The rfb gene cluster of Salmonella LT2 has been cloned and sequenced. The genes rfbA, rfbB, rfbD, rfbF, rfbG, rfbK, rfbM and rfbP were located individually and the gene rfbL was located outside the cluster. Approximately 16 open reading frames were found in the region which is essential for the expression of O antigen. The gene products of rfbB and rfbG were found to have homology with the group of dehydrogenase and related enzymes described previously. Analysis of the G + C ratio of the rfb cluster extended the area of low-G + C composition previously found in the sequence of rfbJ to the whole rfb gene cluster. Three to five segments with discrete G + C contents and codon adaptation indices are present in the rfb region, indicating a heterogeneous origin of these segments. Potential promoters were found near the start of the rfb region, supporting the possibility that the rfb gene cluster is an operon.

Escherichia coli K-12 has long been known not to produce an 0 antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of 0 antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rjb (0 antigen) genes intact which synthesized a variant of 0 antigen 016, giving cross-reaction with anti-017 antibody. We determined the structure of this 0 antigen to be -with an 0-acetyl group on C-2 of the rhamnose and a side chain c-D-Glcp on C-6 of GlcNAc. 0 antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rjb (0 antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rjb region ofEscherichia coli K-12 and those ofE. colh 04 and E. coi Flexneri. All K-12 rjb genes were of low G+C content for E. coil. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rjb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.Escherichia coli K-12 was isolated in 1922 and used as a standard E. coli strain at Stanford University for many years; the strains which survive all derive from cultures given to E. Tatum and others in the 1940s and early 1950s, when E. coli K-12 was first used for the genetic studies which led to its adoption as the major strain for laboratory study. After 50 years of intensive study, E. coli K-12 is arguably the best understood of all organisms, having been used for studies of many facets of living organisms, outlined in a two-volume book on E. coli and Salmonella enterica (45). Currently, 50% of its genome has been sequenced (56), and completion of this task will increase the focus of attention on E. coli K-12 for studies which integrate genomic information and biochemical processes.There are, however, significant gaps in our knowledge of E. coli K-12. During its first 25 to 30 years in the laboratory, it probably accumulated a range of mutations which improved adaptation to a laboratory environment but destroyed its ability to survive in its natural environment. Among them were mutations in the rjb gene cluster which led to loss of 0 antigen synthesis. The 0 antigen, a repeat unit polysaccharide which is a component of lipopolysaccharide (LPS), is the major surface antigen of many gram-negative bacteria (see reference 53 for a review) and, for E. coli, was present in by far the majority of strains when first isolated. However, it is often lost during culture, presumably because it offers no advantage under suc...

A cosmid (pPR1347) carrying both the rjb gene cluster and the rfc gene of a Salmonella group B serovar has been constructed; Escherichia coli K-12 strains carrying this cosmid produce long-chain 0 antigen, are sensitive to phage P22, and can be transduced by P22. Some of the benefits of P22 transduction are now available for studying E. coli and potentially other genera.Transduction was discovered during a search for Escherichia coli K-12-likc conjugation in Salmonella enterica (22). In this first demonstration of transduction, one of the strains used, LT2, and the phage P22 (present as a prophage in the other strain used, LT22) became the mainstay of S. enterica genetic analysis. Note that all Salmonella strains are now considered to belong to onc species, and the old species names are used as serovar names (8): strains LT2 and LT22 both belong to serovar Typhimurium (part of the group B serogroup).Transduction has been used extensively in bacterial genetics and is very useful, for example, in strain construction. In general, transduction within each bacterial species requires use of a specific phage; for example, phage P1 has been used for transduction in E. coli, and phage P22 has been used for transduction in S. enterica sv. Typhimurium. A significant factor in the development of the genetics of S. enterica has been the ease of use of P22 for transductional crosses. The properties of P22 relevant to transduction have been summarized in a review of the Salmonella linkage map (14). In particular, P22 is stable in storage, high-titer stocks are easily obtained, and high-frequency transduction (HT) and integration-deficient mutants have been isolated (16,17 (1) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10), followed by silver staining (19). The results for the JM109 derivative carrying both plasmids (Fig. 1, lane C) show a ladder-like banding pattern characteristic of polymerized 0 antigens of various chain lengths. In the absence of the rfc gene, the LPS has only one 0 unit (indicated by an arrow in Fig. 1, lane B) linked to the LPS core oligosaccharide, and clearly the rfc gene cloned by Collins and Hackett (3) is all that is required to effect polymerization.A restriction-modified P22 stock of CP/I was prepared by propagating the phage on the P22-sensitive JM109 derivative (in L broth containing 1 mM CaCI2 and 10 mM MgCl2) and was used to successfully transduce genetic markers into another P22-sensitive K-12 strain (data not shown). Our next step was to put the rfc gene and the rfb gene cluster into one plasmid, pPR1347 (Fig. 2). A 1.75-kb Hindlll fragment from pADE206, carrying the rfc gene, was ligated into the HindIlI site of pUC1318 (7) to generate the pPR1346 construct. Digestion of pPR1346 with BamHI yielded the above 1.75-kb rfc fragment flanked by two BamHI sites: this fragment was then inserted into the BamHI-digested pPR1028, replacing the 3-kb Cmlr fragment, to give the rfb-rfc-carrying construct pPR1347. When electroporated into JM109, pPR1347 was found ...

The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purity with the 23 kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14 kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit.

The rfb gene cluster which determines the biosynthesis of the O2 O antigen has been cloned from an Escherichia coli O2: K1 strain isolated from a case of septicaemia in chickens. The region required for expression of the O antigen in E. coli K‐12 was localised to a 10.7 to 14.15‐kb segment which was shown to be chromosomal in origin with a close linkage to the gnd and his genetic loci.