Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12: insertion mutagenesis of the rfa locus

Abstract

TnWO insertions were selected on the basis of resistance to the lipopolysaccharide (LPS)-specific bacteriophage U3. The majority of these were located in a 2-kilobase region within the rfa locus, a gene cluster of about 18 kb that contains genes for LPS core biosynthesis. The rfa::TnlO insertions all exhibited a deep rough phenotype that included hypersensitivity to hydrophobic antibiotics, a reduction in major outer membrane proteins, and production of truncated LPS. These mutations were complemented by a Clarke-Carbon plasmid known to complement rfa mutations of Salmonella typhimurium, and analysis of the insert from this plasmid showed that it contained genes for at least six polypeptides which appear to be arranged in the form of a complex operon. Defects in two of these genes were specifically implicated as the cause of the deep rough phenotype. One of these appeared to be rfiaG, which encodes a function required for attachment of the first glucose residue to the heptose region of the core. The other gene did not appear to be directly involved in determination of the sugar composition of the core. We speculate that the product of this gene is involved in the attachment of phosphate or phosphorylethanolamine to the core and that it is the lack of one of these substituents which results in the deep rough phenotype.

The gene coding for 3-deoxy-manno-octulosonic acid transferase and the rfaQ gene are transcribed from divergently arranged promoters in Escherichia coli

“…1) was cloned from pLC17-24 (8, 9) into pBluescript KSII(+), generating plasmid pCL5. The restriction map of the cloned fragment was consistent with that of the same region reported by Austin et al (1). Both strands of the cloned fragment were sequenced after nested deletions were generated in pCL5 as described in Material and Methods.…”

“…Either AMV reverse transcriptase is unable to extend primer K1 the 340 nucleotides necessary to reach the 5' end of this longer transcript or the product found with primer K1 represents a transcript separate from the one detected with primer K2. 'X' characterized by restriction enzyme analysis and by insertion mutagenesis (1,25 That rfaQGP is transcribed in a direction opposite that of kdtA (9,25) suggests the presence of at least two promoters in the intergenic region between rfaQ and kdtA. This suggestion is verified by the restoration of both ,-galactosidase and alkaline phosphatase activities in E. coli CB806 by pCLP310, a plasmid containing the entire intergenic region inserted between the promoterless reporter genes lacZ and phoA in pCB267.…”

“…Only a few of the enzymes encoded by rfa have been characterized at the biochemical level. Schnaitman et al (1,25) have provided evidence for an rfa operon (rfaQGP-BIJYZK) encoding 10 different polypeptides. This operon is suggested to have a complex organization with overlapping transcriptional units, each with its own promoter, and a major promoter at the 5' end of the operon from which a transcript representing all 10 genes is initiated.…”

“…This operon is suggested to have a complex organization with overlapping transcriptional units, each with its own promoter, and a major promoter at the 5' end of the operon from which a transcript representing all 10 genes is initiated. Glycosyltransferases I and II, catalyzing the stepwise transfer of hexose sugars to the distal part of the core oligosaccharide, have been shown to be encoded by the rfaG and rfaI (formerly rfaM in E. coli) genes, respectively (1,11). Several other genes of this operon have been identified as genes involved in the synthesis of the outer core region.…”

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

“…We chose for this purpose the prototrophe strain W3110, since the core region of its LPS has been (together with that of strain W3100) structurally characterized (Holst et al, 1990(Holst et al, , 1991. In addition, a strategy of targeted gene inactivation was used and optimized to exclude polar effects known for insertions within the $a region (Austin et al, 1990). The genes rfuC and rfaF are directly linked within an operon of the rfa locus of E. coli K-12 ( Fig.…”