Examination of spoT, rpoS and rpoN gene expression in response to fatty acid starvation in Borrelia burgdorferi, the Lyme disease spirochete
The purpose of the present investigation is to examine the regulation and expression of the B. burgdorferi spoT/rpoS pathway under normal growth conditions and during serum starvation in wild-type strains and in spoT, rpoS and rpoN mutant strains. Additionally the expression of B. burgdorferi's rpoN gene has been examined within a surrogate Escherichia coli system by using a GFP reporter construct in order to determine if SpoT, RpoS or RpoN affect rpoN expression. ^ In this study, we examined the affects of mutating the spoT, rpoS and rpoN genes. These genes are known to be both affected by starvation and to play a role in gene regulation of the stringent response and starvation. Absence of ppGpp production, as seen in the spoT mutant, suggested that the ability to adapt to and survive transition into stationary phase was compromised by the spoT mutation. Serum starvation induced expression of spoT in wild-type cells and complemented spoT mutants, but not in spoT mutants. Mutations in rpoS and rpoN were found to have little to no effect on spoT expression. During serum starvation, wild-type strains demonstrated patterns of rpoS expression that greatly resembled wild-type expression of spoT. The spoT mutants did not show wild-type changes in rpoS expression; along with a delayed onset, the peak in rpoS expression was approximately 50% that the parental strains. Down regulation of rpoN expression during serum starvation was seen in most strains regardless of spoT or rpoS mutation, suggesting that rpoN expression is not affected by SpoT or RpoS. ^ Examination of the B. burgdorferi rpoN promoter driven GFP expression within wild-type E. coli has demonstrated peaks in GFP fluorescence during time points immediately prior to exponential growth. Absence of ppGpp production negatively influenced the amount of GFP expressed from the BbPrpoN. Fluorescence from BbPrpoN-GFP within the population of relA spoT mutant cells showed the wild-type increase in the rpoN driven GFP expression was delayed by an hour and decreased by approximate 60% when not completely absent. Additionally, mutations in rpoS and rpoN, practically eliminate the wild-type peak in BbPrpoN driven GFP expression at the onset of exponential phase. ^
Wendy Meredith Coy,
"Examination of spoT, rpoS and rpoN gene expression in response to fatty acid starvation in Borrelia burgdorferi, the Lyme disease spirochete"
Dissertations and Master's Theses (Campus Access).