Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used in this study.
Fig 1.
Swimming and twitching motility in L. crescens strain BT‐1.
(A-D) Ultrastructural and (E-H) functional evidence for the presence of flagella and Tad pili in Lcr BT‐1. Five-day-old Lcr cells were cultured for three weeks on BM7A medium containing 0.25% (A-B, E-F) and 0.75% (C-D, G-H) agar for swimming and twitching assays, respectively. Negatively stained cells were analyzed by transmission electron microscopy. Magnification scales are indicated in the images. Note the swimming zone as outward bacterial growth along the surface (E) and clearly downward within the culture medium when the same colony is photographed at an angle (F). By contrast, Tad pilus-mediated twitching appeared as strictly surface restricted growth (G) or along the agar/polystyrene interface when the inoculum was stabbed to the bottom of the plate (H). Relative expression of (I) flagella and (J) Tad pilus biosynthesis genes in Lcr grown in BM7A broth culture and on BM7A plates containing low (0.25%), medium (0.75%) and high (1.5%) agar concentrations. The transcript abundance of each gene was calibrated against expression in liquid BM7A medium and normalized against the expression levels of the chromosomal reference gene gyrB within each sample. Bars represent average ± the standard deviation for three independent experiments with four replications. Asterisks represent significant differences (P < 0.05) in the transcript abundance.
Fig 2.
Mutations in the L. crescens BT‐1 flagellar genes flgF and flgK affected only swimming motility.
(A, B) Swimming and (C, D) twitching motility in the wild-type BT-1, insertional flagellar gene mutants (flgF::pCLL026 and flgK::pCLL027) and complemented (flgF::pCLL026/pCLL038 and flgK::pCLL027/pCLL036) strains on semisolid BM7A plates containing 0.25% and 0.75% agar for swimming and twitching assays, respectively. (E) Swimming and twitching motility zone diameters were measured in three independent assays with three replicates each. The data are average ± standard deviation and the significant differences (P < 0.05, Student’s t test) in swimming behavior are represented by asterisks.
Fig 3.
Tad pilus motor ATPase CpaF is structurally conserved among all Liberibacters.
Sequence alignment of CpaF encoded by L. crescens BT‐1 (WP_015273698.1), ‘Ca. L. asiaticus’ (WP_015452438.1), ‘Ca. L. africanus’ (WP_047264073.1), A. rhizogenes (WP_065114603.1), ‘Ca. L. solanacearum’ (WP_076969128.1), ‘Ca. L. americanus’ (AHA27602.1) and C. crescentus (YP_002518410.1). Invariant residues required for coordination of Mg2+ between β- and γ-phosphate moieties of ATP within the canonical ATP-binding Walker A and Walker B motifs are marked by an asterisk (*). Invariant residues in Asp box and His box motifs are denoted by filled (▲) and open (△) triangles, respectively. Amino acids participating in ATP binding and ATPase activity are denoted by hashtags (#) and those involved in hexamer interface for subunit interactions are denoted by dots (·).
Fig 4.
Mutations in Tad pilus genes affected twitching motility and growth but comEC mutation affected only natural transformation in L. crescens BT-1.
(A-C) Mutation in the Tad pilus motor ATPase gene cpaF had no effect on flagellar swimming (A, C) but reduced twitching motility (B, C) in wild-type BT-1 and the mutant cpaF::pCLL043. (D-F) Mutation in the inner membrane competence protein gene comEC had no effect on flagellar swimming (D, F) or twitching motility (E, F) but completely abolished natural competence for transformation (G). Deficiency in natural competence for transformation in the mutant strain comEC::pMJ054 was fully complemented by full-length comEC gene from Lcr (comEC::pMJ054/pCLL052) but not from CLas strain psy62 (comEC::pMJ054/pCLL053) or CLso strain RS100 (comEC::pMJ054/pCLL056) (G). (H) Insertional mutant strains of Tad pilus genes (cpaA::pMJ081, cpaB::pMJ082, cpaF::pCLL043 and tadC::pMJ083) were severely compromised in growth in culture as compared to wild-type BT‐1, and insertional mutants for flagellar genes (flgF::pCLL026 and flgK::pCLL027) and the competence protein gene (comEC::pMJ054). Motility zone diameters and natural competence for transformation were quantified in three independent experiments with three replicates each. Bacterial growth assays were repeated thrice with five replicates each. The data are average ± standard deviation and the significant differences (P < 0.05, Student’s t test) in bacterial motility, natural transformation and growth are represented by asterisks.
Fig 5.
Tad pilus-mediated uptake of fluorescently labeled dsDNA by L. crescens wild-type BT-1, cpaF and comEC insertional mutant strains.
(A, E and I) Overlay of bright-field and YOYO-1 florescence in Lcr wild-type BT-1, cpaF and comEC insertional mutant strains (cpaF::pCLL043 and comEC::pMJ054, respectively) exposed to YOYO-1-stained dsDNA (1 μg ml-1) for 15 min. Uptake of YOYO-1-stained fluorescent dsDNA by wild-type BT-1, cpaF::pCLL043 and comEC::pMJ054 cells after DNase I exposure for 15 min (B, F and J), 30 min (C, G and K) and 45 min (D, H and L), respectively. Fluorescent DNA foci were transiently observed in cpaF::pCLL043 cells whereas the DNA fluorescence was stable (resistant to DNase I) in wild-type BT-1 and comEC::pMJ054 cells. YOYO-1-stained dsDNA was also pretreated with DNase I for 10 min prior to starting the experiment. Magnification scale = 2 μm.
Fig 6.
Tad pilus-mediated twitching motility is functionally coupled with periplasmic uptake of extracellular dsDNA.
(A) A working model for Tad pilus-mediated uptake of extracellular dsDNA into periplasm and ComEC-mediated cytoplasmic translocation of ssDNA in naturally competent Lcr [26]. Bidirectional motor ATPase CpaF drives pilus extension and retraction via pilin polymerization and depolymerization causing both the twitching motility as well as DNA uptake. Schematic illustration of Tad pilus has been modified from Denise et al. [20]. (B) The structural components of the Tad pilus are named following Andrade and Wang [29]. Essential Tad pilus genes in Lcr encode the biogenesis ATPase (tadZ/cpaE, B488_RS06245), motor ATPase (tadA/cpaF, B488_RS06240), prepilin peptidase (tadV/cpaA, B488_RS06265), inner membrane staging complex (tadB/cpaG, B488_RS06235 and tadC/cpaH, B488_RS06230), secretin (rcpA/cpaC, B488_RS06255), periplasmatic subunits (tadG/cpaB, B488_RS06260 and rcpB/cpaD, B488_RS06250), inner membrane anchor (rcpC/tadG, B488_RS05235) and pilotin (tadD/cpaO, B488_RS05560). The genes encoding the DNA translocation machinery include dsDNA receptor (comEA, B488_RS00175), inner membrane channel protein (comEC, B488_RS05330), ATP-dependent translocase (comF, B488_RS05095), ssDNA binding proteins (ssb, B488_RS01885), DNA processing protein A (dprA, B488_RS04595) and the recombinase protein (recA, B488_RS02195). While Lcr is naturally competent for transformation, in this model, all Liberibacters are presumed to likely assimilate extracellular dsDNA as a food source. Except for comEC, rest of the Tad pilus and natural competence components are well conserved between Lcr and ‘Ca. Liberibacter’ spp. Canonical NTT domain containing nucleotide transporter proteins remain to be identified in Lcr and/or ‘Ca. Liberibacter’ spp. Abbreviations: OM, outer membrane; IM, inner membrane; PP, periplasm; CP, cytoplasm.