bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeography and postglacial dispersal of arctic charr Salvelinus alpinus in North America
Authors: WILSON C. C. , HEBERT P. D. N. , REIST J. D. , DEMPSON J. B.
Published: November 2003

Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) was used to reconstruct postglacial dispersal routes of arctic charr Salvelinus alpinus in North America. Twelve of 35 restriction enzymes detected polymorphisms among representative populations, revealing two distinct lineages with an estimated nucleotide divergence of 1.32%. Subsequent screening of 869 fish from 54 populations with four diagnostic restriction enzymes showed that these lineages have largely allopatric distributions, suggesting their dispersal from separate northern and eastern glacial refugia. In addition, geographical and genetic structure among eastern populations suggested the existence of a second eastern refuge. Among the three lineages, the most divergent (Arctic) lineage occurred from Alaska east to northern Labrador. Quebec, New Brunswick, and New England were colonized by a second (Laurentian) lineage, and Labrador by a third group. Contact between refugial groups was only detected in two Labrador populations. The Arctic lineage was highly differentiated from eastern North American and European haplotypes, and probably diverged during the early Pleistocene. By contrast, the Laurentian and Labrador groups were similar to Old World charr, suggesting a shared ancestry during the mid‐Pleistocene. In addition, the close relationship between Labrador and Laurentian charr indicates their probable divergence during the Wisconsinan glaciation.
Quantification of transgenic plant marker gene persistence in the field
Authors: Widmer F. , Seidler R. J. , Donegan K. K. , Reed G. L.
Published: November 2003

Methods were developed to monitor persistence of genomic DNA in decaying plants in the field. As a model, we used recombinant neomycin phosphotransferase II (rNPT‐II) marker genes present in genetically engineered plants. Polymerase chain reaction (PCR) primers were designed, complementary to 20‐bp sequences of the nopaline synthase promoter in a transgenic tobacco and the cauliflower mosaic virus 35S promoter in a transgenic potato. The PCR reverse primer was complementary to a 20‐bp sequence of the N‐terminal NPT‐II coding region. The PCR protocol allowed for quantification of as few as 10 rNPT‐II genes per reaction. We analysed rNPT‐II marker gene amounts in samples obtained from two field experiments performed at different locations in Oregon. In transgenic tobacco leaves, buried at 10 cm depth in a field plot in Corvallis, marker DNA amount dropped to 0.36% during the first 14 days and was detectable for 77 days at a final level of 0.06% of the initial amount. Monitoring of residual potato plant litter, from the soil surface of a test field in Hermiston, was performed for 137 days. After 84 days marker gene amounts dropped to 2.74% (leaf and stem) and 0.50% (tuber) of the initially detected amount. At the final sample date 1.98% (leaf and stem) and 0.19% (tuber) were detectable. These results represent the first quantitative analysis of plant DNA stability under field conditions and indicate that a proportion of the plant genomic DNA may persist in the field for several months.
PCP degradation is mediated by closely related strains of the genus Sphingomonas
Authors: Ederer M. M. , Crawford R. L. , Herwig R. P. , Orser C. S.
Published: November 2003

There have been numerous reports in the literature of diverse bacteria capable of degrading pentachlorophenol (PCP). In order to gain further insight into the phylogenetic relationships of PCP‐degrading bacteria, we examined four strains: Arthrobacter sp. strain ATCC 33790, Flavobacterium sp. strain ATCC 39723, Pseudomonas sp. strain SR3, and Sphingomonas sp. strain RA2. These organisms were isolated from different geographical locations and all of them degrade high concentrations (100–200 mg/L) of PCP. Southern blot analyses determined that these bacteria all harbour DNA that encodes similar, if not identical, genes involved in PCP degradation. Comparison of the 16S rRNA nucleotide sequences revealed that these organisms were very closely related and, in fact, represent a monophyletic group. The 16S rRNA analyses together with fatty acid and sphingolipid analyses strongly suggest that the four strains are members of the genus Sphingomonas. The close relationship of the four organisms is supported by nucleotide sequence analysis data of the pcpB locus encoding PCP‐4‐monooxygenase, the first enzyme in the PCP degradative pathway.
Nuclear DNA‐based markers for plant evolutionary biology
Authors: Strand A. E. , Leebens‐Mack J , Milligan B. G.
Published: November 2003

While DNA‐based markers can provide a wealth of information for the study of plant evolutionary biology, progress is limited by the lack of primers available for PCR. To overcome this limitation, we outline a protocol for developing oligonucleotide primers targeting regions of low copy‐number nuclear genes. This protocol is intended to lead to universally useful primer sets. To test our approach, we designed eight primer sets and tested their abilities to amplify targets from representatives of each dicot and one monocot subclass. Five of the eight primer sets amplified targets from at least five of the seven taxa and thus exhibited broad taxonomic usefulness; the remaining primers were rather specific, however, and amplified targets from at most three taxa. In only one primer‐taxon combination was a complex multiple‐banded amplification produced. Overall, the protocol outlined proved quite useful at identifying broadly applicable primers targeted to low copy‐number nuclear genes. Wider application of this approach should be effective at greatly increasing the amount of genetic information available for a diversity of plant nuclear genomes.
Paternity assessment in wild groups of toque macaques Macaca sinica at Polonnaruwa, Sri Lanka using molecular markers
Authors: Keane B. , Dittus W. P. J. , Melnick D. J.
Published: November 2003

Genetic variation at four microsatellite loci in conjunction with that at a highly variable allozyme locus was used to analyse paternity over a 12‐year period in 13 social groups of toque macaques Macaca sinica inhabiting a natural forest in Polonnaruwa, Sri Lanka. Paternity exclusion analysis revealed that the set of offspring produced by a female usually consists of half‐siblings because few males father more than one offspring with a particular female. No evidence of offspring produced by matings between first degree relatives was found. The social unit in toque macaques was not identical to the reproductive unit and the possibility of paternity by males outside the social group should be considered when estimating male reproductive output. Although it was common for multiple males to father offspring in a social group each year, reproduction within a group during a breeding season tended to be limited to a few males. The mean number of males reproducing per group per year was independent of the number of males in a group. The paternity data suggests that many males may father relatively few offspring during their entire lives and that the effective population size for toque macaques may be much smaller than indicated by demographic data.
Diagnosis of sibling species of Drosophila involved in the colonization of North America by D. subobscura
Authors: Pascual M. , Balanyà J. , Latorre A. , Serra L.
Published: November 2003

To determine the effects of the recent colonization of the west coast of North America by the Palaearctic species Drosophila subobscura on the dynamics of the Drosophila populations, the sibling species D. athabasca and D. azteca must be classified unambiguously. We have characterized these two species using three molecular techniques: allozymes, mtDNA and RAPDs. All three techniques allow the classification of any individual as belonging to either species. The study of five localities in northern California and southern Oregon show that the area of overlap is larger than previously described.
Chloroplast DNA phylogeography and cytotype geography in autopolyploid Plantago media
Authors: Van Dijk P. , Bakx‐Schotman T.
Published: November 2003

In order to gain insight into the causes of parapatric diploid and tetraploid distributions in Plantago media chloroplast DNA (cpDNA) restriction site polymorphism was studied in 36 European populations. Parapatric distributions are often explained by adaptive differences between cytotypes to an underlying heterogeneity in environmental factors. Alternatively, such distribution patterns may be explained nonadaptively, through frequency‐dependant production of hybrids with low fitness. However, nonadaptive explanations have been neglected in polyploid literature. In this study nine chloroplast haplotypes were found. Their phylogeny suggests that tetraploids arose at least three times from diploids. In general, related haplotypes were also geographically clustered, although there were some marked geographical discontinuities. In the Pyrenees, diploids and tetraploids carried diverged haplotypes throughout their parapatric ranges. At the contact zone the level of cpDNA introgression in a mixed diploid‐tetraploid population was low. It is discussed that the cpDNA phylogeography supports the nonadaptive hypothesis that parapatric cytotype distributions may be explained by postPleistocene range expansions followed by mutual minority cytotype exclusion, due to hybrid unfitness.
Fine‐scale structure of populations of the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune forest ecosystems
Authors: Gryta H. , Debaud J.‐C. , Effosse A. , Gay G. , Marmeisse R.
Published: November 2003

The basidiomycete mushroom Hebeloma cylindrosporum is a frequently found pioneer ectomycorrhizal species naturally associated with Pinus pinaster trees growing in coastal sand dune ecosystems along the Atlantic south‐west coast of France. The genotypic diversity and spatial structure of three populations of this fungal species have been studied. At each site the basidiocarps were mapped, sampled and propagated as pure mycelial cultures. For each of the isolates, we have studied polymorphisms in the mitochondrial genome, polymorphisms at two different nuclear loci and also fingerprints produced with a multicopy DNA probe. The comparison of the different polymorphisms obtained, with each of the four molecular methods used, allowed the identification of several of the different genets present in each site. In two of the studied sites most of the basidiocarps, which often occurred as dense patches of 10–30 in 1 m2 or less, were of a unique genotype, suggesting the below‐ground mycelia to be of a small size (from 50 cm2 to approx. 7 m2 for the larger mycelia) and that the root system of a single Pinus tree can host several genets of the same symbiotic fungus. In the two sites, which were studied again after a 3‐year interval, none of the genotypes identified in the first year of sampling was re‐identified 3 years later. These results contrast with those reported for other species of soilborne homobasidiomycete species, either ectomycorrhizal, parasitic or saprophytic, showing mostly large clones resulting from the vegetative growth and from persistence of below‐ground mycelia. Sexual reproduction through meiospore dispersal seems to play a key role in the structuring of the populations of H. cylindrosporum. Mycelia associated with the root systems seem to be replaced after 1 or a few years, during which basidiocarp differentiation takes place. As opposed to the few other studied ectomycorrhizal species, H. cylindrosporum has the characteristics of ruderal species, with a short life‐span adapted to pioneer situations, e.g. to nutrient‐poor and unstable sandy soils of coastal sand dunes.
A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung
Authors: Paxinos E. , McIntosh C. , Ralls K. , Fleischer R.
Published: November 2003

Endangered San Joaquin kit foxes Vulpes macrotis mutica can be sympatrically distributed with as many as four other canids: red fox, gray fox, coyote and domestic dog. Canid scats are often found during routine fieldwork, but cannot be reliably identified to species. To detect and study the endangered kit fox, we developed mitochondrial DNA markers that can be amplified from small amounts of DNA extracted from scats. We amplified a 412‐bp fragment of the mitochondrial cytochrome‐b gene from scat samples and digested it with three restriction enzymes. The resulting restriction profiles discriminated among all five canid species and correctly identified 10 ‘unknown’ fox scats to species in blind tests. We have applied our technique to identify canids species for an environmental management study and a conservation study. We envision that our protocol, and similar ones developed for other endangered species will be greatly used for conservation management in the future.
Postglacial genetic differentiation of reproductive ecotypes of kokanee Oncorhynchus nerka in Okanagan Lake, British Columbia
Authors: Taylor E. B. , Harvey S. , Pollard S. , Volpe J.
Published: November 2003

Okanagan Lake, south‐central interior of BC, contains two reproductive ecotypes of kokanee Oncorhynchus nerka; individuals spawn in tributary streams (‘stream‐spawners’) as well as on shoreline gravel areas (‘beach‐spawners’). We tested the hypothesis that these sympatric ecotypes comprise a single panmictic population by assaying variation in morphological traits and at allozyme, mitochondrial and minisatellite DNA loci in fish collected from three stream‐spawning and two beach‐spawning sites. No morphological traits consistently distinguished the reproductive ecotypes with the exception of the number of anal fin rays which was greater in stream‐spawning kokanee. Four of 18 allozyme loci screened were polymorphic, but no significant allele frequency differences were detected among populations within ecotypes or between ecotypes. Similarly, allele frequencies at two minisatellite DNA loci were not significantly different among populations or between ecotypes. By contrast, significant differences in the frequencies of mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) haplotypes were detected between stream‐ and beach‐spawners, but not among populations within ecotypes. Further, two RFLPs that distinguished stream‐ and beach‐spawning adults were found in juvenile kokanee sampled from the limnetic zone of Okanagan Lake. The two mtDNA RFLPs and a d‐loop sequence variant appear to be unique to Okanagan Lake kokanee because we did not observe these haplotypes in sockeye salmon and kokanee sampled outside of Okanagan Lake. Our data suggest that: (i) there is restricted female‐mediated gene flow between stream‐ and beach‐spawning kokanee in Okanagan Lake, (ii) the forms have diverged within the lake basin since the retreat of the Wisconsinian glaciers (< ≊ 11 000 years ago), and (iii) distinct reproductive niches may promote divergence in north temperate freshwater fish faunas.
Characterization of Rhizobium etli and other Rhizobium spp. that nodulate Phaseolus vulgaris L. in an Austrian soil
Authors: Sessitsch A. , Hardarson G. , Akkermans A. D. L. , De Vos W. M.
Published: November 2003

Phaseolus vulgaris L. (common bean) is nodulated by rhizobia present in the fields around the Seibersdorf laboratory despite the fact that common bean has not been grown for a long time. Using PCR analysis with repetitive primers, plasmid profiles, nifH profiles, PCR‐RFLP analysis of the 16S rRNA gene and of the 16S rRNA‐23S rRNA intergenic spacer and the nodulation phenotype, two well‐differentiating groups could be distinguished. One group showed high similarity to Rhizobium sp. R602sp, isolated from common bean in France, while the other showed the same characteristics as R. etli. We detected little variation in the symbiotic regions but found higher diversity when using approaches targeting the whole genome. Many isolates obtained in this study might have diverged from a limited number of strains, therefore the Austrian isolates showed high saprophytic and nodulation competence in that particular soil.
Lack of genetic variability in the rare and endangered Limonium cavanillesii (Plumbaginaceae) using RAPD markers
Authors: Palacios C. , González‐Candelas F.
Published: November 2003

Limonium cavanillesii is an extremely endangered plant species endemic to the east Mediterranean region of Spain. Regarded as extinct for several years, the recent discovery of a small population (only 29 individuals) has prompted the adoption of measures for its conservation by official agencies. As part of this effort, we have analysed genetic variation in this population by means of random amplified polymorphic DNA (RAPDs). The analysis of 29 individuals with 11 different primers produced 131 monomorphic bands. To our knowledge, this is the lowest level of genetic variation detected in plants using RAPD markers. This result could be explained both by the apomictic reproductive system of this species and by the passage through a severe bottleneck in recent times, after which there has been no chance for mutation to restore detectable genetic variation.
Increased fitness of transgenic insecticidal rapeseed under insect selection pressure
Authors: Stewart C. N. , All J. N. , Raymer P. L. , Ramachandran S.
Published: November 2003

Rapeseed Brassica napus L. transgenic for a Bacillus thuringiensis (Bt) transgene was developed and was shown to be insecticidal towards certain caterpillars including the diamondback moth Plutella xylostella L. and the corn earworm Helicoverpa zea Boddie. To simulate an escape of the transgenics from cultivation, a field experiment was performed in which transgenic and nontransgenic rapeseed plants were planted in natural vegetation and cultivated plots and subjected to various selection pressures in the form of herbivory from insects. Only two plants, both transgenic, survived the winter to reproduce in the natural‐vegetation plots which were dominated by grasses such as crabgrass. However, in plots that were initially cultivated then allowed to naturalize, medium to high levels of defoliation decreased survivorship of nontransgenic plants relative to Bt‐transgenic plants and increased differential reproduction in favour of Bt plants. Thus, where suitable habitat is readily available, there is a likelihood of enhanced ecological risk associated with the release of certain transgene/crop combinations such as insecticidal rapeseed. This is the first report of a field study demonstrating the effect of a fitness‐increasing transgene in plants.
Population structure and impact of supportive breeding inferred from mitochondrial and microsatellite DNA analyses in land‐locked Atlantic salmon Salmo salar L.
Authors: Tessier N. , Bernatchez L. , Wright J. M.
Published: November 2003

Four tributaries of Lake St‐Jean (Québec, Canada) are used for spawning and juvenile habitat by land‐locked Atlantic salmon. Spawning runs have drastically declined since the mid‐1980s, and consequently, a supportive‐breeding programme was undertaken in 1990. In this study, we analysed seven microsatellite loci and mtDNA, and empirically estimated effective population sizes to test the hypotheses that (i) fish spawning in different tributaries form genetically distinct populations and (ii) the supportive breeding programme causes genetic perturbations on wild populations. Allele frequency distribution, molecular variance and genetic distance estimates all supported the hypothesis of genetic differentiation among salmon from different tributaries. Gene flow among some populations was much more restricted than previously reported for anadromous populations despite the small geographical scale (40 km) involved. Both mtDNA and microsatellites revealed a more pronounced differentiation between populations from two tributaries of a single river compared with their differentiation with a population from a neighbouring river. The comparison of wild and F1‐hatchery fish (produced from breeders originating from the same river) indicated significant changes in allele frequencies and losses of low‐frequency alleles but no reduction in heterozygosity. Estimates of variance and inbreeding population size indicated that susceptibility to genetic drift and inbreeding in one population increased by twofold after only one generation of supplementation.
Genetic population structure of the Greater Bilby Macrotis lagotis, a marsupial in decline
Authors: Moritz C. , Heideman A. , Geffen E. , McRae P.
Published: November 2003

The Greater Bilby has shown a rapid decline in range during this century and now occupies only a small isolated area in south‐western Queensland (QLD) and a larger, but mostly low‐density area in the north‐western deserts of the Northern Territory (NT) and Western Australia (WA). We have examined variation in the control region of mitochondrial DNA (mtDNA) and at nine microsatellite loci in order to investigate the extent of current and historical subdivision across the species range, and to provide a preliminary assessment of genetic structuring and mating system on a finer scale within the QLD population. Both mtDNA and microsatellite loci had substantial variation within and among populations, with mtDNA divergence being greater between QLD and NT than between NT and WA. The QLD population had two unique and divergent mtDNA lineages, but there was no evidence for strong phylogeographical structure across the range. The available evidence suggests that the bilby should be considered as a single Evolutionarily Significant Unit consisting of multiple Management Units. Augmentation of the remnant QLD population from the NT does not appear necessary at this stage, at least not on genetic grounds. Finer‐scale analysis of microsatellite variation for two QLD colonies revealed a deficiency of heterozygotes and significantly greater relatedness within than between colonies. However, structuring was observed only for males; relatedness values for females did not depart from those expected under panmixia. Parentage exclusion analysis for one colony allowed the construction of a partial pedigree which indicated strong polygyny, with one male fathering all but one of the eight offspring assigned. The extent to which fine‐scale genetic structuring and differences between sexes is due to sex‐biased dispersal vs. effects of mating system remain to be determined.
Isolation and characterization of microsatellite loci in Swietenia humilis (Meliaceae): an endangered tropical hardwood species
Authors: WHITE G. , POWELL W.
Published: November 2003

Microsatellites are now firmly established as an informative marker system, with increasing popularity as a tool amongst molecular ecologists. We have developed a method of constructing an enriched microsatellite library for the tropical tree species Swietenia humilis Zucc. (Meliaceae). This method is based on a precloning enrichment of SSRs using synthetic oligonucleotide probes, bound to magnetic beads and hybridizing to complementary microsatellite core sequences in digested genomic DNA. Here we describe the isolation and characterization of 10 microsatellite loci that have been used to survey the genetic diversity within a natural population of S. humilis. A total of 97 alleles were identified with an average of 9.7 alleles over all loci. Very high levels of allelic polymorphism were detected at individual loci, with 23 alleles observed at the most variable. The mean observed heterozygosity was 0.415 (range 0.038‐0.815) exceeding levels of diversity detected in related species which used isozymes as the marker system. Subpopulation differentiation at a microgeographical scale was low (FST= 0.036) and the values of Nm, calculated from the allelic frequencies, were greater than 1 thus reflecting the extent of gene flow occurring between individual trees.
The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N‐acylation of Nod factors by different fatty acids
Authors: Debellé Frédéric , Plazanet Claire , Roche Philippe , Pujol Céline , Savagnac Arlette , Rosenberg Charles , Promé Jean‐Claude , Dénarié Jean
Published: November 2003

Rhizobia synthesize mono‐N‐acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation (nod) genes, including the nodABC genes present in all rhizobial species. The N‐acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti, an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (ω‐1) hydroxylated fatty acid, whereas in Rhizobium tropici, a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non‐polar deletion of nodA, and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N‐acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.
Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa
Authors: Raivio Tracy L. , Hoffer Danita , Prince Robert W. , Vasil Michael L. , Storey Douglas G.
Published: November 2003

RegA is a transcriptional activator that controls exotoxin A (ETA) production in Pseudomonas aeruginosa. To date, functional assays performed with the purified protein have not clearly defined the molecular mechanism of action of RegA. In this study, we sought to identify important coding regions of regA by analysing the sequences around linker insertion mutations in regA that affected toxA transcription. First, we constructed a strain with the regAB locus deleted from the chromosome, PA103ΔregAB::Gm. toxA transcription was obliterated in strain PA103ΔregAB::Gm, demonstrating that the regAB locus is essential for ETA production. Next, we constructed a series of 6 bp linker insertion mutations distributed throughout regA. These regA linker insertion mutants were sequenced and screened in PA103ΔregAB::Gm for their effects on regulation of ETA production. Six linker insertion mutations occurring between amino acids (aa) 53 and 163 of RegA were isolated that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus. One of these linker insertion mutations (pTR53), resulted in a lack of iron‐regulated ETA production and occurred directly upstream from a predicted transmembrane α‐helix. The other five linker mutations (pTR88, pTR124, pTR132, pTR132‐2 and pTR163) occurred within or flanked a region of RegA between aa 87–142 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR. These data suggest the presence of a previously unidentified transcriptional activation domain in RegA between aa 87–142 and implicate the predicted transmembrane α‐helix in the N‐terminus as being involved in sensory transduction.
The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp‐like protein and is not autoregulated
Authors: Cho Kyungyun , Winans Stephen C.
Published: November 2003

The Agrobacterium tumefaciensputA gene, which encodes proline dehydrogenase, is transcriptionally induced by exogenous proline. In contrast to the putA genes of enteric bacteria, the A. tumefaciensputA gene is not regulated by the PutA protein, as the putA promoter remained strongly proline inducible in strains lacking PutA. A putA null mutation increased the expression of the putA promoter under a variety of conditions. However, this mutation is predicted to increase the cytoplasmic concentration of proline, and this alone probably accounts for its effects on putA expression. The putA promoter was also strongly induced by valine, and the putA genotype did not affect expression by this gratuitous inducer. An open reading frame (ORF) encoding an Lrp‐like protein was found transcribed divergently from putA. Disruption of this ORF, designated putR, abolished induction of the putA promoter by proline or valine. In addition to activating putA, PutR also repressed its own transcription, and this autorepression was only slightly affected by exogenous proline. The transcription start sites for the putA and putR genes are separated by 64 nucleotides, suggesting that PutR could regulate both promoters by binding to a single operator.
Localized domains of DNA supercoiling: topological coupling between promoters
Authors: Mojica Francisco J. M. , Higgins Christopher F.
Published: November 2003

According to the twin‐supercoiled domain model, a local domain with a high level of DNA supercoiling can be generated between two divergently transcribed promoters. We have tested this model directly using the rate of DNA photo‐cross‐linking by 4,5′,8‐trimethylpsoralen to measure local regions of unconstrained supercoiling in vivo. We demonstrate that, in plasmids, a localized domain of highly supercoiled DNA is generated upstream of the tetA promoter. The level of supercoiling of this domain depends on the activity of the tetA promoter and on the extent of translation of the tetA gene product. The highest level of supercoiling within this localized domain was observed in a topA strain, although the generation of a localized supercoiling domain could also be detected in a topA+ background, consistent with potential physiological significance. The level of supercoiling in this localized domain correlated with activation of the supercoiling‐sensitive leu‐500 promoter located upstream of tetA. These data provide a direct demonstration that localized domains of increased negative supercoiling can be generated upstream of an actively transcribed promoter, and that this can result in supercoiling‐mediated transcriptional coupling between two promoters.
Identification of proteolytic cleavage sites within the gag‐analogue protein of Ty1 virus‐like particles
Authors: Martin‐Rendon Enca , Hurd Douglas W. , Marfany Gemma , Kingsman Susan M. , Kingsman Alan J.
Published: November 2003

Like retroviruses, the yeast retrotransposon Ty1 produces its proteins as precursors that are subsequently cleaved by a protease encoded by the element. These cleavage events are essential for transposition as they release the active reverse transcriptase and integrase and they modify the structure of the virus‐like particles in a way that is analogous to the morphological changes that occur during retrovirus core maturation. Using a combination of epitope tagging, amino acid analysis and mutagenesis, we have identified the major cleavage sites for the Ty1 protease within the particle‐forming protein, p1, at 407S/408N. In addition, we present evidence indicating that the Ty1 protease may be a 17 kDa protein.
Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis
Authors: Hood Derek W. , Deadman Mary E. , Allen Tina , Masoud Hussein , Martin Adele , Brisson Jean Robert , Fleischmann Robert , Venter J. Craig , Richards James C. , Moxon E. Richard
Published: November 2003

The availability of the complete 1.83‐megabase‐pair sequence of the Haemophilus influenzae strain Rd genome has facilitated significant progress in investigating the biology of H. influenzae lipopolysaccharide (LPS), a major virulence determinant of this human pathogen. By searching the H. influenzae genomic database, with sequences of known LPS biosynthetic genes from other organisms, we identified and then cloned 25 candidate LPS genes. Construction of mutant strains and characterization of the LPS by reactivity with monoclonal antibodies, PAGE fractionation patterns and electrospray mass spectrometry comparative analysis have confirmed a potential role in LPS biosynthesis for the majority of these candidate genes. Virulence studies in the infant rat have allowed us to estimate the minimal LPS structure required for intravascular dissemination. This study is one of the first to demonstrate the rapidity, economy and completeness with which novel biological information can be accessed once the complete genome sequence of an organism is available.
Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway
Authors: Yahr Timothy L. , Goranson Joanne , Frank Dara W.
Published: November 2003

Exoenzyme S is an extracellular ADP‐ribosyltransferase of Pseudomonas aeruginosa. Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for exoenzyme S production. Five Tn5 Tc insertion mutants were isolated which exhibited an exoenzyme S‐deficient phenotype (388::Tn5 Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn5 Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb EcoRI fragment that is not contiguous with the exoenzyme S trans‐regulatory operon. 388::Tn5 Tc 469 and 550 mapped to a region downstream of the trans‐regulatory operon which has been previously shown to contain a promoter region that is co‐ordinately regulated with exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn5 Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB–L proteins of the yersiniaeYop type III export apparatus. RNase‐protection analysis of wild‐type and mutant strains indicated that exsD and pscB–L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino‐ or carboxy‐terminal residues were expressed in P. aeruginosa. Deletion analyses indicated that the amino‐terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of exoenzyme S and potentially other co‐ordinately regulated proteins.
Catabolite repression mediated by the catabolite control protein CcpA in Staphylococcus xylosus
Authors: Egeter Oliver , Brückner Reinhold
Published: November 2003

The gene ccpA encoding the catabolite control protein CcpA of Staphylococcus xylosus has been cloned and characterized. The CcpA protein belongs to the LacI/GalR family of bacterial regulators and is comprised of 329 amino acids, with a molecular mass of 36.3 kDa. It shows 56% identity with the CcpA proteins of Bacillus subtilis and Bacillus megaterium. Inactivation of the ccpA gene in the genome of S. xylosus relieved the activities of three enzymes, α‐glucosidase, β‐glucuronidase, and β‐galactosidase, from catabolite repression by several carbohydrates. Concomitantly, transcription initiation of the maltose‐utilization operon malRA, including the α‐glucosidase gene malA, was no longer subject to glucose‐specific control. Carbon source‐dependent malRA regulation was also lost upon deletion of a palindromic sequence in the malRA promoter region resembling the catabolite‐responsive elements essential for CcpA‐dependent catabolite repression in Bacillus. These results strongly suggest that S. xylosus CcpA controls transcription of catabolite‐repressible genes and operons by binding to catabolite‐responsive operators when rapidly metabolizable carbohydrates are available. Accordingly, the cloned S. xylosusccpA gene could complement the ccpA mutation in B. subtilis. The ccpA gene of S. xylosus is transcribed from two promoters, one of which is subject to autogenous repression by CcpA. Autoregulation results in a slight reduction of CcpA protein in glucose‐grown cells. The characterization of the role of CcpA in carbon catabolite repression in S. xylosus demonstrates that a regulatory mechanism originally detected in Bacillus applies to another Gram‐positive bacterium with low GC content.
The ‘petite‐negative’ yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity
Authors: Bianchi Michele M. , Tizzani Lorenza , Destruelle Monika , Frontali Laura , Wésolowski‐Louvel Micheline
Published: November 2003

We cloned and sequenced the pyruvate decarboxylase (PDC; EC 4.1.1.1) structural gene KIPDCA in the yeast Kluyveromyces lactis and found it to be allelic to the previously isolated rag6 mutation. The putative amino acid sequence of the KIPdcAp appeared to be highly homologous to those of the yeast Pdc proteins identified so far. The disruption of KIPDCA indicated that it is the only PDC structural gene in K. lactis, as evidenced by the lack of PDC activity and ethanol production in the pdcAΔ strains and by the absence of growth on glucose in the presence of respiratory inhibitors. It was observed that expression of the KIPDCA gene is induced by glucose at the transcriptional level. Transcription of the gene was reduced in the rag1, rag2, rag5 and rag8 mutants, which are defective for the low‐affinity glucose permease, phosphoglucose isomerase, hexokinase, and a positive regulator of RAG1 expression, respectively.
Amino‐terminal maturation of the Bordetella pertussis filamentous haemagglutinin
Authors: Jacob‐Dubuisson Françcoise , Buisine Corinne , Mielcarek Nathalie , Clément Eve , Menozzi Franco D. , Locht Camille
Published: November 2003

The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N‐terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC‐dependent manner, indicating that all necessary secre tion signals are localized in the N‐terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell‐associated and secreted FHA polypeptides, indicated that FhaB undergoes N‐terminal maturation by the cleavage of an 8–9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co‐expression of the Fha44‐encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N‐terminal end of Fha44. Regardless of the OmpA signal peptide‐Fha44 fusion point, the E. coli‐secreted Fha44 had the same Mr as that secreted by B. pertussis, indicating that the N‐terminal proteolytic maturation does not require a B. perfussis‐specific factor. Similar to FHA, the B. pertussis‐secreted Fha44 contains an as yet uncharacterized modification at its N‐terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N‐terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in‐frame methionine of FhaB. The N‐terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.
The identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis
Authors: Allen Andrew , Maskell Duncan
Published: November 2003

Bordetella pertussis lipopolysaccharide (LPS) is biologically active, being both toxic and immunogenic. Using transposon mutagenesis we have identified a genetic locus required for the biosynthesis of LPS in B. pertussis, which has been cloned and sequenced. We have also identified equivalent loci in Bordetella bronchiseptica and Bordetella parapertussis and cloned part of it from B. parapertussis. The amino acid sequences derived from most of the genes present in the sequenced B. pertussis locus are similar to proteins required for the biosynthesis of LPS and other complex polysaccharides from a variety of bacteria. The genes are in a unique arrangement in the locus. Several of the genes identified are similar to genes previously shown to play specific roles in LPS O‐antigen biosynthesis. In particular, the amino acid sequence derived from one of the genes is similar to the enzyme encoded by rfbP from Salmonella enterica, which catalyses the transfer of galactose to the undecaprenol phosphate antigen carrier lipid as the first step in building oligosaccharide O‐antigen units, which are subsequently assembled to form polymerized O‐antigen structures. Defined mutation of this gene in the B. pertussis chromosome results in the inability to express band A LPS, possibly suggesting that the trisaccharide comprising band A is a single O‐antigen‐like structure and that B. pertussis LPS is similar to semi‐rough LPS seen in some mutants of enteric bacteria.
Physical mapping and expression of gene families encoding the N‐acetyl d‐galactosamine adherence lectin of Entamoeba histolytica
Authors: Ramakrishnan Girija , Ragland Brian D. , Purdy Jay E. , Mann Barbara J.
Published: November 2003

Adherence of the enteric protozoan parasite Entamoeba histolytica is mediated by an N‐acetyl d‐galactosamine (GaINAc)‐specific lectin, a heterodimer of heavy (170 kDa) and light (35/31 kDa) subunits. The gene families encoding the lectin subunits were characterized using clamped homogeneous electric field (CHEF) gel electrophoresis in the strain HM1:IMSS. The heavy sub‐unit was shown to be encoded by a family of five hgl genes, which were physically mapped to five distinct Hindlll restriction fragments. The light subunit was shown to be encoded by a family of lgl genes located at six loci in the genome. Heavy and light subunit genes did not appear to be linked. Partial sequences of new members of the hgl and lgl gene families were obtained. Several different strains of E histolytica were found to contain multiple hgl loci in their genomes. Expression of hgl and lgl genes in HM1: IMSS trophozoites was examined under different growth conditions using the reverse transcription‐polymerase chain reaction (RT‐PCR). mRNA transcripts were detected from three hgl genes and three lgl genes, with no significant differences between cultured amoebae and amoebae from liver abscesses. The complexity of GaINAc lectin gene expression observed suggests distinct biological functions for the products of the individual genes during pathogenesis.
The FinOP repressor system of plasmid R1: analysis of the antisense RNA control of traJ expression and conjugative DNA transfer
Authors: Koraimann Günther , Teferle Karin , Markolin Gertrude , Woger Wolfgang , Högenauer Gregor
Published: November 2003

A key determinant of the frequency of IncF plasmid‐mediated DNA transfer between enterobacterial cells is the FinOP system. traJ, a positive regulator of the transfer (tra ) genes is controlled at the post‐transcriptional level by two negative elements, finP and finO. FinP is a plasmid‐specific antisense RNA, whereas finO encodes a proteic co‐repressor which is not plasmid specific but exchangeable among F‐like plasmids. We designed a traJ–lacZ test system that allowed us to monitor the effects of FinP and various FinP mutants on traJ expression. Furthermore, the introduction of finO into the test system enabled us to assess the function of FinO in the interaction of FinP with its target, the traJ mRNA. In this test system, FinP, expressed from a single‐copy plasmid, in the absence of FinO, repressed traJ expression six‐fold. When expressed from a pBR322‐derived multicopy plasmid FinP repressed traJ expression approx. 2000‐fold. This result unambiguously demonstrated that FinP is sufficient to repress traJ expression in a gene dosage‐dependent manner. Mutations of finP creating base exchanges either in loop I or loop II of the two stem‐loop structures of the antisense RNA led to a dramatic decrease in the repressor activity. In a combined loop I–loop II mutation the repressor activity was almost completely lost, supporting the model that the first critical interaction between the two RNA molecules occurs via ‘kissing’ of both loops of the RNAs. Addition of finO to the test system enhanced the repression of traJ expression by FinP by up to two orders of magnitude. This effect of FinO on FinP activity in vivo might indicate that FinO, in addition to its function as an RNA stabilizer, promotes complex formation between the target mRNA and the antisense RNA. Such a function of FinO has recently been shown to exist in vitro (van Biesen and Frost (1994) Mol Microbiol14: 427–436).
The specificity of fumarate as a switching factor of the bacterial flagellar motor
Authors: Barak Rina , Giebel Igal , Eisenbach Michael
Published: November 2003

Fumarate restores to flagella of cytoplasm‐free, CheY‐ containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild‐type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching‐promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo‐taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.
A dual role for the Bacillus subtilis glpD leader and the GlpP protein in the regulated expression of glpD: antitermination and control of mRNA stability
Authors: Glatz Elisabeth , Nilsson Rune‐Pär , Rutberg Lars , Rutberg Blanka
Published: November 2003

The Bacillus subtilis glpD gene encodes glycerol‐3‐phosphate dehydrogenase. This gene is preceded by a leader region containing an inverted repeat which acts as a transcription terminator. Expression of glpD is controlled by antitermination of transcription at the inverted repeat. Antitermination is effected by the glpP gene product in conjunction with glycerol‐3‐phosphate and, consequently, GlpP mutants fail to grow on glycerol as a sole carbon and energy source. We have isolated a number of glycerol‐positive revertants of GlpP mutants. Most of these revertants have mutations in the inverted repeat of the glpD leader and produce glycerol‐3‐phosphate dehydrogenase constitutively. Unlike wild‐type bacteria, they are not sensitive to glucose repression of glpD. A few of the revertants are temperature sensitive, i.e. they grow on glycerol at 32°C but not at 45°C and produce glycerol‐3‐phosphate dehydrogenase only at 32°C. Northern blot analyses demonstrated that the temperature‐sensitive expression of glpD is due to destabilization of glpD mRNA. Furthermore, introduction of the wild‐type glpP gene into the revertants stabilized the glpD mRNA. This is probably a result of a direct interaction between the GlpP protein and the leader of glpD mRNA. Besides its function in antitermination of transcription of glpD, it is suggested that GlpP is also involved in controlling glpD mRNA stability. Introduction of the glpP gene into the revertants also restored glucose repression, indicating that this repression is mediated by the GlpP protein.