Genetic characterization of the O4 polysaccharide gene cluster from Escherichia coli

Haraguchi, Gayle E.; Zähringer, Ulrich; Jann, Barbara; Jann, Klaus; Hull, Richard A.; Hull, Sheila I.

doi:10.1016/0882-4010(91)90080-t

Cited by 25 publications

(19 citation statements)

References 47 publications

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“…GH58, which produces 0 antigen with the hybrid main chain, also has the 04 rff region cloned into K-12, but the plasmid, pGH58 (27), has much less 04 DNA than pGH133. We can explain the observations if we assume that GH58 lacks the 04 rhamnose transferase and so cannot complete the main chain of an 04 0-unit.…”

Section: Resultsmentioning

confidence: 99%

“…We can now see that all linkages present in the new structure of strain GH58, but absent in the 04 structure, are found in the K-12 0 antigen: all of the new 0 antigen structures are in effect hybrids between the 04 and K-12 structures. In strain HB1019, in which the rff region of strain K-12 is replaced by that of the 04 strain SH1 (27), half of the 0-units carry a glucose residue on GlcNAc in place of that on rhamnose; this shows both that the 04 Glc transferase gene is in the rff region and that the K-12 Glc transferase gene is not. Strain GH148 has the 04 rfb clone pGH133 (27) in strain K-12 and has 90% of the glucose substitutions on GlcNAc; there is no obvious reason why the K-12 Glc transferase is relatively more effective in this case in which the 04 gene is on a multicopy plasmid.…”

Section: Resultsmentioning

confidence: 99%

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Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster

et al. 1994

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster

et al. 1994

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“…These host genes can 07 (36), and 075 (3), and Shigella flexneri (33), the rfb gene participate with cloned genes in expression of 0 polysacchacluster is located adjacent to gnd, the structural gene for rides and, in some instances, have been reported to modify the 6-phosphogluconate dehydrogenase. The location of the gnd09 structure of 0 polysaccharides produced in recombinant bacgene on pWQ607 was therefore determined, in order to define teria (16,28). To avoid this problem, plasmids carrying rJbog one end of the rfbog gene cluster.…”

Section: Resultsmentioning

confidence: 99%

Cloning and analysis of duplicated rfbM and rfbK genes involved in the formation of GDP-mannose in Escherichia coli O9:K30 and participation of rfb genes in the synthesis of the group I K30 capsular polysaccharide

et al. 1994

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The rfbg,, gene cluster, which is responsible for the synthesis of the lipopolysaccharide 09 antigen, was cloned from Escherichia coi 09:130. The gnd gene, encoding 6-phosphogluconate dehydrogenase, was identified adjacent to the rJb09 cluster, and by DNA sequence analysis the gene order gnd-rJbM-rjbK was established. This order differs from that described for other members of the family Enterobacteriaceae.Nucleotide sequence analysis was used to identify the rftK and rJbM genes, encoding phosphomannomutase and GDP-mannose pyrophosphorylase, respectively. In members of the family Enterobacteriaceae, these enzymes act sequentially to form GDP-mannose, which serves as the activated sugar nucleotide precursor for mannose residues in cell surface polysaccharides. In the E. coli 09:K30 strain, a duplicated rJbM2-rJbK2 region was detected approximately 3 kbp downstream of rJbM'-dbK' and adjacent to the remaining genes of the rib09cluster. The rJbM isogenes differed in upstream flanking DNA but were otherwise highly conserved. In contrast, the rfbK isogenes differed in downstream flanking DNA and in 3'-terminal regions, resulting in slight differences in the sizes of the predicted RfbK proteins. RfbMog and RfbKog are most closely related to CpsB and CpsG, respectively. These are isozymes of GDP-mannose pyrophosphorylase and phosphomannomutase, respectively, which are thought to be involved in the biosynthesis of the slime polysaccharide colanic acid in E. coi K-12 and Salmonella enterica serovar Typhimurium. An E. col 0-:K30 mutant, strain CWG44, lacks rJbM2-rJb0K2 and has adjacent essential rib09 sequences deleted. The remaining chromosomal genes are therefore suflicieift for GDP-mannose formation and K30 capsular polysaccharide synthesis. A mutant of E. coli CWG44, strain CWG152, was found to lack GDP-anannose pyrophosphorylase and lost the ability to synthesize K30 capsular polysaccharide. Wild

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“…Two different mutant strains of the O4 parent E. coli SH1 were constructed. Clone pGH129 (16) containing a large portion of the O4 cluster was used. The cloning strategy used in this experiment is outlined in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Qualitative sugar analyses of LPSs in recombinant strains, as well as boundaries of the complete O4 region, have been established (16). On the basis of available sequence data, a methodology was developed to identify the gene encoding the O4 polymerase.…”

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confidence: 99%

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

Łukomski

Hull

1996

J Bacteriol

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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...

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Genetic characterization of the O4 polysaccharide gene cluster from Escherichia coli

Cited by 25 publications

References 47 publications

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster

Cloning and analysis of duplicated rfbM and rfbK genes involved in the formation of GDP-mannose in Escherichia coli O9:K30 and participation of rfb genes in the synthesis of the group I K30 capsular polysaccharide

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

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