GENOME-WIDE SCREEN FOR THE INDUCTION OF PHOSPHOETHANOLAMINE AND AMINOARABINOSE MODIFICATIONS ON ESCHERICHIA COLI LIPOPOLYSACCHARIDE

Open Access
Author:
Smith, Alexander Brock
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Timothy Meredith, Thesis Supervisor
  • Wendy Hanna-Rose, Honors Advisor
Keywords:
  • Escherichia coli
  • lipopolysaccharide
  • phosphoethanolamine
  • microbiome
  • transposon sequencing
  • metabolic endotoxemia
  • eptA
  • feoB
Abstract:
Chronic inflammation arising from metabolic endotoxemia (ME) is currently a widespread health concern with deep ties to the complex human microbiome. Gram-negative microbiota such as Escherichia coli possess lipopolysaccharide (LPS), a potent endotoxin and mediator of this chronic inflammation. Covalent modification of LPS with phosphoethanolamine (PEtN) has been shown to exacerbate this response, so understanding the genetic regulation of PEtN modifications is essential to fully understanding ME. Different strains of E. coli, however, have a spectrum of basal PEtN expression despite there being 100% DNA sequence conservation within the PEtN modifying gene promoter itself. Methods to identify these strain specific alleles are thus needed. We have constructed a two-component genetic reporter system in E. coli K-12, a strain normally silent for LPS modification, that allows us to introduce and positively select transposon linked alleles from other strains of E. coli that specifically activate this modification pathway. By coupling PEtN activation to cell growth, we have used next-generation sequencing analysis to identify several putative alleles in strain E. coli BL21(DE3) that are responsible for constitutive expression of PEtN modifications. The reporter system that we have developed here can not only be applied to rapidly analyze any E. coli microbiome isolate in order to uncover specific PEtN regulatory alleles, but also to other promoter elements of interest.