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The Functional Significance of Type IV Pilus Proteins for Competence and Effects of Environmental Conditions on Competence in Acinetobacter baylyi

by Leong, Colleen Gong

Abstract

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.

Note

The author has given permission for this work to be deposited in the Digital Archive of Colorado College.

Colorado College Honor Code upheld.

Includes bibliographical references.

Administrative Notes

The author has given permission for this work to be deposited in the Digital Archive of Colorado College.

Colorado College Honor Code upheld.

Copyright
Copyright restrictions apply.
Publisher
Colorado College
PID
coccc:25899
Digital Origin
born digital
Extent
65 pages : illustrations
Thesis
Senior Thesis -- Colorado College
Thesis Advisor
Lostroh, Phoebe Lang, Kristine
Department/Program
Biology
Degree Name
Bachelor of Arts
Degree Type
bachelor
Degree Grantor
Colorado College
Date Issued
2016-05