Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama, United States of America.
Department of Biological Sciences, Auburn University, Auburn, Alabama, United States of America.
PLoS Pathog. 2023 Feb 13;19(2):e1011154. doi: 10.1371/journal.ppat.1011154. eCollection 2023 Feb.
Type IV pilus (TFP) is a multifunctional bacterial structure involved in twitching motility, adhesion, biofilm formation, as well as natural competence. Here, by site-directed mutagenesis and functional analysis, we determined the phenotype conferred by each of the 38 genes known to be required for TFP biosynthesis and regulation in the reemergent plant pathogenic fastidious prokaryote Xylella fastidiosa. This pathogen infects > 650 plant species and causes devastating diseases worldwide in olives, grapes, blueberries, and almonds, among others. This xylem-limited, insect-transmitted pathogen lives constantly under flow conditions and therefore is highly dependent on TFP for host colonization. In addition, TFP-mediated natural transformation is a process that impacts genomic diversity and environmental fitness. Phenotypic characterization of the mutants showed that ten genes were essential for both movement and natural competence. Interestingly, seven sets of paralogs exist, and mutations showed opposing phenotypes, indicating evolutionary neofunctionalization of subunits within TFP. The minor pilin FimT3 was the only protein exclusively required for natural competence. By combining approaches of molecular microbiology, structural biology, and biochemistry, we determined that the minor pilin FimT3 (but not the other two FimT paralogs) is the DNA receptor in TFP of X. fastidiosa and constitutes an example of neofunctionalization. FimT3 is conserved among X. fastidiosa strains and binds DNA non-specifically via an electropositive surface identified by homolog modeling. This protein surface includes two arginine residues that were exchanged with alanine and shown to be involved in DNA binding. Among plant pathogens, fimT3 was found in ~ 10% of the available genomes of the plant associated Xanthomonadaceae family, which are yet to be assessed for natural competence (besides X. fastidiosa). Overall, we highlight here the complex regulation of TFP in X. fastidiosa, providing a blueprint to understand TFP in other bacteria living under flow conditions.
IV 型菌毛（TFP）是一种多功能细菌结构，参与蠕动运动、黏附、生物膜形成以及自然感受态。在这里，通过定点突变和功能分析，我们确定了在重新出现的植物病原严格原核生物韧皮部难养菌（Xylella fastidiosa）中，已知的 38 个基因中每个基因对 TFP 生物合成和调控所赋予的表型。这种病原体感染了>650 种植物物种，并在世界各地的橄榄、葡萄、蓝莓和杏仁等作物中引起破坏性疾病。这种木质部限制、昆虫传播的病原体在不断流动的条件下生存，因此高度依赖 TFP 进行宿主定殖。此外，TFP 介导的自然转化是一个影响基因组多样性和环境适应性的过程。突变体的表型特征表明，有十个基因对运动和自然感受态都是必需的。有趣的是，存在七组旁系同源物，突变表现出相反的表型，表明 TFP 亚基的进化新功能化。次要菌毛 FimT3 是唯一专门用于自然感受态的蛋白质。通过结合分子微生物学、结构生物学和生物化学的方法，我们确定了次要菌毛 FimT3（而不是其他两个 FimT 旁系同源物）是 X. fastidiosa 的 TFP 中的 DNA 受体，并构成了新功能化的一个例子。FimT3 在 X. fastidiosa 菌株中保守，通过同源建模鉴定的带正电荷表面非特异性地结合 DNA。该蛋白质表面包括两个精氨酸残基，它们被替换为丙氨酸，并被证明参与 DNA 结合。在植物病原体中，fimT3 存在于植物相关黄单胞菌科家族的约 10%的可用基因组中，这些基因组尚未评估自然感受态（除了 X. fastidiosa 之外）。总的来说，我们在这里强调了 X. fastidiosa 中 TFP 的复杂调控，为理解在其他在流动条件下生存的细菌中的 TFP 提供了一个蓝图。
