Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12

Abstract

Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion Arfal, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, Arfal resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of 13-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of lrfial with rfaG' DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction ofcps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product ofrfaQ was not required for the functions of fiaG and rifaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented rfial strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.

Down-Regulation of Key Virulence Factors Makes the Salmonella enterica Serovar Typhimurium rfaH Mutant a Promising Live-Attenuated Vaccine Candidate

“…These genes were significantly differently down regulated in the waaG mutant and showed the same tendency for downregulation in the rfaH mutants. These findings correlate with previous reports that deep-rough mutants show a decrease of protein content paralleled by an increase of lipoproteins in the outer membrane (45). Such changes in gene expression may aim to compensate for a "leaky" cell wall evoked by the deeprough phenotype.…”

Exploring the relationship between lipoprotein mislocalization and activation of the Rcs signal transduction system in Escherichia coli

“…These include stresses caused by mutations in: (i) the pgsA gene, encoding phosphatidylglycerophosphate synthase (Shiba et al, 2004); (ii) the mdoH gene for glucosyltransferase, responsible for the synthesis of periplasmic membrane-derived oligosaccharides (Ebel et al, 1997;Shiba et al, 2004); (iii) the tolB gene for a periplasmic component of the Tol system, involved in maintenance of cell envelope integrity (Mouslim & Groisman, 2003); and (iv) the rfaP and rfaG genes, encoding phosphoryl-and glucosyltransferase, respectively, responsible for the assembly of the lipopolysaccharide core (Parker et al, 1992;.…”

Deletion of the Heptosyltransferase Genes rfaC and rfaF in Escherichia Coli K‐12 Results in an Re‐Type Lipopolysaccharide with a High Degree of 2‐Aminoethanol Phosphate Substitution

“…The digoxigenin-I 1-dUTP system (Boehringer) was used for DNA labeling and detection in Southern experiments according to the instructions of the manufacturer. PI transduction with the bacteriophage Plcm" was performed essentially according to the method of Sternberg and Maurer (1991) with the following modifications: since deep rough mutants are partially resistant to PI (Parker et al, 1992), these donor strains were electroporated with Plcm" DNA prepared from a lysogenic strain. A liquid culture of 200 ml (LuriaBertani medium with 5 mM CaCI,, and 10 mM MgSO,) of the lysogenic donor strain was induced for phage production.…”

“…Non-stoichiometric substitutions of these sugars by monophosphate, 2-aminoethyl mono-or diphosphate groups seem to be important for the functional integrity of the outer membrane (Helander et al, 1989;Parker et al, 1992).…”