Briefly, for PD3 and PW with and without BSA, media plates solidified with agar or Gelrite were divided into two halves, 10 to 12 spots of each mutant strain were made using a sterile toothpick, and plates were incubated at 28°C for 4 to 5 days. For XFM with and without BSA, plates solidified with agar were used and incubated for 10 to 12 days before measurements were recorded. Colony peripheral fringes were observed under 10 magnification using a Nikon Eclipse Ti inverted microscope , and fringe widths were measured for six colonies per plate per strain, with at least seven measurements per colony using a Nikon DS-Q1 digital camera connected to a Nikon Eclipse Ti inverted microscope and controlled by NIS-Elements imaging software version 3.0. Twitching experiments were performed at least three times independently for PD3 and PW with and without BSA and once for XFM with and without BSA. Three growth conditions were used: solid agar plates , liquid culture tubes , and continuous liquid flow . PD3 without antibiotics was the medium used, and the initial inocula of the NS1-CmR and pglA-KmR mutants were prepared as described above. Competence in tubes. Twenty-five-milliliter glass test tubes containing 3 ml of PD3 were inoculated with 100 l of each of the OD adjusted strain suspensions as donor and recipient cells . Tubes containing single strain inoculations were included as control treatments.
Tubes were then incubated with shaking . After 3 days, the tubes were vortexed well to mix the biofilm formed on the air-liquid interface with the rest of the suspension and serially diluted and plated as described above. Three independent experiments were performed, nft hydroponic system and three replications were included in each experiment . Competence in MCs. MCs were prepared as previously described . Briefly, two parallel channels with separate inlets for bacterial cells and growing media were etched on a silicon wafer. The channels were modeled into polydimethylsiloxane and sandwiched between the PDMS layer and a glass cover slide. The inlets and outlets were then connected to tubings that were connected to syringes . The syringes were connected to pumps which control the media flow rate in the MC. The MC was mounted onto a Nikon Eclipse Ti inverted microscope to observe cell attachment and biofilm formation using phase-contrast and Nomarski differential interference contrast optics. Time-lapse video was taken using a Nikon DS-Q1 digital camera connected to the microscope and controlled by NIS-Elements imaging software version 3.0. For preparing the inocula for MCs, equal volumes of the strain pairs were mixed and inoculated into the cell inlet syringes, and growing medium was injected in the media syringes. MCs were run for 5 to 7 days with a media flow rate of 0.25 l min 1 until abundant growth of biofilm was observed. At the end of the experiment, the fraction of cells collected in the outlet syringe was harvested, and the fraction formed inside the channels was detached and pushed to the outlet collection syringe by increasing the flow rate to 30 to 40 l min 1 . Serial dilution, plating, CFU counts, and the frequency of recombination calculations were done as described above. Four independent experiments were performed with seven replicates in total .
Competence in MCs with grapevine sap. Grapevine sap was collected from a X. fastidiosa-susceptible variety in Dahlonega, GA, and a tolerant variety in Tallahassee, FL, at the end of the dormant season . A new season cane was pruned, and sap was collected in a 50-ml conical tube, which was stored in ice until it was brought back to the lab. Xylem sap was sterilized by filtering with a 0.22- m vacuum filter and stored at 80°C until used. Sap experiments were performed in the MCs with both pure sap and 50% sap mixed in PD3 . Natural competence assays were same as those for the MC experiment with PD3. Experiments were repeated at least three times for both sap types. Natural competence with heat-killed donor cells and confirmation of homologous recombination. Confirmation of homologous recombination occurring via natural competence was performed by using heat killed donor cells in the solid agar plates. Suspensions of the donor cells were incubated at 90°C for 15 min for heat killing. Complete killing was confirmed by plating an aliquot onto PW plates. The heat-killed donor and live recipients were then spotted on PD3 plates as described above. For confirmation of homologous recombination at the desired genome region, randomly selected recombinant CFU were restreaked onto new double-antibiotic PW plates, and colony PCR was performed using the primers targeting the flanking region of the construct used to generate the mutants according to Kung et al. . Sequences of the flanking regions of antibiotic cassette insertion sites between the parent strains were compared using the muscle pairwise alignment algorithm within the Geneious 9.0.3 platform . Statistical analysis. The number of recombinants, total CFU, and recombination frequency data were analyzed in PROC GLIMMIX , which fits statistical models to data with nonnormal distribution and nonconstant variability.
For the analysis of frequency, the response distribution was used as the binomial distribution of number of recombinants/ total cells. Least-squares differences of means among the treatments were separated by Tukey’s honestly significance difference test at the significance level P 0.05. For the repeated experiments, time factor was used as a random variable. The fringe widths of bacterial colonies among different media also were compared using PROC GLIMMIX in SAS. Accession number. Sequences of the flanking regions of antibiotic cassette insertion sites between the parent strains were deposited in NCBI under accession numbers KU873007 to KU873014.Several hypotheses have been proposed to explain the existence of natural competence in bacteria. One explanation is that starvation signals induce competence, and the incoming DNA serves as a nutrient source under poor nutrient conditions as demonstrated in H. influenzae , Pseudomonas stutzeri , and R. solanacearum . Based on the results with a minimal medium and a rich undefined medium , a previous study speculated that growth in a low-nutrient medium favors natural competence inX. fastidiosa. However, the results of this study with these two media and PD3, another undefined rich medium, demonstrated that growth in PD3 significantly increases the recombination frequency. This suggests that starvation is not necessary to induce competence in X. fastidiosa. Further investigations of the differences between PD3 and PW were performed by either removing or adding these components to/from one another. Initial screening with the components showed a pronounced effect of BSA on the number of recombinants recovered. Additional experiments confirmed that BSA significantly reduces the recombination frequency when present in PD3, PW, and XFM. Since both XFM and PW contain BSA, this may explain the lower recombination frequencies in these media. In a previous study, BSA had been found to reduce the surface attachment and twitching motility of X. fastidiosa . In fact, natural competence and twitching motility are dependent on the activity of type IV pili in X. fastidiosa . Therefore, in this study the correlation between twitching movement and natural competence in different media was investigated. Interestingly, PD3 allowed the highest fringe width, and the presence of BSA significantly reduced twitching motility in all three media. Twitching motility in XFM was lower than in either PD3 or PW as poor growth in XFM resulted in smaller colony sizes. Still the fringe widths of colonies in XFM without BSA were bigger than in XFM with BSA.
Most of the colonies spotted in XFM and XFM-BSA showed very little or no visible growth. This can be expected as XFM is a nutrient-limited minimal medium. Moreover, the pglA-KmR mutant that did not show twitching movement was not competent when tested with heat-killed NS1-CmR mutant and plasmid DNA as the donor. These results of concomitant decreases in natural competence and twitching motility in BSA-supplemented media and non-competency of twitch minus strain suggest that twitching motility is correlated with natural competence in X. fastidiosa. Natural competence in other Gramnegative bacteria is mediated by type IV pili-like structures . In light of the effect of BSA on twitching, hydroponic nft system it remains to be determined if BSA only alters movement or bio-genesis of type IV pili. Our results with different growth settings showed that the recombination frequency is significantly higher in the MC_in fraction than in the MC_out fraction. The MC_in environment closely mimics xylem vessels and the insect foregut with respect to continuous liquid flow, adhesion of cells on channel walls in a fashion similar to adhesion of cells on xylem vessels and the insect foregut, and formation of biofilms. This environment is conducive for both biofilm formation and twitching motility as demonstrated in previous studies . Moreover, expression levels of some of the type IV pili genes were shown to be increased in the MC_in environment compared to those under the other growth conditions , implying that activity of type IV pili is increased in this system, which may explain the higher rates of recombination in the MC_in fraction. The MC_out environment, on the other hand, consists mostly of planktonic cells and some detached biofilm fraction from MC_in, which is washed away with the liquid flow. The differences in recombination frequencies in these two environments suggest that the continuous media flow condition of the xylem vessels and growth in biofilm may increase the chances of recombination. Batch cultures in tubes also allowed recombination but at a lower rate than the continuous flow environment of MC_in and surface-attached condition of solid agar plates. A previous study also showed that growth in solid plates increases recombination compared to the growth in the liquid culture tubes .Recombinantsin theMC_outfraction were recovered when profuse biofilm growth was observed in the MC_in fraction with many recombinants formed. It is possible that the recombinants recovered in the MC_out fraction are due to detachment and washing away of portions of biofilms from the MC_in fractions, supporting the proposition that biofilm formation induces competence. Biofilm formation and quorum sensing signals have been shown to induce natural competence in other naturally competent bacteria such as Vibrio cholerae, Acinetobacter sp. , and Streptococcus mutans . Biofilms, in addition to having dense populations of cells, contain elevated amounts of extracellular DNA , which can be used for transformation by competent cells. Extracellular DNA has been shown to enhance biofilm formation in X. fastidiosa . Moreover, Kung et al. also showed that a knockout mutant on a biosynthetic gene for diffusible signaling factor , a cell-cell communication signal in X. fastidiosa, had a reduced rate of recombination, implying that a cell-cell communication signal also may be involved in regulating natural competence in X. fastidiosa. MC experiments with grapevine sap provide a closer resemblance to the natural habitat than MCs with the artificial culture medium. Previously, we have shown that the biofilm structure in grapevine sap is more similar to the natural biofilm than are the aggregates observed in synthetic medium inside MCs . The experiments with amendments of sap in the MCs detected natural competence, providing an indication that natural competence occurs in the xylem vessels of host plants and possibly in the insect vectors. Although the results with pure sap experiments were not reproducible due to inconsistent growth of one of the strains used, recombinants were recovered once with pure Chardonnay sap as the medium. Recombinants were readily recovered with the 50% sap in PD3 for both tolerant and susceptible varieties. Maintenance of competence with the addition of xylem sap indicates that sap components support DNA acquisition and transformation. Natural competence occurring in environments resembling natural habitats also have been demonstrated in other naturally competent bacteria such as P. stutzeri and V.cholerae, in which artificial medium resembling natural soil extract and natural growth substrate , respectively, induced competence. In R. solanacearum, another xylem-colonizing plant pathogen, natural competence has been demonstrated in planta , and the recipient strains were shown to have increased virulence, acquiring DNA regions as long as 40 kb from donor strains. Findings from competence experiments with grapevine sap and the MCs suggest that when two different strains are established together in the xylem vessels or in the vector foregut, recombination is possible. Noteworthy is the fact that in the experiments reported here, recombination was higher with sap from a tolerant grapevine variety, where infection by X. fastidiosa is symptomless.