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<p>In this study, we elucidated the functions of four previously uncharacterized enzymes, EBN15_09645, EBN15_09650, EBN15_09665, and EBN15_09680, in <i>Xanthomonas cucurbitae</i> (<i>Xc</i>), the causal agent of bacterial spot in cucurbits, and encoded by orthologs of genes that encode novel enzymes initially identified in <i>Bifidobacterium longum</i>, (<i>hypBA1</i>, <i>hypBA2</i>, and <i>hypAA</i>). We used microbial systems to express the four enzymes from <i>Xc</i> to investigate their functions and found that they coordinately degrade arabinofurano-oligosaccharides present on hydroxyproline (Hyp)-rich glycoproteins, or extensin, a component in plant cell walls that is involved in blocking pathogen attack. First, XcHypAA (EBN15_09650) released <span>l</span>-arabinose from L-arabinofuranose (Ara<i>f</i>)-α1,3-Ara<i>f</i>-ß1,2-Ara<i>f</i>-ß1,2-Ara<i>f</i>-ß-Hyp (Ara<sub>4</sub>-Hyp), cleaving its α1,3 bond; second, XcHypBA2 (EBN15_09645) released the disaccharide Ara<i>f</i>-ß1,2-Ara<i>f</i> from Ara<i>f</i>-ß1,2-Ara<i>f</i>-ß1,2-Ara<i>f</i>-ß-Hyp (Ara<sub>3</sub>-Hyp); finally, XcHypBA1-1 (EBN15_09680) and XcHypBA1-2 (EBN15_09665) released <span>l</span>-arabinose from Ara<i>f</i>-ß-Hyp (Ara-Hyp) and XcHypBA1-1 also from Ara<i>f</i>-ß1,2-Ara<i>f</i>-ß-Hyp (Ara<sub>2</sub>-Hyp). In summary, the oligosaccharide structure of Ara<sub>4</sub>-Hyp on extensin was degraded into Ara<sub>3</sub>-Hyp, then to Ara-Hyp, and finally to arabinose monosaccharides by the action of these four enzymes. We also examined RNA expression for the four enzyme-encoding genes in syringe-inoculated cucumber and created a deletion mutant of the operon comprising <i>xchypBA2</i> and <i>xchypAA</i>, which is a major contributor for degrading the arabinofurano-oligosaccharides. The expression of all four genes was highly induced 96&#xa0;h after inoculation, but virulence of the mutant was not lower than the wild type. We discuss the potential reasons for conservation of these unique enzymes in <i>Xanthomonas</i> spp.</p>

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Functional characterization of arabinofuranosidases from Xanthomonas cucurbitae that co-occur in Bifidobacterium longum

  • 期刊论文

  • 发表日期：

    2025年6月10日

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  • Masayuki Nakamura,
  • Taketo Kuratsu,
  • Ryuma Fuchiwaki,
  • Kiyotaka Fujita

摘要

In this study, we elucidated the functions of four previously uncharacterized enzymes, EBN15_09645, EBN15_09650, EBN15_09665, and EBN15_09680, in Xanthomonas cucurbitae (Xc), the causal agent of bacterial spot in cucurbits, and encoded by orthologs of genes that encode novel enzymes initially identified in Bifidobacterium longum, (hypBA1, hypBA2, and hypAA). We used microbial systems to express the four enzymes from Xc to investigate their functions and found that they coordinately degrade arabinofurano-oligosaccharides present on hydroxyproline (Hyp)-rich glycoproteins, or extensin, a component in plant cell walls that is involved in blocking pathogen attack. First, XcHypAA (EBN15_09650) released l-arabinose from L-arabinofuranose (Araf)-α1,3-Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara4-Hyp), cleaving its α1,3 bond; second, XcHypBA2 (EBN15_09645) released the disaccharide Araf-ß1,2-Araf from Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara3-Hyp); finally, XcHypBA1-1 (EBN15_09680) and XcHypBA1-2 (EBN15_09665) released l-arabinose from Araf-ß-Hyp (Ara-Hyp) and XcHypBA1-1 also from Araf-ß1,2-Araf-ß-Hyp (Ara2-Hyp). In summary, the oligosaccharide structure of Ara4-Hyp on extensin was degraded into Ara3-Hyp, then to Ara-Hyp, and finally to arabinose monosaccharides by the action of these four enzymes. We also examined RNA expression for the four enzyme-encoding genes in syringe-inoculated cucumber and created a deletion mutant of the operon comprising xchypBA2 and xchypAA, which is a major contributor for degrading the arabinofurano-oligosaccharides. The expression of all four genes was highly induced 96 h after inoculation, but virulence of the mutant was not lower than the wild type. We discuss the potential reasons for conservation of these unique enzymes in Xanthomonas spp.