The transfer of genetic material between organisms is a fundamental aspect of life. Parent-to-offspring genetic transfer, or vertical gene transfer, is absolutely required for species propagation. Not all genetic transfers occur in this fashion, however. The acquisition of genetic elements from non-parental organisms, or horizontal gene transfer (HGT), is commonplace across the tree of life. Nowhere is this more evident than in the bacterial domain. Here, numerous HGT mechanisms operate to mediate genetic transfers from organism to organism, including phage transduction, conjugation, and natural transformation. While each mechanism is distinct from one another, they all share a common problem: Long, negatively charged, hydrophilic nucleic acid polymers cannot normally cross intact bacterial cell envelopes. Therefore, each of these HGT mechanisms has uniquely evolved to overcome this challenge.The existence of genetically tractable, naturally transformable model organisms has enabled detailed study of the mechanisms involved in mediating DNA transit across the cell envelope during natural transformation. Natural transformation is the uptake of free DNA from the environment into the cytosol and the subsequent integration of this DNA into the recipient cell’s genome, all of which is accomplished by specialized proteins produced by the recipient cell. The vast majority, if not outright all, of the proteins necessary for natural transformation have been identified in both Gram-negative and Gram-positive organisms. How these proteins operate mechanistically to transport DNA across bacterial cell envelopes has been an ongoing question for decades. This thesis will focus on how DNA is transported across the Gram-positive cell wall, the first barrier encountered on the DNA’s path to the cytosol, which is comprised of a dense meshwork of crosslinked peptidoglycan/teichoic acid. In the naturally transformable model organism Bacillus subtilis, expression of only 10 genes is needed for DNA to cross the cell wall barrier in B. subtilis, with 80% of these genes encoding proteins homologous to essential pilus biogenesis proteins. Pili are thin, cytoplasmic-membrane-anchored filaments that span cell envelopes into the extracellular space. Naturally, it has long been hypothesized that B. subtilis (and other Gram-positives) employs a pilus system to breach the cell envelope and mediate DNA entry, but pili have never been observed on the surface of naturally transformable B. subtilis. This thesis seeks to address this shortcoming while simultaneously exploring how these putative pili mediate DNA transport across the cell wall. Recently, a method was developed that allows for the fluorescent labelling of dynamic protein filaments in bacteria, the maleimide labeling method, which is compatible with live cell microscopy. Maleimide labeling involves cysteine substitution mutation of the protein subunits comprising the filament, on a residue that is solvent exposed. During filament production, a fluorescent maleimide conjugate is added to the medium, which will covalently link to the cysteine thiol on each monomer in the filament, making the structure fluorescent. In chapter 2 of this thesis, we detail the production of a comGC (gene encoding the major structural protein of the putative pilus) cysteine substitution panel to search for labelable mutants. After screening ΔcomGCWT complementation strains expressing each of the 41 successfully made comGCCys variants, we found that 9 mutants maintained transformability within 500-fold of wildtype, providing a solid foundation for future maleimide labeling studies. In chapter 3 of this thesis, we demonstrate that strains expressing both comGCWT and comGCE56C or comGCS65C maintained full transformability, improving on the original complementation strains. Both co-expression strains were capable of pilus production during natural competence, as evidenced by maleimide labeling and widefield epifluorescence microscopy, demonstrating for the first time that B. subtilis does produce these structures. In experiments where labeled pili and fluorescently labeled DNA were imaged together, we find that these pilus structures could bind the DNA. During imaging experiments that tracked pili through time, we found that some pili had the ability to dynamically retract back towards the cell bodies. Together, these data suggest a model wherein B. subtilis produces pili during natural competence, the pili bind to environmental DNA, and then pilus retraction brings the DNA across the cell wall, through the pore created by the pilus extending originally.