Identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance cassette

Abstract

Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides. O antigen polymerase, which is encoded by the rfc gene, is a potential membrane protein and therefore should be hydrophobic. To identify the rfc gene, these two ORFs were subjected to insertional mutagenesis. A chloramphenicol resistance cassette was designed which, when properly inserted, does not cause a polar effect in downstream genes. Each of two ORFs, cloned into a plasmid vector, was inactivated with this cassette. Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination. These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis. Only one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide. Therefore, this ORF was identified as the rfc gene. The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a plasmid vector.Lipopolysaccharide (LPS) is the major nonprotein constituent of the outer membrane of gram-negative rod-shaped bacteria (36). It is a complex macromolecule composed of three parts, each of which is synthesized by gene products largely unique to each: lipid A, a complex phospholipid which is embedded in the membrane itself and is responsible for the endotoxic properties of LPS; the core oligosaccharide, composed of an inner and outer core; and the long-chain polysaccharide (PS), also known as the O antigen or O PS, which extends from the surface of the cell and is in contact with the environment (21). Because of its location at the surface of the cell and because of its unique properties, LPS is responsible for many of the surface characteristics of gram-negative bacteria, including resistances to detergents, hydrophobic antibiotics, serum complement, and ingestion by human polymorphonuclear leukocytes (12,14,51). These last two properties are dependent on the O PS, and it appears that the lengths of the chains are critical, at least for resistance to the bactericidal effect of complement (12, 40).The genes responsible for the synthesis of O PS map at the rfb locus, at 44 min on the chromosome of Escherichia coli (1). The O PS is made by the polymerization of O subunits, composed of three to five sugars, which are built upon a special phospholipid carrier called antigen carrier lipid (43). The next step is the polymerization of the subunits into a long-chain PS by the enzyme called O polymerase, which is encoded by the rfc gene. The growing chain is added to the core oligosaccharide, which is made separately from the O PS and linked to the lipid A, by the enzyme called O ligase (29). Both enzymes are thought to be located on the periplasmic face of the cytoplasmic membrane (31). In wild-type strains, the distribution of O-antigen chains is clearly bimodal because of the expression of preferred chain l...

Haemophilus ducreyi Requires the flp Gene Cluster for Microcolony Formation In Vitro

“…5, columns 4 and 9). In contrast, the presence of the promoterless cat cartridge (33) in the flp-1 flp-2 deletion mutant 35000HP.402 did not appreciably affect the levels of the tadB and tadG transcripts (Fig. 5, columns 2 and 7, respectively) in this mutant relative to those levels observed in the wild-type parent strain (Fig.…”

“…However, unlike the previous two mutants described above, this mutation was constructed in the human-passaged H. ducreyi strain 35000HP (6). A promoterless cat cartridge designed to permit transcription and translation of linked downstream ORFs (33) was inserted into a deletion that spanned part of both the flp-1 and flp-2 ORFs. Western blot analysis confirmed that this flp-1 flp-2 mutant lacked the ability to express the Flp1 and Flp2 proteins (Fig.…”

Expression of the Moraxella catarrhalis UspA1 Protein Undergoes Phase Variation and Is Regulated at the Transcriptional Level

“…Plasmid pUSPA1, which contains an incomplete uspA1 ORF from M. catarrhalis O35E (2) inserted into the multiple cloning site downstream from and in the same orientation as the plasmidbased lac promoter, was digested with BglII to remove a 0.6-kb internal fragment, and the BglII ends were then filled in with Pfu DNA polymerase using the manufacturer's recommended conditions. The resulting blunt-ended pUSPA1 plasmid was then ligated with a 706-bp SmaI fragment containing the promoterless cat gene from plasmid pSL1 (29). The ligation mixture was used to electroporate E. coli DH5␣, and recombinant clones were selected for resistance to chloramphenicol; the plasmids in these recombinants had the cat insert oriented in the same direction as the plasmid-based lac promoter.…”

Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1) The GenBank accession number for the DNA sequence reported in this paper is AF125322.

“…The wzx coding sequence has an abundance of codons atypical for E. coli, suggesting that the Wzx protein is poorly translated. Lukomski et al (1996) showed that the wzy gene, which also contains an unusually high number of rare codons, cannot complement a wzy mutation when cloned in high-copynumber vectors. We then cloned the 5n6 kb EcoRI insert of pCM111 into the low-copy-number vector pSF6, resulting in plasmid pCM138 (Fig.…”