Project 467160

The antimicrobial resistance crisis caused by the evolution of multi-drug resistant human pathogens is a prominent issue in healthcare. Staphylococcus aureus is a problematic bacterium in this area, as it has acquired resistance to virtually all commonly used antibiotics and has begun to acquire resistance to the few remaining drugs of last resort. Thus, there is an urgent need for the development of new therapeutics against this pathogen. Naturally-produced antibiotics from bacteria are one avenue to address this issue. The genus Pantoeahas been shown to produce a variety of novel antibiotics antagonizing drug-resistant human pathogens. One strain, P. septica091957A produces a potent antibiotic that inhibits multi-drug resistant S. aureus. The objective of my thesis is to identify the antibiotic gene cluster in this strain and attempt to overproduce the compound so that it can be purified, and its structure determined. First, the gene cluster will be identified by a genetic screen using transposon mutagenesis to create a mutant library of P. septica091957A. Loss of antibiotic production in a mutant will indicate the interruption of an antibiotic-encoding gene, which will be identified by inverse PCR. Once the entire gene cluster is identified, it will be over-expressed by cloning it into an expression vector for E. coli or by assessing appropriate culturing conditions for natural expression in P. septica091957A. Chemical extraction and chromatography will then be used to purify the compound followed by mass spectrometry to determine its chemical structure. The results of this study may lead to the development of a novel therapeutic that can be used for multi-drug resistant S. aureus infections.