bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of reduction in population size using data from microsatellite loci
Authors: Garza J. C. , Williamson E. G.
Published: March 2003

We demonstrate that the mean ratio of the number of alleles to the range in allele size, which we term M, calculated from a population sample of microsatellite loci, can be used to detect reductions in population size. Using simulations, we show that, for a general class of mutation models, the value of M decreases when a population is reduced in size. The magnitude of the decrease is positively correlated with the severity and duration of the reduction in size. We also find that the rate of recovery of M following a reduction in size is positively correlated with post‐reduction population size, but that recovery occurs in both small and large populations. This indicates that M can distinguish between populations that have been recently reduced in size and those which have been small for a long time. We employ M to develop a statistical test for recent reductions in population size that can detect such changes for more than 100 generations with the post‐reduction demographic scenarios we examine. We also compute M for a variety of populations and species using microsatellite data collected from the literature. We find that the value of M consistently predicts the reported demographic history for these populations. This method, and others like it, promises to be an important tool for the conservation and management of populations that are in need of intervention or recovery.
Molecular barcodes for soil nematode identification
Authors: Floyd Robin , Abebe Eyualem , Papert Artemis , Blaxter Mark
Published: March 2003

Using a molecular barcode, derived from single‐specimen polymerase chain reaction (PCR) and sequencing of the 5′ segment of the small subunit ribosomal RNA (SSU) gene, we have developed a molecular operational taxonomic unit (MOTU) scheme for soil nematodes. Individual specimens were considered to belong to the same MOTU when the sequenced segment of 450 bases was > 99.5% identical. A Scottish upland Agrostis‐Festuca grassland soil was sampled, using both culture‐based and random selection methods. One hundred and sixty‐six cultured isolates were sequenced, and clustered into five MOTU. From 74 randomly sampled individuals across the study site, 19 MOTU were defined. A subsequent sample of 18 individuals from a single subplot contained eight MOTU, four of which were unique to the single subplot sample. Interestingly, seven of these MOTU were not present in the culture‐independent sampling. Overall, a total of 23 MOTU were defined from only 240 sequences. Many MOTU could readily be assigned to classical, morphologically defined taxonomic units using a database of SSU sequences from named nematode species. The MOTU technique allows a rapid assessment of nematode taxon diversity in soils. Correlation with a database of sequences from known species offers a route to application of the technique in ecological surveys addressing biological as well as genetic diversity.
Genetic, ecological, and behavioural divergence between two sibling snapping shrimp species (Crustacea: Decapoda:Alpheus)
Authors: Mathews L. M. , Schubart C. D. , Neigel J. E. , Felder D. L.
Published: March 2003

Examination of genetic and ecological relationships within sibling species complexes can provide insights into species diversity and speciation processes.Alpheus angulatusandA. armillatus, two snapping shrimp species with overlapping ranges in the north‐western Atlantic, are similar in morphology, exploit similar ecological niches and appear to represent recently diverged sibling species. We examined phylogenetic and ecological relationships between these two species with: (i) sequence data from two mitochondrial genes (16SrRNA andCOI); (ii) data on potential differences in microhabitat distribution forA. armillatusandA. angulatus; and (iii) data from laboratory experiments on the level of reproductive isolation between the two species. DNA sequence data suggestA. armillatusandA. angulatusare sister species that diverged subsequent to the close of the Isthmus of Panama, and that haplotype diversity is lower inA. armillatusthan inA. angulatus. Both species are distantly related toA. heterochaelisandA. estuariensis, two species with whichA. angulatusshares some similarities in coloration. Ecological data on the distribution ofA. angulatusandA. armillatusfrom two locations revealed differences in distribution of the two species between habitat patches, with each patch dominated by one or the other species. However, there was no apparent difference in distribution of the two species within habitat patches with respect to microhabitat location. Ecological data also revealed that heterospecific individuals often occur in close proximity (i.e. within metres or centimetres) where sympatric. Behavioural data indicated that these species are reproductively isolated, which is consistent with speciation in transient allopatry followed by post‐divergence secondary contact. Our data further resolve taxonomic confusion between the sibling species,A. armillatusandA. angulatus, and suggest that sympatry in areas of range overlap and exploitation of similar ecological niches by these two recently diverged species have selected for high levels of behavioural incompatibility.
Intraspecific molecular variation in the seaweed fly Coelopa frigida consistent with behavioural distinctness of British and Swedish populations
Authors: MacDonald C. , Brookfield J. F. Y.
Published: March 2003

The major aim of this study was to compare the intraspecific variation and genetic structure of the behaviourally distinct British and Swedish populations of the seaweed fly Coelopa frigida. C. frigida has been the subject of intense study into the basis of female choice. The behaviour of British females is consistent with a ‘good genes’ model, whereas that of the Swedish flies suggests a Fisher process, in which the difference between the former and the latter is defined by female choice increasing offspring viability in ‘good genes’ models. Through a study of variability in the mitochondrial cytochrome oxidase II gene from more than 600 flies, we show that there is clear differentiation at the molecular level between the two countries’ populations, with an FST of > 75% and no shared haplotypes. Tajima's test reveals an excess of rare variants relative to expectation, which, if not the result of selective sweep, indicates either a population expansion or purifying selection against weakly deleterious variants. Within the two populations, substantial subpopulation differentiation is observed in the UK, where there is also evidence of isolation by distance. Swedish populations exhibit lower variability, and no evidence of isolation by distance, with the latter result possibly being related to the continuous distribution of suitable habitat. The pattern of intraspecific variation is explainable by a combination of contemporary and also historical factors. British and Swedish populations may have been descended from at least two separate founding populations during the recolonization of these areas following Pleistocene glaciations.
Genealogical exclusivity in geographically proximate populations of Hypochilus thorelli Marx (Araneae, Hypochilidae) on the Cumberland Plateau of North America
Authors: Hedin M. , Wood D. A.
Published: March 2003

The issue of sampling sufficiency is too infrequently explored in phylogeographical analysis, despite both theoretical work and analytical methods that stress the importance of sampling effort. Regarding the evolutionary pattern of reciprocal monophyly, both the probability of recovering this pattern and the possible inferences derived from this pattern, are highly contingent upon the density and geographical scale of sampling. Here, we present an empirical example that relates directly to this issue. We analyse genetic structure in the southern Appalachian spider Hypochilus thorelli, using an average sample of 5 mitochondrial DNA (mtDNA) sequences per location for 19 locations. All sampled sites are reciprocally monophyletic for mtDNA variation, even when separated by geographical distances as small as 5 km. For populations separated by greater geographical distances of 20–50 km, mtDNA sequences are not only exclusive, but are also highly divergent (uncorrected p‐distances exceeding 5%). Although these extreme genealogical patterns are most seemingly consistent with a complete isolation model, both a coalescent method and nested cladistic analysis suggest that other restricted, but nonzero, gene flow models may also apply. Hypochilus thorelli appears to have maintained morphological cohesion despite this limited female‐based gene flow, suggesting a pattern of stasis similar to that observed at higher taxonomic levels in Hypochilus.
Evaluating the effects of historic bottleneck events: an assessment of microsatellite variability in the endangered, North Atlantic right whale
Authors: Waldick R. C. , Kraus S. , Brown M. , White B. N.
Published: March 2003

Commercial exploitation reduced the North Atlantic right whale (Eubalaena glacialis) population from c. 12 000 in the 11th century to around 300 by the 21st century. We examine the effect of this population decline on levels of genetic variation at 16 microsatellite loci and contrast levels of variability to that in a closely related species (E. australis). Of the 13 loci developed from the E. glacialis genome, 100% were polymorphic in E. australis. In contrast, nine loci were polymorphic in E. glacialis and four were fixed. Both allelic diversity (A) and heterozygosity (H) were significantly lower in E. glacialis than E. australis (A = 3.2 ± 2.6 vs. A= 6.9 ± 3.3, P < 0.001; H= 0.31 ± 0.25 vs. H= 0.72 ± 0.23, P < 0.001, respectively). Bottleneck anlayses indicate that the population is in mutation‐drift equilibrium and that a genetic bottleneck did not occur during the most recent decline (18th–20th centuries). Nevertheless, low frequency alleles are relatively uncommon in E. glacialis, suggesting that genetic variability has been reduced in this population. Possible origins of low genetic variability are discussed, including the slow but continual erosion of alleles during the 800‐year period of decline.
History and evolution of the arctic flora: in the footsteps of Eric Hultén
Authors: Abbott Richard J. , Brochmann Christian
Published: March 2003

A major contribution to our initial understanding of the origin, history and biogeography of the present‐day arctic flora was made by Eric Hultén in his landmark book Outline of the History of Arctic and Boreal Biota during the Quarternary Period, published in 1937. Here we review recent molecular and fossil evidence that has tested some of Hultén's proposals. There is now excellent fossil, molecular and phytogeographical evidence to support Hultén's proposal that Beringia was a major northern refugium for arctic plants throughout the Quaternary. In contrast, most molecular evidence fails to support his proposal that contemporary east and west Atlantic populations of circumarctic and amphi‐Atlantic species have been separated throughout the Quaternary. In fact, populations of these species from opposite sides of the Atlantic are normally genetically very similar, thus the North Atlantic does not appear to have been a strong barrier to their dispersal during the Quaternary. Hultén made no detailed proposals on mechanisms of speciation in the Arctic; however, molecular studies have confirmed that many arctic plants are allopolyploid, and some of them most probably originated during the Holocene. Recurrent formation of polyploids from differentiated diploid or more low‐ploid populations provides one explanation for the intriguing taxonomic complexity of the arctic flora, also noted by Hultén. In addition, population fragmentation during glacial periods may have lead to the formation of new sibling species at the diploid level. Despite the progress made since Hultén wrote his book, there remain large gaps in our knowledge of the history of the arctic flora, especially about the origins of the founding stocks of this flora which first appeared in the Arctic at the end of the Pliocene (approximately 3 Ma). Comprehensive analyses of the molecular phylogeography of arctic taxa and their relatives together with detailed fossil studies are required to fill these gaps.
Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently emerged clone
Authors: Morehouse Erica A. , James Timothy Y. , Ganley Austen R. D. , Vilgalys Rytas , Berger Lee , Murphy Peter J. , Longcore Joyce E.
Published: March 2003

Chytridiomycosis is a recently identified fungal disease associated with global population declines of frogs. Although the fungus, Batrachochytrium dendrobatidis, is considered an emerging pathogen, little is known about its population genetics, including the origin of the current epidemic and how this relates to the dispersal ability of the fungus. In this study, we use multilocus sequence typing to examine genetic diversity and relationships among 35 fungal strains from North America, Africa and Australia. Only five variable nucleotide positions were detected among 10 loci (5918 bp). This low level of genetic variation is consistent with the description of B. dendrobatidis as a recently emerged disease agent. Fixed (i.e. 100%) or nearly fixed frequencies of heterozygous genotypes at two loci suggested that B. dendrobatidis is diploid and primarily reproduces clonally. In contrast to the lack of nucleotide polymorphism, electrophoretic karyotyping of multiple strains demonstrated a number of chromosome length polymorphisms.
Genetic instability of the Streptomyces chromosome
Authors: Volff J.‐N. , Altenbuchner J.
Published: March 2003

The Streptomyces wild‐type chromosome is linear in all examples studied. The ends of the chromosome or telomeres consist of terminal inverted repeats of various sizes with proteins covalently bound to their 5′ ends. The chromosome is very unstable and undergoes very large deletions spontaneously at rates higher than 0.1% of spores. Frequently, the telomeres are included in the deletions. Loss of both telomeres leads to circularization of the chromosome. The wild‐type chromosome can also be circularized artificially by targeted recombination. Spontaneously or artificially circularized chromosomes are even more unstable than the linear ones. High‐copy‐number tandem amplifications of specific chromosomal regions are frequently associated with the deletions. RecA seems to be involved in the amplification mechanism and control of genetic instability.
Regulation of transcription initiation at the Escherichia coli nir operon promoter: a new mechanism to account for co‐dependence on two transcription factors
Authors: Wu Hui‐chung , Tyson Kerry L. , Cole Jeffrey A. , Busby Stephen J. W.
Published: March 2003

Expression from the Escherichia coli nir promoter is co‐dependent on Fnr (a transcription factor triggered by oxygen starvation) and on NarL or NarP (transcription factors triggered by nitrite and nitrate ions). Fnr binds to a single DNA site centred between basepairs 41 and 42 upstream from the nir transcript start, whereas NarL and NarP bind to a site upstream, centred between basepairs 69 and 70. A novel mechanism to account for co‐dependence on Fnr and NarL/NarP is suggested from experiments in which the spacing between the DNA sites for Fnr and NarL/NarP was altered. DNA sequence elements located upstream of the NarL/NarP‐binding site are the targets for two or more proteins that act to repress Fnr‐dependent activation of the nir promoter. This inhibition is counteracted by NarL or NarP. The model has been corroborated by the effects of several deletions and single base substitutions in the nir promoter upstream sequences: these deletions and substitutions prevent the binding of the repressor proteins. One of these repressors has been identified as the Fis protein, that binds to a site located 135–149 bp upstream of the nir transcript start: the binding of Fis is suppressed by a single base substitution at position −146. The other repressor protein(s) have yet to be identified, but appear to bind downstream of the DNA site for Fis: binding is suppressed by a single base substitution at position −99.
Leucine alters the interaction of the leucine‐responsive regulatory protein (Lrp) with the fim switch to stimulate site‐specific recombination in Escherichia coli
Authors: Roesch Paula L. , Blomfield Ian C.
Published: March 2003

The leucine‐responsive regulatory protein (Lrp) is a global regulator that controls the expression of numerous operons in Escherichia coli. Lrp can act as a repressor or as an activator of transcription with its effects being potentiated, repressed or unaffected by the presence of exogenous leucine. The phase variation of type 1 fimbria in E. coli provides a unique system in which to investigate the effects of leucine on Lrp, as it is the only known example in which Lrp is a positive regulator and leucine potentiates this effect. Previous studies determined that Lrp binds with high affinity to two sites within the fim switch (fim sites 1 and 2), and binding to these sites stimulates recombination. Here, it is shown that, even though leucine stimulates the fim switch in vivo, it nevertheless causes a slight decrease in Lrp binding to the fim switch in vitro. These contradictory results are explicable by the finding that Lrp binding to a third region adjacent to fim sites 1 and 2 inhibits recombination. According to this model, leucine stimulates recombination by selectively disrupting Lrp binding to this newly characterized region, while having little or no effect on Lrp binding to fim sites 1 and 2.
YopJ of Yersinia pseudotuberculosis is required for the inhibition of macrophage TNF‐α production and downregulation of the MAP kinases p38 and JNK
Authors: Palmer Lance E. , Hobbie Silke , Galán Jorge E. , Bliska James B.
Published: March 2003

Exposure of macrophages to lipopolysaccharide (LPS) leads to production of the pro‐inflammatory cytokine, tumour necrosis factor alpha (TNF‐α). Previous studies have suggested that pathogenic Yersinia spp. inhibit LPS‐mediated production of TNF‐α in macrophages, and that one of the Yop proteins secreted by the plasmid‐encoded type III pathway is required for this activity. We found that TNF‐α production was inhibited when J774A.1 murine macrophages were infected with wild‐type Y. pseudotuberculosis but not with an isogenic ysc mutant defective for Yop secretion. We inactivated multiple yop genes to identify which of these factors are required for the inhibition of TNF‐α production. A mutant unable to express yopJ was defective for the inhibition of TNF‐α production. Production of TNF‐α is regulated at the transcriptional and translational levels by several mitogen‐activated protein (MAP) kinases. The MAP kinases p38 and JNK underwent sustained activation in macrophages infected with the yopJ mutant. Conversely, p38 and JNK were downregulated in macrophages infected with the wild‐type strain. The ability of the yopJ mutant to downregulate p38 and JNK and to inhibit production of TNF‐α was restored by the expression of yopJ+in trans. Therefore, YopJ is required for Y. pseudotuberculosis to downregulate MAP kinases and inhibit the production of TNF‐α in macrophages.
Dominant C‐terminal deletions of FtsZ that affect its ability to localize in Caulobacter and its interaction with FtsA
Authors: Din Neena , Quardokus Ellen M. , Sackett Marcella J. , Brun Yves V.
Published: March 2003

The cell division protein FtsZ is composed of three regions based on sequence similarity: a highly conserved N‐terminal region of ≈320 amino acids; a variable spacer region; and a conserved C‐terminal region of eight amino acids. We show that FtsZ mutants missing different C‐terminal fragments have dominant lethal effects because they block cell division in Caulobacter crescentus by two different mechanisms. Removal of the C‐terminal conserved region, the linker, and 40 amino acids from the end of the N‐terminal conserved region (FtsZΔC281) prevents the localization or the polymerization of FtsZ. Because two‐hybrid analysis indicates that FtsZΔC281 does not interact with FtsZ, we hypothesize that FtsZΔC281 blocks cell division by competing with a factor required for FtsZ localization or that it titrates a factor required for the stability of the FtsZ ring. The removal of 24 amino acids from the C‐terminus of FtsZ (FtsZΔC485) causes a punctate pattern of FtsZ localization and affects its interaction with FtsA. This suggests that the conserved C‐terminal region of FtsZ is required for proper polymerization of FtsZ in Caulobacter and for its interaction with FtsA.
Control of cell shape and elongation by the rodA gene in Bacillus subtilis
Authors: Henriques Adriano O. , Glaser Philippe , Piggot Patrick J. , Moran Jr Charles P.
Published: March 2003

The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively. While rodA and ftsW are essential genes in E. coli, the B. subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan. In this work, we report on the characterization of a B. subtilis gene, designated rodA, encoding a homologue of E. coli RodA. We found that the growth of a B. subtilis strain carrying a fusion of rodA to the IPTG‐inducible Pspac promoter is inducer dependent. Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed. An increase in the level of IPTG induced a sphere‐to‐short rod transition that re‐established viability. Higher levels of inducer restored normal cell length. Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions. Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability. In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell. Electron microscopy observations support these conclusions. The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cell's inability to construct a division septum capable of spanning the enlarged cell. RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation). Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.
Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus
Authors: Lina Gerard , Jarraud Sophie , Ji Guangyong , Greenland Timothy , Pedraza Alicia , Etienne Jerome , Novick Richard P. , Vandenesch François
Published: March 2003

The agr P2 operon in Staphylococcus aureus codes for the elements of a density‐sensing cassette made up of a typical two‐component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post‐translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC, to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr+ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N‐terminal region of AgrC is replaced by the Escherichia coli maltose‐binding protein is also autophosphorylated in response to stimulation by agr+ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two‐component signal system in S. aureus, and that the ligand‐binding site of AgrC is probably located in the third extracellular loop of the protein.
Overexpression and topology of the Shigella flexneri O‐antigen polymerase (Rfc/Wzy)
Authors: Daniels Craig , Vindurampulle Christofer , Morona Renato
Published: March 2003

Lipopolysaccharides (LPS), particularly the O‐antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O‐antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O‐antigen polymerase, an integral membrane protein, which polymerizes the O‐antigen repeat units of the LPS. The wild‐type rfc/wzy gene has no detectable ribosome‐binding site (RBS) and four rare codons in the translation initiation region (TIR). Site‐directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O‐antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O‐antigen chain length regulator (Rol/Wzz) on a low‐copy‐number plasmid. This suggests that the O‐antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM‐PREDICT indicated that Rfc/Wzy had 10–13 transmembrane segments. The computer prediction models were tested by genetically fusing C‐terminal deletions of Rfc/Wzy to alkaline phosphatase and β‐galactosidase. Rfc::PhoA fusion proteins near the amino‐terminal end were detected by Coomassie blue staining and Western blotting using anti‐PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino‐ and carboxy‐termini are located on the cytoplasmic face of the membrane.
Replication and integration of a Vibrio cholerae cryptic plasmid linked to the CTX prophage
Authors: Rubin Eric J. , Lin Wei , Mekalanos John J. , Waldor Matthew K.
Published: March 2003

We identified a 4.7 kb cryptic plasmid in all ctxAB+Vibrio cholerae strains we tested. An isolate of the V. cholerae classical biotype strain O395 that harbours the cryptic plasmid at high copy number was found. Hybridization analysis demonstrated that sequences highly related or identical to this plasmid exist in all toxigenic strains of V. cholerae but were notably absent in all non‐toxigenic environmental isolates that lacked the genes for toxin‐co‐regulated pili and the filamentous CTX prophage. Accordingly, we have named the cryptic plasmid pTLC for toxin‐linked cryptic. The complete nucleotide sequence of pTLC from the high‐copy‐number isolate was determined. The largest open reading frame in the plasmid is predicted to encode a protein similar to the replication initiation protein (pII) of Escherichia coli F‐specific filamentous phages. The nucleotide sequence of pTLC also facilitated the structural characterization of the DNA homologous to pTLC in other strains of V. cholerae. pTLC‐related DNA exists in these strains as both low‐copy‐number, covalently closed circular DNA and tandemly duplicated, chromosomally integrated DNA. Remarkably, the chromosomally integrated form of pTLC is adjacent to the CTX prophage. The strain distribution, chromosomal location and DNA sequence of pTLC suggests that it may be a genetic element that plays some role in the biology of CTXφ, perhaps facilitating either its acquisition or its replication.
Analysis of ToxR‐dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae
Authors: Crawford J. Adam , Kaper James B. , DiRita Victor J.
Published: March 2003

The membrane proteins ToxR and ToxS regulate a variety of genes associated with the virulence of Vibrio cholerae, the agent of human cholera. One of the ToxRS‐regulated genes is the ompU gene, which encodes a porin that may also act as an adhesin. To begin to understand the mechanism of ompU transcription activation by ToxRS, we performed genetic and biochemical studies on the ompU promoter. Deletions with a 5′ end‐point at or downstream of −128, relative to the start site for transcription, did not direct expression of a lacZ reporter gene in wild‐type V. cholerae, although the −128 promoter fragment did direct ToxRS‐dependent reporter gene activity under conditions of ToxR overexpression in E. coli. Consistent with the activation data is that membranes containing ToxR and ToxS caused a gel electrophoretic mobility shift when mixed at low concentrations with deletion fragments whose end‐point is at −211, but not with −128 or −68 fragments. ToxRS membranes did shift the −128 fragment when added at higher concentrations. DNase I footprinting analysis of ompU promoter DNA complexed with ToxRS membranes demonstrated protection of three sites: an upstream site ranging from −238 to −139, and two downstream sites ranging from −116 to −58 and −53 to −24. Within the DNA protected from DNase I digestion by ToxRS membranes, there are no elements bearing similarity to those identified previously within the promoters of two other ToxR‐dependent genes, ctxA and toxT. We suggest a model for transcription activation that involves sequential ToxR‐binding events to distinct regions in the ompU promoter.
Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes
Authors: Occhino Deborah A. , Wyckoff Elizabeth E. , Henderson Douglas P. , Wrona Thomas J. , Payne Shelley M.
Published: March 2003

Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set (tonB1, exbB1, exbD1) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high‐affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild‐type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild‐type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system (tonB2, exbB2, exbD2). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes (hutBCD), and all six genes appeared to be expressed from a single Fur‐regulated promoter upstream of tonB1. A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae, the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae.
A recombinant peptide based on Pf EMP‐1 blocks and reverses adhesion of malaria‐infected red blood cells to CD36 under flow
Authors: Cooke Brian M. , Nicoll Claire L. , Baruch Dror I. , Coppel Ross L.
Published: March 2003

During falciparum malaria infection, severe complications ensue because parasitized red blood cells (PRBCs) adhere to endothelial cells and accumulate in the microvasculature. At the molecular level, adhesion is mediated by interaction of Plasmodium falciparum erythrocyte membrane protein 1 (Pf EMP‐1) on the PRBC surface with receptors on the surface of endothelial cells, including CD36. We have shown that a recombinant 179‐residue subfragment of Pf EMP‐1 (rC1‐2[1–179]), which encompasses the CD36‐binding region, inhibits and reverses adhesion of PRBCs to CD36 under physiologically relevant flow conditions. rC1‐2[1–179] inhibited adhesion in a concentration‐dependent manner over the range 100 pM to 2 μM, with up to 99% of adhesion blocked at the highest concentration tested. The antiadhesive activity of rC1‐2[1–179] was not strain specific and almost totally ablated adhesion of four different parasite lines. Furthermore, rC1‐2[1–179] showed remarkable ability to progressively reverse adhesion when flowed over adherent PRBCs for 2 h. The effect of rC1‐2[1–179] was, however, specific for CD36‐mediated adhesion and had no effect on adhesion mediated by CSA. Interference with binding of PRBCs to the vascular endothelium using rC1‐2[1–179] or smaller organic mimetics may be a useful therapeutic approach to ameliorate severe complications of falciparum malaria.
Ssa1p chaperone interacts with the guanine nucleotide exchange factor of ras Cdc25p and controls the cAMP pathway in Saccharomyces cerevisiae
Authors: Geymonat Marco , Wang Lili , Garreau Hervé , Jacquet Michel
Published: March 2003

We have found that the guanine nucleotide exchange factor for ras, Cdc25p, interacts with Ssa1p in Saccharomyces cerevisiae. This interaction was observed with GST‐fused Cdc25p polypeptides and confirmed by co‐immunoprecipitation with the endogenous Cdc25p. Hsp82 appeared also to be co‐immunoprecipitated with Cdc25p, albeit to a lower level than Hsp70. In a strain deleted for SSA1 and SSA2, we observed a reduced cellular content of Cdc25p. Consistent with a reduced activity of the cAMP‐dependent PKA pathway, the rate of accumulation of both trehalose and glycogen was stimulated in the ssa‐deleted strain. Expression of SSA1 reversed these effects, whereas co‐expression of SSA1 and PDE2 restored high accumulation. The expression of genes repressed by cAMP, GAC1 and TPS1, fused to β‐galactosidase, was also stimulated by deletion of SSA genes. The effect of ssa deletion on glycogen accumulation was lost in a strain deleted for CDC25 rescued by the RAS2ile152 allele. Altogether, these results lead to the conclusion that Ssa1p positively controls the cAMP pathway through Cdc25p. We propose that this connection plays a critical role in the adaptation of cells to stress conditions.
Enteropathogenic and enterohaemorrhagic Escherichia coli : more subversive elements
Authors: Frankel Gad , Phillips Alan D. , Rosenshine Ilan , Dougan Gordon , Kaper James B. , Knutton Stuart
Published: March 2003

Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the ‘attaching and effacing’ (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.
Acid‐induced expression of an LPS‐associated gene in Helicobacter pylori
Authors: McGowan Catherine C. , Necheva Antoaneta , Thompson Stuart A. , Cover Timothy L. , Blaser Martin J.
Published: March 2003

To investigate urease‐independent mechanisms by which Helicobacter pylori resists acid stress, subtractive RNA hybridization was used to identify H. pylori genes whose expression is induced after exposure to acid pH. This approach led to the isolation of a gene that encoded a predicted 34.8 kDa protein (WbcJ), which was homologous to known bacterial O‐antigen biosynthesis proteins involved in the conversion of GDP‐mannose to GDP‐fucose. An isogenic wbcJ null mutant strain failed to express O‐antigen and Lewis X or Lewis Y determinants and was more sensitive to acid stress than was the wild‐type strain. Qualitative differences in LPS profiles were observed in H. pylori cells grown at pH 5 compared with pH 7, which suggests that H. pylori may alter its LPS structure in response to acidic pH. This may be an important adaptation facilitating H. pylori colonization of the acidic gastric environment.
Regulation of autoinducer production in Salmonella typhimurium
Authors: Surette Michael G. , Bassler Bonnie L.
Published: March 2003

Salmonella typhimurium strain LT2 secretes an organic signalling molecule that can be assayed by its ability to activate one of two specific quorum‐sensing systems in Vibrio harveyi. Maximal activity is produced during mid‐ to late exponential phase when S. typhimurium is grown in the presence of glucose or other preferred carbohydrates. The signal is degraded by the onset of stationary phase or when the carbohydrate is depleted from the medium. Presumably, quorum sensing in S. typhimurium is operational during periods of rapid, nutrient‐rich growth. Protein synthesis is required for degradation of the activity, suggesting that a complex regulatory circuitry controls signal production and detection in S. typhimurium. Increased signalling activity is observed if, after growth in the presence of glucose, S. typhimurium is transferred to a high‐osmolarity (0.4 M NaCl) or to a low‐pH (pH 5.0) environment. Degradation of the signal is induced by conditions of low osmolarity (0.1 M NaCl). High osmolarity and low pH are two conditions encountered by S. typhimurium cells when they undergo the transition to a pathogenic existence inside a host organism, suggesting that quorum sensing may have a role in the regulation of virulence in S. typhimurium.
Effects of metabolic flux on stress response pathways in Lactococcus lactis
Authors: Duwat Patrick , Ehrlich S. Dusko , Gruss Alexandra
Published: March 2003

Studies of cellular responses to stress conditions such as heat, oxygen or starvation have revealed the existence of numerous specific or interactive response pathways. We previously observed in Lactococcus lactis that inactivation of the recA gene renders the lactococcal strain sensitive not only to DNA‐damaging agents but also to oxygen and heat. To further examine the stress response pathways in L. lactis, we isolatedthermoresistant insertionalmutants (Trm) of the recA strain. Eighteen independent trm mutations were identified and characterized. We found that mutations map in only seven genes, implicated in purine metabolism (deoB, guaA and tktA), phosphate uptake (pstB and pstS), mRNA stability (pnpA) and in one uncharacterized gene (trmA). All the trm mutations, with the exception of trmA, confer multiple stress resistance to the cell. Some of the mutations confer improved heat stress resistance not only in the recA but also in the wild‐type context. Our results reveal that cellular metabolic pathways are intimately related to stress response and that the flux of particular metabolites, notably guanine and phosphate, may be implicated in stress response in lactococci.
Phosphorylation of tyrosine 474 of the enteropathogenic Escherichia coli (EPEC) Tir receptor molecule is essential for actin nucleating activity and is preceded by additional host modifications
Authors: Kenny Brendan
Published: March 2003

The enteropathogenic Escherichia coli (EPEC) Tir protein becomes tyrosine phosphorylated in host cells and displays an increase in apparent molecular mass. The interaction of Tir with the EPEC outer membrane protein, intimin, triggers actin nucleation beneath the adherent bacteria. The enterohaemorrhagic E. coli 0157:H7 (EHEC) Tir molecule is not tyrosine phosphorylated. In this paper, Tir tyrosine phosphorylation is shown to be essential for actin nucleation activity, but not for the increase in apparent molecular mass observed in target cells. Tyrosine phosphorylation had no role in Tir molecular mass shift, indicating additional host modifications. Analysis of Tir intermediates indicates that tyrosine‐independent modification functions to direct Tir's correct insertion from the cytoplasm into the host membrane. Deletion analysis identified Tir domains participating in translocation, association with the host membrane, modification and antibody recognition. Intimin was found to bind a 55‐amino‐acid region (TIBA) within Tir that topological and sequence analysis suggests is located in an extracellular loop. Homologous TIBA sequences exist in integrins, which also bind intimin. Collectively, this study provides definitive evidence for the importance of tyrosine phosphorylation for EPEC Tir function and reveals differences in the pathogenicity of EPEC and EHEC. The data also suggest a mechanism for Tir insertion into the host membrane, as well as providing clues to the mode of intimin–integrin interaction.
DNA restriction dependent on two recognition sites: activities of the Sfi I restriction–modification system in Escherichia coli
Authors: Bilcock Denzil T. , Halford Stephen E.
Published: March 2003

In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4–6 bp long, the Sfi I endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites. The mode of action of the Sfi I endonuclease thus seems more appropriate for DNA rearrangements than for restriction. To elucidate its biological function, strains of Escherichia coli expressing the Sfi I restriction–modification system were transformed with plasmids carrying Sfi I sites. The Sfi I system often failed to restrict the survival of a plasmid with one Sfi I site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated Sfi I sites were not restricted. No rearrangements of the plasmids carrying Sfi I sites were detected among the transformants. Hence, provided the target DNA contains at least two recognition sites, Sfi I displays all of the hallmarks of a restriction–modification system as opposed to a recombination system in E. coli cells. The properties of the system in vivo match those of the enzyme in vitro. For both restriction in vivo and DNA cleavage in vitro, Sfi I operates best with two recognition sites on the same DNA.
Identification of UreR binding sites in the Enterobacteriaceae plasmid‐encoded and Proteus mirabilis urease gene operons
Authors: Thomas Venetta J. , Collins Carleen M.
Published: March 2003

The closely related Proteus mirabilis and Enterobacteriaceae plasmid‐encoded urease genes are positively regulated by the AraC‐like transcriptional activator UreR. In the presence of the effector molecule urea, UreR promotes transcription of ureD, the initial gene in the urease operon, and increases transcription of the divergently transcribed ureR. Here, we identify UreR‐specific binding sites in the ureRp–ureDp intergenic regions. Recombinant UreR (rUreR) was expressed and purified, and gel shift and DNase I protection assays were performed with this protein. These analyses indicated that there are two distinct rUreR binding sites in both the plasmid‐encoded and P. mirabilis ureRp–ureDp intergenic regions. A consensus binding site of TA/GT/CA/TT/GC/TTA/TT/AATTG was predicted from the DNase I protection assays. Although rUreR bound to the specific DNA binding site in both the presence and the absence of urea, the dissociation rate constant k–1 of the rUreR–DNA complex interaction was measurably different when urea was present. In the absence of urea, the dissociation of the protein–DNA complexes, for both ureRp and ureDp, was complete at the earliest time point, and it was not possible to determine a rate. In the presence of urea, dissociation was measurable with a k–1 for the rUreR–ureRp interaction of 1.2 ± 0.2 × 10−2 s−1 and a k–1 for the rUreR–ureDp interaction of 2.6 ± 0.1 × 10−3 s−1. This corresponds to a half‐life of the ureRp–rUreR interaction of 58 s, and a half‐life of the ureDp–rUreR interaction of 4 min 26 s. A model describing a potential role for urea in the activation of these promoters is proposed.
Characterization of Escherichia coli cspE, whose product negatively regulates transcription of cspA, the gene for the major cold shock protein
Authors: Bae Weonhye , Phadtare Sangita , Severinov Konstantin , Inouye Masayori
Published: March 2003

Escherichia coli contains nine members of the CspA protein family from CspA to CspI. To elucidate the cellular function of CspE, we constructed a ΔcspE strain. CspE is highly produced at 37°C. The synthesis level of CspE transiently increased during the growth lag period after dilution of stationary‐phase cells into the fresh medium at 37°C. This is consistent with the ΔcspE phenotype of the longer growth lag period after dilution. The protein synthesis patterns of the ΔcspE strain and the wild‐type strain were compared using two‐dimensional gel electrophoresis. In the ΔcspE strain, the synthesis of a number of proteins at 37°C was found to be altered and cspA was derepressed. The derepression of cspA in the ΔcspE strain was at the level of transcription in a promoter‐independent fashion but was not caused by stabilization of the cspA mRNA, which was shown to be a major cause of CspA induction after cold shock. In vitro transcription assays demonstrated that both CspE and CspA enhanced transcription pause at the region immediately downstream of the cold box, a putative repressor binding site on the cspA mRNA. In a cell‐free protein synthesis system using S‐30 cell extracts, CspA production was specifically inhibited by the addition of CspE. These results indicate that CspE functions as a negative regulator for cspA expression at 37°C, probably by interacting with the transcription elongation complex at the cspA cold box region.
Modelling in Escherichia coli of mutations in mitoribosomal protein S12: novel mutant phenotypes of rpsL
Authors: Toivonen Janne M. , Boocock Martin R. , Jacobs Howard T.
Published: March 2003

The rpsL gene of Escherichia coli encodes the highly conserved rps12 protein of the ribosomal accuracy centre. We have used the E. coli gene to model the phenotypic effects of specific substitutions found in the mitochondrial gene for rps12. Variants created by in vitro mutagenesis were tested in two different plasmid vector systems, in both streptomycin‐sensitive and streptomycin‐resistant hosts. A substitution with respect to eubacterial rps12 (K87→Q), found in all metazoan and fungal mitochondrial orthologues thus far studied, is associated with low‐level resistance to streptomycin and a modest (15%) drop in translational elongation rate, but without significant effects on translational accuracy. An amino‐acid replacement at a highly conserved leucine residue (L56→H), associated with the phenotypes of sensitivity to mechanical vibration and hemizygous female lethality in Drosophila, creates a functionally inactive but structurally stable protein that is not assembled into ribosomes. The presence in the cell of the mutant, but not wild‐type, rpsL greatly downregulates the level of a prominent polypeptide of ≈ 50 kDa. These results indicate novel structure–function relationships in rps12 genes affecting translational function, ribosome assembly and drug sensitivity, and indicate a novel regulatory pathway that may influence ribosome biogenesis.