bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphic molecular markers from anonymous nuclear DNA for genetic analysis of populations
Authors: Bagley M. J. , Medrano J. F. , Gall G. A. E.
Published: November 2003

A simple method is presented for developing polymorphic, anonymous DNA markers suitable for population genetic studies. Anonymous DNA fragments are screened for sequence variability using a common mutation detection technique (single strand conformation polymorphism analysis; SSCP) and locus‐specific PCR primers are designed for polymorphic DNA fragments. Detection of the markers by SSCP analysis coupled with sequence analysis of SSCP variants allows rapid screening while retaining information about the genealogical relationship among alleles. Variability detected for six markers was assessed in rainbow trout Oncorhynchus mykiss and was compared with variability detected by similar analysis of intron loci. Between three and 12 distinct alleles were observed at each marker locus, and average within‐population heterozygosity ranged from 0.12 to 0.44. Advantages and limitations of the methodology for population genetic analysis are discussed.
Comparative molecular phylogeography of two Xenopus species, X. gilli and X. laevis, in the south‐western Cape Province, South Africa
Authors: Evans B. J. , Morales J. C. , Picker M. D. , Kelley D. B. , Melnick D. J.
Published: November 2003

Xenopus gilli is a vulnerable anuran with a patchy distribution along the south‐western coast of the Cape Province, South Africa. This species is sympatric with Xenopus laevis laevis, a widespread relative found over much of southern Africa. We examined the molecular phylogeography and population structure of the contact zone between these species to obtain information about historical biogeography and conservation management of this region. Analyses of the distribution, frequency, and cladistic and phenetic relationships among mitochondrial DNA haplotypes indicate that population subdivision is present in both taxa but that long‐term isolation of sets of populations has occurred in X. gilli only. Haplotype and nucleotide diversity are also considerably higher within and among X. gilli ponds than X. l. laevis ponds in this region. We attribute the genetic segregation of X. gilli populations to ancient habitat fragmentation by ocean transgression into X. gilli habitat and to continued habitat alteration by human activity. The lower level of genetic diversity in X. l. laevis in this region is likely a result of a recent arrival of this taxon to the south‐western Cape region relative to X. gilli. Population structure in X. l. laevis may be a result of isolation by distance. Clear evidence exists for at least two management units within X. gilli and strongly supports the establishment of protective measures east of False Bay in order to conserve a substantial portion of this species' extant genetic diversity.
Detection and isolation of nuclear haplotypes by PCR‐SSCP
Authors: Ortí G. , Hare M. P. , Avise J. C.
Published: November 2003

SSCP (single‐strand conformational polymorphism) is used widely in the field of human biomedicine, but its potential as a population genetics tool for the recovery of nuclear gene genealogies remains to be realized. We describe and illustrate a use for SSCP in the physical isolation of nuclear haplotypes that circumvents several difficulties associated with more conventional cloning procedures. The DNA sequence can be determined directly from the isolated haplotypes and used for phylogenetic inference. SSCP provides a convenient first step toward generating nuclear genealogies for population studies.
Nucleotide polymorphism in the chalcone synthase‐A locus and evolution of the chalcone synthase multigene family of common morning glory Ipomoea purpurea
Authors: Huttley G. A. , Durbin M. L. , Glover D. E. , Clegg M. T.
Published: November 2003

Chalcone synthase (CHS) is a small multigene family with at least four members (CHS‐A, B, C and PS) in common morning glory Ipomoea purpurea ROTH. The chalcone synthase enzyme performs the initial condensation reaction that results in the 15‐carbon three‐ring structure that is the backbone of flavonoid biosynthesis. The biochemical pathway that commences with CHS is important in plant disease defence, pigment biosynthesis and UV protection. Accordingly, it is of substantial interest to characterize levels and patterns of molecular diversity for genes that encode this important enzyme. We report the sequence of 19 CHS‐A alleles from Mexican and American populations of common morning glory. American populations of this annual self‐compatible vine are believed to have been introduced from Mexico, where the species is native. Individual plants were sampled from populations of common morning glory throughout Mexico and the south‐eastern USA. Four American alleles were sequenced and these, together with one allele from Mexico City, were identical in primary nucleotide sequence. These data suggest a restricted origin for the American population, probably as a consequence of selection for domestication by pre‐Columbian peoples. Additionally the Mitontic (Chiapas, Mexico) population is significantly more homogeneous than expected by chance indicating that this population may also have experienced a recent population bottleneck. Estimates of nucleotide diversity from the Mexican CHS‐A alleles were high. We present evidence that these estimates may, in part, result from low to moderate levels of interlocus recombination/gene conversion. We also present evidence that the ancient duplication of the CHS gene family, preceding the origin of the genus Ipomoea, was associated with heterogeneity in the rate of substitution between the resulting gene family members. The group of gene family members whose sequences possess a signature amino acid of the closely related Stilbene synthase exhibit a significantly faster proportional rate of nonsynonymous substitution.
Genetic heterogeneity, reproductive isolation and host preferences in mealy aphids of the Hyalopterus pruni complex (Homoptera, Aphidoidea)
Authors: Mosco M. C. , Arduino P. , Bullini L. , Barbagallo S.
Published: November 2003

Three taxa were detected by allozyme markers within the mealy aphids of the Hyalopterus pruni complex, having different cultivated Prunus species as main primary hosts. The genetically closer H. pruni and H. amygdali A (DNei= 0.10) never share primary hosts, whereas H. amygdali A and B (DNei= 0.32) may occasionally share them, producing few F1 hybrids. The three species proved reproductively isolated in the field, with no gene exchange. Their speciation seem to have occurred long before the agricultural revolution, crop colonization representing a host range expansion rather than a host shift, as in sympatric speciation.
Effective number of breeding adults in Bufo bufo estimated from age‐specific variation at minisatellite loci
Authors: Scribner K. T. , Arntzen J. W. , Burke T.
Published: November 2003

Estimates of the effective number of breeding adults were derived for three semi‐isolated populations of the common toad Bufo bufo based on temporal (i.e. adult‐progeny) variance in allele frequency for three highly polymorphic minisatellite loci. Estimates of spatial variance in allele frequency among populations and of age‐specific measures of genetic variability are also described. Each population was characterized by a low effective adult breeding number (Nb) based on a large age‐specific variance in mini‐satellite allele frequency. Estimates of Nb (range 21–46 for population means across three loci) were ≊ 55–230‐fold lower than estimates of total adult census size. The implications of low effective breeding numbers for long‐term maintenance of genetic variability and population viability are discussed relative to the species' reproductive ecology, current land‐use practices, and present and historical habitat modification and loss. The utility of indirect measures of population parameters such as Nb and Ne based on time‐series data of minisatellite allele frequencies is discussed relative to similar measures estimated from commonly used genetic markers such as protein allozymes.
Genetic diversity within and among populations of a threatened species: Erodium paularense Fern. Gonz. & Izco
Authors: Martín C. , González‐Benito M. E. , Iriondo J. M.
Published: November 2003

Erodium paularense Fern. Gonz. & Izco (Geraniaceae) is a perennial, endemic species of central Spain. The restricted distribution of its three populations has made it necessary to consider measures for conservation. Random amplified polymorphic DNA markers were used to determine genetic variation among and within populations. Sixty‐three per cent of the bands were polymorphic. No population‐specific marker was identified. Cluster analysis based on Jaccard's coefficient of all the individuals grouped most of them according to the present populations. Within‐population variability was of a similar order in the three populations. Highly significant genetic differences between populations were detected through AMOVA. Within population diversity contributed 80–85% to the species diversity, a value typical of an outbreeding plant.
Noninvasive genetic tracking of the endangered Pyrenean brown bear population
Authors: Taberlet P. , Camarra J.‐J. , Griffin S. , Uhrès E. , Hanotte O. , Waits L. P. , Dubois‐Paganon C. , Burke T. , Bouvet J.
Published: November 2003

Pyrenean brown bears Ursus arctos are threatened with extinction. Management efforts to preserve this population require a comprehensive knowledge of the number and sex of the remaining individuals and their respective home ranges. This goal has been achieved using a combination of noninvasive genetic sampling of hair and faeces collected in the field and corresponding track size data. Genotypic data were collected at 24 microsatellite loci using a rigorous multiple‐tubes approach to avoid genotyping errors associated with low quantities of DNA. Based on field and genetic data, the Pyrenean population was shown to be composed at least of one yearling, three adult males, and one adult female. These data indicate that extinction of the Pyrenean brown bear population is imminent without population augmentation. To preserve the remaining Pyrenean gene pool and increase genetic diversity, we suggest that managers consider population augmentation using only females. This study demonstrates that comprehensive knowledge of endangered small populations of mammals can be obtained using noninvasive genetic sampling.
Phylogeography, population structure and dispersal patterns of the beluga whale Delphinapterus leucas in the western Nearctic revealed by mitochondrial DNA
Authors: O'Corry‐Crowe G. M. , Suydam R. S. , Rosenberg A. , Frost K. J. , Dizon A. E.
Published: November 2003

The recent evolutionary history, population structure and movement patterns of beluga whales in the western Nearctic were inferred from an analysis of mitochondrial DNA control region sequence variation of 324 whales from 32 locations representing five summer concentration areas in Alaska and north‐west Canada. Phylogenetic relationships among haplotypes were inferred from parsimonious networks, and genetic subdivision was examined using haplotypic frequency‐based indices and an analysis of variance method modified for use with interhaplotypic distance data. MtDNA relationships were characterized by a series of star‐like phylogenies which, when viewed in conjunction with information on haplotype frequency and distribution, suggested a rapid radiation of beluga whales into the western Nearctic following the Pleistocene, and an early divergence of the Beaufort Sea from the Chukchi and Bering Seas subpopulations. Overall nucleotide diversity was low (0.51%) yet all major summering concentrations were significantly differentiated (ΦST= 0.33) from one another. Stratification of samples by gender and age from the three northernmost subpopulations suggested that female cohorts from neighbouring subpopulations were more differentiated than males. Further stratification of adult animals by age revealed that older adults were substantially less subdivided among locations than younger adults, particularly for males, suggesting that dispersal, although limited, is biased toward older adult males. Overall, the patterns of mtDNA variation in beluga whales indicated that the summering concentrations are demographically, if not phyletically distinct. Population structure appears to be maintained primarily by natal homing behaviour, while asymmetries in dispersal may be associated with the type of mating system.
Intron variation in marbled murrelets detected using analyses of single‐stranded conformational polymorphisms
Authors: Friesen V. L. , Congdon B. C. , Walsh H. E. , Birt T. P.
Published: November 2003

Combination of the targeted amplification of nuclear introns and the analysis of single‐stranded conformational polymorphisms has the potential to provide an inexpensive, rapid, versatile and sensitive genetic assay for evolutionary studies and conservation. We are developing primers and protocols to analyse nuclear introns in vertebrates, and are testing them in a population genetic study of marbled murrelets Brachyramphus marmoratus. Here we present protocols and results for introns for aldolase B, α‐enolase, glyceraldehyde‐3‐phosphate dehydrogenase and lamin A. Results suggest that this approach presents a potentially powerful method for detecting genetic variation within and among local populations and species of animals: (i) a variety of genes can be surveyed, including genes of special interest such as those involved in disease resistance; (ii) assays are rapid and relatively inexpensive; (iii) large numbers of genes can be assayed, enabling accurate estimation of variation in the total genome; (iv) almost any mutation can be detected in the genes amplified; (v) the exact nature of variation can be investigated by sequence analysis if desired; (vi) statistical methods previously developed for proteins and/or sequence data can be used; (vii) protocols can be easily transferred to other species and other laboratories; and (viii) assays can be performed on old or degraded samples, blood or museum skins, so that animals need not be killed. Results of analyses for murrelets support earlier evidence that North American and Asiatic subspecies represent reproductively isolated species, and that genetic differences exist among murrelets from different sites within North America.
Microsatellite loci reveal highly significant genetic differentiation among Atlantic salmon (Salmo salar L.) stocks from the east coast of Canada
Authors: McConnell S. K. J. , Ruzzante D. E. , O'Reilly P. T. , Hamilton L. , Wright J. M.
Published: November 2003

Allele frequency data from eight microsatellite loci provide evidence of highly significant genetic differentiation among stocks of Atlantic salmon Salmo salar L. from the Bay of Fundy, eastern and north‐western Nova Scotia and Newfoundland. Estimates of genetic structure (RST and θ) were significant both among all samples taken from the different geographical locations and among samples from geographical regions for which more than one stock was sampled. Samples from the Bay of Fundy taken from stocks which are phenotypically and behaviourally diverse showed particularly high levels of genetic structure. Rogers', allele sharing and (δμ)2 distances also revealed significant differences among stock samples and were significantly correlated [Rogers' and (δμ)2] with sea distance between rivers. Results suggest that stocks of Atlantic salmon in eastern Canada are highly diverse genetically and that this should be an important consideration in any management programme for stocks in the area.
Analysis of population genetic structure and variability using RAPD markers in the endemic and endangered Limonium dufourii (Plumbaginaceae)
Authors: Palacios C. , González‐Candelas F.
Published: November 2003

Limonium dufourii (Plumbaginaceae) is a triploid species, with apomictic reproduction, endemic to the east mediterranean coast of Spain, where it is present in only six populations with a few individuals in most of them. L. dufourii is included in the Red List of Endangered Species by the IUCN. Genetic variation and population structure in this species has been studied using RAPDs. Twelve different primers provided 124 reliable bands of which 33 were polymorphic among the 165 individuals analysed. Those polymorphic bands were able to define 44 different patterns, of which all but six were present in only one population. Several methods for statistical evaluation have been used for intra‐ and interpopulation analysis of genetic variability. Relationships among patterns have led to the identification of four main clusters. Two of them show a perfect correspondence to the population of origin of those individuals that present them (Cullera and Torreblanca), and the other two (Groups A and B) include patterns found in individuals coexisting in the same populations (Marjal del Moro populations) and in El Saler. Most of the variation found in this species is due to differences among populations as shown by the analysis of molecular variance. This agrees with the expectation for an apomictic species such as L. dufourii. The analysis of homogeneity of variance shows that substantial differences in the amount of genetic variability present in the six populations exist. These results have been used to understand the evolutionary and demographic history of L. dufourii, which is a requisite in order to establish efficient conservation measures for this species.
Conservation of (GA)n microsatellite loci between Quercus species
Authors: Steinkellner H. , Lexer C. , Turetschek E. , Glössl J.
Published: November 2003

Microsatellite (simple sequence repeat, SSR) amplification was performed in eight different members of the Fagaceae family by using sets of primers developed from sessile oak, Quercus petraea. In total, 136 cases of heterologous amplification were carried out, and 66% resulted in interpretable amplification products. From these, 12 PCR amplification products were sequenced and all 12 contained a sequence homologous to the original locus from Q. petraea. Although SSR primers worked even across different genera, with increasing evolutionary distance there was a clear tendency for decreasing ability to successfully amplify loci and a decreasing proportion of polymorphism amongst those markers which could be amplified. Two of the loci, ssrQpZAG46 and ssrQpZAG110, were polymorphic in all Quercus species tested. Only at one locus, ssrQpZAG58, a specific PCR product could be amplified in all species analysed. For four loci found in two species, we observed significant interspecies differences in the size range of the amplified alleles. Sequence analysis of two alleles showed that the size differences are not only due to variations in the number of (GA) repeats but also to an insertion of approximately 80 nucleotides in the flanking region. Our findings prove the usefulness of SSR markers within and amongst closely related genera of plants.
Genetic population structure in the yellow mongoose, Cynictis penicillata
Authors: Van Vuuren B. Jansen , Robinson T. J.
Published: November 2003

Phylogeographic structure was determined for the yellow mongoose, Cynictis penicillata, using mtDNA RFLPs and control region sequences. The RFLP analysis revealed 13 haplotypes which showed weak geographical patterning consistent with a recent range expansion from a refugial population(s). An analysis of molecular variance (AMOVA) revealed no correspondence between mtDNA phylogeography and subspecies delimitation, nor between matrilines and areas characterized by a high incidence of the viverrid‐type rabies, of which the yellow mongoose is the principal vector. The lack of structure was also shown by control region sequences although four of the maternal lineages shared a near‐perfect 81 bp repeat. We speculate that regional hot spots of the viverrid rabies biotype reflect population density differences in the yellow mongoose that are not underscored by genetic partitioning, at least at the level of resolution provided by our analyses.
Development of multiple genetic markers for studies of genetic variation in arbuscular mycorrhizal fungi using AFLP™
Authors: Rosendahl S. , Taylor J. W.
Published: November 2003

Intraspecific and interspecific genetic variation was studied among arbuscular mycorrhizal fungi. DNA was extracted from single spores and variation was analysed by AFLP (Amplified Fragment Length Polymorphism). The patterns of amplified fragments revealed substantial genetic variation between spores from the same isolates. Possible recombination in the fungi was studied by comparing the obtained data with data generated by artificial recombination of the data sets. No evidence for recombination was found by the analysis, suggesting that the fungi reproduce clonally. The AFLP technique generated a large number of fragments, and the potential for using the technique in population genetic studies of these important unculturable biotrophic fungi is discussed.
Microsatellite polymorphism and population subdivision in natural populations of European sea bass Dicentrarchus labrax (Linnaeus, 1758)
Authors: García De León F. J. , Chikhi L. , Bonhomme F.
Published: November 2003

Polymorphism of microsatellite markers was used to study the genetic variability and structure in natural populations of European sea bass Dicentrarchus labrax. The data consisted of six microsatellite loci analysed for 172 individuals from three samples collected in the Golfe‐du‐Lion (France) and one sample collected in the Golfo‐de‐Valencia (Spain). Our goals were (i) to assess the level of genetic variability as revealed by these markers, (ii) to estimate the genetic differentiation among natural populations within a restricted area, and (iii) to evaluate how microsatellite loci fit the predictions of the two most widely used mutation models (the infinite allele model and the stepwise mutation model). As expected, our results indicate that the genetic polymorphism is very high when compared with previously used genetic markers, the mean expected heterozygosity per locus ranging between 0.69 and 0.93. We also found that all loci but one fitted the infinite allele model better. Using this model as a lower limit, we could extrapolate from the observed diversity effective population sizes on the order of 35 000 individuals. Our results also suggest that there may be a slight genetic differentiation between the two gulfs (FST= 0.007, P < 0.05), indicating that the corresponding populations are likely to be dynamically independent. This finding for a species with high dispersal abilities, if confirmed, has important beatings on fish‐stock assessment.
Male mating success, reproductive success and multiple paternity in a natural population of sand lizards: behavioural and molecular genetics data
Authors: Gullberg A. , Olsson M. , Tegelström H.
Published: November 2003

Sand lizard Lacerta agilis females characteristically mate with several males which, in staged mating experiments, results in multiple paternity of the offspring. In order to investigate multiple paternity in a natural population and interpret male reproductive behaviours in terms of sired young, we sampled the blood of females, potential fathers and hatchlings, and determined paternity using multilocus DNA fingerprinting as well as the variation at a single locus detected by the probe (TC)n. The paternity analyses were preceded by a laboratory experiment in which we established that the parental alleles identified by the single locus probe were inherited in a Mendelian way. Our molecular data demonstrated that 12 out of 13 males (92%) that sired offspring were correctly identified from the 56 sexually mature males in the population. Also smaller males were accepted as sexual partners by the females, but sired fewer young in competition with larger males and were less able to maintain prolonged post‐copulatory mate guarding. This may result in that some sexually successful males are only observed inside a female's home range, but never in pair‐association with the female.
Phylogeography and host association in a pollinating seed parasite Greya politella (Lepidoptera: Prodoxidae)
Authors: Brown J. M. , Leebens‐Mack J. H. , Thompson J. N. , Pellmyr O. , Harrison R. G.
Published: November 2003

Inferring the historical context of ecological diversification is an important step in understanding the way that population‐level processes result in a diversity of species and interactions in communities. We performed a phylogeographic analysis of mitochondrial DNA haplotypes from the pollinating seed parasite Greya politella (Lepidoptera: Prodoxidae) in order to determine the degree to which populations were structured according to geographical location and host‐plant association. Ninety‐eight individuals were sampled from 29 locations ranging from southern California to western Idaho. Restriction‐site variation in 87 individuals (27 populations) was screened by digestion with 11 endonucleases, followed by Southern blotting; 38 restriction‐site positions were mapped by double digests. Haplotypes were further defined by generating fragments 251 bases in length via PCR, screening them for sequence variation using denaturing gel gradient electrophoresis (DGGE), and sequencing the resulting variants. Parsimony analysis of the resulting 12 restriction‐site and 15 sequence haplotypes indicated strong geographical structuring of populations: (i) most populations were monomorphic for haplotype; (ii) haplotypes from California and the Pacific Northwest (Oregon, Washington and Idaho) formed robust monophyletic groups. Population structure was significant both within and between the two regions, as reflected by NST. Patterns of host‐plant association and haplotype phylogeny suggest that populations have recently undergone host‐plant shifts in many different parts of the species range, although the direction and number of host shifts cannot be determined at the present level of sampling resolution.
Migration between nests in the Australian arid‐zone ant Rhytidoponera sp. 12 revealed by DGGE analyses of mitochondrial DNA
Authors: Tek Tay W. , Cook J. M. , Rowe D. J. , Crozier R. H.
Published: November 2003

Denaturing gradient gel electrophoresis (DGGE) of a PCR‐amplified region of the mitochondrial DNA (mtDNA) including a large part of the cytochrome b gene revealed four haplotypes among worker ants from 75 colonies of the queenless ant Rhytidoponera sp. 12. The DGGE results were checked by sequencing examples of the haplotypes; all changes were transitions and two haplotypes differed by only a single substitution. Previous work (e.g. Crozier et al. 1984) showed that intranest relatedness is low for nuclear genes yet neighbouring nests are related; gene flow via winged males appeared the best explanation for this phenomenon. Two mtDNA haplotypes were found in 34.7% of the colonies studied, showing that female movement also occurs between nests. Migration of mated individuals on such a large scale when the number of reproductives is relatively small is unexpected (Crozier & Pamilo 1996). An observed tendency to clumping of the haplotypes is in accordance with the wingless nature of the female dispersers.
Conversion of an extracellular cytolysin into a phagosome‐specific lysin which supports the growth of an intracellular pathogen
Authors: Jones Siân , Preiter Karen , Portnoy Daniel A.
Published: November 2003

Listeria monocytogenes is a facultative intracellular pathogen which secretes a pore‐forming cytolysin, listeriolysin O (LLO), necessary for intracellular growth. Clostridium perfringens is an extracellular pathogen which secretes a related cytolysin, perfrlngolysln O (PFO). When PFO is secreted by intracellular L. monocytogenes, it is toxic to the infected host cell. PFO‐mediated toxicity renders the infected host cell permeable to gentamicin and leads to the death of the intracellular bacteria. In this study, we selected for L. monocytogenes mutants in which PFO supported the intracellular growth of L. monocytogenes. Six independent mutants were isolated, each containing a single amino acid change within the PFO protein. Three classes of PFO mutations were identified, all capable of mediating lysis of the vacuole but without a toxic effect upon the infected host cell. The first class had a severe defect in haemolytic activity. The second class had a change in the pH optimum of PFO. The third class had nearly wild‐type levels of haemolytic activity, but had a decrease in protein half‐life in the host‐cell cytosol. Acquisition of single amino acid changes in PFO were sufficient to convert an extracellular cytolysin into a vacuole‐specific lysin which mediated growth of L. monocytogenes in cultured cells.
Construction of derivatives of the F plasmid pOX–tra715: characterization of traY and traD mutants that can be complemented in trans
Authors: Maneewannakul Kesmanee , Kathir Pushpa , Endley Seema , Moore Deanna , Manchak Jan , Frost Laura , Ippen‐Ihler Karin
Published: November 2003

A derivative of the F plasmid, pOX38–tra715, expresses the entire F tra operon from a foreign promoter (PT7) derived from phage T7. A series of plasmids related to pOX38–tra715 were constructed which carry either deletion mutations or point mutations in traY. When the PT7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied in trans from compatible plasmids. Insertion of a kanamycin‐resistance cassette in the traY gene of the pOX38 plasmid, which contains the wild‐type PY promoter, resulted in loss of F piliation and transfer ability. Introduction of TraY in trans partially restored piliation and transfer suggesting that TraY has a role in positively regulating the PY promoter. pOX38–tra719–traD411, which contains a chloramphenicol‐resistance cassette in place of the kanamycin‐resistance cassette in pOX38–tra715 and a mutation in traD, was constructed to demonstrate the utility of this series of plasmids in studying the long (30 kb) F tra operon.
System to study horizontal gene exchange among microorganisms without cultivation of recipients
Authors: Strätz Michael , Mau Margit , Timmis Kenneth N.
Published: November 2003

Ribosomal RNA genes are characterized by highly conserved sequences and are present in multiple copies in most prokaryotic chromosomes. In principle, therefore, they might serve as sites for homologous recombination between unrelated microorganisms. Plasmids containing 23S ribosomal gene sequences, from different bacteria, which had been interrupted by insertion of a kanamycin‐resistance gene, were used to transform Acinetobacter sp. DSM587 (former name: Acinetobacter calcoaceticus BD413‐ivl10). In all cases, homologies between the 23S rRNA genes of phylogenetically distant bacteria and Acinetobac‐ter sp. DSM587 were sufficient for replacement recombination events. The integration events, resulting in inactivation of any one of the seven rrn operons of Acinetobacter sp. DSM587, had no observable influence on cell growth. These results suggest the possibility of rRNA genes serving as natural vehicles for horizontal gene transfer. They also provide the basis of a novel strategy to analyse gene transfer without selection or cultivation of recipient cells. Because of the highly conserved structure of bacterial rrn operons, recombination events subsequent to gene transfer can be readily identified by polymerase chain reaction amplification of the recombinant sequence using a universal forward primer for the 16S rRNA gene and a reverse primer specific for the integrated marker gene.
Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue
Authors: Binet Rachel , Wandersman Cécile
Published: November 2003

The Serratia marcescens haemophore HasA is secreted by an ABC exporter comprising three envelope proteins. The ABC protein (ATP‐binding cassette) HasD and the MFP protein (membrane fusion protein) HasE but not the outer membrane component have been isolated previously. In Escherichia coli, TolC, the outer membrane component of the haemolysin transporter, can form a hybrid exporter with HasD and HasE. This hybrid secretes HasA and the very similar metalloproteases from S. marcescens and Erwinia chrysanthemi. By analogy, the genuine exporter was predicted to secrete metalloproteases. The hasF gene was thus cloned from S. marcescens into an E. coli tolC mutant carrying hasD and hasE genes, by screening for a proteolytic phenotype on skimmed‐milk plates. hasF encodes a protein sharing 74% identity with the E. coli TolC protein. Anti‐TolC antibodies cross‐reacted with a protein with an apparent molecular weight of 53 kDa in E. coli expressing hasF and in S. marcescens. hasF is unlinked to the has cluster and, unlike the has operon, is not iron regulated. hasF complements some of the tolC phenotypes, including drug‐ and detergent sensitivities and haemolysin secretion but not colicin E1 uptake. This suggests that the various functions of TolC could correspond to distinct domains on the protein.
Co‐ordination between membrane oriC sequestration factors and a chromosome partitioning protein, TolC (MukA)
Authors: Bahloul Abdelkader , Meury Jean , Kern Renée , Garwood Jeremy , Guha Sibajyoti , Kohiyama Masamichi
Published: November 2003

oriC DNA in the hemimethylated (but not in the fully methylated) state reacts with an Escherichia coli K‐12 outer membrane preparation. This reaction is drastically reduced when the membrane preparation of a seqA null mutant is used. An in vitro reconstitution of the activity was undertaken by adding a partially purified SeqA protein to a seqA mutant membrane without success. A possible reason for this failure might be a profound modification of the outer membrane of the seqA mutant (as revealed by the fact that membrane from the mutant sediments more slowly than that from the wild type during ultracentrifugation). There is also a reduction in the content of OmpF protein. Moreover, one of the minor outer membrane proteins involved in partitioning of newly synthesized chromosomes, the TolC (MukA) protein, was also found to be downregulated in the seqA mutant. This is also true of the hobH mutant grown in a high‐osmolarity medium. Mutants of both seqA and hobH stop dividing after hyperosmotic shock, forming filaments (as observed in dam mutants).
YbtA, an AraC‐type regulator of the Yersinia pestis pesticin/yersiniabactin receptor
Authors: Fetherston Jacqueline D. , Bearden Scott W. , Perry Robert D.
Published: November 2003

The pesticin receptor (Psn) of Yersinia pestis confers sensitivity to the bacteriocin, pesticin, and is an integral component of an inorganic‐iron‐transport system that functions at 37°C. Synthesis of Psn is under the control of its own promoter and is regulated by iron and probably by the presence of its cognate siderophore. We have used a psn promoter fusion with lacZ to identify cis‐ and trans‐acting factors which affect transcription of the psn gene. As expected, expression of lacZ from this construct was iron regulated and repressed by Fur. Mutations within a putative siderophore biosynthetic gene (irp2 ) also decreased expression. A set of repeats adjacent to the −35 region of the psn promoter was required for maximum expression of the psn::lacZ gene. Sequence analysis of the region upstream of irp2 revealed the presence of a gene (ybt A) with homology to the AraC family of transcriptional regulators. Insertional inactivation of ybt A resulted in decreased synthesis of Psn and proteins encoded by the irp2 operon as well as decreased expression from the psn::lacZ promoter fusion, indicating that Ybt A is a transcriptional activator for psn and the putative siderophore biosynthetic genes. Ybt A also represses its own transcription.
The N‐terminal ‐barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization
Authors: Mol Olaf , Oudhuis Wendy C. , Fokkema Heleen , Oudega Bauke
Published: November 2003

The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer. The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit‐binding specificity were investigated by analysis of mutant chaperones. FaeE contains a C‐terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones. A C‐terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C‐terminal amino acid residues. Expression and complementation experiments revealed that this C‐terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis. Two hybrid chaperones were constructed. Each hybrid protein contained one ‐barrel domain of FaeE and the other ‐barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively). Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae. The Fan/FaeE hybrid chaperone was active in the biosynthesis of K99 fimbriae. The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG. Cross‐linking experiments suggested that the C‐terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild‐type K88 chaperone. Altogether, the data suggested that the N‐terminal ‐barrel domain of a fimbrial chaperone determines subunit specificity. In the case of the K88 periplasmic chaperone, this N‐terminal domain also determines dimerization of the protein.
Splicing of a group II intron in a functional transfer gene of Lactococcus lactis
Authors: Shearman Claire , Godon Jean‐Jacques , Gasson Michael
Published: November 2003

A chromosomally located sex factor that controls conjugation in Lactococcus lactis 712 has been cloned and sequenced, leading to the discovery of an open reading frame with homology to the maturases of group II self‐splicing introns. Reverse transcriptase polymerase chain reaction amplification was used to demonstrate that the intron was spliced out of mRNA in vivo, and sequence analysis revealed the site of splicing. The intron was inserted within a sex‐factor gene which encodes a protein with homology to proteins involved in rolling‐circle DNA replication. Gene‐disruption experiments were used to demonstrate that this mobA gene was essential for sex‐factor transfer and this suggests that intron splicing is a necessary part of the conjugation process. The sequence of the intron was modelled to produce a secondary structure that exhibited several features characteristic of the IIA subgroup. Here we report the characterization of a new group II intron in the Gram‐positive bacterium L. lactis and demonstrate for the first time in bacteria both splicing in vivo and an active role for the gene carrying the intron.
Circadian clocks in prokaryotes
Authors: Johnson Carl Hirschie , Golden Susan S. , Ishiura Masahiro , Kondo Takao
Published: November 2003

Prokaryotes have long been thought incapable of expressing circadian (daily) rhythms. Recently, however, such biological ‘clocks’ have been discovered in several species of cyanobacteria. These endogenous timekeepers control gene expression on a global level in cyanobacteria. Even in cyanobacterial cultures that are growing with average doubling times more rapid than one per 24 h, the circadian clock controls gene expression and cell division. We have isolated mutants of the cyanobacterial circadian pacemaker and are currently characterizing the loci responsible for their altered period phenotypes.
An isogenic haemolysin‐deficient mutant of Haemophilus ducreyi lacks the ability to produce cytopathic effects on human foreskin fibroblasts
Authors: Palmer Katherine L. , Goldman William E. , Munson, Jr Robert S.
Published: November 2003

The haemolysin of Haemophilus ducreyi is the newest member of the Proteus/Serratia family of pore‐forming toxins. In order to assess the role of the haemolysin in virulence, we constructed an isogenic haemolysin‐deficient mutant of H. ducreyi strain 35000. This strain, designated 35000‐3, lacks detectable haemolytic activity. We tested H. ducreyi strains 35000 and 35000‐3 for their cytopathic activity against human foreskin fibroblasts (HFFs). We observed strong cytopathic activity when strain 35000 was co‐cultured with HFFs. In contrast, cytopathic activity was not observed when strain 35000‐3 was co‐cultured with HFF cells. We also analysed the isogenic pair of H. ducreyi strains for cytopathic activity against HeLa cells and the keratinocyte cell line HaCaT. Strains 35000 and 35000‐3 were strongly cytotoxic when co‐cultured with HeLa cells. HaCaT monolayers were slightly damaged by cocultivation with strain 35000‐3 but this damage was much less than that observed when HaCaT cells were cocultured with strain 35000. These results indicate that the H. ducreyi haemolysin is responsible for the previously observed cytotoxic activity against HFF cells and is partially responsible for the activity observed with HaCaT cells. The haemolysin, however, is not responsible for the activity observed with HeLa cells.
NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals
Authors: López‐Lara Isabel M. , Blok‐Tip Leonore , Quinto Carmen , Garcia Minviluz L. , Stacey Gary , Bloemberg Guido V. , Lamers Gerda E. M. , Lugtenberg Ben J. J. , Thomas‐Oates Jane E. , Spaink Herman P.
Published: November 2003

The nodulation genes of rhizobia are involved in the production of the lipo‐chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild‐type 2‐O‐methylfucose residue at the reducing‐terminal N‐acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro (Macroptilium atropurpureum). Here we show that transfer of nodZ to Rhizobium leguminosarum biovar (bv) viciae, which produces LCOs that are not modified at the reducing‐terminal N‐acetylglucosamine, results in production of LCOs with a fucosyl residue on C‐6 of the reducing‐terminal N‐acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureumGlycine sojaVigna unguiculata and Leucaena leucocephala. Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen‐fixing symbiosis with this plant.
The clp (CS31A) operon is negatively controlled by Lrp, ClpB, and l‐alanine at the transcriptional level
Authors: Martin Christine
Published: November 2003

Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase‐variation control and repressed by l‐alanine. Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, revealed the presence of a regulatory gene, clpB. The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa‐3 fimbriae, respectively. The clp regulatory region contained two deoxyadenosine methylase sites (GATC‐I and GATC‐II). The leucine‐responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC‐II site. The activity of the clp promoter was monitored in a clp–lacZYA single‐copy fusion.The cloned DNA used in this study did not contain a related papI gene. In these conditions, we showed, as expected, that phase variation did not occur. However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase. In the presence of AfaF, a PapI equivalent, the phase‐variation control was restored. We concluded that two regulatory mechanisms were superimposed to control the clp expression. Phase variation, mediated by Lrp and a PapI equivalent, controlled the number of cells producing CS31A in a single colony. The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell. Furthermore, we showed that l‐alanine reduced, by about twofold, the clp promoter activity independently of a PapI equivalent, ClpB, Lrp or Dam methylase. In addition, the presence of l‐alanine prevented the phase‐variation control mediated by AfaF.
Molecular cloning and functional analysis of a novel macrolide‐resistance determinant, mefA, from Streptococcus pyogenes
Authors: Clancy Joanna , Petitpas Joan , Dib‐Hajj Fadia , Yuan Wei , Cronan Melissa , Kamath Ajith V. , Bergeron Jay , Retsema James A.
Published: November 2003

Several streptococcal strains had an uncharacterized mechanism of macrolide resistance that differed from those that had been reported previously in the literature. This novel mechanism conveyed resistance to 14‐ and 15‐membered macrolides, but not to 16‐membered macrolides, lincosamides or analogues of streptogramin B. The gene encoding this phenotype was cloned by standard methods from total genomic digests of Streptococcus pyogenes 02C1064 as a 4.7 kb heterologous insert into the low‐copy vector, pACYC177, and expressed in several Escherichia coli K‐12 strains. The location of the macrolide‐ resistance determinant was established by functional analysis of deletion derivatives and sequencing. A search for homologues in the genetic databases confirmed that the gene is a novel one with homology to membrane‐associated pump proteins. The macrolide‐resistance coding sequence was subcloned into a pET23a vector and expressed from the inducible T7 promoter on the plasmid in E. coli BL21(DE3). Physiological studies of the cloned determinant, which has been named mefA for macrolide efflux, provide evidence for its mechanism of action in host bacteria. E. coli strains containing the cloned determinant maintain lower levels of intracellular erythromycin when this compound is added to the external medium than isogenic clones without mefA. Furthermore, intracellular accumulation of [14C]‐erythromycin in the original S. pyogenes strain was always lower than that observed in erythromycin‐sensitive strains. This is consistent with a hypothesis that the gene encodes a novel antiporter function which pumps erythromycin out of the cell. The gene appears to be widely distributed in S. pyogenes strains, as demonstrated by primer‐specific synthesis using the polymerase chain reaction.
Streptomyces linear plasmids that contain a phage‐like, centrally located, replication origin
Authors: Chang Poa‐Chun , Kim Eung‐Soo , Cohen Stanley N.
Published: November 2003

Unlike previously studied linear replicons containing 5′ DNA termini covalently bound to protein, pSLA2, a 17 kb linear plasmid of Streptomyces rochei, initiates replication internally rather than at the telomeres (Chang and Cohen, 1994). Here we identify and characterize the replication origin of pSLA2, showing that it contains a series of direct repeats (iterons) within a centrally located gene encoding an essential DNA‐binding protein (Rep1); a second essential protein (Rep2), which resembles prokaryotic DNA helicases and has ATPase activity stimulated by single‐stranded DNA, is expressed from the same transcript. A 430 bp locus separated by almost 2 kb from the iterons of the origin specifies an as yet undefined additional function required in cis for plasmid replication. pSCL, a 12 kb linear plasmid of Streptomyces clavuligerus, contains, near the centre of the plasmid, a region configured like the pSLA2 origin. The replication regions of pSLA2 and pSCL, which are capable of propagating plasmid DNA in either a circular or linear form (Shiffman and Cohen, 1992; Chang and Cohen, 1994) resemble those of temperate bacteriophages of the Enterobacteriacae and Bacillus. Our observations suggest that Streptomyces linear plasmids may occupy an evolutionarily intermediate position between circular plasmids and linear phage replicons.