A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring

Crystallization and preliminary diffraction analysis of the catalytic domain of major extracellular endoglucanase from Xanthomonas campestris pv. campestris

Cellulases, such as endoglucanases, exoglucanases and β-glucosidases, are important enzymes used in the process of enzymatic hydrolysis of plant biomass. The bacteria Xanthomonas campestris pv. campestris expresses a large number of hydrolases and the major endoglucanase (XccEG), a member of glycoside hydrolase family 5 (GH5), is the most strongly secreted extracellularly. In this work, the native XccEG was purified from the extracellular extract and crystallization assays were performed on its catalytic domain. A complete data set was collected on an in-house X-ray source. The crystal diffracted to 2.7Å resolution and belonged to space group C2, with unit-cell parameters a = 174.66, b = 141.53, c = 108.00Å, β= 110.49°. The Matthews coefficient suggests a solvent content of 70.1% and the presence of four protein subunits in the asymmetric unit.FAPESP (08/56255-9, 07/08706-9, 10/52362-5, 09/05349-6)CAPESCNPq / INCT do Bioetanol (471834/2009-2, 301981/2011-6

PILZ Protein Structure and Interactions with PILB and the FIMX EAL Domain: Implications for Control of Type IV Pilus Biogenesis

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3`-> 5`)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv Citri (PilZ(XAC1133), this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ(XAC1133) shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which NZ proteins regulate specific processes is not clear. In this study, we show that PilZ(XAC1133) binds to PilB, an ATPase required for TV polymerization, and to the EAL domain of FiMX(XAC2398), which regulates TV biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimXX(AC2398) in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ(XAC1133). Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ(XAC1133) interactions with FimX(XAC2398) and PilB(XAC3239) are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of ""degenerate"" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery. (C) 2009 Elsevier Ltd. All rights reserved.FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq, BrazilConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Ectopic expression of <i>hrpG</i> under control of a constitutive promoter restores full pathogenicity and HR of the <i>rsmA</i> mutant of <i>Xanthomonas citri</i> subsp. citri.

<p><b>A</b>) XCC strains 306 (WT) and the <i>rsmA</i> mutant carrying the empty plasmid pBBR5 (ΔrsmA) or pBBR5 with <i>hrpG</i> wild-type (hrpG) or <i>hrpG</i> alleles with E44K and D60N mutations were inoculated into <b>A</b>) sweet orange (<i>Citrus sinensis</i>) leaves or <b>B</b>) tobacco leaves by infiltrating bacterial cells at a concentration of 10⁶ CFU/ml. Sweet orange leaves inoculated with WT and <i>rsmA</i> mutant strains harboring both <i>hrpG</i> and hrpG-E44K alleles showed canker symptoms 7 days after inoculation, while a strong HR was observed in tobacco leaves only 2 days after infiltration. However, constitutive expression of the hrpGD60N allele was not able to recover the pathogenicity and HR in the <i>rsmA</i> mutant. <b>C</b>) <i>In plant</i> growth curve experiments confirmed that the <i>rsmA</i> mutant transformed with both empty pBBR5 (ΔrsmA) and pBBR5Lac-<i>hrp</i>GD60N-6His (D60N) have impaired growth in host plants. Error bars represent standard deviations. <b>D</b>) Western-blotting analysis of HrpG-His protein levels in the wild-type and <i>rsmA</i> mutant strains carrying the <i>hrpG</i> (hrpG) or mutated <i>hrpG</i> alleles (E44K and D60N) under control of a constitutive promoter. Equal amounts of total cell extracts were analyzed by immunoblotting with anti-6HisTag antibodies (MBL, USA).</p

RsmA regulates protein levels of T3SS in <i>X. citri</i> subsp. citri.

<p>Immunoblotting analyses of the total protein extracts of the wild-type (Wt), the <i>rsmA</i> mutant (Δ<i>rsmA</i>) harboring the empty plasmid pUFR047 and the complemented strain (pUFRrsmA) are shown. Bacterial cells were grown in the XVM2 medium and collected at OD_{600 nm} = 0.5. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The blots were probed with <b>A</b>) HrpB1, <b>B</b>) HrpD6 or <b>C</b>) HrcU and HrpB2 polyclonal antibodies, respectively. Protein-A conjugated with horseradish peroxidase was used to detect the blots. Beneath the panels are presented the values of the relative levels of detected proteins in <i>rsmA</i> mutant and complemented strains which were estimated according to wild-type results. The estimated values were normalized with the values obtained to unspecific protein bands also recognized by the antibodies. <b>D</b>) and <b>E</b>) GUS assays using translational fusion constructs. The different constructs used in this assay are represented by diagrams bellow of the graphics. D) Wild-type and <i>rsmA</i> mutant cells harboring plasmid-borne promoterless <i>gusA</i> in-frame fused to the native promoters and the first codons of the <i>hrp</i> genes. E) Wild-type and <i>rsmA</i> mutant cells transformed with translational fusions driven by the constitutive P<i>lac</i> promoter. Values presented are means ± standard deviations of three independent experiments. * represents the significant difference between the wild-type and Δ<i>rsmA</i> values by using ANOVA. The GUS assay was repeated twice with similar results.</p

RNA mobility shift assays with purified 6HisRsmA of <i>X. citri</i> subsp. citri

<p><b>A</b>) 6HisRsmAxcc (65 nM) binds to the high affinity RNA target R9-43. Biotin 3′-end-labeled R9-43 (6.25 nM) was incubated with 6HisRsmAxcc (65 nM) for 30 minutes at room temperature, followed by analysis on a 5% native polyacrylamide gel. A competitive assay in which unlabeled R9-43 RNA (6.25 nM) was added to the reaction reduced the signal resulting from the biotinylated nucleotide. <b>B</b>) 6HisRsmAxcc directly interacts with the 5′ UTRs of <i>hrpG</i> and <i>hrpD</i>. The leader sequences of <i>hrpD</i>, <i>hrpE</i> and <i>hrpG</i> cloned were transcribed <i>in vitro</i> and biotinylated with RNA Labeling kit (Roche). Biotinylated RNA probes were incubated with 6HisRsmAxcc and resolved in a 5% native polyacrylamide gel. The addition of unlabeled competitor R9-43 to the reactions reduced the intensity of the shifted band, which confirmed the specificity of the RsmAxcc-<i>hrpG</i> and RsmAxcc-<i>hrpD</i> interactions. <b>C</b>) 3′-end-biotin-labeled RNA probes encoding the leader sequences of <i>hrpB</i>, <i>hrpC</i>, <i>hrpF</i> and <i>hrpX</i> were tested for interactions with 6HisRsmAxcc (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945.s007" target="_blank">Table S4</a>). In addition, 3′-end-biotin–labeled RNA probes <i>hrpG1</i> and <i>hrpG2</i>, which bear the GGA motifs encoded by the 5′ leader sequence of <i>hrpG</i>, were used to map the interaction RsmAxcc-<i>hrpG</i>. Only the GGA motif between nucleotides 80 and 120 in the <i>hrpG</i> leader sequence (<i>hrpG</i>2 probe) interacted with 6HisRsmAxcc. <b>D</b>) To determinate the apparent equilibrium binding constant (K_d), 3′ end-labeled <i>hrpG</i>2 RNA (6.25 nM) was incubated with increasing concentrations of 6HisRsmAxcc as noted at the bottom of each lane. The binding curve for the 6HisRsmAxcc-<i>hrpG</i>2 interaction was determined as a function of 6HisRsmAxcc concentration and shifted band intensity. The average pixel value of each shifted band was calculated with ImageJ software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Girish1" target="_blank">[68]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Hartig1" target="_blank">[69]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Jensen1" target="_blank">[112]</a>. The apparent equilibrium binding constant (K_d) for this reaction was 0.18±0.2 µM 6HisRsmAxcc. Samples were loaded and resolved onto a 5% native polyacrylamide gel. All probes were transferred and cross-linked to a nylon membrane, incubated with streptavidin conjugated with horseradish peroxidase, and detected according to manufacturer's instructions (LightShiftChemiluminescent RNA EMSA Kit, Thermo Scientific). Signals + and − correspond to the presence and absence in the reaction, respectively. Positions of bound and free probes are shown.</p

Analysis of <i>hrpG</i> and <i>hrpD</i> mRNA stability in the wild-type and <i>rsmA</i> mutant strains by RT-PCR.

<p><b>A</b>) Cells of the XCC wild-type and Δ<i>rsmA</i> strains were grown in XVM2 medium to OD_{600 nm} = 0.6, treated with 10 µg/µL ciprofloxacin and harvested at several time points after treatment. Total RNA was isolated, and 2 µg of RNA was used for One Step RT-PCR (Qiagen) in 25 µL reactions. Reactions were subjected to PCR amplification for 26 cycles. Ten microliters of each reaction were resolved on a 1.5% agarose gel. The stability of the <i>hrpD</i> transcript was evaluated using primers annealing within the first orf <i>hrpQ</i>. The 16S RNA was analyzed as a control for normalizing the <i>hrpG</i> and <i>hrpD</i> amplification products. <b>B</b>) The relative values of <i>hrpG</i> and <i>hrpD</i> mRNA half-lives were estimated by determinating the average pixel value of each amplified product and subtracting the background using ImageJ software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Girish1" target="_blank">[68]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Jensen1" target="_blank">[112]</a>. The mean values were normalized to the corresponding 16S amplification product. Mean values derived from two independent experiments are shown. <b>C</b>) Model for the predicted secondary structure of the <i>hrpG</i> and <i>hrpD</i> leader sequences obtained with MFold software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Zuker1" target="_blank">[66]</a>. The positions of GGA motifs in the structures are indicated with arrows. AUG is shown in an open box.</p

Determination of <i>hrp/hrc</i> transcriptional start sites of <i>X. citri</i> subsp. citri.

<p>The transcriptional start sites for <i>the genes hrpG, hrpX and hrpF, and the operons hrpB, hrpC, hrpD and hrpE</i> of XCC were determined by 5′RACE. <b>A</b>) Specific PCR products were detected after the amplification of reverse-transcribed cDNA with the gene-specific primers for <i>hrp/hrc genes</i> together with an adapter primer (Roche), respectively. <b>B</b>) Sequencing of the PCR products identified the nucleotides indicated by arrow as the transcription start sites of <i>hrpG, hrpX, hrpB, hrpC, hrpD, hrpE and hrpF</i> of XCC. Analysis of the 5′ leader sequences of the <i>hrp/hrc</i> transcripts suggests potential RsmA binding sites (highlighted) in <i>hrpG</i>, <i>hrpC</i>, <i>hrpD</i> and <i>hrpE</i>. However, the 5′ leader regions of the <i>hrpB</i>, <i>hrpF</i> and <i>hrpX</i> do not contain the GGA motifs. The +1 nucleotide is indicated with an arrow, putative RsmA binding sites (GGA) in the leader sequences are highlighted in red color, and the PIP-box motif within each promoter are in bold. The −35 and −10 sequences are shown in bold and italics. ATG or GTG are indicated in bold and underlined. The specific primers used to amplify the fragments are underlined.</p

<i>In vivo</i> phosphorylation of HrpG Asp60 residue is critical to restore the virulence in the Δ<i>rsmA</i> mutant of XCC.

<p><b>A</b>) Western-blotting analysis of XCC lysates on a 25 µM Phos-tag acrylamide gel (Wako, USA). Bacteria cells were grown in nutrient broth or XVM2 medium (T3SS inducible medium) to OD_{600 nm} = 0.6, and equal amounts of total cell extracts were analyzed by immunoblotting with anti-6HisTag antibodies (MBL, USA). <b>B</b>) Western-blotting analysis to determine HrpG-His protein levels in XCC cell extracts resolved in a 12% acrylamide gel system without Manganese(II)-Phos-tag. Strains analyzed: WT harboring the wild-type <i>hrpG</i>-6His allele; Δ<i>rsmA</i> carrying the wild-type <i>hrp</i>G-6His allele and the mutated <i>hrpG</i>-6His alleles D41N, E44K and D60N. All <i>hrpG</i>-6His constructs were cloned into the pBBR5 plasmid and placed under the control of a constitutive promoter.</p