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P R Reeves

Publications and source records attributed to P R Reeves.

At least 19 recordsLinked to original sources
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↗
PubMed1995

Genetic organisation and evolution of Yersinia pseudotuberculosis 3,6-dideoxyhexose biosynthetic genes.

3,6-dideoxyhexose (DDH) sugars occur in some of the O antigens of Salmonella enterica and Yersinia pseudotuberculosis, but are otherwise rarely found in nature. Y. pseudotuberculosis DDH biosynthetic genes rfbS (encoding CDP-paratose synthetase) and rfbE (encoding CDP-tyvelose epimerase) were amplified and cloned, and their sequences determined. Comparisons with the equivalent genes of S. enterica show that the genetic arrangement of DDH genes is very similar; however, in Y. pseudotuberculosis there is no suggestion that paratose producing strains are derived from tyvelose-producing strains by inactivation of rfbE, which is the case in S. enterica. The previously determined DNA sequence of the rfb region of an abequose-producing strain was re-examined. It contains the remnants of an insertion sequence (IS) adjacent to a truncated and non-functional rfbE gene. This suggests that the IS was involved in recombination events contributing to O-antigen antigenic diversity in Y. pseudotuberculosis.

Amino Acid Sequence↗
PubMed1995

Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111.

The O antigens found in Salmonella enterica (Se) and Escherichia coli (Ec) show a great deal of diversity, and only three structures are known to be common to both genera. Two of them contain the 3,6-dideoxyheoxse colitose, not found in other serogroups of the two species. The first of these is common to Ec O111 and Se O:35 (sv Adelaide); the other is found in both Ec O55 and Se O:50 (sv Greenside). The genes specific for the synthesis of O antigen are generally located in the rfb gene cluster at map position 45 min in Ec and 42 min in Se. The rfb (O antigen) gene cluster of an Ec O111 strain M92 had been cloned earlier and hybridisation analysis suggested that the rfb clusters of Ec M92 and a Se sv Adelaide strain had been acquired separately by the two species since their divergence. We have now sequenced part of the rfb cluster from Ec M92. We identify two genes of the GDP-colitose pathway, rfbM and rfbK, and show that several other ORFs have similarity to the rfb and cps (capsular polysaccharide) genes. Downstream of this block of genes is an ORF which encodes a protein with predicted transmembrane segments which is presumed to correspond to the rfbX gene. The % G+C values of the Ec M92 rfb sequence are extremely low, indicating that the rfb evolved in a low % G+C species of bacteria before transfer into Ec.

Amino Acid Sequence↗
PubMed1995

A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants.

We identify a function-controlling O antigen chain length for a plasmid-borne gene, cldpHS-2, harboured by Flexneri strains of Escherichia coli known to cause reactive arthritis. The predicted amino acid sequence of the gene product is very similar to those of other cld genes and that of fepE, thought to be part of the enterobactin iron uptake system of E. coli. The predicted proteins are compared with rfb-associated chain length determinants as a family of related genes.

Amino Acid Sequence↗
PubMed1995

The sixth and seventh cholera pandemics are due to independent clones separately derived from environmental, nontoxigenic, non-O1 Vibrio cholerae.

The DNA sequences of the asd genes from 45 isolates of Vibrio cholerae (19 clinical O1 isolates, 2 environmental nontoxigenic O1 isolates, and 24 isolates with different non-O1 antigens) were determined. No differences were found within either sixth- or seventh-pandemic isolates; however, variation was found between the two forms and among the non-O1 isolates. O139 isolates had sequences identical to those of seventh-pandemic isolates. Phylogenetic trees with Vibrio mimicus as the outgroup suggest that the sixth-pandemic, seventh-pandemic, and U.S. Gulf isolates are three clones that have evolved independently from different lineages of environmental, nontoxigenic, non-O1 V. cholerae isolates. There is evidence for horizontal transfer of O antigen, since isolates with nearly identical asd sequences had different O antigens, and isolates with the O1 antigen did not cluster together but were found in different lineages. We also found evidence for recombination events within the asd gene of V. cholerae. V. cholerae may have a higher level of genetic exchange and a lower level of clonality than species such as Salmonella enterica and Escherichia coli.

Bacterial Vaccines↗
PubMed1995

Transferases of O-antigen biosynthesis in Salmonella enterica: dideoxyhexosyltransferases of groups B and C2 and acetyltransferase of group C2.

The O antigen is a polymer of oligosaccharide units. O antigens differ in their sugar composition and glycosidic linkages, and genes responsible for O-antigen-specific biosynthesis are grouped in the rfb gene cluster. In this study, we identified two abequosyltransferase genes and an acetyltransferase gene in Salmonella enterica groups B and C2 by in vitro assay and identified paratosyl-, tyvelosyl-, and abequosyltransferase genes from S. enterica groups A and D and Yersinia pseudotuberculosis serovar IIA, respectively, by comparison.

Acetyltransferases↗
PubMed1994

In vitro synthesis of CDP-d-abequose using Salmonella enzymes of cloned rfb genes. Production of CDP-6-deoxy-D-xylo-4-hexulose, CDP-3,6-dideoxy-D-xylo-4-hexulose and CDP-3,6-dideoxy-D-galactose, and isolation by HPLC.

In vitro enzymic synthesis of CDP-D-[U-14C]abequose, CDP-6-deoxy-D-xylo-4-hexulose and CDP-3,6-dideoxy-D-xylo-4-hexulose was achieved using enzymes from cell extracts of cultures of Escherichia coli strains harbouring and expressing genes of the rfb gene cluster of Salmonella enterica LT2. From an initial synthesis step, CDP-6-deoxy-D-xylo-4-hexulose was isolated after 30 min reaction, using CDP-D-glucose, NAD and CDP-glucose 4.6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 90.6%). From that intermediate, CDP-3,6-dideoxy-D-xylo-4-hexulose was produced in a reaction using NADH and a crude extract containing the required enzymes. CDP-D-abequose synthesis was performed either in the presence of excess NADH and NADPH or using an enzymic system which regenerates low concentrations of the coenzymes. In a two-step reaction, CDP-D-glucose was first converted to CDP-6-deoxy-D-xylo-4-hexulose, then, following addition of the required coenzymes and enzymes, CDP-D-abequose was formed from this intermediary product in a 1-h incubation. Starting from 250 mg CDP-D-glucose, the molar yield of CDP-D-abequose after protein precipitation and HPLC was 82%, corresponding to more than 200 mg. CDP-D-[U-14C]abequose was synthesised from alpha-D-[U14C]glucose 1-phosphate and CTP using purified glucose-1-phosphate cytidylyltransferase in a reaction preceding the later steps. GC-MS and NMR revealed that the hexose part of the end product was 3.6-dideoxy-D-galactose (abequose) and that the corresponding intermediates were 4-keto-6-deoxy-D-xylo-hexose and 4-keto-3,6-dideoxy-D-xylo-hexose, respectively. The synthesized CDP-6-deoxy-D-xylo-4-hexulose exhibited the characteristic ultraviolet light absorption at 318 nm but no corresponding absorption was found for CDP-3,6-dideoxy-D-xylo-4-hexulose. A HPLC technique, where the four CDP-sugars were baseline separated, was developed and used for enzyme assays and for the analysis of synthesized products.

Carbohydrate Epimerases↗
PubMed1994

Purification, characterization, and high performance liquid chromatography assay of Salmonella glucose-1-phosphate cytidylyltransferase from the cloned rfbF gene.

We report the purification and characterization of glucose-1-phosphate cytidylyltransferase, the first of five enzymes committed to biosynthesis of CDP-D-abequose from Salmonella enterica strain LT2. The purification was greatly facilitated by using a cloned rfbF gene encoding this enzyme. Pure enzyme was obtained by 64-fold enrichment in three chromatography steps. The NH2-terminal sequence of the purified enzyme was in agreement with the sequence predicted from the nucleotide sequence of the rfbF gene. The SDS-polyacrylamide gel electrophoresis estimated subunit M(r) of 31,000 agrees well with the M(r) of 29,035 calculated from the amino acid composition deduced from the nucleotide sequence of the rfbF gene. The glucose-1-phosphate cytidylyltransferase catalyzes a reversible bimolecular group transfer reaction and steady-state kinetic measurements, including product inhibition patterns, indicate that this reaction proceeds by a "ping-pong" type of mechanism. The Km values for CTP, alpha-D-glucose 1-phosphate, CDP-D-glucose, and pyrophosphate are 0.28, 0.64, 0.11, and 1.89 mM, respectively.

Amino Acid Sequence↗
PubMed1994

MULTICOMP: a program for preparing sequence data for phylogenetic analysis.

MULTICOMP is a program that assists in the phylogenetic analysis of DNA sequences. It streamlines sequence handling and analysis. Input is from either individual sequence files or a file of aligned sequences. It produces data on variation at DNA and amino acid sequence level and can also convert sequences to data formats suitable for PHYLIP, PAUP and MacClade phylogenetic inference programs. Further, two tree-building programs, NEIGHBOR and DNAPARS, of PHYLIP can be directly run from within it. MULTICOMP performs Sawyer's algorithm for detection of gene conversion. The program facilitates analysis using only part of the data of a data set or using two or more combined data sets.

Algorithms↗
PubMed1994

Molecular evolution in the gnd locus of Salmonella enterica.

The gnd gene, the structural gene for 6-phosphogluconate dehydrogenase, was sequenced and analyzed in 34 isolates from different serovars of the seven subspecies of Salmonella enterica to provide comparative information on the evolution in this gene, which has been studied extensively in Escherichia coli. The gene tree obtained by the neighbor-joining method in general gave separate branches for each subspecies, with the few exceptions readily explained by recombination. There is evidence of recombination involving transfer of long (more than 400 bp) and short (30-150 bp) segments of DNA. Four of the six long-segment transfers detected are at the 5' end of the gene, and in all four cases a variant of the chi sequence is located close to the recombination junction and appears to have mediated the recombination events. We suggest that in these four cases and in a fifth case with intersubspecies transfer of the whole gnd gene, the adjacent rfb (O antigen) locus may have been transferred in the same event. The estimates of the number of synonymous substitutions per synonymous site, KS, and the number of nonsynonymous substitutions per nonsynonymous site, KA, within the E. coli and S. enterica gnd genes, and also between the two species show an interesting distribution, with KS being lower toward the ends of the gene and KA in particular being lower in the first than in the second domain. In S. enterica, synonymous sites also seem to be subjected to negative selection. The ratio of KA to KS was higher within S. enterica and E. coli than between them, which may indicate that intraspecies variation is essentially between clones and that mildly deleterious mutations can be fixed within clones, which would thus raise KA within species.

Amino Acid Sequence↗
PubMed1994

Evidence for effect of random genetic drift on G+C content after lateral transfer of fucose pathway genes to Escherichia coli K-12.

The cps cluster of Escherichia coli K-12 comprises genes involved in synthesis of capsular polysaccharide colanic acid. Part of the E. coli K-12 cps region has been cloned and sequenced and compared to its Salmonella enterica LT2 counterpart. The cps genes from the two organisms are homologous; in the case of the LT2 genes, with G+C content of 0.61 and codons characteristic of high G+C species, it seems clear that they have been acquired relatively recently by lateral transfer from a high G+C species. The K-12 form of these cps genes is closely related to those of LT2 so must derive from the same high G+C species, but it appears to have transferred much earlier such that random genetic drift has brought P3 (the corrected G+C content of codon base 3) down from 0.77 to 0.64, more than halfway to the E. coli average of 0.57. We estimate, using an equation developed by Sueoka, that the lateral transfer to E. coli took place approximately 45 million years ago. This is the first report we are aware of demonstrating the expected adjustment of P3 after lateral transfer between species with different G+C content DNA.

Amino Acid Sequence↗
PubMed1994

Escherichia coli K12 regains its O antigen.

Extant Escherichia coli K12 strains are phenotypically rough, their lipopolysaccharide having a complete core structure, but no O antigen. We used DNA hybridization and DNA sequencing to show that the rough phenotype of this strain is due to the presence of one of two independent mutations in the rfb gene cluster. The rfb-50 mutation, consisting of an IS5 insertion at the downstream end of rfb, is present in strain EMG2, which is representative of most K12 derivatives. The rfb-51 mutation is a deletion at the upstream end of rfb, and was found in strain WG1. A gene cloned from strain WG1 could complement the rfb-50 mutation in strain EMG2, and the complemented strain produced O antigen which was typed as O16 with cross reaction to O17.

Amino Acid Sequence↗
PubMed1994

Involvement of the galactosyl-1-phosphate transferase encoded by the Salmonella enterica rfbP gene in O-antigen subunit processing.

rfbT of Salmonella enterica LT2 was previously thought, together with rfaL, to be involved in the ligation of polymerized O antigen to core-lipid A, and three mutants were known. We report the mapping of the mutations to rfbP, the galactosyl-1-phosphate transferase gene, which is now shown to encode a bifunctional protein. The mutations which have the former rfbT phenotype are referred to as rfbP(T). We also show that rfbP(T) mutants are not blocked in the ligation step as previously believed but in an earlier step, possibly in flipping the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic to periplasmic face of the cytoplasmic membrane.

Amino Acid Sequence↗