Characterization of unknown Bacillus subtilis genes with transposon sequencing

The exact essentials for maintaining a living bacterial cell are still unclear. out of over 4000 genes in the model bacteria Bacillus subtilis and Escherichia coli, only approximately 260 genes have been identified as essential. However, this number does not accurately reflect the bacterial requirements due to the presence of many redundant pathways. To overcome this problem, we utilizes a genome-reduced strain of B. subtilis, PG10, that lacks 35% of the original chromosome, and likely many redundant pathways. Through extensive Tn-seq analysis, described in Chapter 2, we revealed 133 new essential genes in PG10, 69 of which remain functionally uncharacterized. In this thesis we describe the functional characterization of the unknown genes ytiB, ywnA, and ywgA. Chapter 3 reveals that the absence of ytiB leads to the overexpression of mtlR and subsequent upregulation of the mtlA operon involved in mannitol utilization. In Chapter 4, we explore the role of YwnA in oxidative stress response, and its regulatory impact on the ywnAB operon. Chapter 5 describes the discovery of a new dNTP triphosphatase that is controlled by YwgA. Finally, Chapter 6 investigates a phenomenon that we encountered throughout our research; a possible confounding effect on the transcriptome due to presence of the erythromycin resistance marker ermC. In conclusion, this PhD thesis elucidates the activity of several unknown genes in B. subtilis, thereby further our understanding of this important bacterial model system, and provides useful insights in the use of genome reduction to help uncover the minimal necessities for a bacterial cell.