Biomedical subjects

Peter R Reeves

Publications and source records attributed to Peter R Reeves.

29 records · Page 2Linked to original sources

PubMed2003

Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence.

All Shigella and enteroinvasive Escherichia coli (EIEC) strains carry a 230-kb virulence plasmid (pINV) which is essential for their invasiveness. There are two sequence forms, pINV A and pINV B, of the plasmid (R. Lan, B. Lumb, D. Ryan, and P. R. Reeves, Infect. Immun. 69:6303-6309, 2001), and the recently sequenced pINV plasmid from Shigella flexneri serotype 5 is a pINV B form. In this study we sequenced the majority of the coding region of the pINV A form from S. flexneri serotype 6 other than insertion sequence or related sequences and compared it with the pINV B form. More than half of the genes sequenced appear to be under positive selection based on their low ratio of synonymous to nonsynonymous substitutions. This high proportion of selected differences indicates that the two pINV forms have functional differences, and comparative studies of pathogenicity in different Shigella-EIEC strains could be informative. There are also genes absent in the S. flexneri serotype 6 plasmid, including the sepA gene encoding serine protease, the major secreted protein of S. flexneri serotype 2a, and the stbAB genes, which encode one of the two partition systems found in S. flexneri serotype 5. The incompatibility of the two pINV forms appears to be due to either small differences in the mvpAT postsegregational killing system or the presence of an unknown system in pINVA.

Amino Acid Sequence↗

PubMed2003

Species-wide variation in the Escherichia coli flagellin (H-antigen) gene.

Escherichia coli is a clonal species. The best-understood components of its clonal variation are the flagellar (H) and polysaccharide (O) antigens, both well documented since the mid-1930s because of their use in serotyping. Flagellin is the protein subunit of the flagellum that carries H-antigen specificity. We show that 43 of the 54 H-antigen specificities of E. coli map to the flagellin gene at fliC and sequenced all 43 forms and confirmed specificity of each by cloning and expression. This is, to our knowledge, the first time that all known forms of such a highly polymorphic gene have been fully sequenced and characterized for any species. The established distinction between a highly variable central region and more conserved flanking regions is upheld. The sequences fall into two groups, one of which may be derived from the fliC gene of the E. coli/Salmonella enterica common ancestor, the other perhaps obtained by lateral transfer since species divergence. Comparison of sequences revealed that both horizontal DNA transfer and fixation of mutations under diversifying selection pressure contributed to polymorphism in this locus.

Antigens, Bacterial↗

PubMed2002

Escherichia coli in disguise: molecular origins of Shigella.

Shigella, which still stands as a genus with four species today, in reality belongs to the extremely diverse species Escherichia coli. There are several lineages of Shigella strains derived through independent acquisition of the pINV virulence plasmid. The chromosomally determined phenotypic properties of Shigella result from convergent evolution during niche adaptation, most due to loss of function, some from negative selection pressure.

Adaptation, Biological↗

PubMed2002

Relationship of Yersinia pseudotuberculosis O antigens IA, IIA, and IVB: the IIA gene cluster was derived from that of IVB.

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria and is highly polymorphic. In this study, we obtained sequences of the O-antigen gene clusters for the Yersinia pseudotuberculosis antigens IA, IIA, and IVB. We propose that the IIA gene cluster was derived from the IVB cluster, one of the very few cases in which a parent gene cluster is identified, and that the IA gene cluster could be a hybrid of the IVB and IB gene clusters. All three O antigens contain 6-deoxy-D-mannoheptose, and we identified six genes for the biosynthetic pathway for the precursor of this sugar, GDP-6-deoxy-D-mannoheptose.

Bacterial Proteins↗

PubMed2002

The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene.

Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E. coli clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E. coli O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the E. coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.

Base Sequence↗

PubMed2002

Extensive variation in the O-antigen gene cluster within one Salmonella enterica serogroup reveals an unexpected complex history.

The 46 serogroups of Salmonella enterica have different O-antigens, and each is thought to have a specific form of the O-antigen cluster. Comparison of the 145 serovars of serogroup B revealed much more intraserogroup genetic diversity than expected. The O27 factor, due to an alpha 1-6 linkage between O units in place of the more common alpha 1-2 linkage and previously thought to be due to a converting bacteriophage, is now shown to be due to a wzy(alpha(1-6)) gene located within the major gene cluster. Surprisingly a remnant of this gene in all O27(-) serovars shows that the ancestor was O27(+). There are six distinct gene cluster forms, five apparently derived by a series of deletions and one by an insertion from an ancestral O27(+) form present in 57 serovars. The history of the gene cluster and movement between subspecies I and II can be traced. Two of the derivative forms still have a functional wzy(alpha(1-6)) gene, while in three it has been inactivated by deletion or insertion. Two of the forms lacking a functional wzy(alpha(1-6)) gene have the wzy(alpha(1-2)) gene first described for strain LT2 as rfc, whereas for the third the wzy gene has not been located.

Cloning, Molecular↗

PubMed2002

Pandemic spread of cholera: genetic diversity and relationships within the seventh pandemic clone of Vibrio cholerae determined by amplified fragment length polymorphism.

The seventh cholera pandemic started in 1961 and continues today. A collection of 45 seventh pandemic isolates of V. cholerae sampled over a 33-year period were analyzed by amplified fragment length polymorphism (AFLP) fingerprinting. All but four pairs and one set of three isolates were distinguished. AFLP revealed far more variation than ribotyping, which was until now the most useful method of revealing variation within the pandemic clone. Unfortunately, the ribotype variation observed is mainly due to recombination between the multiple copies of the rrn genes (R. Lan and P. R. Reeves, Microbiology 144:1213-1221, 1998), which makes changes susceptible to repeat occurrences and reversion. This AFLP study shows that particularly for the common ribotypes G and H, such events have indeed occurred. AFLP grouped most of the 45 isolates into two clusters. Cluster I consists mainly of strains from the 1960s and 1970s, while cluster II contains mainly strains from the 1980s and 1990s, revealing a temporal pattern of change in the clone. This is best seen in the relationships of the strains from Africa, which correlate with the epidemiology of epidemics on that continent. The data confirm independent introductions to Africa during the 1970s outbreak and reveal several other African introductions. In the 1991 cholera upsurge, isolates from the Southern and Eastern African epidemic focus are markedly different from those from the West African epidemic focus. An isolate from 1987 in Algeria was identical to the West epidemic isolates, suggesting that the strain was present in Africa at least 3 years before causing large outbreaks. These observations have major implications for our understanding of cholera epidemiology.

Cholera↗

PubMed2002

Fluorescent amplified fragment length polymorphism analysis of Salmonella enterica serovar typhimurium reveals phage-type- specific markers and potential for microarray typing.

Fluorescent amplified fragment length polymorphism (AFLP) was applied to 46 Salmonella enterica serovar Typhimurium isolates of Australian origin comprising nine phage types, by using the restriction enzymes MseI and EcoRI and all 16 possible MseI +1-EcoRI +1 primer pair combinations. AFLP in the present study showed a very good discrimination power with a Simpson index of diversity of 0.98, and 35 different AFLP patterns were observed in the 46 isolates. AFLP grouped most serovar Typhimurium isolates by phage type and enabled differentiation of phage types. Furthermore, 84 phage-type-specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage type specificity) were observed in the 46 strains studied. Eighteen phage-type-specific AFLP fragments were cloned and sequenced. Fifteen are of known genes or have a homologue in the databases. Three sequences are plasmid related, eight are phage related, and four relate to chromosomal genes. Twelve of the 18 fragments are polymorphic because the DNA is present or absent as indicated by Southern hybridization, and we see good potential to use sequences of these fragments as the basis for multiplex PCR and development of a microarray-based molecular phage-typing method for serovar Typhimurium.

Animals↗

PubMed2001

Molecular evolution of the GDP-mannose pathway genes (manB and manC) in Salmonella enterica.

The evolutionary history of the GDP-mannose pathway in Salmonella enterica was studied via sequencing manB and manC genes from 13 representative strains for O antigens containing mannose and/or sugar derivatives of GDP-D-mannose. In addition, colanic acid (CA) manB and manC genes were sequenced from selected strains, as the basis for a detailed comparison. Interestingly, including the eight previously characterized O antigen gene clusters, 12 of the 21 S. enterica strains studied in total (each representing a different O antigen structure) possess a manB gene which displays DNA identity, ranging from 93 to 99%, to the CA manB gene of S. enterica LT2. Furthermore, the CA-like manB genes (as well as the CA manB and manC genes) display subspecies specificity, and the CA and CA-like manB genes (for individual strains) appear to be evolving in concert via gene conversion events. In comparison, the manC genes were generally not CA-like, a situation also apparent in Escherichia coli,and therefore most strongly reflected the evolutionary history of the S. enterica O antigen GDP-mannose pathway. It appears that, in relatively recent times, gene capture from a distant source has occurred infrequently, and that groups of manB and manC genes have been maintained and are continuing to evolve within S. enterica and more closely related species.

Bacterial Proteins↗

PubMed2000

Genetic variation of dTDP-L-rhamnose pathway genes in Salmonella enterica.

The genetic variation in the dTDP-L-rhamnose pathway gene set (rmlB, rmlD, rmlA, rmlC) in Salmonella enterica was examined after sequencing the four genes from 11 rml-containing gene clusters encoding seven O antigens, and a 903 bp rmlB segment from another 23 strains representing the seven subspecies. There was considerable sequence variation and strong polarity in the nature and level of variation among rml genes. The 5' end of the rml gene set, including rmlB, rmlD and most of rmlA, is in general subspecies specific. In contrast, the 3' end, including part of rmlA and all of rmlC, is O antigen specific. The G+C content of the 3' end is lower than that of the 5' end. The variation in the 3' end of the gene set is much greater than that of the 5' end. It is apparent that the rml gene set of S. enterica includes genes with two different evolutionary histories. In addition, there has been extensive recombination in the gene set, probably related to O antigen transfer between subspecies. These findings provide evidence for the lateral transfer of O antigen genes between species and among subspecies of S. enterica. The results have also shown that conserved genes at the end of an O antigen gene cluster play a major role in mediating exchange of the central serogroup-specific regions.

Bacterial Proteins↗

PubMed1998

Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae.

Individual rrn operons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone of Vibrio cholerae. The genome of an early isolate of the seventh pandemic clone had nine rrn operons of which two were in tandem with other rrn operons. The site for BglI, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate. This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation. Additional rrn recombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon. Spacers of 431, 509, 607 and 711 bp were found. A total of at least eight rrn recombination events were detected. Three rrn loci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G. In addition there was variation due to deletion of tandem operons. The frequency of recombination between rrn operons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study. This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study. Recombination between rrn operons may be a factor in ribotype variation in all systems. The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved.

Bacterial Typing Techniques↗