Co-regulation of Iron Metabolism and Virulence Associated Functions by Iron and XibR, a Novel Iron Binding Transcription Factor, in the Plant Pathogen <i>Xanthomonas</i>

<div><p>Abilities of bacterial pathogens to adapt to the iron limitation present in hosts is critical to their virulence. Bacterial pathogens have evolved diverse strategies to coordinately regulate iron metabolism and virulence associated functions to maintain iron homeostasis in response to changing iron availability in the environment. In many bacteria the ferric uptake regulator (Fur) functions as transcription factor that utilize ferrous form of iron as cofactor to regulate transcription of iron metabolism and many cellular functions. However, mechanisms of fine-tuning and coordinated regulation of virulence associated function beyond iron and Fur-Fe²⁺ remain undefined. In this study, we show that a novel transcriptional regulator XibR (named <u><i>X</i></u><i>anthomonas</i> <u>i</u>ron <u>b</u>inding <u>r</u>egulator) of the NtrC family, is required for fine-tuning and co-coordinately regulating the expression of several iron regulated genes and virulence associated functions in phytopathogen <i>Xanthomonas campestris</i> pv. <i>campestris</i> (Xcc). Genome wide expression analysis of iron-starvation stimulon and XibR regulon, GUS assays, genetic and functional studies of <i>xibR</i> mutant revealed that XibR positively regulates functions involved in iron storage and uptake, chemotaxis, motility and negatively regulates siderophore production, in response to iron. Furthermore, chromatin immunoprecipitation followed by quantitative real-time PCR indicated that iron promoted binding of the XibR to the upstream regulatory sequence of operon’s involved in chemotaxis and motility. Circular dichroism spectroscopy showed that purified XibR bound ferric form of iron. Electrophoretic mobility shift assay revealed that iron positively affected the binding of XibR to the upstream regulatory sequences of the target virulence genes, an effect that was reversed by ferric iron chelator deferoxamine. Taken together, these data revealed that how XibR coordinately regulates virulence associated and iron metabolism functions in Xanthomonads in response to iron availability. Our results provide insight of the complex regulatory mechanism of fine-tuning of virulence associated functions with iron availability in this important group of phytopathogen.</p></div

The Xcc XibR and NtrC are two functionally distinct homologs of NtrC family proteins.

<p>(A) Multiple sequence alignment of XibR and NtrC of Xcc. Sequence alignment was performed by using CLUSTALW. Asterisks indicate identical amino acids; (:) indicate highly conserved and (.) less conserved. (*) indicate the putative conserved aspartate residue phosphorylation site (D55). Region inside the green box indicate the N-terminal receiver (Rec) domain; region inside the red box indicate the central σ54 interacting domain or AAA+ domain; and the region inside the blue box indicate the C-terminal DNA binding domain or HTH domain Conserved motifs and amino acids are underlined. (B) Homology model of XibR and GlnG (NtrC) of Xcc 8004 showing structural similarity and both having N-terminal Rec domain, middle σ54 interacting domain and C-terminal DNA binding domain. Homology modeling was performed by using the SWISS-MODEL ProMod Version 3.70. (C) Schematic representation of XibR and GlnG (NtrC) domain swapped hybrid constructs in the plasmid vector pHM1. (1) pSSP30 (wild-type <i>xibR</i> allele in pHM1; XibR), (2) pSSP34 (wild-type <i>glnG</i> or <i>ntrC</i> allele; NtrC), (3) pSS61 (<i>xibR</i> with the swapped Rec domain from NtrC; XibR Swp ^Rec), (4) pSS62 (<i>xibR</i> allele with the swapped σ54 interacting domain from NtrC; XibR Swp ^σ54), (5) pSS63 (<i>xibR</i> with the swapped DNA binding HTH domain from NtrC; XibR Swp ^HTH), (7) pSS64 (<i>ntrC</i> with the swapped Rec domain from XibR; NtrC Swp ^Rec), (8) pSS65 (<i>ntrC</i> with the swapped σ54 interacting domain from XibR; NtrC Swp ^σ54), and (9) pSS66 (<i>ntrC</i> with the swapped DNA binding HTH domain from XibR; NtrCSwp ^HTH). (D) Quantification of siderophore production. Average ratio of siderophore halo to colony diameter for different strains of Xcc grown on PSA-CAS-DP plate. Xcc strains: Xcc 8004 (wild-type), Δ<i>xibR</i> (<i>xibR</i> deletion mutant), Δ<i>glnG</i> (<i>glnG</i> deletion mutant), Δ<i>xibR</i>/pSSP30 (Δ<i>xibR</i> mutant harboring the plasmid containing the wild-type <i>xibR</i> allele; XibR), Δ<i>glnG</i>/pSSP34 (Δ<i>glnG</i> mutant harboring the plasmid containing wild-type <i>glnG</i> or <i>ntrC</i> allele; NtrC), Δ<i>xibR</i> (pSS61; XibR Swp ^Rec), Δ<i>xibR</i> (pSS62; XibR Swp ^σ54), Δ<i>xibR</i> (pSS63; XibR Swp^HTH), Δ<i>glnG</i> (pSS64; NtrC Swp ^Rec), Δ<i>glnG</i> (pSS65; NtrC Swp ^σ54) and Δ<i>glnG</i> (pSS66; NtrC Swp^HTH). Error bars represent SD of the mean (n = 3).</p

<i>xibR</i> is required for optimal virulence.

<p>(A) Infected cabbage leaves with different Xcc strain showing symptoms as a lesion at 15 days postinoculation. 30 days old plants were inoculated with bacterial cultures (1 X 10⁹ cells/ml suspension) of different Xcc strains by clip method. (B) <i>In planta</i> growth assays of Xcc 8004, Δ<i>xibR</i>, Δ<i>xibR</i>/pSSP30 and Δ<i>xibR</i>/pSSP39 strains. Bacterial populations were measured by crushing the leaves of 1cm² areas for each and serial dilution plating at the indicated post inoculation days. Data are shown as mean ± S.E. (n = 3). (C) <i>In planta</i> bacterial migration assay was performed by inoculating 1cm pieces of infected leave, cut from base to tip with sterile scissors on rich PS medium with respective antibiotics. Migration was estimated by observing colonies formed after 1 to 3 days by the bacterial ooze from the cut ends of cabbage leaf pieces. (D) Quantification of lesion length at 15 days post inoculation. Data shown as mean ± S.E. (n = 25). (E) Relative quantification of the expression of different Type III secretion system <i>hrp</i> genes of Xcc 8004, Δ<i>xibR</i>, and Δ<i>xibR</i>/pSSP30 strains by real-time qRT-PCR. * Indicating p-value < 0.05, **indicating p-value < 0.01 and *** indicating p-value < 0.001 statistically significance by paired student t-test.</p

Chemotaxis and motility are regulated by <i>xibR</i> and induced under iron limitation.

<p>(A) Quantitative chemotaxis capillary assay in response to D-(+)-Xylose with different Xcc strains grown under PS, PS + 100 μM DP and PS + 100 μM DP + 100 μM FeSO₄. Cells were incubated at 28°C with capillaries containing D-(+)-Xylose (1.2 mM) and PBS. Relative chemotaxis response was determined by migrated bacterial cells in capillary containing D-(+)-Xylose over the migrated bacterial cells in capillary containing PBS. Data are shown as mean ± S.E. (n = 3). The experiment was repeated two times. (B) Relative quantification of the expression of chemotaxis histidine protein kinase (cheA3) of Xcc grown under PS, PS + 100 μM DP, and PS + 100 μM DP + 100 μM FeSO₄ by real-time qRT-PCR. 16S ribosomal RNA was used as an endogenous control to normalize the RNA for cellular abundance. (C) Expression analysis of <i>motA</i> operon in wild-type Xcc 8004 and Δ<i>xibR</i> mutant grown under PS, PS + 100 μM DP, and PS + 100 μM DP + 100 μM FeSO₄ by monitoring the β-glucuronidase (GUS) activity. (D) Swim plate motility assay for different Xcc strains; Xcc 8004, Δ<i>xibR</i>, Δ<i>xibR</i>/pSSP30 and Δ<i>xibR</i>/pSSP39. (E) Motility zone diameter quantification from semisolid swim plate motility assay. (F) Relative quantification of the expression of flagellar biosynthesis gene for a cell-distal portion of basal-body rod (XC_2239) of Xcc grown under PS, PS + 100 μM DP, and PS + 100 μM DP + 100 μM FeSO₄ by real-time qRT-PCR. (G) Bacterial velocity measurement from the live cell imaging of bacterial movement at single cell level by using manual tracking and chemotaxis tools with ImageJ software. Cells were grown in PS, low-iron (PS + 100 μM 2,2′-dipyridyl) and PS + 100 μM 2,2′-dipyridyl + 100 μM FeSO₄ at 28°C up to mid-exponential phase, stained with Syto9 and incubated for 10 min at 28°C. The stained cells were loaded into a chamber of sterile glass bottom plates containing PS medium with 0.3% agar and visualized on epifluorescence microscope. Values are mean of at least 25 bacteria up to 20 frames. The experiment was repeated three times. Error bars are SEM. Data shown in the graphs are mean ± S.E. (n = 3).* Indicating p-value < 0.05, **indicating p-value < 0.01 and *** indicating p-value < 0.001 statistically significance by paired student t-test.</p

Δ<i>xibR</i> mutant of Xcc exhibit altered ferric iron uptake and defect in iron storage.

<p>(A) Δ<i>xibR</i> mutant exhibits defect in ferric iron uptake. Transport was initiated by addition of 0.5 μM ⁵⁵FeCl₃ to cell suspensions of Xcc 8004, Δ<i>xibR</i>, Δ<i>xibR</i>/pSSP30 and Δ<i>xibR</i>/pSSP39 grown under low-iron condition. Low-iron was made by addition of 150 μM DP to PS medium. Incorporation of radiolabelled Fe³⁺ was detected by scintillation counter. (B) Δ<i>xibR</i> mutant exhibits enhanced uptake of ferric iron-vibrioferrin complex. Transport was initiated by the addition of 0.5 μM 1:1 ratios of ⁵⁵FeCl₃ and vibrioferrin to the cell suspensions of different Xcc strains grown under low-iron condition. (C and D) Relative quantification of the expression of <i>Xanthomonas</i> siderophore uptake gene (<i>xsuA</i>) and outer membrane receptor for ferric iron (XC_0925) of Xcc grown under PS, PS + 100 μM DP, and PS + 100 μM DP + 100 μM FeSO₄ by real-time qRT-PCR. 16S ribosomal RNA was used as an endogenous control to normalize the RNA for cellular abundance. (E) Streptonigrin (SNG) sensitivity plate assay. Different Xcc strains were grown in PS media at a density of 1 × 10⁹ cells/ml. 4 μL of cultures from each serial dilution was spotted on PSA plates containing 1μg/ml SNG and 0.01 M sodium citrate. Plates were incubated for 72 h at 28°C to observe bacterial growth. (F) Intracellular iron content quantification determined by atomic absorption spectrophotometry. Different Xcc strains were grown at a density of 1.2 OD₆₀₀ in PS medium. Cells were harvested, freeze dried, and determined the iron content by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). (G and H) Relative quantification of the expression of <i>Xanthomonas</i> putative ferritin-like protein (XC_2164) and putative ferritin related protein (XC_2190) of Xcc grown under PS, PS + 100 μM DP, and PS + 100 μM DP + 100 μM FeSO₄ by real-time qRT-PCR. Data shown in the graphs are mean ± S.E. (n = 3). * Indicating p-value < 0.05, **indicating p-value < 0.01 and *** indicating p-value < 0.001 statistically significance by paired student t-test.</p

Genome-wide expression analysis of the iron-starvation and/or XibR regulon in Xcc.

<p>(A) The map of differentially expressed genes in response to iron limitation and/or <i>xibR</i> mutation in Xcc represented by using circos plot. From the outer to the inner circle, Track 1 shows circular genome of Xcc 8004 (~5.15Mb) with scale in Mb; Track 2, loci presentation of Xcc 8004 circular genome; Track 3, differentially expressed genes in Δ<i>xibR</i> mutant versus wild-type Xcc 8004 strain grown under iron-replete condition (PS medium); Track 4, differentially expressed genes in Δ<i>xibR</i> mutant grown under low-iron condition (PS + DP) versus iron-replete condition; Track 5, differentially expressed genes in Δ<i>xibR</i> mutant versus wild-type Xcc 8004 strain, both grown under low-iron condition; and Track 6, differentially expressed genes in the wild-type strain grown under low-iron condition versus iron-replete condition. Color scale indicates log₂ –fold change of expression (from green for downregulated to red for upregulated). Low-iron was made by addition of 100 μM DP to PS medium. (B) Venn diagram showing the overlap and unique subset of genes belonging to different functional groups of Xcc whose expression is upregulated or downregulated under low-iron condition and/or XibR. For detail list of genes please see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006019#ppat.1006019.s004" target="_blank">S3</a> to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006019#ppat.1006019.s011" target="_blank">S10</a> Tables. (C) Expression analysis by microarray and real-time qRT-PCR indicating <i>xibR</i> and/or iron limitation regulated genes involved in flagellar biogenesis and regulation, metabolism, chemotaxis, and virulence. The y-axis represents log_2-fold change in expression. For RT-PCR, data were normalized to an internal 16S rRNA control, and the relative changes in the transcriptional level were calculated as a ratio of transcript levels of Δ<i>xibR</i> versus wild-type Xcc 8004 strain grown in PS medium (iron-replete condition), and Xcc 8004 grown under low-iron condition (PS + DP) versus that grown in PS medium (iron-replete condition) using log₂ of fold difference method. I, II and III represent set of genes which are affected by <i>xibR</i> only (I), influenced by both <i>xibR</i> and iron limitation (II) and genes affected by iron limitation only (III). Data represents the means ± S.E. (n = 3).</p

A proposed model for the role XibR in the regulation of iron homeostasis, chemotaxis, motility, biofilm formation, and virulence in Xcc.

<p>XibR is phosphorylated by a yet-unknown sensor kinase in response to change in environmental condition such as iron availability or host environment. Under iron-replete condition, holo-XibR (XibR-Fe³⁺) represses expression of <i>Xanthomonas</i> siderophore synthesis (<i>xss</i>) cluster along with Fur-Fe²⁺. XibR positively regulates chemotaxis and motility in Xcc. Under iron-deplete condition, wherein, the apo-XibR may be the predominant form in the cell, may act as a strong activator of motility and chemotaxis genes. The apo-XibR positively regulates expression of outer membrane receptors for ferric iron uptake, iron storage proteins (ferritin). XibR regulates the expression of several cellular functions such as biofilm formation and production of virulence associated functions (Type III effectors and regulators).</p

XibR binds to the upstream promoter region of <i>mot</i>, <i>flg</i> and xss operons.

<p>(A) Schematic representation of ChIP-qPCR primer locations (indicated by arrows) relative to the transcriptional start sites of <i>mot</i>, <i>flg</i>, and <i>xss</i> operons. (B, C, and D) ChIP-qPCR was performed to assess XibR occupancy on the upstream promoter region of <i>mot</i>, <i>flg</i>, and <i>xss</i> operons. Δ<i>xibR</i>/pSSP80 encodes full-length XibR with C-terminal HA-tag as a test and Δ<i>xibR</i>/pSSP30 encodes full-length XibR without tag as ChIP control grown in rich PS medium, iron-replete (PS + 100 μM FeSO₄) and iron-deplete (PS + 100 μM DP) media and then immunoprecipitated with anti-HA antibodies. Data are shown as mean ± S.E. (n = 3). *p-value < 0.05, **p-value < 0.01 an ***p-value < 0.001 indicated statistically significant difference than ChIP control strain by paired student t-test. (E) Electrophoretic mobility shift assay (EMSA) showing binding of XibR to a ³²P-labeled <i>motA</i> (-525 to +28) probe. More DNA-protein binary complex was observed while increasing the concentration of XibR. (F) Cold probe competition with unlabelled <i>motA</i> and non-specific DNA probe. Specific binding is indicated by a loss of XibR binding to the radiolabelled probe in the presence of excess of cold probe (indicated by CP). In the presence of a nonspecific probe (negative control), XibR did not exhibit binding. (G) Circular dichroism spectrum of XibR in absence or presence of 250 μM FeCl₃. Y-axis indicates molar ellipticity.</p

<i>xibR</i> promotes Biofilm formation.

<p>(A) Biofilm formation by Xcc 8004, Δ<i>xibR</i>, Δ<i>xibR</i>/pSSP30 and Δ<i>xibR</i>/pSSP39 strains in the static biofilm after 24 hrs of growth and staining with 0.1% Crystal Violet. (B) Quantification of attached cells of different Xcc strains in the static biofilm after 24 hours of growth. Attached cells were stained with Crystal Violet (CV), dissolved in ethanol and quantified by measuring absorbance at 570 nm. Data are shown as mean ± S.E. (n = 3). (C) Representative confocal laser-scanning microscopy (CLSM) images of biofilms formed on glass slides at the air–media interface by different Xcc strains grown in PS medium for 24h, and stained with BacLight LIVE/DEAD stain. Each 3D image represents the layer in the Z-stack. (D) Average biofilm thickness of different strains of Xcc formed on the glass slide at the air-media interphase. For quantification of the thickness, five independent biofilms were scanned with CLSM at ten randomly selected positions and thickness was determined through height of the biofilm. Data are shown as mean ± S.E. (n = 3). ** Indicating p-value < 0.01 and *** indicating p-value < 0.001 statistically significance by paired student t-test. (E) Relative quantification of the expression of pili assembly chaperone of Xcc grown under PS, PS + 100 μM DP, and PS + 100 μM DP + 100 μM FeSO₄ by real-time qRT-PCR. 16S ribosomal RNA was used as an endogenous control to normalize the RNA for cellular abundance. Data are shown as mean ± S.E. (n = 3).</p

Chemical Tools for Study of Phosphohistidine: Generation of Selective τ-Phosphohistidine and π-Phosphohistidine Antibodies

Non-hydrolysable stable analogues of τ-pHis and π-pHis have been designed using electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as haptens to generate pHis polyclonal antibodies. Both τ-pHis and π-pHis conjugates in the form of a BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis were synthesized and characterized by 31P NMR spectroscopy. Commercially available τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA-G-τ-pHis and BSA-G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the generated pHis antibodies generated using phosphopyrazole 8 and pyridyl amino amide 13 as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, τ-pHis and π-pHis. Antibodies generated using the phosphopyrazole 8 as a hapten were found to be selective for τ-pHis, and antibodies generated using the pyridyl amino amide 13 were found to be selective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The τ-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHi