Aconitase B Is Required for Optimal Growth of Xanthomonas campestris pv. vesicatoria in Pepper Plants

The aerobic plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) colonizes the intercellular spaces of pepper and tomato. One enzyme that might contribute to the successful proliferation of Xcv in the host is the iron-sulfur protein aconitase, which catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle and might also sense reactive oxygen species (ROS) and changes in cellular iron levels. Xcv contains three putative aconitases, two of which, acnA and acnB, are encoded by a single chromosomal locus. The focus of this study is aconitase B (AcnB). acnB is co-transcribed with two genes, XCV1925 and XCV1926, encoding putative nucleic acid-binding proteins. In vitro growth of acnB mutants was like wild type, whereas in planta growth and symptom formation in pepper plants were impaired. While acnA, XCV1925 or XCV1926 mutants showed a wild-type phenotype with respect to bacterial growth and in planta symptom formation, proliferation of the acnB mutant in susceptible pepper plants was significantly impaired. Furthermore, the deletion of acnB led to reduced HR induction in resistant pepper plants and an increased susceptibility to the superoxide-generating compound menadione. As AcnB complemented the growth deficiency of an Escherichia coli aconitase mutant, it is likely to be an active aconitase. We therefore propose that optimal growth and survival of Xcv in pepper plants depends on AcnB, which might be required for the utilization of citrate as carbon source and could also help protect the bacterium against oxidative stress

<i>X. campestris</i> pv. <i>vesicatoria</i> strain 85-10Δ<i>acnB</i> has increased sensitivity to the superoxide-generating agent menadione.

<p>Dilutions of 10⁻⁶ of exponential phase cultures (OD₆₀₀ =  0.6) of the <i>Xanthomonas</i> strains indicated were spotted on NYG agar plates containing 50 µM and 100 µM menadione. Bacterial colonies surviving the treatment were counted after 24 h and 48 h of incubation at 30°C and CFU were expressed as surviving fraction in percent. Strain 85-10 (black columns), strain 85-10Δ<i>acnB</i> (white columns) and strain 85-10ΔXCV1925<i>-26acnB</i> (gray columns).</p

Co-transcription of <i>xcv1925, xcv1926</i> and <i>acnB</i>.

<p>A. Schematic representation of the deletions introduced in the genes at the <i>acnB</i> locus. 1. represents the extent of the deletion in strain 85-10Δ<i>acnB</i>; 2. represents the deletion in 85-10ΔXCV1925-26<i>acnB</i>; 3. represents the deletion in strain 85-10ΔXCV1925-26; and 4. represents the deletion in strain 85-10Δ<i>acnA</i>. B. RT-PCR analysis of the XCV1925-XCV1926<i>-acnB</i> transcript. Total RNA was isolated and analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034941#s4" target="_blank">Methods</a> using oligonucleotide primers r-secacnB and f-sec3565 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034941#pone.0034941.s001" target="_blank">Table S1</a>). Lane 1, DNA size standards; lane 2, PCR product with cDNA; lane 3, control in which RT was omitted from the cDNA synthesis reaction; lane 4, control in which total RNA was omitted from the cDNA synthesis reaction; lane 5, PCR using genomic DNA as template.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Schematic overview of the <i>acnA-xcv1925-xcv1926-acnB</i> locus in different <i>Xanthomonas</i> species.

<p>Shown is the organisation of the <i>acnA</i> and <i>acnB</i> genes, together with XCV1925 and XCV1926 which encode proteins of unknown function. The colour key for the genes is shown in the upper right of the figure. The synteny of the <i>acnAB</i> locus of <i>Xcv</i> was used as a comparator with other members of the <i>Xanthomonadales</i>. The genes represented by white arrows indicate genes with products unrelated to aconitases or XCV1925 or XCV1926 and the parallel, sloping lines in the <i>E. coli</i> genome representation denote physical separation of the two loci. Note that the genes are not drawn to scale. The accession numbers of the strains are as follows: <i>Xcv</i> strain 85-10, AM039952; <i>X. campestris pv. campestris</i> str. 8004, CP000050; <i>X. oryzae</i> pv. <i>oryzae</i> str. KACC10331, AE013598; <i>X. axonopodis</i> pv. <i>citri</i> str. 306, AE008923; <i>X. albilineans</i> str. GPE PC73, FP565176; <i>X. gardneri</i> ATCC 19865, AEQX00000000 annotation incomplete <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034941#pone.0034941-Potnis1" target="_blank">[61]</a>; <i>Xylella fastidiosa</i> str. M-23, CP000941; <i>Stenotrophomonas maltophilia</i> str. K279a, AM743169; <i>E. coli</i>, CP001509.</p

Strain 85-10Δ<i>acnB</i> shows restricted growth <i>in vitro</i> with citrate as a carbon source.

<p>The indicated strains were grown as follows: A. Aerobic growth <i>in vitro</i> in MA minimal medium with sucrose as carbon source, where filled circles represent strain 85-10, open circles represent strain 85-10Δ<i>acnB</i> and filled inverted triangles represent strain 85-10ΔXCV1925-26<i>acnB</i>; B. Growth of strain 85-10 (filled squares) and 85-10Δ<i>acnB</i> (filled circles) aerobically in MA minimal medium with 15 mM citrate as sole carbon source. The standard error is shown for each experiment.</p

Analysis of aconitase transcripts and AcnB protein levels at different stages of growth.

<p>A. Semi-quantitative RT-PCR analysis of <i>acnA, acnB</i> and <i>acnA2</i> transcripts was performed using total RNA isolated from exponential and stationary phase cultures of strain 85-10 grown <i>in vitro</i> in MA minimal medium. Equivalent amounts of RNA were used for cDNA synthesis using oligonucleotide primers r-acnB-RT1, r-acnA-RT1 and r-acnA2-RT1 for <i>acnB</i>, <i>acnA</i> and <i>acnA2</i> transcripts, respectively. PCR (33 cycles) was subsequently performed with the respective primer pairs of f-acnB-RT/r-acnB-RT2, f-acnA-RT/r-acnA-RT2 and f-acnA2-RT/r-acnA2-RT2. The lanes represents minus cDNA (lane 1), including cDNA (lane 2) and minus reverse transcriptase (lane 3). The lane labelled C represents the products of a PCR with genomic DNA as template and the primer pairs described above. The lane on the left of each gel segment shows a DNA size standards. Analysis of 16S rRNA revealed equvalent loading (data not shown). B. A Western blot is shown in which 25 µg of protein derived from crude extracts of the strains indicated were separated in 8% SDS-PAGE and transferred to nitrocellulose membranes and subsequently probed with antibodies raised against <i>E. coli</i> AcnB. The location of AcnB is indicated and Exp. and Stat. represent samples from exponential and stationary phase cultures, respectively. The location of molecular mass markers in kDa is indicated on the right of the Figure.</p

Aconitase B synthesis is unaffected by the T3SS regulators HrpG and HrpX.

<p>Western blot analysis of crude extracts (25 µg of protein in each) derived from the strains indicated separated on 8% SDS-PAGE and transferred to nitrocellulose. Aconitase B was detected by antibodies raised against <i>E. coli</i> AcnB. Lane 1, 85-10; lane 2, 85-10Δ<i>hrpG</i>; lane 3 85*Δ<i>hrpX</i>; 85-10Δ<i>acnB</i>; <i>E. coli</i> W3110 (5 µg of protein from a crude extract). Because no change in protein levels was observed no loading control was shown. Molecular mass markers (Pageruler Prestained Protein Ladder, Thermo Scientific) are given in kDa.</p

Aconitase B from <i>Xcv</i> functionally complements an <i>E. coli acnB</i> mutation.

<p>The <i>E. coli</i> wild type W3110 (filled squares), the <i>acnB</i> mutant JRG3258 (filled circles) and JRG3258 transformed with plasmid pBRM<i>acnB</i> (filled triangles) were grown aerobically in LB broth as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034941#s4" target="_blank">Methods</a> section. The standard error is shown for each experiment.</p

Strain 85-10Δ<i>acnB</i> shows restricted growth <i>in planta</i>.

<p>The indicated strains were grown as follows: A. Growth <i>in planta</i> where filled squares represent strain 85-10, filled circles represent strain 85-10Δ<i>acnA</i>, filled triangles represent strain 85-10Δ<i>acnB</i> and filled inverted triangles represent strain 85*Δ<i>hrcN</i> B. Growth <i>in planta</i> where filled squares represent strain 85-10, filled triangles represent strain 85-10ΔXCV1925-26, filled inverted triangles represent strain 85-10ΔXCV1925-26<i>acnB</i> and filled circles represent strain 85*Δ<i>hrcN</i>; C. Growth <i>in planta</i> where filled squares represent strain 85-10, open squares represent strain 85-10/pLAFR6, filled circles represent strain 85-10Δ<i>acnB</i>/pL6<i>acnB</i>, open circles represent strain 85-10Δ<i>acnB</i>/pLAFR6 and filled inverted triangles represent 85*Δ<i>hrcN</i>. The standard error is shown for each experiment.</p