This Is AuburnElectronic Theses and Dissertations

Fitness strategies of fastidious prokaryotes




Merfa e Silva, Marcus Vinicius

Type of Degree

PhD Dissertation


Entomology and Plant Pathology


Fastidious prokaryotes are slow-growing bacteria that either require specific media to grow or are not able to grow axenically. ‘Candidatus Liberibacter asiaticus’ (CLas) and Xylella fastidiosa are important fastidious plant pathogenic bacteria that threaten agriculture worldwide. CLas is associated with Huanglongbing, the most devastating disease of citrus globally, whereas X. fastidiosa harms many economically important crops worldwide, including grapevine, citrus, and olive. In this study, we sought to understand the fitness of CLas in its natural environments based on published studies and aimed to replicate these conditions in vitro to increase its culturability, since CLas is hitherto unculturable axenically. Commercial grapefruit juice (GJ), which was used as base culture medium, was amended with a wide range of compounds, and incubated under different conditions to evaluate the optimization of CLas growth. A reproducible growth behavior in which CLas growth ratios were inversely proportional to the initial inoculum concentration was observed. Additionally, strategies to reduce the cell density allowed CLas to reach maximum growth as fast as 3 days post inoculation, but with no sustained exponential growth over time. Conversely, we performed a complete functional analysis of the molecular components of the type IV pili (TFP) machinery of X. fastidiosa that mediates both its twitching motility and natural competence, an important mechanism to generate genetic diversity and likely modulate its fitness within natural hosts. We determined the core components of TFP involved in these two traits, as well as screened the role for virulence in planta for some of these components. Remarkably, we identified a novel minor pilin, FimT3, which is the DNA receptor of the X. fastidiosa TFP, being thus essential for its natural competence by binding DNA. Moreover, we identified recently recombined genes within X. fastidiosa strains by performing whole genome analyses. Identified genes are involved in host colonization, regulation and signaling, host evasion, and nutrient acquisition, which are all important for the ecology of X. fastidiosa. We hope our results may help to better understand the fitness of these two pathogens, which may aid the development of strategies for controlling them.