Genetic determinants of the synthesis of the polysaccharide capsular antigen K27(A) of Escherichia coli.

Most of the his+ hybrids from crosses between the Escherichia coli donor Hfr45(O8:K27) and different E. coli O9 recipients expressed the donor O8 antigen specificity and produced the capsular antigen K27. Therefore these hybrids must have inherited the his-linked donor rfb region determining the synthesis of O8- specific polysaccharides as well as his-linked genes involved in K27 antigen synthesis. In the living state these hybrids were inagglutinable in O8 antiserum like the donor cells. However, when E. coli K12 and O8:K42- were used as recipients most of the his+ hybrids were agglutinable in O8 and K27 antisera. The amounts of K27 antigen present in these hybrids, designated as K27i (intermediate) forms, were sufficient to evoke the production of K27 antibodies in rabbits, but insufficient to inhibit O-agglutination of the respective cells. The additional transfer of the trp region of E. coli O8:K27 into such K27i forms frequently resulted in O-inagglutinable K27+ hybrids. This is attributed to the introduction of trp-linked genes which apparently play a role in the synthesis of K27 capsular antigen. Tus it is concluded that at least two gene loci, one close to his and the other close to trp, are required for the synthesis of the complete capsular antigen K27.     

Chemical and structural analysis of the capsular polysaccharide from Escherichia coli O9:K28(A):H- (K28 antigen)

The structure of the capsular polysaccharide from Escherichia coli O9:K28(A):H- (K28 antigen) has been determined by using the techniques of methylation, periodate oxidation, and partial hydrolysis. N.m.r. spectroscopy (1H and 13C) was used to establish the nature of the anomeric linkages. O-Acetyl groups were determined spectrophotometrically and were located using methyl vinyl ether as a protective reagent. The polysaccharide is comprised of repeating units of the tetrasaccharide shown (three-plus-one type) with 70% of the fucosyl residues carrying an O-acetyl substituent. (formula; see text) This structure resembles that of E. coli K27 and has the structural pattern of Klebsiella K54 polysaccharide.     

Structure of the 2-keto-3-deoxy-D-manno-octonic-acid-containing capsular polysaccharide (K12 antigen) of the urinary-tract-infective Escherichia coli O4:K12:H-

The primary structure of the K12 antigenic capsular polysaccharide (K12 antigen) of Escherichia coli O4:K12:H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation and oligosaccharide analysis. The polysaccharide consists of repeating trisaccharide alpha-rhamnosyl-1,2-alpha-rhamnosyl-1,5-dOclA units (dOclA = 2-keto-3-deoxy-D-manno-octonic acid) which are joined through beta-2,3-linkages. About 50% of the dOclA units are O-acetylated at C7 or C8. The sequence of acetylated and non-acetylated dOclA residues is not known. As had been reported before, the polysaccharide is linked to a phosphatidic acid at the reducing end (dOclA) via a phosphodiester bridge. The serologically specific part of the K12 antigen is its polysaccharide moiety.     

Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.     

Formation of sugar phosphates in colicin K-treated Escherichia coli.

Colicin K greatly decreased the incorporation of 32P-labeled inorganic orthophosphate into nucleotides and nucleic acids, causing a concomitant increase in the formation of 32P-labeled sugar phosphates in sensitive cells of Escherichia coli. These sugar phosphates were formed in aerobically growing cells, as well as in cells under stringent control of ribonucleic acid synthesis. The main 32P-labeled product was identified as sedoheptulose 7-phosphate in two strains (B1 and K-12 MK-1) and fructose 1,6-diphosphate in one strain (K-12 CP78). The formation of sugar phosphates induced by colicin K was inhibited by carbonyl cyanide m-chlorophenylhydrazone. It was also not observed in N,N'-dicyclohexylcarbodiimide-treated cells or Mg2+-(Ca2+)-adenosine triphosphatase-less mutant (strain K-12 AN120) cells. Thus, the formation of sugar phosphates in colicin K-treated cells is dependent on the formation of adenosine 5'-triphosphate by oxidative phosphorylation.     

Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major

Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDK) are involved in the salvage pathway by which free purines are converted to nucleosides and subsequently to nucleotides. In this report, we describe the cloning of NDK coding-sequence from Leishmania major, the expression of the enzyme containing a His(6)-tag in Escherichia coli, and purification of the catalytically active native protein by affinity chromatography using Ni-NTA resin.     

Expression and morphology of enterotoxigenic Escherichia coli surface antigen CS31A in E. coli K12 and Vibrio cholerae

Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cell's surface than strains from other orders or genera such as V. cholerae.     

Transfer and expression of the genetic determinants for O and K antigen synthesis in Escherichia coli O9:K(A)30 and Klebsiella sp. O1:K20, in Escherichia coli K12

Escherichia coli serotype O9:K(A)30 and Klebsiella O1:K20 produce thermostable capsular polysaccharides or K antigens, which are chemically and serologically indistinguishable. Plasmid pULB113 (RP4::mini-Mu) has been used to mediate chromosomal transfer from E. coli O9:K30 and Klebsiella O1:K20 to a multiply marked, unencapsulated, E. coli K12 recipient. Analysis of the cell surface antigens of the transconjugants confirmed previous reports that the genetic determinants for the E. coli K(A) antigens are located near the his and rfb (O antigen) loci on the E. coli linkage map. The Klebsiella K20 capsule genes were also found to be in close proximity to the his and rfb loci. Electron microscopy revealed significant differences in the structural organization of capsular polysaccharides in these two microorganisms and the morphological differences were also readily apparent in transconjugants expressing the respective K antigens. These results are consistent with the interpretation that at least some of the organizational properties of capsular polysaccharides may be genetically determined, rather than being a function of the outer membrane to which the capsular polysaccharides are ultimately attached.     

Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface

Monoclonal antibodies were produced against the capsular antigen of Escherichia coli serotype K(A)30, using a mouse hybridoma system. The antibodies also recognised the chemically identical capsular polysaccharide produced by Klebsiella K20. Chemical modification of the K30 polysaccharide indicated that the glucuronic acid residues found in the E. coli K30 capsular antigen were important in the epitope recognised by these antibodies. Use of the antibodies as molecular probes revealed the presence of two discrete forms of the K30 antigen. One form was comprised of high molecular weight polysaccharide, present as a surface capsular layer. The second form of the antigen was of low molecular weight and was associated with lipopolysaccharide fractions from cell surface polysaccharide extracts. Separation of lipopolysaccharide fractions using gel chromatography in the presence of detergent showed that the low molecular weight K-antigenic fraction comigrated with a lipopolysaccharide lipid A core fraction present in encapsulated E. coli K30 bacteria but absent in acapsular mutants.