Horizontal gene transfer through natural competence and recombination in the generalist plant pathogen Xylella fastidiosa

Horizontal gene transfer has been implicated as a contributing factor towards the diversity and adaptation of pathogens, and the emergence of new diseases. For naturally competent bacteria, DNA acquired through transformation and recombined into the genome could provide a means for this genetic transfer to occur. The work presented here illustrates that Xylella fastidiosa, a bacterial pathogen responsible for several important plant diseases, is naturally competent and able to homologously recombine acquired DNA into its genome. Xylella fastidiosa is vector-transmitted and often exists in natural environments as an endophyte, but causes disease when it multiplies to high levels inside the xylem vessels of its host plants, causing symptoms such as leaf scorching and stunted growth. Several factors were identified that affect the competence of X. fastidiosa, including nutrient availability, growth stage, and methylation state and size of transforming DNA. Recombination efficiencies for X. fastidiosa were at least 1000-fold higher when cells were grown in a defined nutrient medium compared to cells grown in a rich medium. In addition, surface-attached cells transformed and recombined DNA at efficiencies approximately two orders of magnitude higher than their planktonic counterparts. Maximum recombination efficiencies, defined as the number of recombinant cells recovered divided by the total number of cells, were approximately 10-3 when high concentrations of exogenous plasmid DNA were added to cells, and 10-5 when strains harboring different antibiotic markers were co-cultured on solid medium. For planktonic cells, maximum recombination efficiencies were only approximately 10-5 when DNA was added and 10-7 when different strains were co-cultured. Cells appeared most competent while undergoing exponential growth. For planktonic cells, competence peaked after two days of growth and then rapidly declined, with no recombination observed after 8 days. In biofilms, however, cells remained highly competent for at least five days, with recombination events observed even after 21 days of growth. The transformation mechanism in X. fastidiosa is likely similar to that of other naturally competent bacteria, with mutations in type IV pili, competence-related genes (com genes), and cell-cell signaling genes impacting competence./nIt was also experimentally determined that flanking homologous sequences as short as 96bp in transforming DNA is sufficient to initiate recombination, with efficiencies increasing exponentially with length of the homologous region up to 1kb. In addition, recombination efficiencies decreased exponentially with the size of non-homologous insert. Integration of up to 4kb of non-homologous DNA was observed experimentally. An in silico analysis of genomic sequences confirmed that the experimental data was consistent with events detected in natural populations, with an estimated mean size of recombination events of 1,906 bp. Each recombination event also modified, on average, 1.79% of the nucleotides in the recombined region. Based on sequence similarity of shared coding regions, it appears that recombination between different subspecies of X. fastidiosa could frequently occur./nOriginally, it was thought that X. fastidiosa was primarily clonal, but recent studies have suggested that recombination plays a significant role in generating genetic diversity in this bacterium. The work presented here illustrates that X. fastidiosa is naturally competent and that DNA acquired through natural transformation could be a substantial source of donor DNA for recombination. Understanding how this process is regulated and what factors affect its efficiency could provide insight into the genetic diversity of this organism and how new diseases emerge.

Advisor: Almeida, Rodrigo P.P.