Proteomic analysis of L-forms and investigation of mutations to potentially enhance L-form growth and stability for synthetic biology applications
Abstract
Many bacteria, both gram-positive and gram-negative, are able to transition to a phenotype where they lack a peptidoglycan cell wall. When bacteria exist in this cell wall-deficient state, they are called L-form bacteria or L-forms. In this state, they can resist to antibiotics targeting the biosynthesis of peptidoglycan. In addition, they represent a model for the study of primitive cells, before the cell wall originated. They are also of interest for synthetic biology due to potential advantages conferred by the absence of a cell wall, such as the potential ability to avoid or limit immune responses (if there is complete absence of peptidoglycan), or their potential as better producers of therapeutic compounds without a barrier for protein secretion. In this line, they could serve as Òliving pillsÓ. We are particularly interested in the use of L-forms to design hybrid prokaryotic-eukaryotic hybrid systems. This project combines two approaches to better understand the L-form phenotype at the molecular level with the ultimate goal of being able to induce it temporarily (unstable L-forms) or permanently (stable L-forms) at will. The first approach aims to conduct proteomic analysis of E. coli cells in walled and wall-deficient state (L-form). We would like to characterize the proteome of L-form E.coli grown in osmotically protective media and induced by a cell wall-targeted antibiotic (cefsulodin). The L-form proteome has never been characterized with advanced high-throughput proteomic techniques. We will compare this proteome with the wild type proteome when cells are grown in osmotically protective media and in widely used LB media (to serve as a reference). We have already conducted a preliminary proteomic analysis and introduced several improvements aimed to maximize the coverage of the proteome. The second approach is aimed to investigate the genetic underpinnings of L-form formation, growth, and stability. Our ultimate interest lies in the identification of mutants that enhance L-form growth, and the ones that lead to the formation of stable, non-reversible, L-forms. We propose to design a mutant library according to the CREATE (CRISPR-EnAbled Trackable genome Engineering) methodology and customized to interrogate the most relevant pathways and genes associated to L-form transformation and growth. Previously, we compiled a list of pathways, genes, and mutations associated to published work on the L-form phenotype in E.coli, as well as mutations in genes associated to biological processes that have proved to be relevant to L-form in other organisms, mainly B.subtilis. These include mutations that disrupt the synthesis of peptidoglycan, mutations that up-regulate fatty acid biosynthesis, mutations that disrupt the synthesis of components of the electron transport chain, and mutations that increase the response to several types of stresses, mostly to reactive oxygen species. We will also include designs for off-mutants of all genes in the E.coli genome for hard-to-predict mutant effects. Finally, we will incorporate designs associated to the obtained L-form proteomic data. We suggest to complete the library design by rationally selecting and predicting sites and types of mutagenesis to be conducted for each gene; we will use a variety of sophisticated bioinformatic predictive tools to select those sites and domains that are more likely to have an effect of the phenotype of interest and we will carefully design a subpool distribution of the selected designs. We expect the resulting library design and synthesized oligo library proposed here will set the basis to enable future screens of the resulting CREATE library for the identification of mutants that enhance or stabilize L-forms. We expect the results will improve our understanding of the molecular mechanisms underlying the L-form phenotype in E.coli and will pave the way to the use of L-forms as part of the development of prokaryotic-eukaryotic hybrid systems.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jun 25, 2021
- Source ID
- W911NF2110228
Entities
People
- Maria Isabel Martinez Ferrando
Organizations
- Army Contracting Command
- Centre for Genomic Regulation
- United States Army