TRS-based PCR as a potential tool for inter-serovar discrimination of Salmonella Enteritidis,S. Typhimurium,S. Infantis,S. Virchow,S. Hadar,S. Newport and S. Anatum

Salmonella enterica subsp. enterica comprises a number of serovars, many of which pose an epidemiological threat to humans and are a worldwide cause of morbidity and mortality. Most reported food infection outbreaks involve the serovars Salmonella Enteritidis and Salmonella Typhimurium. Rapid identification to determine the primary sources of the bacterial contamination is important to the improvement of public health. In recent years, many DNA-based techniques have been applied to genotype Salmonella. Herein, we report the use of a manual TRS-PCR approach for the differentiation of the Salmonella enterica subspecies enterica serovars in a single-tube assay. One hundred seventy Salmonella strains were examined in this work. These consisted of serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum. Five of the TRS-primers, N₆(GTG)₄, N₆(CAC)₄, N₆(CGG)₄, N₆(CCG)₄ and N₆(CTG)₄, perfectly distinguished the S. Enteritidis and S. Typhimurium serovars, and the N₆(GTG)₄ primer additionally grouped the other five frequently isolated serovars. In our opinion, the TRS-PCR methodology could be recommended for a quick and simple DNA-based test for inter-serovar discrimination of Salmonella strains.     

Neutron-scattering studies of the ribosome of Escherichia coli: a provisional map of the locations of proteins S3, S4, S5, S7, S8 and S9 in the 30 S subunit.

Results of neutron-scattering experiments to determine the distances between seven pairs of proteins within the 30 S ribosomal subunit are presented. These results, combined with earlier data (Engelman et al., 1975; Moore et al., 1977) lead to the construction of a three-dimensional map of the positions of the centers of mass of proteins S3, S4, S5, S7, S8 and S9. The properties of this map and its relationship to other information on the structure of the 30 S subunit are discussed.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

Comparative Xylose Metabolism among the Ascomycetes C. albicans,S. stipitis and S. cerevisiae

The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source.     

Solubility of hemoglobin S by sedminetation, equilibrium, and antisickling compounds.

The effect of the compounds guanidine, arginine, lysine, and aspartic acid and the salt arginine aspartate on the solubility of deoxyhemoglobin S (Hb S) was studied by sedimentation equilibrium at 20–22 °C. Guanidine and arginine were found to be most effective, whereas aspartic acid and lysine had only a small effect. The effectiveness of these compounds in solubilizing Hb S is relatively pH independent. It is unlikely that the small effect of lysine and aspartic acid on the solubility of Hb S can account for the antisickling properties of lysine and aspartic acid previously reported (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118). The effect of guanidine and arginine is large enough to account for a large part of such antisickling properties (Sophianopoulos et al. (1974). The nonideality of concentrated hemoglobin solutions (up to 0.3 g cm^?3) has been studied in detail. By using the liganded as well as the unliganded forms of both Hb S and Hb A, it was found that the magnitude of the virial (nonideality) coefficients can change with varying solution conditions. A comparison of pure Hb S with hemolysates is made using viscosity and sedimentation velocity.     

Karyological studies in IberianSonchus (Asteraceae: Lactuceae): S. oleraceus, S. microcephalus andS. asper and a general discussion

Chromosome numbers and karyotypes ofSonchus oleraceus (n=16, 2n=32),S. asper (n=9, 2n=18 for subsp.asper and subsp.glaucescens, andS. microcephalus (n=15, 2n=30) are studied; for the first time their idiograms are given. Karyotypes mainly comprise small chromosomes with a degree of asymmetry 2B inS. oleraceus andS. microcephalus and 2A or 2A-2B inS. asper. No karyological differences can be pointed out betweenS. asper subspecies. Data presented support the amphiploid character ofS. oleraceus, and the origin ofS. microcephalus through a dysploid process involving the former taxon. In Iberian representatives of the genus, diagrams of karyotype asymmetry indices show a cluster grouping for species, with the detached exceptions ofS. maritimus andS. crassifolius, which spontaneously hybridize in central Spain. A review of available karyological data shows that in the evolution of the genusSonchus s.l. and relatives, the basic chromosome number x=9 has generally been maintained. Dysploidy is restricted to the seriesS. bourgeaui (n=8) —S. tenerrimus (n=7) andS. oleraceus (n=16) —S. microcephalus (n=15), evolutionarily related and included in the present subgenusSonchus. Polyploidy has been detected in a total of nine taxa ofSonchus and in the generaEmbergeria, Kirkianella, andDendroseris, being more common in peripheral regions of the distribution area of the group. Five diversification centers are proposed for the whole group, of which the Western Mediterranean area, including the Iberian Peninsula, is related to diversification of the present subgenusSonchus.     

Distribution of 45S and 5S rDNA sites in 23 species of Eleocharis (Cyperaceae)

Studies of rDNA location in holocentric chromosomes of the Cyperaceae are scarce, but a few reports have indicated the occurrence of multiple 45S rDNA sites at terminal positions, and in the decondensed state of these regions in prometaphase/metaphase. To extend our knowledge of the number 45S and 5S rDNA sites and distribution in holocentric chromosomes of the Cyperaceae, 23 Brazilian species of Eleocharis were studied. FISH showed 45S rDNA signals always located in terminal regions, which varied from two (E. bonariensis with 2n = 20) to ten (E. flavescens with 2n = 10 and E. laeviglumis with 2n = 60). 5S rDNA showed less variation, with 16 species exhibiting two sites and 7 species four sites, preferentially at terminal positions, except for four species (E. subarticulata, E. flavescens, E. sellowiana and E. geniculata) that showed interstitial sites. The results are discussed in order to understand the predominance of terminal rDNA sites, the mechanisms involved in the interstitial positioning of 5S rDNA sites in some species, and the events of amplification and dispersion of 45S rDNA terminal sites.     

Flow cytometry and GISH reveal mixed ploidy populations and Spartina nonaploids with genomes of S. alterniflora and S. maritima origin

Background

The genus Spartina exhibits extensive hybridization and includes classic examples of recent speciation by allopolyploidy. In the UK there are two hexaploid species, S. maritima and S. alterniflora, as well as the homoploid hybrid S. × townsendii (2n = 60) and a derived allododecaploid S. anglica (2n = 120, 122, 124); the latter two are considered to have originated in Hythe, southern England at the end of the 19th century.

Methods

Genomic in situ hybridization (GISH) and flow cytometry were used to characterize the genomic composition and distribution of these species and their ploidy levels at Eling Marchwood and Hythe, both near Southampton, southern England.

Key Results

GISH identified approx. 60 chromosomes each of S. maritima and S. alterniflora origin in S. anglica and 62 chromosomes from S. alterniflora and 30 chromosomes from S. maritima in a nonaploid individual from Eling Marchwood, UK. GISH and flow cytometry also revealed that most (94 %) individuals examined at Hythe were hexaploid (the remaining two individuals (6 %) were dodedcaploid; n = 34), whereas hexaploid (approx. 36 % of plants), nonaploid (approx. 27 %) and dodecaploid (approx. 36 %) individuals were found at Eling Marchwood (n = 22).

Conclusions

Nonaploid individuals indicate the potential for introgression between hexaploid and dodecaploid species, complicating the picture of polyploid-induced speciation within the genus. Despite the aggressive ecological habit of S. anglica, it has not out-competed S. × townsendii at Hythe (homoploid hybrids at a frequency of 94 %, n = 34), despite >100 years of coexistence. The success of GISH opens up the potential for future studies of polyploid-induced genome restructuring in this genus.     

The alkenic unreactivity of mono- and bi-cyclic derivatives of 3,6-dihydro-2-(methylthio)-2H-thiopyran S,S,S′,S′-tetraoxide

The inertness of the alkenic bond towards electrophilic additions in 3-exocyano-3-(methylthio)-2-thiabicyclo[2.2.1]hept-5-ene S,S,S′,S′-tetraoxide (5), 3,6-dihydro-2-(methylthio)-2H-thiopyran-2-carbonitrile S,S,S′,S′-tetraoxide (3), and 2-(acetamidomethyl)-3,6-dihydro-2-(methylthio)-2H-thiopyran S,S,S′,S′-tetraoxide (4) is attributed to the “supra-annular effect” and field effects. Conformational analysis of a pentadeuterated derivative of 4 (10) is reported. On the basis of the 220-MHz ¹H n.m.r.-spectral data of 10, the compound was concluded to adopt the ⁰H₂ conformation in chloroform solution.     

Morphological, phytochemical and genetic variation in mixed stands and a hybrid swarm of Senecio germanicus and S. ovatus (Compositae, Senecioneae)

Mixed stands of Senecio ovatus subsp. ovatus and S. germanicus subsp. germanicus occur in the colline belt of central and eastern Europe. The latter species is adapted to more continental climate conditions and shows a later flowering time (August?CSeptember) than the widespread S. ovatus (July?CAugust) that grows in more oceanic climates. We have surveyed 253 plants from 15 populations north of Regensburg (south-eastern Germany) using 16 qualitative and quantitative morphological characters and molecular markers [amplified fragment length polymorphisms (AFLP)] to detect introgressive hybridisation between these two species. Both multivariate statistical analyses based on morphological characters and the Bayesian clustering based on AFLP fingerprint data show that in most populations under study the two species form distinct entities and do not hybridise with each other. However, in one population from the Upper Palatine Forest a high number of intermediate individuals were found. A more detailed genetic (AFLP) and phytochemical (pyrrolizidine alkaloid, PA) analysis based on 125 individuals from this hybrid swarm indicated that these intermediate individuals are backcrosses towards S. germanicus. It is shown that the two species differ considerably concerning the qualitative and quantitative PA patterns and that backcrossed individuals either show an additive PA pattern or a PA pattern similar to S. germanicus, while in quantitative respects all of these individuals are approaching S. germanicus. These findings are discussed in terms of differential selection regimes influencing the fitness of pure and hybrid plants in an area which is an eco-climatological optimum for the more oceanic S. ovatus but which forms a distributional edge for the more continental S. germanicus.     

Sedum plumbizincicola X.H. Guo et S.B. Zhou ex L.H. Wu (Crassulaceae): a new species from Zhejiang Province, China

Sedum plumbizincicola X.H. Guo et S.B. Zhou ex L.H. Wu (Crassulaceae), a new species restricted to lead–zinc mining areas in Zhejiang Province, China, is described and illustrated. This taxon belongs to sect. Sedum (H. Ohba) S.H. Fu based on the adaxially gibbous carpels and follicles. It superficially resembles S. alfredii Hance and three other Sedum species found in the same area, but differs from these other taxa in bearing 4-merous flowers. Differences in geographical distribution, growth habit, phenology, macromorphology, leaf and stem anatomy, as well as seed micromorphology among S. plumbizincicola, S. alfredii and other related taxa in the genus Sedum are also reported. nrDNA internal transcribed spacers (ITS) sequences from seven populations of S. plumbizincicola support the recognition of this as a taxonomic entity distinct from S. alfredii.     

Disease Isolates of Streptococcus pseudopneumoniae and Non-Typeable S. pneumoniae Presumptively Identified as Atypical S. pneumoniae in Spain

We aimed to obtain insights on the nature of a collection of isolates presumptively identified as atypical Streptococcus pneumoniae recovered from invasive and non-invasive infections in Spain. One-hundred and thirty-two isolates were characterized by: optochin susceptibility in ambient and CO₂-enriched atmosphere; bile solubility; PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802, aliB-like ORF2, and a specific 16S rRNA region; multilocus sequence analysis; and antimicrobial susceptibility. By multilocus sequence analysis, 61 isolates were S. pseudopneumoniae, 34 were pneumococci, 13 were S. mitis, and 24 remained unclassified as non-pneumococci. Among S. pseudopneumoniae isolates, 51 (83.6%) were collected from respiratory tract samples; eight isolates were obtained from sterile sources. High frequency of non-susceptibility to penicillin (60.7%) and erythromycin (42.6%) was found. Only 50.8% of the S. pseudopneumoniae isolates displayed the typical optochin phenotype originally described for this species. None harbored the cpsA gene or the pneumococcal typical lytA restriction fragment length polymorphism. The Spn9802 and the specific 16S rRNA regions were detected among the majority of the S. pseudopneumoniae isolates (n = 59 and n = 49, respectively). The ply and pspA genes were rarely found. A high genetic diversity was found and 59 profiles were identified. Among the S. pneumoniae, 23 were capsulated and 11 were non-typeable. Three non-typeable isolates, associated to international non-capsulated lineages, were recovered from invasive disease sources. In conclusion, half of the atypical pneumococcal clinical isolates were, in fact, S. pseudopneumoniae and one-fourth were other streptococci. We identified S. pseudopneumoniae and non-typeable pneumococci as cause of disease in Spain including invasive disease.     

The biology ofSclerotinia sclerotiorum,S. trifoliorum,andS. minor with emphasis on specific nomenclature

The separation ofSclerotinia sclerotiorum (Lib.) de Bary, 5.trifoliorum Erikss., andS. minor Jagger into three distinct species has been based on traditional morphological and physiological criteria such as gross cultural characteristics, sclerotial size, ascus and ascospore dimensions, time of apothecial development in the field, and host association. However, these characteristics tend to be variable and some workers have concluded that the three fungi should be included in one species, 5.sclerotiorum. Recently, new data have been published on morphological, cultural, physiological, ontogenetic, enzyme pattern, mycelial interaction, and cytological characteristics of isolates ofSclerotinia spp. This information supplements, but does not replace, that available from more traditional taxonomic methods and helps to resolve the controversy on the taxonomy and nomenclature of these fungi. This article reviews the relevant literature on the biology of 5.sclerotiorum, S. trifoliorum, and 5.minor, with particular emphasis given to those differences between them that could be of significance regarding their specific nomenclature. After an introduction, mycelia, microconidia, sclerotia, apothecia, infection, control, and taxonomy and nomenclature are discussed. The authors conclude thatS. sclerotiorum, S. trifoliorum, andS. minor are distinct species. The characteristics used to distinguish between them are summarized in table form.     

Positions of proteins S6, S11 and S15 in the 30 S ribosomal subunit of Escherichia coli

A map of the positions of 12 of the 21 proteins of the 30 S ribosomal subunit of Escherichia coli (S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 and S15), based on neutron scattering, is presented and discussed. Estimates for the radii of gyration of these proteins in situ are also obtained. It appears that many ribosomal proteins have compact configurations in the particle.     

An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species,Sonneratia alba,S. caseolaris and S. ovata: development of a bioassay method for allelopathy,the protoplast co-culture method

A bioassay method for allelopathy, the ‘protoplast co-culture method’ was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl₂, MgSO₄ and CaCl₂) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog’s (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3 % sucrose and 0.6–0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the ‘sandwich method’ with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.     

Distribution and Frequency of kdr Mutations within Anopheles gambiae s.l. Populations and First Report of the Ace.1G119S Mutation in Anopheles arabiensis from Burkina Faso (West Africa)

An entomological survey was carried out at 15 sites dispersed throughout the three eco-climatic regions of Burkina Faso (West Africa) in order to assess the current distribution and frequency of mutations that confer resistance to insecticides in An. gambiae s.l. populations in the country. Both knockdown (kdr) resistance mutation variants (L1014F and L1014S), that confer resistance to pyrethroid insecticides, were identified concomitant with the ace-1 G119S mutation confirming the presence of multiple resistance mechanisms in the An. gambiae complex in Burkina Faso. Compared to the last survey, the frequency of the L1014F kdr mutation appears to have remained largely stable and relatively high in all species. In contrast, the distribution and frequency of the L1014S mutation has increased significantly in An. gambiae s.l. across much of the country. Furthermore we report, for the first time, the identification of the ace.1 G116S mutation in An. arabiensis populations collected at 8 sites. This mutation, which confers resistance to organophosphate and carbamate insecticides, has been reported previously only in the An. gambiae S and M molecular forms. This finding is significant as organophosphates and carbamates are used in indoor residual sprays (IRS) to control malaria vectors as complementary strategies to the use of pyrethroid impregnated bednets. The occurrence of the three target-site resistance mutations in both An. gambiae molecular forms and now An. arabiensis has significant implications for the control of malaria vector populations in Burkina Faso and for resistance management strategies based on the rotation of insecticides with different modes of action.