Specificity traits consistent with legume-rhizobia coevolution displayed by Ensifer meliloti rhizosphere colonization

Rhizobia are α‐ and ß‐proteobacteria that associate with legumes in symbiosis to fix atmospheric nitrogen. The chemical communication between roots and rhizobia begins in the rhizosphere. Using signature‐tagged‐Tn5 mutagenesis (STM) we performed a genome‐wide screening for Ensifer meliloti genes that participate in colonizing the rhizospheres of alfalfa and other legumes. The analysis of ca. 6,000 mutants indicated that genes relevant for rhizosphere colonization account for nearly 2% of the rhizobial genome and that most (ca. 80%) are chromosomally located, pointing to the relevance and ancestral origin of the bacterial ability to colonize plant roots. The identified genes were related to metabolic functions, transcription, signal transduction, and motility/chemotaxis among other categories; with several ORFs of yet‐unknown function. Most remarkably, we identified a subset of genes that impacted more severely the colonization of the roots of alfalfa than of pea. Further analyses using other plant species revealed that such early differential phenotype could be extended to other members of the Trifoliae tribe (Trigonella, Trifolium), but not the Fabeae and Phaseoleae tribes. The results suggest that consolidation of E. meliloti into its current symbiotic state should have occurred in a rhizobacterium that had already been adapted to rhizospheres of the Trifoliae tribe.Instituto de Biotecnologia y Biologia Molecula

Transkriptomische Analysen zur Xanthanproduktion in Xanthomonas campestris pv. campestris

Serrania Vallejo J. Transkriptomische Analysen zur Xanthanproduktion in Xanthomonas campestris pv. campestris. Bielefeld (Germany): Bielefeld University; 2008.Xanthomonas campestris pv. campestris (Xcc) wird industriell zur Produktion des Exopolysaccharids Xanthan verwendet, das z.B. in der Lebensmittelindustrie als Verdickungsmittel eingesetzt wird. Xcc ist ebenfalls der Erreger der Aderschwärze bei der Pflanzenfamilie der Kreuzblütengewächse. In dieser Arbeit wurde ein genomweites transkriptomisches Profil einer Kultivierung in einem 10 L Bioreaktor erstellt. Zur Transkriptionsanalyse wurde der kürzlich entwickelte Microarray Xcc5kOLI verwendet. Es konnten zwei Genregionen identifiziert werden, die ein wachstumsphasenspezifisches Transkriptionsprofil aufwiesen. Eine der zwei Genregionen beinhaltet hauptsächlich Gene ribosomaler Proteine und zeigte eine Induktion in der exponentiellen und eine Repression in der stationären Wachstumsphase. Dieses Profil spiegelt wahrscheinlich die allgemeine metabolische Aktivität der Bakterienkultur wieder. Die zweite Genregion beinhaltet hauptsächlich Gene, die in die Flagellar-Biosynthese involviert sind. Diese Gene zeichnen sich durch eine Induktion in der exponentiellen und eine kurze erneute Induktion zu Beginn der stationären Phase aus. Zu Zwecken der Stammoptimierung des Produktionsstammes Xcc LMG 8031* wurde untersucht, ob die Inaktivierung der Motilität zu einer Erhöhung der Biomasse- oder Xanthanproduktion führt. Hierfür wurden Stämme produziert, die Deletionen in den Genen fleQ und flgE tragen. Das Gen fleQ kodiert für einen Regulator der Flagellar-Biosynthese und flgE für das Strukturprotein des Flagellar-Ankers. Beide Deletionen führten zu einem nicht motilen Phänotyp. Das transkriptomische Profil des Gens galU wies eine Reduktion der Genexpression im Übergang zur stationären Phase auf. GalU katalysiert die Umwandlung von Glucose-1-Phosphat zu UDP-Glucose, welches für die Xanthanproduktion benötigt wird. Durch Klonierung eines konstitutiv hoch exprimierten Promotors vor das galU-Gen wurde der Einfluss einer Überexpression auf die Xanthanproduktion untersucht. Weder die Verwendung der nicht motilen Stämme, mit den Deletionen in den Genen fleQ bzw. flgE, noch die Überexpression des galU-Gens führten zu einer erhöhten Biomasse- oder Xanthanproduktion. Ein weiterer Schwerpunkt dieser Arbeit war die Analyse der Kohlenstoffverwertung in Xcc. Hierfür wurden transkriptomische Analysen zur Kurzzeit- und Langzeitantwort des Stammes Xcc B100 auf die Anwesenheit von Galactose durchgeführt. Es konnten in dieser Arbeit drei Genregionen identifiziert werden, die sowohl im Kurzzeit- als auch Langzeitwachstum in Galactose eine Induktion der Gene im Vergleich zum Wachstum in Glucose aufweisen. Diese Genregionen beinhalteten unter anderem Gene für Glycosidasen, die in das Periplasma oder ins Medium exportiert werden. Ebenfalls sind die Gene zweier TonB-abhängiger Rezeptoren in den Genregionen lokalisiert, die für den Transport von Galactose durch die äußere Membran zuständig sein können. Das Genprodukt des Gens sglT konnte als möglicher Galactosetransporter durch die innere Membran identifiziert werden. Auch liegen Gene für Enzyme des Galactoseabbaus im Cytoplasma in den Galactose-Verwertungsregionen. Somit war es möglich, mit den Genen der drei identifizierten Galactose-Verwertungsregionen ein Modell der Galactoseverwertung in Xcc zu entwickeln

Fine-tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential wggR (expG)-, PhoB-, and mucR-dependent regulation of two promoters

Bahlawane C, Baumgarth B, Serrania J, Rueberg S, Becker A. Fine-tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential wggR (expG)-, PhoB-, and mucR-dependent regulation of two promoters. JOURNAL OF BACTERIOLOGY. 2008;190(10):3456-3466

Identification of Xanthomonas campestris pv. campestris galactose utilization genes from transcriptome data

Serrania J, Vorhölter F-J, Niehaus K, Pühler A, Becker A. Identification of Xanthomonas campestris pv. campestris galactose utilization genes from transcriptome data. Journal of Biotechnology. 2008;135(3):309-317.A 70 met oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Xanthomonas campestris pv. campestris B100, a plant-pathogenic bacterium that is industrially employed to produce the exopolysaccharide xanthan gum which has many applications as a stabilizing, thickening, gelling, and emulsifying agent in food, pharmaceutical, and cosmetic industries. As an application example, global changes of gene expression were monitored during growth of X. campestris pv. campestris B100 on two different carbon sources. Exponential growing bacterial cultures were incubated either for 1 h or permanently in minimal medium supplemented with 1% galactose in comparison to growth in minimal medium supplemented with 1% glucose. Six genes were identified that were significantly increased in gene expression under both growth conditions. These genes were located in three distinguished chromosomal regions in operon-like gene clusters. Genes from these clusters encode secreted glycosidases, which were predicted to be specific for galactose-containing carbohydrates, as well as transport proteins probably located in the outer and inner cell membrane. Finally genes from one cluster code for cytoplasmic enzymes of a metabolic pathway specific for the breakdown of galactose to intermediates of glycolysis. (C) 2008 Elsevier B.V. All rights reserved

<i>sX13</i> contributes to bacterial growth in culture and virulence.

<p>Growth of <i>Xcv</i> wild type 85-10 (wt), the <i>sX13</i> deletion mutant (Δ<i>sX13</i>) and Δ<i>sX13</i> containing chromosomally re-integrated <i>sX13</i> (Δ<i>sX13</i>+<i>sX13</i>_ch) in (A) complex medium NYG and (B) minimal medium MMA, respectively. Error bars represent standard deviations. Asterisks indicate statistically significant differences compared to wt (<i>t</i>-test; <i>P</i><0.05). (C) Growth of <i>Xcv</i> 85-10 (wt) and Δ<i>sX13</i> in leaves of susceptible ECW pepper plants. Data points represent the mean of three different samples from three different plants of one experiment. Standard deviations are indicated by error bars. (D) Plant infection assay. <i>Xcv</i> strains 85-10 (wt) and Δ<i>sX13</i> carrying the empty vector (pB) or the sX13 expression construct (p<i>sX13</i>) and strains additionally expressing HrpG* (p<i>hrpG*</i>) were inoculated at a density of 4×10⁸ (left panel) and 10⁸ cfu/ml (right panel), respectively, into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms in ECW were photographed 9 days post inoculation (dpi). The HR was visualized by ethanol bleaching of the leaves 3 dpi (left panel) and 18 hours post inoculation (right panel), respectively. Dashed lines indicate the inoculated areas. All experiments were performed at least three times with similar results.</p

Small RNA sX13: A Multifaceted Regulator of Virulence in the Plant Pathogen <i>Xanthomonas</i>

<div><p>Small noncoding RNAs (sRNAs) are ubiquitous posttranscriptional regulators of gene expression. Using the model plant-pathogenic bacterium <i>Xanthomonas campestris</i> pv. <i>vesicatoria</i> (<i>Xcv</i>), we investigated the highly expressed and conserved sRNA sX13 in detail. Deletion of <i>sX13</i> impinged on <i>Xcv</i> virulence and the expression of genes encoding components and substrates of the Hrp type III secretion (T3S) system. qRT-PCR analyses revealed that sX13 promotes mRNA accumulation of HrpX, a key regulator of the T3S system, whereas the mRNA level of the master regulator HrpG was unaffected. Complementation studies suggest that sX13 acts upstream of HrpG. Microarray analyses identified 63 sX13-regulated genes, which are involved in signal transduction, motility, transcriptional and posttranscriptional regulation and virulence. Structure analyses of <i>in vitro</i> transcribed sX13 revealed a structure with three stable stems and three apical C-rich loops. A computational search for putative regulatory motifs revealed that sX13-repressed mRNAs predominantly harbor G-rich motifs in proximity of translation start sites. Mutation of sX13 loops differentially affected <i>Xcv</i> virulence and the mRNA abundance of putative targets. Using a GFP-based reporter system, we demonstrated that sX13-mediated repression of protein synthesis requires both the C-rich motifs in sX13 and G-rich motifs in potential target mRNAs. Although the RNA-binding protein Hfq was dispensable for sX13 activity, the <i>hfq</i> mRNA and Hfq::GFP abundance were negatively regulated by sX13. In addition, we found that G-rich motifs in sX13-repressed mRNAs can serve as translational enhancers and are located at the ribosome-binding site in 5% of all protein-coding <i>Xcv</i> genes. Our study revealed that sX13 represents a novel class of virulence regulators and provides insights into sRNA-mediated modulation of adaptive processes in the plant pathogen <i>Xanthomonas</i>.</p></div

sX13 accumulation is altered under stress conditions in <i>Xcv</i> 85-10.

<p>(A) Northern blot analysis of sX13. Exponential phase cultures of NYG-grown <i>Xcv</i> 85-10 were transferred to NYG medium or MMA containing the indicated additives or lacking a nitrogen or carbon source (ΔN; ΔC). Cultures were shaken for three hours at 30°C unless otherwise indicated. 5S rRNA was probed as loading control. (B) sX13 and selected sX13-regulated genes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat-1003626-t001" target="_blank">Table 1</a>) were analyzed by qRT-PCR using RNA from <i>Xcv</i> 85-10 (wt) cultures shown in (A) and NYG-grown Δ<i>sX13</i>. Bars represent fold-changes (log₁₀) of mRNA amounts compared to <i>Xcv</i> 85-10 grown in NYG at 30°C. Experiments were performed twice with similar results.</p

Selected sX13-regulated genes validated by qRT-PCR analysis.

a<p>, bold letters indicate genes with known TSS <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat.1003626-Schmidtke1" target="_blank">[16]</a>.</p>b<p>, refers to Thieme <i>et al.</i> (2005) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat.1003626-Thieme1" target="_blank">[32]</a>.</p>c<p>, presence of a 4G-motif within the 5′-UTR or 100 bp upstream of translation start codon if TSS is unknown (a) and within 100 bp downstream of start codon (b) (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat.1003626.s004" target="_blank">Figure S4</a>).</p>d<p>, genes not detected as expressed are marked with —.</p>e<p>, values represent mean fold-change and standard deviation (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat-1003626-g004" target="_blank">Figure 4</a>);</p><p>n.t. - not tested.</p

sX13 loops differentially contribute to abundance of putative mRNA targets.

<p>Relative transcript levels of (A) <i>XCV2821</i>, (B) <i>XCV3927</i>, (C) <i>hfq</i>, (D) <i>pilH</i>, (E) <i>XCV3572</i> and (F) <i>XCV0612</i> were analyzed by qRT-PCR in total RNA of NYG-grown <i>Xcv</i> strains 85-10 (wt) and Δ<i>sX13</i> carrying pBRS (pB), p<i>sX13</i> or mutated sX13-derivatives (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat-1003626-g006" target="_blank">Figure 6</a>). The mRNA abundance in the wt was set to 1. Data points and error bars represent mean values and standard deviations obtained with at least three independent biological samples. Statistically significant differences are indicated (<i>t</i>-test; <i>P</i><0.015).</p

Deletion of <i>sX13</i> derogates virulence gene expression.

<p>(A) <i>Xcv</i> strains 85-10 (wt) and the <i>sX13</i> deletion mutant (Δ<i>sX13</i>) carrying the empty vector (pB) or the sX13 expression construct (p<i>sX13</i>) and strains additionally expressing HrpG* (p<i>hrpG*</i>) were incubated for 3.5 hours in <i>hrp</i>-gene inducing medium XVM2. Total protein extracts were analyzed by immunoblotting using antibodies directed against HrpF, HrcN, HrcJ and GroEL. The experiment was repeated twice with similar results. (B) <i>Xcv</i> 85-10 (wt), Δ<i>sX13</i> and Δ<i>sX13</i>+<i>sX13</i>_ch and strains additionally expressing HrpG* were incubated for 3.5 hours in <i>hrp</i>-gene inducing medium XVM2. Total protein extracts were analyzed by immunoblotting using antibodies directed against HrcJ and GroEL. The experiment was repeated twice with similar results. (C) Indicated genes were analyzed by qRT-PCR using RNA from cultures described in (B). The amount of each RNA in <i>Xcv</i> 85-10 was set to 1. Data points and error bars represent mean values and standard deviations obtained with three independent biological samples. Asterisks indicate statistically significant differences compared to wt (<i>t</i>-test; <i>P</i><0.03).</p