Pleiotropic effects of rfa-gene mutations on Escherichia coli envelope properties

Christophe Pagnout,Jérôme F L Duval,Pascale Bauda,Céline Caillet,Angélina Razafitianamaharavo,Marc Offroy,Stéphane Jomini,Audrey Beaussart,Héloïse Gendre,Bénédicte Sohm,Marjorie Leduc

doi:10.1038/s41598-019-46100-3

Abstract

Mutations in the rfa operon leading to severely truncated lipopolysaccharide (LPS) structures are associated with pleiotropic effects on bacterial cells, which in turn generates a complex phenotype termed deep-rough. Literature reports distinct behavior of these mutants in terms of susceptibility to bacteriophages and to several antibacterial substances. There is so far a critical lack of understanding of such peculiar structure-reactivity relationships mainly due to a paucity of thorough biophysical and biochemical characterizations of the surfaces of these mutants. In the current study, the biophysicochemical features of the envelopes of Escherichia coli deep-rough mutants are identified from the molecular to the single cell and population levels using a suite of complementary techniques, namely microelectrophoresis, Atomic Force Microscopy (AFM) and Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) for quantitative proteomics. Electrokinetic, nanomechanical and proteomic analyses evidence enhanced mutant membrane destabilization/permeability, and differentiated abundances of outer membrane proteins involved in the susceptibility phenotypes of LPS-truncated mutants towards bacteriophages, antimicrobial peptides and hydrophobic antibiotics. In particular, inner-core LPS altered mutants exhibit the most pronounced heterogeneity in the spatial distribution of their Young modulus and stiffness, which is symptomatic of deep damages on cell envelope likely to mediate phage infection process and antibiotic action.

Highlights

Lipopolysaccharides (LPS) cover surface of the outer membrane of Gram-negative bacteria
Recent studies based on large-scale phenotypic screening of the Keio collection (i.e. 3985 isogenic mutants of E. coli K-12) are consistent with these observations, highlighting a distinctive rfa-gene mutation phenotype compared to the wild-type strain with respect to e.g. susceptibility to bacteriophages[13,14,15] and antibiotics[16,17] and in terms of their ability to become non-motile[18] or to form biofilms[19]
The mobility μ of the four strains of interest is negative over the whole range of electrolyte concentrations, which is in line with the protolytic properties of the functional groups carried by the outer membrane and the LPS structures

Summary

Introduction

Lipopolysaccharides (LPS) cover surface of the outer membrane of Gram-negative bacteria. The pleiotropic effects associated with single rfa-gene mutations leading to inner (ΔrfaC and ΔrfaG) or outer (ΔrfaJ) core truncated LPS on E. coli K-12 membrane (Fig. 1B) are addressed by a suite of complementary experimental techniques probing cell envelope properties at different scales, from the molecular level, the single cell level up to the cell population, namely (i) iTRAQ-based quantitative proteomics for analysis of the cell envelope protein content, (ii) Atomic Force Microscopy (AFM) and Spectroscopy for cell imaging and evaluation of membrane elasticity and internal Turgor pressure (which is related to cell stiffness), and (iii) electrokinetics (electrophoretic mobility), which provides information on the bacterial surface charge density and on the permeability of the biosurface to the tangential electroosmotic flow developed during cell migration under action of an applied electric field These data are thoroughly discussed in relation with the peculiar reactivity of deep-rough mutants of E. coli within the contexts of resistance/sensitivity to bacteriophages, antimicrobial peptides and hydrophobic antibiotics

Methods

Results

Conclusion