Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.
AUTOR(ES)
Parker, C T
RESUMO
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 delta rfa1, 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, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-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 delta rfa1 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 of cps-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 of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=205891Documentos Relacionados
- Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12.
- Structures of the rfaB, rfaI, rfaJ, and rfaS genes of Escherichia coli K-12 and their roles in assembly of the lipopolysaccharide core.
- Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium.
- Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen.
- Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12.