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Identifying the Functions of Uncharacterized Genes in Bacillus Subtilis
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Bacteria encode a variety of ways to sense and respond to their dynamic environments. Consequently, in order to implement the appropriate response to execute changes in metabolism or subcellular organization, gene expression is tightly coordinated with the cell’s ability to recognize a perturbation. For instance, the devastating effects of a toxic compound can be mitigated by expressing gene products to export or metabolize the compound. The Gram-positive model organism Bacillus subtilis utilizes a variety of gene expression programs in order to survive the diverse environments it encounters. Intriguingly however, nearly 40% of the genes in B. subtilis are either unannotated or annotated without experimental validation. Genes that play important roles in regulating gene expression, metabolism and regulating essential cell processes are likely found within this set. The lack of gene characterization is due in part to our inability to obtain tractable phenotypes from which to form testable hypotheses regarding gene function. To associate gene products with phenotypes, we utilized a gene misexpression library comprising more than 800 strains as a tool to uncover gene products that perturb growth and/or subcellular organization when artificially expressed. From this set, 10 DNA-binding proteins predicted to be transcription factors were selected for transcriptomic analysis and uncovered candidate regulatory targets for 8/10 of the DNA-binding proteins. Artificial expression of one of the transcriptional regulators, YxaD, resulted in cells unable to properly segregate chromosomes or replicate DNA, and confirmed a prior observation that YxaD is capable of interaction with ScpA, a subunit of the Structural Maintenance of Chromosome (SMC) complex. Additionally, we showed that YxaD acts as a repressor of its own promoter as well as a divergently transcribed operon encoding the cid/lrg homologs, yxaKC. Transcriptional profiling revealed that yxaKC is expressed both in a specific region of a biofilm, and in glucose-containing medium, with expression peaking during stationary phase growth just as glucose is depleted. Using untargeted NMR-based metabolomics, we showed that cells artificially expressing YxaKC show enhanced export of the overflow metabolite 2-acetolactate. We hypothesize that YxaKC helps to maintain cellular homeostasis possibly by acting as a passive transporter of 2-acetolactate.
Hartman, Sarah (2019). Identifying the Functions of Uncharacterized Genes in Bacillus Subtilis. Doctoral dissertation, Texas A&M University. Available electronically from