Role of the Extracytoplasmic Function Protein Family Sigma Factor RpoE in Metal Resistance of Escherichia coli

RpoE of Escherichia coli is a sigma factor of the extracytoplasmic function protein family and is required for the expression of proteins involved in maintaining the integrity of periplasmic and outer membrane components. RpoE of E. coli was needed for full resistance to Zn(II), Cd(II), and Cu(II). Promoter gene fusion and quantitative real time reverse transcription (RT)-PCR (qRT-PCR) assays demonstrated that expression of RpoE was induced by metals. Global gene expression profiles upon metal treatment of a ΔrpoE mutant strain and its wild-type strain were analyzed with microarrays, and selected genes were confirmed by qRT-PCR. The absolute number of genes that were changed in their expression upon metal stress was similar in both strains, but the increase or decrease in transcript levels upon metal treatment was smaller in the ΔrpoE mutant strain than in the wild type. Genes showing increased expression in the ΔrpoE mutant strain encoded proteins that belong to general defense systems against protein-denaturing agents. Genes showing decreased expression were part of the RpoE modulon itself plus the ompC gene, encoding a major outer membrane protein. A ΔompC deletion strain was as sensitive to Cu(II) and Cd(II) as the ΔrpoE mutant or a ΔrpoE ΔompC double mutant strain. In the case of Zn(II), the double mutant was more sensitive than either single mutant. This indicates that increased expression of OmpC contributes to the RpoE modulon-mediated response to metals

A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture

<div><p>Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen <em>Xanthomonas campestris</em> pv. <em>vesicatoria</em> exhibits E3 ubiquitin ligase activity <em>in vitro</em> and <em>in planta</em>, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the <em>Xanthomonas</em> XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.</p> </div

Analysis of cell death induction by XopL in <i>Nicotiana benthamiana</i>.

<p><i>Agrobacterium</i>-strains carrying binary constructs encoding XopL (WT), XopL_D502A (D502A), XopL_K578A (K578A), XopL_A579W (A579W), XopL_Q612A (Q612A), XopL_L619A (L619A), XopL[aa 1–449] (leucine-rich repeat, LRR), XopL[aa 450–660] (C-terminal domain, CTD), both XopL[aa 1–449] and XopL[aa 450–660] expressed in trans (LRR+CTD) or GFP under control of the <i>Cauliflower mosaic virus</i> (CaMV) 35S promoter, were inoculated into <i>N. benthamiana</i> leaves (8×10⁸ cfu/ml). (<b>A</b>) Phenotypes of the inoculated leaf area were documented 6 days post inoculation (dpi). (<b>B</b>) Cell death quantification using electrolyte leakage measurements. Measurements were carried out 2 dpi (light grey bars) and 4 dpi (dark grey bars), respectively. Bars represent the average of triplicates of 5 leaf discs each, error bars represent standard deviations. Asterisks indicate statistically significant differences compared to GFP control (<i>t</i>-test, <i>P</i><0.01). (<b>C</b>) Leaf tissue was harvested 2 dpi, and protein extracts were analyzsed by western blot using a <i>Strep</i>-tag (α-strep) and ubiquitin-specific antibody (α-Ub), respectively. Signals specific for full-length XopL, XopL[aa 1–449] (XopL_LRR)and XopL[aa 450–660] (XopL_CTD) are labeled. Polyubiquitination is indicated by (Ub)_n. Equal loading is shown by Ponceau staining of Rubisco. The experiments were performed three times with similar results.</p

Structure of the N-terminal LRR domain of XopL.

<p>(<b>A</b>) The left panel shows the ribbon diagram of the XopL[aa 144–450] structure (green). N- and C-termini and the secondary structure elements (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003121#ppat.1003121.s001" target="_blank">Figure S1</a>) are labeled. In comparison, the IpaH3 LRR domain (PDB code 3CVR), represented by aa residues 25–268, is shown in the right panel as a ribbon diagram (purple) with labeled N- and C-termini. Disordered regions in the protein are represented as grey dashed lines. (<b>B</b>) Sequence alignment of the nine leucine-rich repeats of XopL[aa 145–450], showing their consensus and relationship to the plant-specific (PS)-LRR subclass of LRRs. The positions of the helical turn (red box) and β- strand (blue arrow) in the “typical” LRR of XopL are given.</p

The C-terminal domain of XopL shows E3 ubiquitin ligase activity.

<p>(<b>A</b>) <i>In vitro</i> ubiquitin ligase assay in presence of E1, UBE2D2, ATP, ubiquitin and His₆-XopL full-length protein (1–660) or derivatives thereof (numbers indicate amino acid positions corresponding to full-length protein). The western blots were reacted with antibodies against ubiquitin (α-Ub, left panel) and polyhistidine (α-His, middle panel), respectively, while the right panel shows the reaction mixture via Coomassie Blue staining of the SDS-PAGE. (Ub)_n indicates polyubiquitination. Asterisks indicate His₆-XopL derivatives. Unspecific signals are labeled by †. (<b>B</b>) Ubiquitin polymerization reaction at different time points in the presence (+) or absence (−) of E1, AtUBC11 (E2), ubiquitin and His₆-XopL[aa 144–660]. Polyubiquitination was determined by western blot (left panel) using ubiquitin antibodies. The right panel shows the state of modification of the proteins via Coomassie Blue staining of the 10–15% step-gradient SDS-PAGE gel. Components of the reactions (XopL[aa 144–660], ubiquitin (Ub) and AtUBC11) on western blots or Coomassie-stained gels are labeled. (<b>C</b>) <i>In vitro</i> ubiquitination assay in the presence of ATP, E1, AtUBC11, His₆-XopL[aa 474–660], ubiquitin (WT) and lysine (K) to arginine (R) mutant derivatives thereof. The left panel shows the western blots probed against ubiquitin (α-Ub) of the <i>in vitro</i> reactions, run on a 10–15% step-gradient SDS-PAGE using ubiquitin mutant derivatives in which only the indicated K residues were substituted by R. The right panel shows the starting material of the <i>in vitro</i> reactions in the left panel via Coomassie Blue staining of the SDS-PAGE. Polyubiquitination is indicated by (Ub)_n; the components of the reactions and the di-ubiquitin (Ub)2 on the western blot or Coomassie-stained SDS-PAGE are labeled.</p

Data collection, phasing and refinement statistics for SAD (SeMet) structures.

*<p>Values in parentheses are for highest-resolution shell.</p>a<p><i>R</i>_merge = Σ|<i>I</i>−<<i>I</i>>|/Σ <i>I</i>.</p>b<p><i>R</i>_work = Σ | <i>F</i>_obs−<i>F</i>_calc |/Σ |<i>F</i>_obs|, where <i>F</i>_obs and <i>F</i>_calc are observed and calculated structure factors, respectively.</p>c<p><i>R</i>_free calculated using 5% of total reflections randomly chosen and excluded from the refinement.</p

XopL displays E2 specificity <i>in vitro</i>.

<p>(<b>A</b>) <i>In vitro</i> ubiquitin ligase assay with ATP, ubiquitin, E1, human UBE2D2 (E2D2) or different <i>Arabidopsis thaliana</i> E2s (ATUBC28, 11, 13 or 19) in the presence (+) or absence (−) of His₆-XopL[aa 1–660]. The left panel shows the western blot reacted with ubiquitin antibodies (α-Ub) after 5 hours incubation, while the right panel shows the Coomassie stained gel of the reactants at the start of the reaction. Polyubiquitination is indicated by (Ub)_n. A lower-molecular weight impurity or degradation product in the full-length XopL protein purification is denoted by †. (<b>B</b>) Ubiquitin ligase assay described in (A) using His₆-XopL[aa 474–660], AtUBC28 and mutant derivatives R5A, F62A, K63A and A96D. Reaction times are indicated. The left panel shows the western blot reacted with ubiquitin antibodies (α-Ub), while the right panel shows the Coomassie-stained gel at the equivalent time points. (Ub)_n indicates polyubiquitination, and positions on the western blot or Coomassie-stained gels corresponding to ubiquitin (Ub), di-ubiquitin (Ub)2, AtUBC28, mono-ubiquitinated AtUBC28 (AtUBC28-Ub) and His₆-XopL[aa 474–660] (E3) are labeled.</p

XopL inhibits pathogen-associated molecular pattern (PTI)-induced defense gene expression.

<p><i>Arabidopsis thaliana</i> Col-0 protoplasts were co-transformed with <i>pNHL10-LUC</i> (luciferase) as reporter, the <i>p35S-</i>effector gene constructs <i>xopL</i>, <i>xopL_Q612A</i>, <i>xopL_LRR</i> and <i>xopL_CTD</i> or <i>p35S-cfp</i> and p<i>35S</i>-<i>avrPto</i> (negative and positive control, respectively), and <i>pUBQ10-GUS</i> (β-glucuronidase) for normalization. 14 h after transformation, protoplasts were treated with H₂O (<b>A</b>), 100 nM elf18 (<b>B</b>) and 100 nM flg22 (<b>C</b>), and luciferase activity was monitored for 3 h. Results are depicted as LUC/GUS ratios (with the zero timepoint, H₂O-treated sample set at a reference value of one). (<b>D</b>) Protein extracts of transformed protoplasts were taken 10 min after treatment and analyzed by immunoblotting using a pTepY-antibody (specific for activated MAP-Kinases) and HA-specific antibodies for detection of HA-tagged effector-or CFP-fusion proteins. MPK3, 4, 6, 11: mitogen activated protein kinase 3, 4, 6, 11. The experiments were performed three times with similar results.</p