Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiologyExperimental work performed at the Centre for Molecular Bacteriology and Infection, Imperial College LondonLipoteichoic acid (LTA) is an important polymer present in the envelope of various Gram-positive bacteria. In Staphylococcus aureus it is composed of a polyglycerolphosphate chain synthesised by the enzyme LtaS, and is anchored to the membrane via a glycolipid anchor. Depletion of LTA is known to cause growth arrest, aberrant positioning of septa and enlargement of cells leading to eventual cell lysis. This highlights the importance of LTA for bacterial growth and also suggests a possible link between LTA synthesis and cell division. Although key enzymes required for LTA synthesis have been identified, how this process is regulated has not been elucidated. This study investigates the mechanisms of regulation of LTA synthesis, as well as provides further insights into the roles of LTA in S. aureus. In this study, nuclear magnetic resonance (NMR) was used to experimentally confirm a function for LTA in the incorporation of D-alanines into a second polymer, wall teichoic acid (WTA). LTA was also shown to be essential not only for bacterial growth, but also for bacterial survival, as cells become bactericidal in its absence. Furthermore, fluorescence microscopy using a GFP-tagged version of the synthase enzyme indicated that LtaS localises to the septum, suggesting a possible role for LTA in cell division. Regarding LTA regulation, this work has shown that the ltaS gene has two promoters and a large upstream UTR, but that only one promoter is essential and sufficient for expression. Studying the effect of salt stress on LTA production has also revealed that an increase the NaCl concentration in the media causes a reduction in the levels of LTA produced, which in turn has an effect on growth. However this is not due to a decrease in the levels of LtaS, reflecting a possible ionic or osmotic effect on LtaS enzymatic activity

A fluorescent reporter for single cell analysis of gene expression in clostridium difficile

Genetically identical cells growing under homogeneous growth conditions often display cell-cell variation in gene expression. This variation stems from noise in gene expression and can be adaptive allowing for division of labor and bet-hedging strategies. In particular, for bacterial pathogens, the expression of phenotypes related to virulence can show cell-cell variation. Therefore, understanding virulence-related gene expression requires knowledge of gene expression patterns at the single cell level. We describe protocols for the use of fluorescence reporters for single cell analysis of gene expression in the human enteric pathogen Clostridium difficile, a strict anaerobe. The reporters are based on modified versions of the human DNA repair enzyme O ( 6)-alkylguanine-DNA alkyltransferase, called SNAP-tag and CLIP-tag. SNAP becomes covalently labeled upon reaction with O ( 6)-benzylguanine conjugated to a fluorophore, whereas CLIP is labeled by O ( 6)-benzylcytosine conjugates. SNAP and CLIP labeling is orthogonal allowing for dual labeling in the same cells. SNAP and CLIP cassettes optimized for C. difficile can be used for quantitative studies of gene expression at the single cell level. Both the SNAP and CLIP reporters can also be used for studies of protein subcellular localization in C. difficile.</p