Using small molecule probes to study the induction and mitigation of vancomycin resistance

The problem of antibiotic resistance is becoming a serious threat to human health. In the struggle against infection, vancomycin has been the subject of much research as various bacterial species develop resistance to this drug. In the course of designing new antibiotics, it was discovered that appending a lipid on to the sugar moiety of vancomycin increased its activity against resistant strains. This change allowed this molecule to be used as a probe to specifically expand the phenotypic space of microorganisms to explore the novelty of this new activity.; In Chapter Two, glycolipid derivatives of vancomycin were used to probe the induction of resistance response in Streptomyces coelicolor. In these investigations it was demonstrated that the vancomycin aglycone was required to induce resistance. Lipidating this aglycone led to an increased bactericidality of the compound and a decreased ability to induce resistance.; In Chapter Three, chlorobiphenyl vancomycin was used to generate E. coli mutants that were resistant to the increased activity of the drug. These mutants were found to reside in the gene yfgL. Loss of yfgL alone was shown to be sufficient for resistance. Since yfgL was a gene of unknown function, further experimentation was necessary to determine its role.; In Chapter Four, additional resistant mutants were constructed and examined. Nonsense mutants in yfgL that prevented the synthesis of the YfgL protein were obtained. Mutants in Gly-210 of yfgL were also identified. Finally, mutations were found in the imp-4213 allele. Double mutants in yfgL and imp-4213 were not additive, suggesting that these two genes resided in a common pathway. Along with biochemical data, these results may implicate YfgL as having a role in the biosynthesis of the bacterial outer membrane.; The work in this thesis presents a new strategy in the fledgling field of chemical genetics. Small molecule probes were used to specifically make increases in the phenotypic space of microorganisms, thus allowing for progress to be made in annotating the wealth of genetic information gained from the recent sequencing revolution.