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Integrative analysis of the native ethanolamine utilization bacterial microcompartment system in Escherichia coli.

ABSTRACT: Bacterial microcompartments (BMCs) represent prokaryotic counterparts to the eukaryotic organelles. BMCs act as intracellular microbioreactors that spatially insulate targeted pathways and enhance their kinetics through metabolic channelling effects. Some strains of E. coli natively bear an ethanolamine utilization (eut) operon with all the genetic elements required to produce Eut BMCs. The E. coli Eut BMCs could represent powerful tools for synthetic biology applications, such as encapsulating heterologous pathways in vivo to improve their yields. However, they remain poorly characterized. We have thus implemented a systems biology approach to evaluate whether E. coli can produce well-assembled and functional Eut BMCs, focusing on the laboratory strain E. coli K12 W3110. Using 13C-tracer fluxomics data and modelling based approaches, we first characterized the ethanolamine metabolism. We then observed the Eut BMCs by transmission electron microscopy as well as cryotomography. Moreover, we generated novel insights into the Eut BMCs composition through a proteomics analysis. Altogether, these results allowed us to understand how the E. coli Eut BMC operates and how to hack its function while maintaining its integrity.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Alexandre STELLA

LAB HEAD: Odile Schiltz

PROVIDER: PXD048973 | Pride | 2024-07-02

REPOSITORIES: Pride

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Project description:Studies of the RNA polymerase-binding molecule ppGpp in bacteria and plants have shown that changes to the kinetics of the RNA polymerase can have dramatic biological effects in the short-term as a stress response. Here we describe the reprogramming of the kinetic parameters of the RNAP through mutations arising during laboratory adaptive evolution of Escherichia coli in minimal media. The mutations cause a 10- to 30-fold decrease in open complex stability at a ribosomal promoter and approximately a 10-fold decrease in transcriptional pausing in the his operon. The kinetic changes coincide with large scale transcriptional changes, including strong downregulation of motility, acid-resistance, fimbria, and curlin genes which are observed in site-directed mutants containing the RNA polymerase mutations as well as the evolved strains harboring the mutations. Site-directed mutants also grow 60% faster than the parent strain and convert the carbon-source 15% to 35% more efficiently to biomass. The results show that long-term adjustment of the kinetic parameters of RNA polymerase through mutation can be important for adaptation to a condition. Mutations in the RNA polymerase beta prime subunit (rpoC) were discovered in E. coli following adaptation to continual logarithmic growth (OD <= 0.3) in glycerol M9 minimal media at 30 C. We used site-directed mutagenesis to make strains of E. coli isogenic to wild-type except for single adaptive rpoC mutations. We found they increase growth rate in this condition by 60%. In order to understand how the mutations affect gene expression in this condition, we extracted total mRNA from the strains, which had been growing in the adaptive evolution condition, at OD=0.3. There were three RNAP mutants and the wild-type (E. coli K-12 MG1655). Each strain had three flasks from which RNA was extracted (three biological replicates). There were no technical replicates. The mRNA was synthesized into cDNA, labeled, and hybridized to an Affymetrix E. coli 2.0 GeneChip.

			Action	DRS
	F122869.dat	Other
	F122870.dat	Other
	F122871.dat	Other
	F122872.dat	Other
	F122873.dat	Other

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Integrative analysis of the native ethanolamine utilization bacterial microcompartment system in Escherichia coli.

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