Thiomicrospira crunogena is a chemolithoautotrophic gamma-proteobacterium that has the ability to survive in fluctuating levels of inorganic carbon due to its carbon concentrating mechanism (CCM). The purpose of this study was to construct a genetic mutant of T. crunogena that would be unable to survive in low levels of inorganic carbon, in order to identify gene(s) involved in T. crunogena's CCM. First, Escherichia coli (BW 20767) was conjugated with T. crunogena so that the Tn5 transposon in this E. coli strain would randomly insert into the T. crunogena genome and interrupt the coding region of a gene involved in the CCM of T. crunogena. Then, mutants were selected based on the ability to survive in different amounts of inorganic carbon. Next, the DNA from the T. crunogena mutants was isolated and a genomic library of junction plasmids created, with some plasmids containing the Tn5 transposon and genomic DNA surrounding the insertion site. E. coli (DH5α/λpir) was transformed with the junction plasmids. Finally, the DNA from the Kanamycin-selected E. coli transformants was isolated and sequenced using outward-directed primers. Two non-CCM T. crunogena mutants were created and characterized. After DNA sequencing it was discovered that the Tn5 transposon of one T. crunogena mutant was inserted into the cardiolipin gene. Future experiments into the role of cardiolipin can be used to understand stress-related processes in T. crunogena. Future work will generate CCM mutants of T. crunogena, which will be used to analyze and understand T. crunogena's distinctive CCM.