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...
We here report on the purification and characterization of glucose-1-phosphate thymidylyltransferase, the first of four enzymes commited to biosynthesis of dTDP-L-rhamnose from Salmonella enterica strain LT2. The purification was greatly facilitated by the cloning of the IjhA gene encoding this enzyme. Pure enzyme was obtained by 109-fold enrichment in three chromatography steps.The glucose-1 -phosphate thymidylyltransferase catalyzes a reversible bimolecular group transfer reaction and kinetic measurements indicate that it acts by a 'ping-pong' mechanism. The K , values for dTTP and a-D-glucose 1-phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the K , values for dTDP-D-glucose and diphosphate are 0.083 mM and 0.15 mM, respectively. The enzyme also accepts UTP and UDP-D-glucose and a-D-glucosamine 1-phosphate is accepted equally as well as a-D-glucose 1-phosphate.The NH,-terminal sequence of glucose-1 -phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the 0lf6.l gene of the rj& gene cluster. The SDS/ PAGE estimated subunit mass of 31 kDa agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the 0rf6.l gene (32453 Da).Saccharides are important surface structures in eukaryotic and prokaryotic cells and involved in cell recognition phenomena. They occur as free saccharides and as glycoconjugates linked to proteins and lipids. One of the problems in studies of their activities is the difficulty in obtaining them in pure form in sufficient quantities. In recent years advances have been made in the chemical synthesis of saccharides [l]. However, saccharide synthesis is still a relatively complicated procedure, in particular when larger saccharides or large quantities are desirable. In the last few years we have been investigating the feasibility of in vitro enzymatic synthesis of the Salmonella enterica 0-antigen-specific oligosaccharide and the nucleotide sugar precursors using enzymes overexpressed by cloned genes [Z].The 0-antigen part of S. enterica sero-group B strains is a repeating tetramer oligosaccharide composed of abequose, mannose, rhamnose and galactose. This saccharide is assembled from appropriate nucleoside &phosphate monosaccharides. The enzymes which participate in the biosynthesis of S. enterica 0-antigen polysaccharide are encoded by genes Abbreviations. Buffer A, 50mM Tris/HCl pH7.6, 10mM MgCl, and 1 mM EDTA; buffer B, 20 mM Tris/HC pH 8.0, 1 mM MgC1, and 22% glycerol; buffer C, 50mM sodium phosphate pH 7.0, 1 M ammonium sulphate and 22% glycerol.Enzymes. Glucose-I-phosphate thymidylyltransferase (EC 2.7.7.24), inorganic pyrophoshatase (EC 3.6.1.1).which are located mostly in the rj8 gene cluster [3]. The rj& gene cluster has been cloned [4-61, and Escherichia coli K12 strains harboring plasmids containing different parts of the rj8 gene cluster of S. enten'ca LT2 was the source of the enzymes used for in vitro enzymatic synthesis of dTDP-Lrhamnose, ...
An epidemic of mumps in Lithuania started in December 1998 and continued until May 2000. The total registered number of cases was about 11.000 of a total of 3,7 million inhabitants in Lithuania (29,7 cases/10,000). Virus-containing samples were collected from 80 patients treated at the hospital of Kaunas from October 1999 until the end of the epidemic. Out of the 80 patients with parotitis, meningitis was observed in 11 patients and orchitis in 22 of 69 male patients. Twenty-seven virus strains were genotyped by nucleotide sequencing of the small hydrophobic (SH) protein gene, and the 57 amino acid sequences of the gene were deduced. Twenty-five virus strains belonged to the C genotype and two were of the D genotype. By phylogenetic analysis the virus strains causing meningitis grouped in a separate cluster, designated C1, within the C genotype. Another group of ten of the 25 genotype C strains exhibited an amino acid triplet at amino acid positions 28 to 30 of the protein, consisting of valine, alanine and serine, instead of the previously recognised valine, valine and serine combination of genotype C. The amino acid alanine at position 29 was found in combination with the amino acid serine at position 48. This variant was designated C2 and it was associated with parotitis. The amino acid alanine at position 29 and serine in position 48 of the C2 genotype may constitute a marker of low neurovirulence compared to other genotype C strains.
The virus safety of blood derivatives continues to be of concern, especially with respect to nonenveloped and/or heat-stable viruses. Previously, we demonstrated that treatment of whole plasma, AHF concentrate or fibrinogen with short wavelength ultraviolet light (UVC) results in the inactivation of > or = 10(6) infectious doses (ID) of encephalomyocarditis virus (EMCV), hepatitis A virus (HAV) and porcine parvovirus (PPV), each of which is nonenveloped. Protein recovery was enhanced greatly by inclusion of the flavonoid, rutin, added prior to UVC exposure to quench reactive oxygen species. We now report on the treatment of albumin and intravenous immune globulin (IVIG) isolated by a previously described, integrated chromatographic method. Albumin was treated with either 0.1 or 0.2 J/cm2 UVC in the presence of 0.8 or 1.6 mM rutin; IVIG was treated with either 0.05 or 0.1 J/cm2 UVC in the presence of 0.5 or 1.0 mM rutin. Our results show that > or = 10(6.9) ID of EMCV and PPV were inactivated under each of the conditions studied except the treatment of albumin with 0.1 J/cm2 UVC in the presence of 1.6 mM rutin where 10(4.3) ID of EMCV and > or = 10(6.9) ID of PPV were killed. It appears that the sensitivity of PPV to UVC exceeds that of EMCV and that virus kill with UVC is higher in IVIG than in albumin. In the absence of rutin, UVC increased the extent of aggregation of both albumin and IVIG by two- to three-fold. With rutin present, the increase in albumin aggregation was reduced, and it was virtually eliminated by subsequent processing on Sephacryl S-200, a step in the existing procedure designed to remove aggregates. The increase in aggregation of IVIG appeared to be eliminated on inclusion of either 0.5 mM or 1 mM rutin. We conclude that both albumin and IVIG can be treated with UVC to inactivate > or = 10(6) ID of nonenveloped viruses. The inclusion of rutin during treatment helps protect against protein aggregation.
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