Biomedical subjects

P R Reeves

Publications and source records attributed to P R Reeves.

At least 37 records · Page 2Linked to original sources

PubMed1998

A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains.

The bacterial species Vibrio cholerae includes harmless aquatic strains as well as strains capable of causing epidemics and global pandemics of cholera. While investigating the relationship between pathogenic and nonpathogenic strains, we identified a chromosomal pathogenicity island (PAI) that is present in epidemic and pandemic strains but absent from nonpathogenic strains. Initially, two ToxR-regulated genes (aldA and tagA) were studied and were found to be associated with epidemic and pandemic strains but absent in nontoxigenic strains. The region containing aldA and tagA comprises 13 kb of previously unidentified DNA and is part of a PAI that contains a regulator of virulence genes (ToxT) and a gene cluster encoding an essential colonization factor and the cholera toxin phage receptor (toxin-coregulated pilus; TCP). The PAI is 39.5 kb in size, has low %G+C (35%), contains putative integrase and transposase genes, is flanked by att sites, and inserts near a 10Sa RNA gene (ssrA), suggesting it may be of bacteriophage origin. We found this PAI in two clinical non-O1/non-O139 cholera toxin-positive strains, suggesting that it can be transferred within V. cholerae. The sequence within this PAI includes an ORF with homology to a gene associated with the type IV pilus gene cluster of enteropathogenic Escherichia coli, a transposase from Vibrio anguillarum, and several ORFs with no known homology. As the PAI contains the CTXPhi receptor, it may represent the initial genetic factor required for the emergence of epidemic and pandemic cholera. We propose to call this island VPI (V. cholerae pathogenicity island).

Aldehyde Dehydrogenase↗

PubMed1998

Domain organisation in phosphomannose isomerases (types I and II).

Phosphomannose isomerase (PMI) types I and II were found to possess a conserved protein motif. This motif coincides with the catalytic site of the Candida albicans type I PMI, indicating a common catalytic process for both PMI types. The type II PMI are bifunctional enzymes possessing PMI and guanosine diphospho-D-mannose pyrophosphorylase (GMP) activity in separate catalytic domains, which in some species may function as separate proteins.

Amino Acid Sequence↗

PubMed1998

Cholera in the 1990s.

Two strains of Vibrio cholerae are currently significant in cholera: a remnant from the sixth pandemic (1899-1923) still present in South Asia and the seventh pandemic strain which emerged in 1961. The 1990s were marked by spread of the seventh pandemic to South America in 1991 and appearance of an O139 form of the seventh pandemic strain in 1992 (or possibly 1991), which in 1993 predominated in some areas but then declined. Molecular analysis showed that the sixth and the seventh pandemic clones are related, but have a different TCP pathogenicity island and possibly different CTX phages, suggesting independent derivation from related environmental strains. Upsurges of the seventh pandemic were accompanied by increased genetic variation enabling the relationships between strains to be studied, but the basis for variation in pathogenicity is not known. There is clearly a risk of new forms arising and a strategy for speedy development of vaccines needs to be established.

Cholera↗

PubMed1998

Organization of Escherichia coli O157 O antigen gene cluster and identification of its specific genes.

The O157:H7 clone of Escherichia coli, which causes major, often prolonged outbreaks of gastroenteritis with hemolytic-uremic syndrome (HUS) such as those in Japan, Scotland, and the United States recently, is thought to be resident normally in cattle or other domestic animals. This clone is of major significance for public health and the food industry. We have developed a fast method for sequencing a given O antigen gene cluster and applied it to O157. The O157 O antigen gene cluster is 14 kb in length, comprising 12 genes and a remnant H-repeat unit. Based on sequence similarity, we have identified all the necessary O antigen genes, including five sugar biosynthetic pathway genes, four transferase genes, the O unit flippase gene, and the O antigen polymerase gene. By PCR testing against all 166 E. coli O serogroups and a range of gram-negative bacterial strains, including some that cross-react serologically with E. coli O157 antisera, we have found that certain O antigen genes are highly specific to O157 E. coli. This work provides the basis for a sensitive test for rapid detection of O157 E. coli. This is important both for decisions on patient care, since early treatment may reduce the risk of life-threatening complications, and for detection of sources of contamination. The method for fast sequencing of O antigen gene clusters plus an ability to predict which genes will be O antigen specific will enable PCR tests to be developed as needed for other clones of E. coli or, once flanking genes are identified, clones of any gram-negative bacterium.

Base Sequence↗

PubMed1998

The wzz (cld) protein in Escherichia coli: amino acid sequence variation determines O-antigen chain length specificity.

The O antigen is a polymer with a repeated unit. The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz. It is concluded that the O-antigen chain length heterogeneity in E. coli strains is the result of amino acid sequence variation of the Wzz protein.

Amino Acid Sequence↗

PubMed1998

Escherichia coli clone Sonnei (Shigella sonnei) had a chromosomal O-antigen gene cluster prior to gaining its current plasmid-borne O-antigen genes.

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. The surface-exposed O antigen is subject to selection by the host immune system, which may account for the maintenance of many different O-antigen forms. Characteristically, all genes specific to O-antigen synthesis are clustered in a region close to the his and gnd genes on the chromosome of Escherichia coli and related species. Shigella sonnei, essentially a clone of E. coli (E. coli clone Sonnei), is an important human pathogen and is unusual in that its O-antigen gene cluster is located on a plasmid. Our results suggest that it once had a normal chromosomal O-antigen gene cluster which has been largely deleted. We suggest that the O antigen encoded by the plasmid-borne genes offered a selective advantage in adapting to a new environment and that the chromosomal O-antigen genes were eventually inactivated. We also identified, by PCR and sequencing, a potential ancestor of E. coli Sonnei among the 166 known E. coli serotype strains.

Bacterial Proteins↗

PubMed1998

Relationships among the O-antigen gene clusters of Salmonella enterica groups B, D1, D2, and D3.

The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of Salmonella enterica group D3. This segment includes a novel O-antigen polymerase gene (wzyD3). The polymerase gives alpha(1-->6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-like wzy gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original wzy gene of these O-antigen gene clusters.

Antibody Specificity↗

PubMed1998

Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12.

GDP-L-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP-D-mannose. L-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced the E. coli colanic acid gene cluster and identified one of the GDP-L-fucose biosynthetic pathway genes, gmd. We report here the identification of the gene (fcl), located downstream of gmd, encoding the fucose synthetase.

Amino Acid Sequence↗

PubMed1998

Variation of the ribosomal operon 16S-23S gene spacer region in representatives of Salmonella enterica subspecies.

The 16S-23S spacer regions of two ribosomal operons (rrnA and rrnE) have been sequenced in seven representatives of the Salmonella enterica subspecies. Isolated nucleotide substitutions were found at the same sites as in Escherichia coli but the number of polymorphic sites was much larger, as could be expected for a more heterogeneous species. Still, as in E. coli, most of the variation found was due to insertions and/or deletions affecting blocks of nucleotides generally located at equivalent regions of the putative secondary structure for both species. Isolated polymorphic sites generated phylogenetic trees generally consistent with the subspecies structure and the accepted relationships among the subspecies. However, the sequences of rrnE put subspecies I closer to E. coli K-12 than to the other S. enterica subspecies. The distribution of polymorphisms affecting blocks of nucleotides was much more random, and the presence of equivalent sequences in distantly related subspecies, and even in E. coli, could reflect relatively frequent horizontal transfer. The smallest 16S-23S spacers in other genera of the family Enterobacteriaceae were also sequenced. As expected, the level of variation was much larger. Still, the phylogenetic tree inferred is consistent with those of 16S rRNA or housekeeping genes.

Base Sequence↗

PubMed1998

Sequencing of Escherichia coli O111 O-antigen gene cluster and identification of O111-specific genes.

Shiga toxin (Stx)-producing Escherichia coli strains of serogroup O111 are the most frequently isolated non-O157 strains causing outbreaks of gastroenteritis with hemolytic-uremic syndrome. The O111 O-antigen gene cluster had been cloned and about half of it has been sequenced; we have now sequenced the remainder of the gene cluster, which is 12.5 kb in length and which comprises 11 genes. On the basis of sequence similarity, we have identified all the O-antigen genes expected, including five sugar biosynthetic pathway genes, three transferase genes, the O-unit flippase gene, and the O-antigen polymerase gene. By PCR testing with E. coli strains representing all 166 O-antigen forms, some randomly selected gram-negative bacteria, and Salmonella enterica serovar Adelaide, we showed that four O-antigen genes are highly specific to O111. This work provides the basis for a sensitive test for the rapid detection of E. coli O111. This is important both for decisions related to patient care, because early treatment may reduce the risk of life-threatening complications, and for the detection of sources of contamination.

Antigens, Bacterial↗

PubMed1997

Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies.

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.

Biological Evolution↗

PubMed1996

Periplasmic expression of part of the major rotavirus capsid protein VP7 containing all the three antigenic regions in Escherichia coli.

Part of the porcine rotavirus outer capsid protein VP7 containing all the three antigenic regions was expressed as a chimeric protein with bacterial alkaline phosphatase (AP) in E. coli. The construct contains an ompF promoter, the DNA encoding the signal sequence and the first 12 amino acids of mature OmpF, part of vp7 and the DNA encoding mature AP. The chimeric protein is stable, retains the biological property of AP and ability to react with polyclonal antiserum against the virus, and can be exported through the bacterial inner membrane into the periplasm.

Alkaline Phosphatase↗

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

Gene transfer is a major factor in bacterial evolution.

Lateral gene transfer in four strains of Salmonella enterica has been assessed using genomic subtraction. Strain LT2 (subspecies I serovar Typhimurium) chromosomal DNA was used as target and subtracted by three subspecies I strains of serovars Typhimurium (S21), Muenchen (S71), Typhi (M229), and a subspecies V strain (M321). Data from probing random cosmids of LT2 DNA with preparations of the residual LT2 DNA after subtraction were used to estimate the amounts of LT2 DNA not able to hybridize to strains S21, S71, M229, and M321 to be in the range of 84-106, 191-355, 305-629, and 778-1,286 kb, respectively. Several lines of evidence indicate that most of this DNA is from genes not present in strain M321 and not from genes that have diverged in sequence. The amounts correlate with the divergence of the four strains as revealed by multilocus enzyme electrophoresis and sequence variation of housekeeping genes. Sequence of 39 of the fragments from the M321 subtracted residual LT2 DNA revealed only six inserts of known gene function with evidence of both gain and loss of genes during the development of S. enterica clones. Sixteen of the 39 segments have 45% or lower G+C content, below the species average, but over half are within the normal range for the species. We conclude that even within a species, clones may differ by up to 20% of chromosomal DNA, indicating a major role for lateral transfer, and that on the basis of G+C content, a significant proportion of the DNA is from distantly related species.

Bacteria↗

PubMed1996

Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid.

Colanic acid (CA) is an extracellular polysaccharide produced by most Escherichia coli strains as well as by other species of the family Enterobacteriaceae. We have determined the sequence of a 23-kb segment of the E. coli K-12 chromosome which includes the cluster of genes necessary for production of CA. The CA cluster comprises 19 genes. Two other sequenced genes (orf1.3 and galF), which are situated between the CA cluster and the O-antigen cluster, were shown to be unnecessary for CA production. The CA cluster includes genes for synthesis of GDP-L-fucose, one of the precursors of CA, and the gene for one of the enzymes in this pathway (GDP-D-mannose 4,6-dehydratase) was identified by biochemical assay. Six of the inferred proteins show sequence similarity to glycosyl transferases, and two others have sequence similarity to acetyl transferases. Another gene (wzx) is predicted to encode a protein with multiple transmembrane segments and may function in export of the CA repeat unit from the cytoplasm into the periplasm in a process analogous to O-unit export. The first three genes of the cluster are predicted to encode an outer membrane lipoprotein, a phosphatase, and an inner membrane protein with an ATP-binding domain. Since homologs of these genes are found in other extracellular polysaccharide gene clusters, they may have a common function, such as export of polysaccharide from the cell.

Acetyltransferases↗

PubMed1996

A Wzz (Cld) protein determines the chain length of K lipopolysaccharide in Escherichia coli O8 and O9 strains.

The modal distribution of O-antigen chain length is determined by the Wzz (Cld/Rol) protein in those cases in which it has been studied. The system of O-antigen synthesis in Escherichia coli serotypes O8 and O9 is different from that reported for most other bacteria, and chain length distribution is thought not to be determined by a Wzz protein. We report the existence in E. coli O8 and O9 strains of wzz genes which are very similar to and have sequences within the range of variation of those which determine the chain length of typical O antigens. We also find that wzz genes previously identified by their effect on O-antigen chain length, when cloned and transferred to O8 and O9 strains, affect the chain length of a capsule-related form of LPS, K(LPS). We conclude that in at least some O8 and O9 strains there is a wzz gene which controls the chain length of K(LPS) but has no effect on the O8 or O9 antigen.

Bacterial Proteins↗

PubMed1996

An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase.

O antigen is the major cell surface antigen of gram-negative bacteria, and the genes responsible for its synthesis are located in a single gene cluster. The wzx (rbfX) gene, which is characteristic of the major class of O-antigen gene clusters, encodes a hydrophobic protein with 12 potential transmembrane segments. We demonstrate that a wzx mutant accumulates undecaprenol pyrophosphate-linked O units which appear to be on the cytoplasmic side of the cytoplasmic membrane, suggesting that the wzx gene encodes a flippase for O-unit translocation across that membrane.

Bacterial Proteins↗

PubMed1996

C-terminal half of Salmonella enterica WbaP (RfbP) is the galactosyl-1-phosphate transferase domain catalyzing the first step of O-antigen synthesis.

We previously showed that the product of the wbaP gene of Salmonella enterica serovar Typhimurium has two functions: it is involved in the first step of O-antigen synthesis (the galactosyltransferase [GT] function) and in a later step (the T function), first thought to be the flipping of the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic face to the periplasmic face of the cytoplasmic membrane. We now locate two wbaP(T) mutations within the first half of the wbaP gene by sequencing. Both mutants retain GT activity, although one was a frameshift mutation resulting in a stop codon 10 codons after the frameshift to give an open reading frame containing only 138 of the 476 codons in WbaP. We also show that there is a secondary translation starting within the wbaP gene resulting in the synthesis of a polypeptide with GT activity. These results indicate that the N- and C-terminal halves of WbaP are the T and GT functional domains, respectively. We now propose that the T block operates prior to the flippase function, probably at the release of undecaprenyl pyrophosphate-linked galactose from WbaP.

Amino Acid Sequence↗