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  • Thumbnail for The Functional Significance of Type IV Pilus Proteins for Competence and Effects of Environmental Conditions on Competence in Acinetobacter baylyi
    The Functional Significance of Type IV Pilus Proteins for Competence and Effects of Environmental Conditions on Competence in Acinetobacter baylyi by Leong, Colleen Gong

    Acinetobacter baylyi are naturally competent soil bacteria. Natural transformation is the acquisition of new genetic material via the uptake of foreign, exogenous DNA. Competence is the physiological state some bacterial species may realize in order for natural transformation to occur. Natural transformation, and therefore competence, is clinically relevant, as natural transformation serves as a chief method by which antibiotic-resistant genes are dispersed amongst the bacterial population. A. baylyi serves as an ideal model organism to model natural transformation. A. baylyi are easy to cultivate in vitro, may be genetically modified with ease, and there is a complete library of single gene deletion mutants available for research use. Our first goal was to test the role of Type IV pilus (T4P) proteins in competence using a novel surface-associated quantitative protocol. From the French collection, we obtained knockout mutants lacking proteins predicted to be important for comprising a T4P or for uptake of DNA across the inner membrane. Transformation of cells on a nutritive agar surface allowed for quantitative determination of transformation efficiency over nine orders of magnitude. Using this method we determined which genes were necessary for competence. Under the conditions we tested, genes absolutely required for transformation in A. baylyi include genes encoding the basal apparatus of a T4P (comM, pilF, pilC, pilU, and pilT), the gene encoding the inner membrane DNA translocation protein (comA), the gene encoding the major pilin (comP), genes encoding minor pilins (pilV, fimT), and the gene encoding the pilus tip protein (comC). Mutations in genes encoding for a periplasmic protein that helps target DNA to the comA channel (comEA), conserved hypothetical protein (CHP), genes encoding for a signal transduction response and regulatory receiver (pilG, pilR, and pilS), and a gene encoding for a minor pilins, comF and comE, resulted in a 2-4 log loss in competence. Using transformation on a surface instead of in liquid, we have discovered that a T4P, including its major pilin, is required for transformation A. baylyi. Further, in order to determine conditions under which A. baylyi are most competent in LB media, a simple broth comprised of three ingredients in an easy ratio, we tested additives to LB broth. Though A. baylyi may be grown in a variety of different medias, our laboratory chooses to grow ADP1 cells in LB because of its ubiquity in bacterial labs, low cost, and high rates of natural transformation. Our experiments address whether we may increase the efficacy of LB by altering growing temperature or infusing it with a variety of experimental additives. Overall, we found that A. baylyi cells are competent in nearly all LB conditions tested, but notably, rates of transformation slightly increase in LB+succinate but drastically decrease in LBK+Fe-deficient.

  • Thumbnail for A Nanoscopic Investigation of Pili and Pilus Production in Response to Environmental DNA in Acinetobacter baylyi
    A Nanoscopic Investigation of Pili and Pilus Production in Response to Environmental DNA in Acinetobacter baylyi by Boyd, Caroline Mallory

    Competence is the type of horizontal gene transfer in which a bacterium acquires DNA from the environment and includes this new genetic material in its genome. While this is a common phenomenon in many bacterial species, the mechanism used to ensnare and internalize environmental DNA is unknown. Other studies have shown that type IV pili (T4P), hair-like appendages, are somehow involved in competence but their role is unclear. A highly competent species, Acinetobacter baylyi (ADP1), expresses proteins that comprise the T4P and produce pili on its surface that are morphologically similar to T4P in their diameter and propensity to bundle. To examine the role these pili may play in the process of competence, we exposed ADP1 cells to a variety of environmental DNA concentrations and evaluated pilus production. We expected an increase in abundance or distribution of pili in the presence of more DNA. Pilus production was measured quantitatively by imaging the cells and their pili with atomic force microscopy and semi-automatically tracing the pili. Both the total length of all pili and the number of pili per cell were calculated. Our data demonstrate that ADP1 did not increase pilus production when exposed to more DNA. The cells consistently produced an average of 0.15 +/- 0.05 µm of pili per µm^2 of open mica with a few outliers. Additionally, the Primary pili produced per µm of shoreline was similar across the tested DNA concentrations and minor variations did not follow a trend of increasing Primary pili with increasing DNA concentrations. Slight increases in pilus production were seen at DNA concentrations of 200:1 and 1800:1 DNA molecules:cells and slight decreases seen at 100:1 and 400:1 DNA molecules:cells. Overall, our results do not demonstrate the involvement of pili in the acquisition of environmental DNA as measured by an increase in pilus production when cells were exposed to more DNA. Therefore, more research is needed to elucidate the role of pili in competence.