Role of carbohydrate metabolism in cell wall biosynthesis and multicellular development in Bacillus subtilis

Abstract

Biofilms are complex multicellular bacterial communities encapsulated in a self-produced matrix which play a large role in the survival of bacteria within its natural environment. Matrix produced by the Gram-positive model organism Bacillus subtilis is comprised of a protein and a carbohydrate exopolysaccharide (EPS) fraction. Although the exact composition of the EPS is poorly characterized, the generation of uridine-diphosphate-galactose (UDP-galactose) is essential for the formation of robust biofilms. To this end, the gan operon has previously been shown to be linked to galactose metabolism, though the exact regulation of this operon and involvement in biofilm formation are unknown. Although required for matrix formation, accumulation of UDP-galactose resulting from improper metabolism due to defect in the highly conserved Leloir pathway is toxic and forms a condition known as Galactosemia, known to impact all domains of life. In B. subtilis, a deletion of the final Leloir pathway gene, galE, causes severe structural defects which lead to cell death, with the mechanism of toxicity in this case and all others unknown. Thus, understanding the manner in which galactose is acquired, its role in biofilm formation, and the mechanism by which it causes toxicity through metabolism is vital to better understanding the lifecycle of B. subtilis in its natural environment. In chapter 1, I review what is currently known about the importance of biofilms within the rhizosphere, the region of soil containing plant root and the bacteria that colonize them. I review the complex regulation of biofilm formation and matrix production in B. subtilis, and provide the current literature on the formation of the Gram-positive bacterial cell wall and envelope. Finally, I discuss Galactosemia and the role of the conserved Leloir pathway in this disease. In chapter 2, I characterize the gan operon of B. subtilis, involved in the use of galactan, a complex carbohydrate found as a primary component of plant cell walls within the rhizosphere. I demonstrate that regulation of this operon is performed in part by a LacI-like transcriptional regulator GanR. I show that β-1,4-galactobiose acts to both induce expression of this operon through derepression of GanR binding to a DNA motif, as well as inhibit the activity of the encoded β-galactosidase GanA upon formation of the final catalytic product galactose. I finally show that this operon plays an important role in in situ root colonization, thus providing a mechanism by which B. subtilis can sense, regulate, and utilize plant-based carbohydrates in the formation of biofilms. In chapter 3, I characterize the mechanism of toxicity in B. subtilis due to improper metabolism of galactose. I show that upon accumulation of UDP-galactose due to defect in galE, cell wall biosynthesis is inhibited due to accumulation of essential peptidoglycan precursor. I show that this inhibition is partially rescued by overexpression of the essential glycotransferase MurG involved in generation of lipid II, which is catalytically inhibited by UDP-galactose in vitro. This toxicity and the rescue imparted by MurG is also observed in multiple species including E. coli and V. cholerae, suggesting that this mechanism may be conserved to other glycotransferases. Finally, in chapter 4, I summarize my findings, discuss their impact, and provide future directions.