Regulation of Lasso Peptide Expression in Alphaproteobacteria

Abstract

Lasso peptides are a class of ribosomally-synthesized and post-translationally modified peptides (RiPPs). Lasso peptides are produced across a diversity of organisms, and are characterized by their knot-like structure, which confers enhanced structural stability and resistance to protease degradation. Lassoes have a broad range of activity, such as having narrowspectrum antimicrobial activity against Gram-negative bacteria, however the functional roles that particular lasso peptides fulfill are largely uncharacterized. Like all proteins, hypothesis about the function of lasso peptides can be inferred from their structure, but also arguably from the regulation of their expression. Understanding the conditions in which the lasso peptides are natively expressed can be essential to the characterization of novel lasso peptides. However, the regulation of lasso peptide expression in their native hosts is poorly studied. Although the regulation of lasso peptides is largely uncharacterized, GntR-family regulators, one of the largest classes of DNA-binding transcription factors that is widespread and conserved across bacteria, are frequently observed in lasso BGCs upstream of the canonical genes for lasso biosynthesis and are suggested to regulate lasso clusters. This thesis aims to determine whether the observed GntR homologs might play a role in the regulation of lasso peptide production. This question has been approached with analysis of potential binding sites near or within the BGCs of lasso peptides, harnessing a computational approach and the conservation of GntR-family DNA-binding sequences. Lasso peptide expression has also been tested under varied conditions that have been proposed to correlate to lasso function, such as iron limitation in the medium that is often used to induce secondary metabolite production. Heterologous expression of lasso peptides has been readily achieved in the Link Laboratory via the transformation of E. coli with plasmids containing key genes for lasso peptide synthesis. The experimental and computational work of this thesis has focused on the three lasso peptides (astexin-1, -2, and -3) produced by the Gram-negative bacterium Asticcacaulis excentricus. Astexin-2 species were determined to be natively expressed under normal growth conditions and iron-limiting conditions that do not inhibit A. excentricus growth. Neither the astexin-1 or -3 species appear to be natively expressed under any conditions. GntR regulation of the astexin lassos could be achieved through the binding of the astexin GntR homologs to putative binding sites in the astexin BGCs that have been identified in this thesis. The influence of various environmental conditions on A. excentricus growth as well as astexin expression and the putative binding sites of GntR transcription factor homologs in the astexin BGCs together contribute to the knowledge surrounding lasso peptide regulation and biosynthesis.