Putative DNA-Binding Proteins Affecting the Subcellular Organization of Bacillus Subtilis

Bacteria lack compartmentalization and therefore rely on subcellular organization to temporally and spatially localize major cellular processes. As one of the major structures in the cell, the nucleoid possesses positional cues that help direct important functions such as DNA replication and cell division. While some of the factors that contribute to maintaining subcellular organization are characterized, many of the underlying determinants are still unknown. In Bacillus subtilis, there remains a class of putative DNA-binding proteins currently uncharacterized, and I hypothesize that some of these proteins use DNA to localize functions important in maintaining subcellular organization. Since many uncharacterized genes lack easily discernable knockout phenotypes, I utilized a gene misexpression approach to identify DNA-binding proteins capable of perturbing some aspect of cellular organization. The misexpression library employed comprises over 800 strains, each containing an uncharacterized gene under the control of an inducible promoter on the B. subtilis chromosome. Genes for 98 putative DNA-binding proteins were misexpressed and analyzed for their ability to grow in the presence of inducer. Of these strains, nine exhibited reduced growth and 17 strains did not grow. When analyzed by fluorescence microscopy, 19 of these strains could be associated with a distinct phenotype, including defects in the structure and placement of the chromosome, abnormal cell division, and loss of cell shape. To further characterize strains with the most severe phenotypes we performed RNA-seq experiments to determine if gene misexpression alters the transcription profile. We identified four genes that do not affect transcription, suggesting that the misexpression phenotype is not due to transcriptional regulation. We hypothesize that this class of DNA-binding proteins act as positional cues that help maintain subcellular organization.