Implementation of a Leaky Outer Membrane E. Coli Cell Line for Characterization of Peptidoglycan Recycling Pathways and Improved Cell Wall Labeling

• Liam-Michael Sandles, Biochemistry, University of Delaware

• Catherine Grimes, Chemistry & Biochemistry, University of Delaware

In recent years, the bacterial microbiota of the gut has been recognized for its vital role in the body. Commensal bacteria are essential to a healthy lifestyle, while pathogenic bacteria are one of the leading causes of death worldwide. Both share the distinct feature of a Peptidoglycan (PG) cell wall surrounding a cellular membrane; however, many mysteries surrounding the nature of PG remain unsolved, such as the exact structure of PG fragments that are known to elicit an immune response. To elucidate these fragments, the Grimes lab developed a method for labeling the PG of bacterial cells using N-acetyl muramic acid (NAM) probes that hijack bacterial PG recycling pathways. Earlier work assisted in engineering Escherichia coli cell lines containing recycling enzymes anomeric NAM/N-acetyl glucosamine (NAG) kinase (AmgK) and α-1-phosphate uridylyl transferase (MurU) from either Pseudomonas Putida or Tannerella Forsythia that demonstrate exogenously administered NAM can be incorporated under lethal fosfomycin conditions. Recent projects implementing a leaky outer membrane (OM) E. Coli strain, RFM795, with a T. Forsythia AmgK/MurU plasmid (RFM795 Tf-KU) in tandem with various click chemistries, has led to the improved efficiency of bacterial labeling and visualization. Leaky OM cells are more susceptible to molecules that would otherwise be unable to enter the periplasm. Current work is focused on developing a plate reader assay for identifying NAM recycling inhibitors and analyzing growth patterns using this now-improved library of molecules. Promising candidates will then be tested for enzyme inhibition through in vitro kinetics experiments. Many known pathogens, such as Pseudomonas Aeruginosa and T. Forsythia, contain AmgK/MurU homologs that assist in bacterial survival under adverse conditions, making NAM recycling inhibitors a potential target for antibiotics in the future.