and Secondary Root Density (C). The strains are grouped according to the clusters defined earlier, based on PC. Within each cluster, the strains are ranked in ascending mean value order. Presented values are means of the four experimental replicates (64 or 128 biological replicates +/− confidence interval (α = 5 %) for each strain and the control, respectively). Significant changes compared with the control without bacteria are marked with an asterisk (*). (DOCX 374 kb