Abstract
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.
Highlights
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Based on (a) the conservation of Tad pilus and DNA uptake genes, (b) our earlier observation of DNA uptake and natural competence in L. crescens (Lcr) [26] and (c) the fact that all Liberibacters are deficient in nucleotide biosynthesis [30], we propose that Tad pilus-mediated uptake of extracellular dsDNA for food is likely essential for survival and colonization of ‘Ca. Liberibacter’ spp. in both plant and insect hosts
The fluorescence data were captured using an Olympus IX81-DSU Spinning Disc Confocal Microscope (Olympus Corporation, Tokyo, Japan) fitted with Hamamatsu ORCA-Flash 4.0LT + camera (Hamamatsu Photonics, K.K., Hamamatsu, Japan). Both flagella and much smaller Tad pili were observed by Transmission electron microscopy (TEM) of L. crescens BT-1
Summary
E. coli was grown in Luria-Bertani (LB) medium at 37 ̊C. Lcr BT-1 was maintained on BM7A medium containing 20 g l-1 N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (Sigma-Aldrich, St. Louis, MO, USA) [26] with gentle shaking at 110 rpm at 28 ̊C. Antibiotics were used as needed at the following concentrations (in μg/ml): 100 ampicillin (Amp) and 50 kanamycin (Kn) for E. coli; 4.5 Kn, 2.0 chloramphenicol (Cm) and 2.0 gentamycin (Gm) for Lcr
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