Plant Pathology Department, University of Florida, Gainesville, Florida, United States of America.
Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.
PLoS One. 2021 Oct 13;16(10):e0258583. doi: 10.1371/journal.pone.0258583. eCollection 2021.
Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured plant pathogenic species of the genus Liberibacter, including 'Candidatus L. asiaticus' (CLas) and 'Ca. L. solanacearum' (CLso). All Liberibacters encode a completely conserved gene repertoire for both flagella and Tad (Tight Adherence) pili and all are missing genes critical for nucleotide biosynthesis. Both flagellar swimming and Tad pilus-mediated twitching motility in Lcr were demonstrated for the first time. A role for Tad pili in the uptake of extracellular dsDNA for food in Liberibacters was suspected because both twitching and DNA uptake are impossible without repetitive pilus extension and retraction, and no genes encoding other pilus assemblages or mechanisms for DNA uptake were predicted to be even partially present in any of the 35 fully sequenced Liberibacter genomes. Insertional mutations of the Lcr Tad pilus genes cpaA, cpaB, cpaE, cpaF and tadC all displayed such severely reduced growth and viability that none could be complemented. A mutation affecting cpaF (motor ATPase) was further characterized and the strain displayed concomitant loss of twitching, viability and reduced periplasmic uptake of extracellular dsDNA. Mutations of comEC, encoding the inner membrane competence channel, had no effect on either motility or growth but completely abolished natural transformation in Lcr. The comEC mutation was restored by complementation using comEC from Lcr but not from CLas strain psy62 or CLso strain RS100, indicating that unlike Lcr, these pathogens were not naturally competent for transformation. This report provides the first evidence that the Liberibacter Tad pili are dynamic and essential for both motility and DNA uptake, thus extending their role beyond surface adherence.
无菌培养的韧皮部杆菌属 crescens（Lcr）是一种与未培养的韧皮部杆菌属植物病原种密切相关的替代物，包括“候选菌属 L. asiaticus”（CLas）和“候选菌属 L. solanacearum”（CLso）。所有韧皮部杆菌属都编码了一套完整的鞭毛和 Tad（紧密附着）菌毛的保守基因，并且都缺失了核苷酸生物合成所必需的基因。首次证明了 Lcr 中鞭毛的游动和 Tad 菌毛介导的蠕动运动。由于没有重复的菌毛延伸和缩回，Tad 菌毛在韧皮部杆菌属中摄取细胞外双链 DNA 作为食物的作用就成了怀疑对象，因为蠕动和 DNA 摄取都是不可能的，并且没有预测到在 35 个完全测序的 Liberibacter 基因组中甚至存在部分其他菌毛组装或 DNA 摄取机制的基因。Lcr Tad 菌毛基因 cpaA、cpaB、cpaE、cpaF 和 tadC 的插入突变都显示出严重的生长和活力降低，以至于没有一个能够得到互补。影响 cpaF（马达 ATP 酶）的突变进一步进行了表征，该菌株显示出协同的蠕动、活力丧失和减少细胞外双链 DNA 的周质摄取。编码内膜感受通道的 comEC 突变对运动或生长都没有影响，但完全消除了 Lcr 中的自然转化。使用来自 Lcr 的 comEC 对 comEC 突变进行了互补，恢复了 Lcr 的自然转化能力，但不能恢复来自 CLas 菌株 psy62 或 CLso 菌株 RS100 的自然转化能力，这表明与 Lcr 不同，这些病原体不具有自然转化能力。本报告首次提供了证据，证明了 Liberibacter Tad 菌毛是动态的，对运动和 DNA 摄取都是必不可少的，从而扩展了它们在表面附着之外的作用。
