Biomedical subjects

C Whitfield

Publications and source records attributed to C Whitfield.

At least 37 records · Page 2Linked to original sources

PubMed1999

Overexpression, purification, crystallization and preliminary structural study of dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD), the fourth enzyme of the dTDP-L-rhamnose synthesis pathway, from Salmonella enterica serovar Typhimurium.

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precursor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlD catalyses the terminal step of this pathway by converting dTDP-6-deoxy-L-lyxo-4-hexulose to dTDP-L-rhamnose. RmlD from -Salmonella enterica serovar Typhimurium has been overexpressed in Escherichia coli. The recombinant protein was purified by a two--step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals of native and selenomethionine-enriched RmlD have been obtained using the sitting-drop vapour-diffusion method with polyethylene glycol as precipitant. Diffraction data have been collected from orthorhombic crystals of both native and selenomethionyl-derivatized protein, allowing tracing of the protein structure.

Carbohydrate Dehydrogenases↗

PubMed1999

Assembly of the K40 antigen in Escherichia coli: identification of a novel enzyme responsible for addition of L-serine residues to the glycan backbone and its requirement for K40 polymerization.

Escherichia coli O8:K40 coexpresses two distinct lipopolysaccharide (LPS) structures on its surface. The O8 polysaccharide is a mannose homopolymer with a trisaccharide repeat unit and is synthesized by an ABC-2 transport-dependent pathway. The K40LPS backbone structure is composed of a trisaccharide repeating unit of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) and has an uncommon substitution, an L-serine moiety attached to glucuronic acid. The gene cluster responsible for synthesis of the K40 polysaccharide has previously been cloned and sequenced and was found to contain six open reading frames (ORFs) (P. A. Amor and C. Whitfield, Mol. Microbiol. 26:145-161, 1997). Here, we demonstrate that insertional inactivation of orf1 results in the accumulation of a semirough (SR)-K40LPS form which retains reactivity with specific polyclonal serum in Western immunoblots. Structural and compositional analysis of the SR-K40LPS reveals that it comprises a single K40 repeat unit attached to lipid A core. The lack of polymerization of the K40 polysaccharide indicates that orf1 encodes the K40 polymerase (Wzy) and that assembly of the K40 polysaccharide occurs via a Wzy-dependent pathway (in contrast to that of the O8 polysaccharide). Inactivation of orf3 also results in the accumulation of an SR-LPS form which fails to react with specific polyclonal K40 serum in Western immunoblots. Methylation linkage analysis and fast atom bombardment-mass spectrometry of this SR-LPS reveals that the biological repeat unit of the K40 polysaccharide is GlcNAc-GlcA-GlcNAc. Additionally, this structure lacks the L-serine substitution of GlcA. These results show that (i) orf3 encodes the enzyme responsible for the addition of the L-serine residue to the K40 backbone and (ii) substitution of individual K40 repeats with L-serine is essential for their recognition and polymerization into the K40 polysaccharide by Wzy.

Acetylglucosamine↗

PubMed1999

Conserved organization in the cps gene clusters for expression of Escherichia coli group 1 K antigens: relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae.

Group 1 capsules of Escherichia coli are similar to the capsules produced by strains of Klebsiella spp. in terms of structure, genetics, and patterns of expression. The striking similarities between the capsules of these organisms prompted a more detailed investigation of the cps loci encoding group 1 capsule synthesis. Six strains of K. pneumoniae and 12 strains of E. coli were examined. PCR analysis showed that the clusters in these strains are conserved in their chromosomal locations. A highly conserved block of four genes, orfX-wza-wzb-wzc, was identified in all of the strains. The wza and wzc genes are required for translocation and surface assembly of E. coli K30 antigen. The conservation of these genes points to a common pathway for capsule translocation. A characteristic JUMPstart sequence was identified upstream of each cluster which may function in conjunction with RfaH to inhibit transcriptional termination at a stem-loop structure found immediately downstream of the "translocation-surface assembly" region of the cluster. Interestingly, the sequence upstream of the cps clusters in five E. coli strains and one Klebsiella strain indicated the presence of IS elements. We propose that the IS elements were responsible for the transfer of the cps locus between organisms and that they may continue to mediate recombination between strains.

Antigens, Bacterial↗

PubMed1998

The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides.

The major core oligosaccharide biosynthesis operons from prototype Escherichia coli strains displaying R1 and R4 lipopolysaccharide core types were polymerase chain reaction-amplified and analyzed. Comparison of deduced products of the open reading frames between the two regions indicate that all but two share total similarities of 94% or greater. Core oligosaccharide structures resulting from nonpolar insertion mutations in each gene of the core OS biosynthesis operon in the R1 strain allowed assignment of all of the glycosyltransferase enzymes required for outer core assembly. The difference between the R1 and R4 core oligosaccharides results from the specificity of the WaaV protein (a beta1, 3-glucosyltransferase) in R1 and WaaX (a beta1, 4-galactosyltransferase) in R4. Complementation of the waaV mutant of the R1 prototype strain with the waaX gene of the R4 strain converted the core oligosaccharide from an R1- to an R4-type lipopolysaccharide core molecule. Aside from generating core oligosaccharide specificity, the unique beta-linked glucopyranosyl residue of the R1 core plays a crucial role in organization of the lipopolysaccharide. This residue provides a novel attachment site for lipid A-core-linked polysaccharides and distinguishes the R1-type LPS from existing models for enterobacterial lipopolysaccharides.

Amino Acid Sequence↗

PubMed1998

Involvement of waaY, waaQ, and waaP in the modification of Escherichia coli lipopolysaccharide and their role in the formation of a stable outer membrane.

The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli. This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule. In the R1 core prototype strain, E. coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function. In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy. All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability. Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII. Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY. Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity. Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations.

Base Sequence↗

PubMed1998

The assembly system for the lipopolysaccharide R2 core-type of Escherichia coli is a hybrid of those found in Escherichia coli K-12 and Salmonella enterica. Structure and function of the R2 WaaK and WaaL homologs.

In Escherichia coli F632, the 14-kilobase pair chromosomal region located between waaC (formerly rfaC) and waaA (kdtA) contains genes encoding enzymes required for the synthesis of the type R2 core oligosaccharide portion of lipopolysaccharide. Ten of the 13 open reading frames encode predicted products sharing greater than 90% total similarity with homologs in E. coli K-12. However, the products of waaK (rfaK) and waaL (rfaL) each resemble homologs in Salmonella enterica serovar Typhimurium but share little similarity with E. coli K-12. The F632 WaaK and WaaL proteins therefore define differences between the type R2 and K-12 outer core oligosaccharides of E. coli lipopolysaccharides. Based on the chemical structure of the core oligosaccharide of an E. coli F632 waaK::aacC1 mutant and in vitro glycosyltransferase analyses, waaK encodes UDP-N-acetylglucosamine:(glucose) lipopolysaccharide alpha1, 2-N-acetylglucosaminyltransferase. The WaaK enzyme adds a terminal GlcNAc side branch substituent that is crucial for the recognition of core oligosaccharide acceptor by the O-polysaccharide ligase, WaaL. Results of complementation analyses of E. coli K-12 and F632 waaL mutants suggest that structural differences between the WaaL proteins play a role in recognition of, and interaction with, terminal lipopolysaccharide core moieties.

Amino Acid Sequence↗

PubMed1998

Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica.

Bacterial lipopolysaccharides (LPS) are unique and complex glycolipids that provide characteristic components of the outer membranes of Gram-negative bacteria. In LPS of the Enterobacteriaceae, the core oligosaccharide links a highly conserved lipid A to the antigenic O-polysaccharide. Structural diversity in the core oligosaccharide is limited by the constraints imposed by its essential role in outer membrane stability and provides a contrast to the hypervariable O-antigen. The genetics of core oligosaccharide biosynthesis in Salmonella and Escherichia coli K-12 have served as prototypes for studies on the LPS and lipo-oligosaccharides from a growing range of bacteria. However, despite the wealth of knowledge, there remains a number of unanswered questions, and direct experimental data are not yet available to define the precise mechanism of action of many gene products. Here we present a comparative analysis of the recently completed sequences of the major core oligosaccharide biosynthesis gene clusters from the five known core types in E. coli and the Ra core type of Salmonella enterica serovar Typhimurium and discuss advances in the understanding of the related biosynthetic pathways. Differences in these clusters reflect important structural variations in the outer core oligosaccharides and provide a basis for ascribing functions to the genes in these model clusters, whereas highly conserved regions within these clusters suggest a critical and unalterable function for the inner region of the core.

Amino Acid Sequence↗

PubMed1997

UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbDKPO1 gene.

The O-side-chain polysaccharide in the lipopolysaccharide of Klebsiella pneumoniae O1 is based on a backbone structure of repeat units of [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]; this structure is termed D-galactan I. The rfb (O-antigen biosynthesis) gene cluster directs the synthesis of D-galactan I and consists of six genes termed rfbA-FKPO1. In this paper we show that rfbDKPO1 encodes a UDP-galactopyranose mutase (NAD(P)H-requiring) (EC 5.4.99. 9), which forms uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactofuranosyl ester (UDP-Galf), the biosynthetic precursor of galactofuranosyl residues. The deduced amino acid sequence of rfbDKPO1 shows 85% and 37.5% identity to the rfbDKPO8 gene of K. pneumoniae serotype O8 and the glf gene of Escherichia coli, respectively. The molecular mass of the purified RfbDKPO1 enzyme is 45 kDa as determined by SDS-polyacrylamide gel electrophoresis, while gel filtration revealed a molecular mass of 92 kDa, suggesting a dimeric structure for the native protein. The rfbDKPO1 gene product interconverts uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactopyranosyl ester (UDP-Galp) and UDP-Galf. Unlike Glf, RfbDKPO1 showed a requirement for NADH or NADPH, which could not be replaced by NAD or NADP. RfbDKPO1 was used to synthesize milligram quantities of UDP-Galf, allowing this compound to be purified and fully characterized in an intact form for the first time. The structure of UDP-Galf was proven by NMR spectroscopy.

Amino Acid Sequence↗

PubMed1997

Modulation of the surface architecture of gram-negative bacteria by the action of surface polymer:lipid A-core ligase and by determinants of polymer chain length.

Lipopolysaccharides (LPSs) are complex glycolipids found in the outer membrane of Gram-negative bacteria. The lipid A-core component of the LPS molecule provides a versatile anchor to which a surface polymer:lipid A-core ligase enzyme can attach one or more structurally distinct surface polymers in a single bacterial strain. In some cases the same polymer can be found on the cell surface in both lipid A-core-linked and -unlinked forms. Analysis by SDS-PAGE of populations of LPS molecules extracted from bacterial cells indicates that there is extensive heterogeneity in their size distribution. Much of the heterogeneity results from complex modal distributions in the chain length of the polymers which are attached to lipid A-core. This is the result of preferential ligation of polymers with specific degrees of polymerization during the assembly of the LPS molecule. The surface architecture of the Gram-negative bacterial cell is therefore profoundly affected by the activities of the surface polymer:lipid A-core ligase and by molecular determinants of polymer chain length. Because of the involvement of cell-surface polymers in interactions between pathogenic bacteria and their hosts, these enzymatic activities also have an important impact on virulence. In this review, the organization of LPSs and related surface polymers will be described and the current understanding of the molecular mechanisms involved in surface diversity will be discussed. Emphasis is placed on the Enterobacteriaceae, but similarities to other bacteria suggest that aspects of the enterobacterial system will have broader significance.

ATP-Binding Cassette Transporters↗

PubMed1997

Molecular and functional analysis of genes required for expression of group IB K antigens in Escherichia coli: characterization of the his-region containing gene clusters for multiple cell-surface polysaccharides.

Escherichia coli group I capsular K antigens are found in two forms on the cell surface. The K(LPS) form is linked to lipopolysaccharide lipid A core, whereas the high-molecular-weight capsular form is assembled independently of lipid A core. Subgroup IB K antigens are generally co-expressed with either the O8 or O9 antigen and, under the appropriate conditions, with the exopolysaccharide, colanic acid. To examine the relationships between the genetic loci and the synthetic pathways for these various cell-surface polymers, the gene cluster responsible for expression of a prototype group IB K antigen (serotype K40) was cloned and the flanking chromosomal regions characterized. Analysis of the six orfs within the cluster indicates features typical of Wzy (Rfc)-dependent O antigens. Synthesis of group IB K antigens is initiated by WecA (Rfe), a UDP-GlcNAc::undecaprenylphosphate GlcNAc-1-phosphate transferase, and the chain length of K40LPS is determined by the wzz gene product. The his-region of the E. coli O8:K40 prototype is almost exclusively devoted to the expression of three different surface polysaccharides. The rfbK40 cluster is located adjacent to the cps (colanic acid synthesis) and rfbO8 (O8 antigen synthesis) loci in the gene order: his-rfbO8/O9-wzz-ugd-gnd-rfbK40-galF-cps. Thus, rfbK40 is in the location occupied by other Wzy-dependent rfb gene clusters, and rfbO8/O9 represents an additional locus.

Antigens, Bacterial↗

PubMed1997

O-antigen seroepidemiology of Klebsiella clinical isolates and implications for immunoprophylaxis of Klebsiella infections.

To provide a database for the development of an O-antigen-polysaccharide-containing vaccine against Klebsiella spp., we examined the O-antigen seroepidemiology of 378 Klebsiella clinical isolates collected prospectively in two university centers. Strains were typed by competitive enzyme-linked immunosorbent assay with rabbit antisera specific for serogroups O1 to O12 and monoclonal antibodies (MAbs) specific for serogroups O1, O2ab, O2ac, and the genus-specific core antigen. The numbers of isolates (percentages) of individual O serogroups were as follows: 148 (39.2) for serogroup O1, 40 (10.6) for serogroup O2ab, 4 (1.1) for serogroup O2ac, 89 (23.6) for serogroup O3, 2 (0.5) for serogroup O4, 32 (8.5) for serogroup O5, none for serogroups O7, O9, and O12, and 21 (5.6) for serogroup O11. Forty-two (11.1) of the strains were non-O-typeable. O-serogroup distributions were virtually identical between isolates from invasive infections and those from noninvasive infections or colonizations. A vaccine containing the O-specific polysaccharides of serogroups O1, O2ab, O3, and O5 would cover 82% of clinically occurring O-antigen specificities. Three hundred thirty-eight of 378 isolates (89.4%) reacted with the genus-specific MAb V/9-5, which recognizes an epitope of the outer core region of Klebsiella lipopolysaccharide. Antibodies directed against this epitope may represent a further alternative for O-antigen-targeted immunoprophylaxis of Klebsiella infections. These data support further experimental investigations on the protective potential of O-antigen-based vaccines and/or hyperimmune globulins in Klebsiella infection.

Antibodies, Bacterial↗

PubMed1997

Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens.

Individual Escherichia coli strains produce several cell surface polysaccharides. In E. coli E69, the his region of the chromosome contains the rfb (serotype O9 lipopolysaccharide O-antigen biosynthesis) and cps (serotype K30 group IA capsular polysaccharide biosynthesis) loci. Polymorphisms in this region of the Escherichia coli chromosome reflect extensive antigenic diversity in the species. Previously, we reported a duplication of the manC-manB genes, encoding enzymes involved in GDP-mannose formation, upstream of rfb in strain E69 (P. Jayaratne et al., J. Bacteriol. 176:3126-3139, 1994). Here we show that one of the manC-manB copies is flanked by IS1 elements, providing a potential mechanism for the gene duplication. Adjacent to manB1 on the IS1-flanked segment is a further open reading frame (ugd), encoding uridine-5'-diphosphoglucose dehydrogenase. The Ugd enzyme is responsible for the production of UDP-glucuronic acid, a precursor required for K30 antigen synthesis. Construction of a chromosomal ugd::Gm(r) insertion mutation demonstrated the essential role for Ugd in the biosynthesis of the K30 antigen and confirmed that there is no additional functional ugd copy in strain E69. PCR amplification and Southern hybridization were used to examine the distribution of IS1 elements and ugd genes in the vicinity of rfb in other E. coli strains, producing different group IA K antigens. The relative order of genes and, where present, IS1 elements was established in these strains. The regions adjacent to rfb in these strains are highly variable in both size and gene order, but in all cases where a ugd homolog was present, it was found near rfb. The presence of IS1 elements in the rfb regions of several of these strains provides a potential mechanism for recombination and deletion events which could contribute to the antigenic diversity seen in surface polysaccharides.

Antigens, Bacterial↗

PubMed1996

A novel pathway for O-polysaccharide biosynthesis in Salmonella enterica serovar Borreze.

The plasmid-encoded gene cluster for O:54 O-polysaccharide synthesis in Salmonella enterica serovar Borreze (rfbO:54) contains three genes that direct synthesis of a ManNAc homopolymer with alternating beta1,3 and beta1,4 linkages. In Escherichia coli K-12, RfbAO:54 adds the first ManNAc residue to the Rfe (UDP-GlcpNAc::undecaprenylphosphate GlcpNAc-1-phosphate transferase)- modified lipopolysaccharide core. Hydrophobic cluster analysis of RfbAO:54 indicates this protein belongs to the ExoU family of nonprocessive beta-glycosyltransferases. Two putative catalytic residues and a potential substrate-binding motif were identified in RfbAO:54. Topological analysis of RfbBO:54 predicts four transmembrane domains and a large central cytoplasmic domain. The latter shares homology with a similar domain in the processive beta-glycosyltransferases Cps3S of Streptococcus pneumoniae and HasA of Streptococcus pyogenes. Hydrophobic cluster analysis of RfbBO:54 and Cps3S indicates both possess the structural features characteristic of the HasA family of processive beta-glycosyltransferases. Four potential catalytic residues and a putative substrate-binding motif were identified in RfbBO:54. In Deltarfb E. coli K-12, RfbAO:54 and RfbBO:54 direct synthesis of smooth O:54 lipopolysaccharide, indicating that this O-polysaccharide involves a novel pathway for O-antigen transport. Based on sequence and structural conservation, 15 new ExoU-related and 17 new HasA-related transferases were identified.

Amino Acid Sequence↗

PubMed1996

Bacterial polysaccharide synthesis and gene nomenclature.

Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.

Computer Communication Networks↗

PubMed1996

Clonally diverse rfb gene clusters are involved in expression of a family of related D-galactan O antigens in Klebsiella species.

Klebsiella species express a family of structurally related lipopolysaccharide O antigens which share a common backbone known as D-galactan I. Serotype specificity results from modification of D-galactan I by addition of domains of altered structure or by substitution with O-acetyl and/or alpha-D-Galp side groups with various linkages and stoichiometries. In the prototype, Klebsiella serotype O1, the his-linked rfb gene cluster is required for synthesis of D-galactan I, but genes conferring serotype specificity are unlinked. The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8. By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysaccharide synthesized in Escherichia coli K-12 hosts, we show that, like rfbO1, the rfbO8 region directs formation of unmodified D-galactan I. The rfbAB genes encode an ATP-binding cassette transporter required for export of polymeric D-galactan I across the plasma membrane prior to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core. Complementation experiments show that the rfbAB gene products in serotypes O1 and O8 are functionally equivalent and interchangeable. Hybridization experiments and physical mapping of the rfb regions in related Klebsiella serotypes suggest the existence of shared rfb genes with a common organization. However, despite the functional equivalence of these rfb gene clusters, at least three distinct clonal groups were detected in different Klebsiella species and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions. The clonal groups cannot be predicted by features of the O-antigen structure. To examine the relationships in more detail, the complete nucleotide sequence of the serotype O8 rfb cluster was determined and compared with that of the serotype O1 prototype. The nucleotide sequences for the six rfb genes showed variations in moles percent G+C values and in the values for nucleotide sequence identity, which ranged from 66.9 to 79.7%. The predicted polypeptides ranged from 64.3% identity (78.4% total similarity) to 94.3% identity (98.0% similarity). The results presented here are not consistent with dissemination of the Klebsiella D-galactan I rfb genes through recent lateral transfer events.

Antigens, Bacterial↗

PubMed1996

Structure of the core oligosaccharide in the serotype O8 lipopolysaccharide from Klebsiella pneumoniae.

Two classes of mutants with O-antigen-deficient lipopolysaccharides were isolated from the serotype O8 reference strain, belonging to Klebsiella pneumoniae subspecies ozaenae. These mutants were selected by resistance to bacteriophage KO1-2, which recognizes and lyses strains with lipopolysaccharide molecules containing the D-galactan II O antigen. Strain RFK-11 contains a defect in O-antigen synthesis and has a complete core, including the attachment site for O antigen. This mutation is complemented by a plasmid carrying the rfb (O-antigen biosynthesis) gene cluster from the related K. pneumoniae serotype O1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide from strain RFK-9 has a mobility typical of deep-rough lipopolysaccharide. RFK-9 lipopolysaccharide lacks the attachment site for O antigen. Lipopolysaccharides from strains RFK-9 and RFK-11 were isolated, and their structures were determined by methylation analyses, muclear magnetic resonance spectroscopy, and mass spectroscopy. The deduced O8 core oligosaccharide includes the partial core structure reported for the K. pneumoniae subspecies pneumoniae serotype O1 lipopolysaccharide (M. Süsskind, S. Müller-Leonnies, W. Nimmich, H. Brade, and O. Holst, Carbohydr. Res. 269:C1-7, 1995), consistent with the possibility of a conserved core structure within the species. The core oligosaccharide differs from those of the genera Salmonella and Escherichia by the absence of a hexose-containing outer core, the lack of phosphate residues in the inner core, and the presence of galacturonic acid residues.

Antigens, Bacterial↗

PubMed1996

Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens.

The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K(LPS). By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K(LPS) antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K(LPS), as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K(LPS) with longer polysaccharide chains. Collectively, these results suggested that group IA K(LPS) is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K(LPS) results from the absence of Rol activity in these strains.

Antigens, Bacterial↗

PubMed1996

Risk factors in heart transplantation. A statistical study of New Zealand cases.

The first heart transplantation in New Zealand was performed on December 2, 1987. This study covers all forty-five transplantations that were performed in New Zealand at Green Lane Hospital from that time through January 1994. Eleven patients from that group have died. The 30 day and 1, 2 and 4 year survival rates were found to be 91.1, 86.7, 80.0 and 75.6%, respectively. The mean waiting period of the patients for heart transplantation was 61.9 days. Only pulmonary vascular resistance was a statistically significant risk factor for early death after cardiac transplantation. Ischemic time was not a significant risk factor in this study.

Adult↗