EMBL Accession Number Research Articles

'Massilibacteroïdes vaginae' gen. nov., sp. nov., a new genus within the family of Bacteroïdaceae isolated from a patient with vaginosis.

‘Massilibacteroïdes vaginae’ sp. nov. strain MV12T is the type strain of ‘Massilibacteroïdes’ gen. nov., a new genus within the family of Porphyromonadaceae. This strain was isolated in Marseille from a patient with vaginosis. ‘Massilibacteroïdes vaginae’ is a Gram-negative, aero-anaerobic bipolar bacillus. Here we describe the phenotypic characteristics and complete genome sequence of this bacterium. The EMBL accession number is FXDJ00000000 (FXDJ01000001–FXDJ01000009), and its genome consists of nine scaffolds. The strain was deposited in two culture collections under the numbers CSUR P1477 and DSM 100704.

The IgE-diagnostic impact of a recombinant serine protease inhibitor from Hevea brasiliensis in latex-allergic health care workers

» Amplifi cation of the SPI-sequence from a cDNA of Hevea brasiliensis » Identifi cation of the sequence in a pDrive vector system (EMBL accession number: HF937118) » Expression of the SPI variant in the pMAL system » Preparative isolation of a soluble, recombinant maltose-binding protein (MBP)-SPI fusion protein » Biotinylation of the SPI-hybrid and coupling onto StreptavidineImmunoCAPs (ThermoFisher Scientifi c, Uppsala , Sweden) » 21 sera of health care workers (HCW) with symptoms to natural rubber latex (NRL) were tested for SPI and all available single latex allergens

Cloning and Characterization of a Candidate Auxin Plant Growth Regulator-Activated Cell Surface Hydroquinone (NADH) Oxidase

ENOX (ECTO-NOX) proteins of the external surface of the plasma membrane catalyze oxidation of both NADH and hydroquinones and protein disulfide-thiol interchange. They exhibit both prion-like and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate a regular period of 24 min in length. Here we report the cloning, expression and characterization of a constitutive plant ENOX protein activated by both natural (Indole-3-acetic acid, IAA) and synthetic (2,4-dichlorophenoxyacetic acid, 2,4-D) auxin plant growth regulators with an optimum of about 1 μM, higher concentrations being less effective. The gene encoding the 213 amino acid protein (ABP20) is found in EMBL accession number U81162. Functional motifs characteristic of ENOX1 proteins, previously identified by site-directed mutagenesis, are present in the candidate auxin-activated ENOX (dNOX, ENOX5), including adenine nucleotide and copper binding motifs along with essential cysteines and a motif having homology with a previously identified auxin-binding motif. Periodicity was exhibited by both the oxidative and protein disulfide-thiol inter-change activities as is characteristic for other ENOX proteins. Activity was blocked by the ENOX2-specific quassinoid inhibitor glaucarubolone and other ENOX2 inhibitors but not by the ENOX1-specific quassinoid inhibitor simalikalactone D. Activity required both auxin and bound copper. The inactive auxin 2,3-D was without effects.

Capnobotryella isiloglui, a new rock-inhabiting fungus from Austria

A new rock-inhabiting fungal taxon was isolated from St. Margarethen Church in Vienna, Austria. Based on small subunit and internal transcribed regions of rDNA sequencing, the new taxon was described as Capnobotryella isiloglui Sert & Sterflinger. The type culture of the taxa is in the ACBR culture collection (Vienna, Austria, MA 3619, EMBL Accession Number: AM746201.1).

Two new species of Capnobotryella from historical monuments

In this paper, two new species of Capnobotryella, melanized fungi with meristematic development, are described. On the basis of small subunit and internal transcribed sequence regions of ribosomal DNA sequencing, both of the new species are accommodated in the genus Capnobotryella: C. erdogani and C. kiziroglui. The type cultures of the taxa are in the ACBR culture collection (MA 4625, EMBL Accession Number: AJ972857.1; MA 4899, EMBL Accession Number: AJ972859.1).

Computational studies of some DNA-based photonic multilayered structures

Using detrended fluctuation analysis and wavelet transform multifractal analysis, introns from the human transketolase pseudogene (EMBL accession number: Z49258) were found to exhibit persistent long-range correlations (LRCs) with homogeneous fractal fluctuations. DNA-intron-based photonic multilayers were then constructed from the intron sequences by identifying their purine/pyrimidine components as homogeneous dielectric layers and the normal-incidence propagation of electromagnetic waves through them was examined. The richness of their transmittance spectra, in terms of resonant peaks, was found to be directly related to the strength of LRCs and to the number of layers in them. Photon distributions that are visually neither extended nor strictly localized appeared in some intron multilayers. The DNA-intron-based multilayers studied here seem to be materials situated in the regime between absolute disorder and Fibonacci quasi-periodicity.

Novel genetic environment of plasmid-mediated quinolone resistance gene qnrB2 in Salmonella Bredeney from poultry

Novel genetic environment of plasmid-mediated quinolone resistance gene qnrB2 in Salmonella Bredeney from poultry

First report of Tomato chlorosis virus on tomato crops in Cyprus

In the summer of 2004, yellowing symptoms similar to those causedby nitrogen and/or magnesium deficiency were observed in field- andglasshouse-grown tomatoes ( Lycopersicon esculentum ), in the Parekklisiaarea of Cyprus. Initially, lower leaves showed extensive interveinal yellowingwith necrotic flecks, brittleness and occasional upward leaf rolling, beforefinally the whole plant turned yellow. Similar symptoms were observedduring 2005 in glasshouse tomatoes grown in areas located on the south-west coastal region of the island. The abundance of whiteflies on theaffected plants suggested the involvement of the whitefly-transmittedTomato chlorosis virus (ToCV) and/or Tomato infectious chlorosis virus(TICV), both of the genus Crinivirus (Wisler et al., 1998).Leaves of 18 affected plants were collected, total RNA was isolated andRT-PCR was performed in a single tube using primers HS-11 and HS-12,followed by a multiplex nested-PCR with primers TIC-3/TIC-4 and ToC-5/ToC-6, for the detection of TICV and ToCV, respectively (Dovas et al.,2002). A PCR product of 463 bp, corresponding to the HSP 70 gene ofToCV, was amplified for all tested samples. The sequences of four clonedPCR products were identical (EMBL accession number AM158958) andshowed 99% nucleotide identity to a ToCV isolate from Florida (accessionnumber AY903448). ToCV is vectored by Bemisia tabaci (biotypes A whitefly-transmitted, phloem-limited, bipartite closterovirus of tomato. andB), Trialeurodes vaporariorum and T. abutilonea. Although there havebeen no systematic studies on whitefly incidence and distribution in Cyprus,it seems that B. tabaci is the predominant species present, as Tomatoyellow leaf curl virus (Ioannou, 1985) and Cucurbit yellow stunting disordervirus (Papayiannis et al., 2005), vectored by this species, are prevalent intomatoes and cucurbit crops, respectively. On the other hand, theincidence of Beet pseudo-yellows virus (transmitted by T. vaporariorum)is much lower. This is the first report of ToCV in Cyprus.ReferencesDovas CI, Katis NI, Avgelis AD, 2002. Multiplex detection of criniviruses associated with epidemics of a yellowing disease of tomato in Greece.

Occurrence of Sweet potato leaf curl virus in Sicily

The Ipomoea -infecting begomoviruses are an evolutionary curiosity. Two such viruses have been identified: Sweet potato leaf curl virus (SPLCV; Lotrakul & Valverde, 1999) and Sweet potato leaf curl Georgia virus (SPLCGV), originating from the USA (SPLCV and SPLCGV) and Spain [SPLCV-Ipo, previously known as Ipomoea yellow vein virus (Banks et al ., 1999) but now recognized as a distinct strain of SPLCV]. These viruses are monopartite (no DNA B or DNA β component has been identified for any of the viruses), have a genome organization typical of Old World begomoviruses, and are distinct from all other begomoviruses. Cuttings of Ipomoea indica (Convolvulaceae) showing yellow vein symptoms were collected in the vicinity of Catania, Sicily in 1999. A full-length clone of the begomovirus associated with the disease was produced by PCR-mediated amplification with a pair of primers abutting at their 5 ′ ends and spanning (for SPLCV-Ipo) nucleotides 697–758 (virion-sense primer 5 ′ -GGATCCGCTGAACTTTGGCCAGATCTTCACTATG-3 ′ ; complementary-sense primer 5 ′ -GGATCCTTATTGGGCCTTGTATCACGAATCAACC-3 ′ ). These were designed to the sequence of SPLCV-Ipo (EMBL accession number AJ132548) and span a unique Bam HI restriction endonuclease site. The full-length PCR product was cloned into the T-Easy vector (Promega) and the sequence of a single clone was determined. This sequence (accession no. AJ586885) is 2830 nucleotides long and shows the arrangement of genes typical of the genomes (or DNA A components) of Old World begomoviruses. The virus is closely related to other begomoviruses isolated from Ipomoea spp., including SPLCGV (84·2% nucleotide sequence identity), SPLCV (89·9%) and SPLCV-Ipo (90·9%), with which it clusters in phylogenetic analyses. We conclude that the isolate originating from Sicily is a strain of SPLCV which is provisionally designated SPLCV-[Sicily]. The occurrence of this begomovirus species in Sicily indicates that its geographical range extends further across the Mediterranean basin than previously identified.

Purification, cloning and functional expression of hydroxyphenylpyruvate reductase involved in rosmarinic acid biosynthesis in cell cultures of Coleus blumei.

Hydroxyphenylpyruvate reductase (HPPR) is an enzyme involved in the biosynthesis of rosmarinic acid in Lamiaceae reducing hydroxyphenylpyruvates in dependence of NAD(P)H to the corresponding hydroxyphenyllactates. The HPPR protein was purified from suspension cells of Coleus blumei accumulating high levels of rosmarinic acid by ammonium sulfate precipitation, anion exchange chromatography, hydroxylapatite chromatography, chromatography on 2',5'-ADP-Sepharose 4B and SDS-polyacrylamide gel electrophoresis. The protein was tryptically digested and the peptides sequenced. Sequence information was used to isolate a full-length cDNA-clone for HPPR (EMBL accession number AJ507733) by RT-PCR, screening of a C. blumei cDNA-library and 5'-RACE-PCR. The open reading frame of the HPPR-cDNA consists of 939 nucleotides encoding a protein of 313 amino acid residues. The sequence showed that HPPR belongs to the family of D-isomer-specific 2-hydroxyacid dehydrogenases. The HPPR-cDNA was heterologously expressed in Escherichia coli and the protein was shown to catalyse the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenylpyruvate to 3,4-dihydroxyphenyllactate.

Two subfamilies of plant purple acid phosphatases

The cDNA sequences of two purple acid phosphatases (previously purified AcPase1 and putative AcPase2) from yellow lupin (Lupinus luteus L) have been determined. AcPase1 mRNA is 1755 nt in length, including a single open reading frame of 1434 nt that encodes 477 amino acid residues (EMBL accession number AJ458943). The AcPase2 mRNA is a 170‐nt molecule, including a single open reading frame of 1392 nt that encodes 463 amino acid residues (EMBL accession number AJ505579). Both sequences were compared with known purple acid phosphatases (PAPs). The analysis of all known PAP sequences showed that they are divided into two subfamilies. The characteristic feature of the first subfamily containing AcPase1 is the lack of cysteine residues in the C‐terminal region of the polypeptide around position 350. AcPase2 belongs to the second subfamily which contains a disulphide bridge formed of cysteines present around position 350. A possible relationship between structure and substrate specificity in these two PAP subfamilies is presented. Expression of AcPase1 and AcPase2 shows that the first enzyme is produced in the early stage of germination (its mRNA in polyadenylated form is present already in dry seeds), whereas the second phosphatase is produced mainly in the roots of mature plants. The early expression of AcPase1 was confirmed by a comparative analysis of a cotranslational N‐glycosylation of AcPase1 and diphosphonucleotide phosphatase/phosphodiesterase (PPD1) known to be expressed in the final stage of germination.

Cyclic AMP-dependent protein kinase is involved in morphogenesis of Aspergillus niger.

The cAMP signal transduction pathway controls many processes in fungi. The pkaR gene, encoding the regulatory subunit (PKA-R) of cAMP-dependent protein kinase (PKA), was cloned from the industrially important filamentous fungus Aspergillus niger. To investigate the involvement of PKA in morphology of A. niger, a set of transformants which overexpressed pkaR or pkaC (encoding the catalytic subunit of PKA) either individually or simultaneously was prepared as well as mutants in which pkaR and/or pkaC were disrupted. Strains overexpressing pkaR or both pkaC and pkaR could not be distinguished from the wild-type, suggesting that regulation of PKA activity is normal in these strains. Absence of PKA activity resulted in a two- to threefold reduction in colony diameter on plates. The most severe phenotype was observed in the absence of PKA-R, i.e., very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Suppressor mutations easily developed in the DeltapkaR mutant and one of these mutants appeared to lack PKA-C activity. These data suggest that cAMP-dependent protein phosphorylation in A. niger regulates growth polarity and formation of conidiospores.

Swarming motility in Bacillus cereus and characterization of a fliY mutant impaired in swarm cell differentiation.

This report describes a new behavioural response of Bacillus cereus that consists of a surface-induced differentiation of elongated and hyperflagellated swarm cells exhibiting the ability to move collectively across the surface of the medium. The discovery of swarming motility in B. cereus paralleled the isolation of a spontaneous non-swarming mutant that was found to carry a deletion of fliY, the homologue of which, in Bacillus subtilis, encodes an essential component of the flagellar motor-switch complex. However, in contrast to B. subtilis, the fliY mutant of B. cereus was flagellated and motile, thus suggesting a different role for FliY in this organism. The B. cereus mutant was completely deficient in chemotaxis and in the secretion of the L2 component of the tripartite pore-forming necrotizing toxin, haemolysin BL, which was produced exclusively by the wild-type strain during swarm-cell differentiation. All the defects in the fliY mutant of B. cereus could be complemented by a plasmid harbouring the B. cereus fliY gene. These results demonstrate that the activity of fliY is required for swarming and chemotaxis in B. cereus, and suggest that swarm-cell differentiation is coupled with virulence in this organism.

Adhesion to cellulose of the Gram-positive bacterium Ruminococcus albus involves type IV pili.

This study was aimed at characterizing a cell-surface 25 kDa glycoprotein (GP25) that was previously shown to be underproduced by a spontaneous adhesion-defective mutant D5 of Ruminococcus albus 20. An antiserum against wild-type strain 20 was adsorbed with the mutant D5 to enrich it in antibodies 'specific' to adhesion structures of R. albus 20. The resulting antiserum, called anti-Adh serum, blocked adhesion of R. albus 20 and reacted mainly with GP25 in bacterial and extracellular protein fractions of R. albus 20. The N-terminal sequence of purified GP25 was identical to that of CbpC, a 21 kDa cellulose-binding protein (CBP) of R. albus 8. The nucleotide sequence of the gp25 gene was determined by PCR and genomic walking procedures. The gp25 gene encoded a protein of 165 aa with a calculated molecular mass of 16940 Da that showed 72.9% identity with CbpC and presented homologies with type IV pilins of Gram-negative pathogenic bacteria. Negative-staining electron microscopy revealed fine and flexible pili surrounding R. albus 20 cells while mutant cells were not piliated. In addition, immunoelectron microscopy showed that the anti-Adh serum probing mainly GP25, completely decorated the pili surrounding R. albus 20, thereby showing that GP25 was a major pilus subunit. This study shows for the first time the presence of pili at the surface of R. albus and identifies GP25 as their major protein subunit. Though GP25 was not identified as a CBP, isolated pili were shown to bind cellulose. In conclusion, these pili, which belong to the family of type IV pili, mediate adhesion of R. albus 20 to cellulose.

Disruption in Candida albicans of the TPS2 gene encoding trehalose-6-phosphate phosphatase affects cell integrity and decreases infectivity.

The gene CaTPS2 encoding trehalose-6-phosphate (T6P) phosphatase from Candida albicans has been cloned and disrupted in this organism. The Catps2/Catps2 mutant did not accumulate trehalose but accumulated high levels of T6P. Disruption of the two copies of the CaTPS2 gene did not abolish growth even at 42 degrees C, but decreased the growth rate. In the stationary phase, the Catps2/Catps2 mutant aggregated, more than 50% of its cells became permeable to propidium iodide and a large amount of protein was found in the culture medium. Aggregation occurred only at pH values higher than 7 and was avoided by osmoprotectants; it was never observed during the exponential phase of growth. The mutant formed colonies with a smooth border on Spider medium. Mice inoculated with 1.5 x 10(6) c.f.u. of wild-type cells died after 8 days, while 80% of those inoculated with the same number of c.f.u. of the Catps2/Catps2 mutant survived for at least 1 month. Reintroduction of the wild-type CaTPS2 gene in the Catps2/Catps2 mutant abolished the phenotypes described. It is hypothesized that the accumulation of T6P interferes with the assembly of a normal cell wall.

Transport Activity and Surface Expression of the Na+-Ca2+ Exchanger NCX1 Are Inhibited by the Immunosuppressive Agent Cyclosporin A and by the Nonimmunosuppressive Agent PSC833

Cyclosporin A (CsA) treatment of HEK 293 cells expressing the rat heart RHE-1 (NCX1.1, EMBL accession number ) or the rat brain RBE-2 (NCX1.5, GenBank(TM) accession number ) Na(+)-Ca(2+) exchanger inhibited their transport activity in a concentration-dependent manner. The inhibition was detectable at 2 microm CsA, and exposure of the cells to 20 microm CsA resulted in a decrease of the Na(+)-dependent Ca(2+) uptake to about 20% relative to that of untreated cells. Determination of the surface expression of the exchanger protein revealed a parallel concentration-dependent reduction in the amount of the immunoreactive protein. No reduction was detected in the amount of total immunoreactive exchanger protein in CsA-treated cells relative to untreated ones. Among the different drugs tested, only PSC833, an analog of cyclosporin D, mimicked the effects of CsA. Exposure of the transfected cells to the chemically related cyclosporin D and macrolide drugs (FK506 or rapamycin) had no effect on the transport activity or the surface expression of the Na(+)-Ca(2+) exchanger. Co-expression of the human multidrug transporter P-glycoprotein (of which both drugs are modulators) with the cloned Na(+)-Ca(2+) exchanger revealed that transport activity and surface expression of each transporter in the co-transfected system were similar to those of each transporter alone in both the presence and absence of CsA or PSC833. CsA and PSC833 inhibited the surface expression of the NCX1 protein but did not alter the surface expression of P-glycoprotein. Unlike some P-glycoprotein endoplasmic reticulum-retained mutants (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 709-712), CsA did not rescue RBE-2/F913-->Stop, an endoplasmic reticulum-retained function-competent mutant of the Na(+)-Ca(2+) exchanger (Kasir, J., Ren, X., Furman, I., and Rahamimoff, H. (1999) J. Biol. Chem. 274, 24873-24880) and did not induce its kinesis to the surface membrane, further demonstrating molecular differences between P-glycoprotein and NCX1 mutants for interaction with CsA.

Engineering of a single chain variable fragment antibody specific for the Citrus tristeza virus and its expression in Escherichia coli and Nicotiana tabacum

Citrus tristeza virus (CTV) is one of the most destructive citrus virus diseases in the world. The construction of an engineered antibody, EMBL accession number AJ278109, able to specifically recognize its antigen, i.e. the coat protein of CTV, directly on infected plant material without any purification or manipulation of the entire woody plant. The potential uses of this engineered antibody are discussed.

Sunflower necrosis disease from India is caused by an ilarvirus related to Tobacco streak virus

A serious disease of sunflower (Helianthus annuus) with putative virus aetiology was found occurring in all major sunflower-growing regions of India. Symptoms of this sunflower necrosis disease (SND) comprised chlorotic and necrotic ringspots and leaf distortion. A general leaf and stem necrosis extending to midveins, petioles and flower bracts eventually results in stunting and dieback, especially when plants become infected in the early stages of development. Ilarvirus-like particles were detected by electron microscopy in crude sap of SND-affected sunflower, and in Chenopodium quinoa plants inoculated with leaf extracts prepared from SND-affected sunflower plants. In addition to several other herbaceous virus indicator plants, groundnut, cowpea and cotton, significant crops in India, also became infected. Back-transmission to healthy sunflower seedlings with leaf extracts of systemically infected indicator plants resulted in identical symptoms of SND, confirming the ilarvirus-like component as the causative agent of sunflower necrosis disease. An antiserum (DSMZ AS-0615, Braunschweig, Germany) raised against purified virus preparations reacted well in DAS±ELISA and proved reliable for field diagnosis of Sunflower necrosis virus (SNV). In a comparative serological study using plants infected with other ilarviruses, a reaction was demonstrated only with Tobacco streak virus (TSV). This serological relationship was confirmed by Western blot analysis and by immunosorbent electron microscopy decoration assays using SNV and TSV antisera in reciprocal tests. In RT±PCR, using oligonucleotide primers deduced from conserved sequences within TSV RNA3 and flanking the entire coat protein region, an approximately 1000 bp dsDNA fragment was amplified from SNV-infected sunflowers. Sequence analysis of cloned SNV PCR fragments revealed nucleotide identities of approximately 90% with TSV RNA3 (EMBL accession number X00435) and a coat protein amino acid homology between SNV and TSV of more than 90%. The variability of nucleotide sequences retrieved from three sunflower isolates from different locations was less than 3%. These experimental data unequivocally prove that the virus causing sunflower necrosis disease in many sunflower varieties and in many different sunflowergrowing regions of India is a strain of Tobacco streak virus. It is now important to establish whether TSV is associated with diseases of other significant crops, such as groundnut, in India. Plant Pathology (2001) 50, 800

Sequence and structure of the mouse connexin45 gene.

The connexin45 (Cx45) gene was cloned from a mouse genomic Bacterial Artificial Chromosome library. Approximately 8.4 kb of the genomic DNA was sequenced, and the structure of the Cx45 gene was determined. The mouse Cx45 gene is composed of 3 exons, with the entire coding sequence contained within exon III (EMBL Accession Number AJ300716). This structure is unique for the Cx45 gene, since all other members of the connexin family have only two exons. In addition, computer analysis reveals a potential TATA box and two putative AP-1 binding sites in the 5' region of the gene. Sequence alignment with connexin43 indicates substantial homology in the intronic sequences upstream of the 3' exons of the two genes, suggesting that the Cx45 gene is inherently similar to the rest of the connexin family, and that it probably evolved from an ancestor common to the other connexins.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis.

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.