Natural transformation is one of the mechanisms of horizontal gene transfer, wherein bacteria acquire exogenous DNA and integrate it into their genome. For this process to occur, the bacteria must be competent, meaning they express proteins necessary for DNA uptake and internalization. Type IV pili (T4P) constitute one such protein complex that aids bacteria in twitching motility, enabling them to move through their environment by extending and retracting the pili. Additionally, T4P can bind to and take up exogenous DNA for further internalization, making them a crucial component of natural transformation. Acinetobacter baylyi ADP1 is known to be highly competent and thus serves as a good model organism for studying genes and proteins necessary for DNA uptake and natural transformation. In this study, three A. baylyi mutants created by random transposon insertions were tested for twitching motility and transformation efficiency. All mutants were previously found to exhibit altered twitching motility to varying degrees, leading to the hypothesis that they may also have altered transformation efficiency since both motility and transformation are dependent upon the same molecular structure. In determining transformation efficiency, mutant cells are cultured in LB broth along with isolated streptomycin resistance DNA. The three mutant strains each exhibit distinct natural transformation phenotypes: lower, equivalent, and higher than the ADP1 wild type. This suggests that the transposon may interrupt different sites in the genome for each mutant, resulting in loss, gain, or neutral function of the competence protein. Transposon Insertion Sequencing could determine the exact location of the interrupted gene. Due to time constraints, sequencing results are still inconclusive, but this lays the groundwork for further experimentation. To further investigate whether altered transformation efficiency is due to malfunctioning T4P or the mechanism by which bacteria integrate DNA, we examined the presence of ComP, the main protein composing the extracellular part of T4P, using Western blotting. This experiment provided inconclusive results due to the presence of a protein band in the negative control. Alternatively, we also examined the pili structure under a scanning electron microscope, which was observable in all mutants. These results suggest that although T4P may remain physically intact, transposon interruption affects either the DNA integration mechanism in some mutants or the T4P DNA uptake system in others, resulting in altered natural transformation. This research serves as a good starting point for testing the phenotypes of ADP1 mutants generated through transposon mutagenesis and developing experimental procedures for more conclusive results.