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

International audience; 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. Lipopolysaccharides (LPS) cover surface of the outer membrane of Gram-negative bacteria. They are tripar-tite molecules composed of lipid A, core oligosaccharides usually containing glucose, heptose, galactose, 2-keto-3-deoxyoctonate (KDO), and a highly variable O-antigen component (O-antigen is missing in Escherichia coli K-12). LPS act as a protective and permeable barrier against large molecules and hydrophobic compounds from the environment. They are positioned among phospholipids and proteins of the outer membrane, and contribute to the structural properties of the latter. In Escherichia coli, genes involved in the LPS synthesis are organized according to three operons in the rfa (also known as waa) locus (Fig. 1A). The first operon contains rfaD (or gmhD), rfaF, rfaC and rfaL genes. The three first genes encode proteins involved in the biosynthesis and transfer of the two first heptose residues in the inner core of LPS, whereas rfaL encodes a ligase required for the attachment of O-antigen. The second operon contains (i) rfaQ and rfaK (or waaU) that encodes the heptosyltransferases adding the third and fourth heptose residues, respectively, (ii) genes rfaG (or waaG), rfaI (or waaO) and rfaJ (or warR /waaJ) encoding the gluco-syltransferases that add the three glucose residues in the LPS outer core, (iii) rfaB that encodes the galactosyl-transferase adding the galactose residue to the first glucose, (iv) rfaY and rfaP that encodes kinases responsible for phosphorylation of heptoses, (v) rfaZ involved in the KDO attachment during LPS core biosynthesis, and finally (vi) rfaS that encodes a protein necessary for the attachment of rhamnose to the LPS core by linkage to the KDOII residue. The short kdtA operon contains kdtA (or waaA) that encodes the KDO transferase adding the two KDO residues to the lipid A and kdtB (or coaD) that is not involved in the LPS synthesis 1-3. A defining feature of E. coli LPS is the presence of phosphoryl substituents on the LPS core-heptose residues, essential for