Proteome analysis of the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae

The plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight, which is one of the most serious diseases of rice. Xoo has been studied for over one century, and much has been learned about it, but proteomic investigation has been neglected. In this study, proteome reference maps of Xoo were constructed by two-dimensional gel electrophoresis, and 628 spots in the gels representing 469 different protein species were identified with MALDI-TOF/TOF MS. The identified spots were assigned to 15 functional categories according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the annotations from the National Center for Biotechnology Information (NCBI) database. The data set has been deposited in the World-2DPAGE database (Database ID: 0044). In addition, comparative proteomic analysis revealed that proteins related to the TonB-dependent transportation system and energy metabolism are involved in the phenazine-1-carboxylic acid resistance in Xoo. In conclusion, we have established a proteome database for Xoo and have used this database in a comparative proteomic analysis that identified proteins potentially contributing to phenazine-1-carboxylic acid resistance in Xoo.     

Construction and characterization of a BAC library from a gynogenetic channel catfish Ictalurus punctatus

A bacterial artificial chromosome (BAC) library was constructed by cloning HindIII-digested high molecular weight DNA from a gynogenetic channel catfish, Ictalurus punctatus, into the vector pBeloBAC11. Approximately 53 500 clones were arrayed in 384-well plates and stored at -80°C (CCBL1), while clones from a smaller insert size fraction were stored at -80°C without arraying (CCBL2). Pulsed-field gel electrophoresis of 100 clones after NotI digestion revealed an average insert size of 165 kb for CCBL1 and 113 kb for CCBL2. Further characterization of CCBL1 demonstrated that 10% of the clones did not contain an insert. CCBL1 provides a 7.2-fold coverage of the channel catfish haploid genome. PCR-based screening demonstrated that 68 out of 74 unique loci were present in the library. This represents a 92% chance to find a unique sequence. These libraries will be useful for physical mapping of the channel catfish genome, and identification of genes controlling major traits in this economically important species.     

Construction of a BAC library of Korean ginseng and initial analysis of BAC-end sequences

We estimated the genome size of Korean ginseng ( Panax ginseng C.A. Meyer), a medicinal herb, constructed a Hin dIII BAC library, and analyzed BAC-end sequences to provide an initial characterization of the library. The 1C nuclear DNA content of Korean ginseng was estimated to be 3.33 pg (3.12×10³ Mb). The BAC library consists of 106,368 clones with an average size of 98.61 kb, amounting to 3.34 genome equivalents. Sequencing of 2167 BAC clones generated 2492 BAC-end sequences with an average length of 400 bp. Analysis using BLAST and motif searches revealed that 10.2%, 20.9% and 3.8% of the BAC-end sequences contained protein-coding regions, transposable elements and microsatellites, respectively. A comparison of the functional categories represented by the protein-coding regions found in BAC-end sequences with those of Arabidopsis revealed that proteins pertaining to energy metabolism, subcellular localization, cofactor requirement and transport facilitation were more highly represented in the P. ginseng sample. In addition, a sequence encoding a glucosyltransferase-like protein implicated in the ginsenoside biosynthesis pathway was also found. The majority of the transposable element sequences found belonged to the gypsy type (67.6%), followed by copia (11.7%) and LINE (8.0%) retrotransposons, whereas DNA transposons accounted for only 2.1% of the total in our sequence sample. Higher levels of transposable elements than protein-coding regions suggest that mobile elements have played an important role in the evolution of the genome of Korean ginseng, and contributed significantly to its complexity. We also identified 103 microsatellites with 3–38 repeats in their motifs. The BAC library and BAC-end sequences will serve as a useful resource for physical mapping, positional cloning and genome sequencing of P. ginseng.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. Grandbastien     

Comprehensive DNA copy number profile and BAC library construction of an Indian individual

Bacterial artificial chromosomes (BACs) are used in genomic variation studies due to their capacity to carry a large insert, their high clonal stability, low rate of chimerism and ease of manipulation. In the present study, an attempt was made to create the first genomic BAC library of an anonymous Indian male (IMBL4) consisting of 100,224 clones covering the human genome more than three times. Restriction mapping of 255 BAC clones by pulse field gel electrophoresis confirmed an average insert size of 120 kb. The library was screened by PCR using SHANK3 (SH3 and multiple ankyrin repeat domains 3) and OLFM3 (olfactomedin 3) specific primers. A selection of clones was analyzed by fluorescent in situ hybridization (FISH) and sequencing. Fine mapping of copy number variable regions by array based comparative genomic hybridization identified 467 CNVRs in the IMBL4 genome. The IMBL4 BAC library represents the first cataloged Indian genome resource for applications in basic and clinical research.     

Construction and Characterization of aMagnaporthe griseaBacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aMagnaporthe griseabacterial artificial chromosome library.Fungal Genet. Biol.20,280–288. A bacterial artificial chromosome (BAC) library ofMagnaporthe griseacontaining 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents ofM. griseaand has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using alys1-3auxotroph, we have shown that BAC clones at least 113 kb can be transformed intoM. griseato screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Molecular characterization and expression patterns of the actinin-associated LIM protein (ALP) subfamily genes in porcine skeletal muscle

The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development.     

Identification and characterization of PorH, a new cell wall channel of Corynebacterium glutamicum

The cell wall of Corynebacterium glutamicum contains the cation-selective channel (porin) PorA_C.glut and the anion-selective channel PorB_C.glut for the passage of hydrophilic solutes. Lipid bilayer experiments with organic solvent extracts of whole C. glutamicum cells cultivated in minimal medium suggested that also another cation-selective channel-forming protein, named PorH_C.glut, is present in C. glutamicum. The protein was purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The pure protein had an apparent molecular mass of about 12 kDa on SDS-PAGE. Western blot analysis suggested that the cell wall channel is presumably formed by protein oligomers. The purified protein forms cation-selective channels with an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. The PorH_C.glut protein was partially sequenced, and based on the resulting amino acid sequence, the corresponding gene, designated as porH_C.glut, was identified in the published genome sequence of C. glutamicum ATCC13032. PorH_C.glut contains only the inducer methionine but no N-terminal extension, which suggests that the export and assembly of the protein follow a yet unknown pathway. PorH_C.glut is coded in the bacterial chromosome by a gene that is localized in the vicinity of porA_C.glut, within a putative operon of 13 genes. RT-PCR revealed that both porins are cotranscribed. They coexist according to immunological detection experiments in the cell wall of C. glutamicum together with PorB_C.glut and PorC_C.glut.     

Functional characterization of a novel tropinone reductase-like gene in Dendrobium nobile Lindl

Dendrobium nobile, a herbal medicine plant, contains many important alkaloids and other secondary metabolites with pharmacological and clinical effects. However, the biosynthetic pathway of these secondary metabolites is largely unknown. In present study, a cDNA sequence (DnTR2) that encodes a peptide with high similarity to known tropinone reductase (TR) was cloned from D. nobile Lindl. Sequence comparison and phylogenetic analysis showed that DnTR2 was evolutionarily distant from those well-characterized subgroups of TRs. qRT-PCR revealed that DnTR2 was expressed constitutively in all three vegetative organs (leaves, stems and roots) and was regulated by methyl jasmonate (MeJA), salicylic acid (SA) and nitrogen oxide (NO). Catalytic activity analysis using recombinant protein found that DnTR2 was not able to reduce tropinone, but reduced the two structural analogs of tropinone, 3-quinuclidinone hydrochloride and 4-methylcyclohexanone. Structural modeling and comparison suggested that the substrate specificity of TRs may not be determined by their phylogenetic relationships but by the amino acids that compose the substrate binding pocket. To verify this hypothesis, a site-directed mutagenesis was performed and it successfully restored the DnTR2 with tropinone reduction activity. Our results also showed that the substrate specificity of TRs was determined by a few residues that compose the substrate binding pocket which may have an important role for directed selecting of TRs with designated substrate specificities.     

Construction and analysis of a BAC library in the grass<Emphasis Type="Italic"> Brachypodium sylvaticum</Emphasis>: its use as a tool to bridge the gap between rice and wheat in elucidating gene content

A BAC library of 30,228 clones with an average insert size of 102 kb was constructed in the grass Brachypodium sylvaticum. Brachypodium has a simple genome, similar in size and repetitive DNA content to that of rice, and is more closely related than rice both to the major temperate cereals wheat and barley, and to the forage grasses. The library represents 6.6 genome equivalents, implying a 99.9% probability of recovering any specific sequence. The library was arrayed onto two high-density colony filters, which were screened with heterologous DNA probes from rice chromosome nine and from syntenous regions of wheat, barley, maize and oat. The construction of Brachypodium BAC contigs revealed that synteny between rice, wheat and Brachypodium was largely maintained over several regions of rice chromosome nine. This suggests that Brachypodium will be a useful tool in the elucidation of gene content in agronomically important cereal crops, complementing rice as a grass genome model.     

Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite

A single-chain antibody library against Eimeria acervulina merozoites was constructed by phage display approach. Antibody-displaying phage was selected in four panning rounds against cryopreserved E. acervulina merozoites. Five clones were randomly selected from the fourth panning round, and their nucleotide sequences were aligned and compared to mouse germ-line sequences. Soluble antibody was produced in a non-suppressor Escherichia coli strain, purified by protein A affinity chromatography, and characterized by Western-blotting. Immunofluorescence assay showed localization of the produced recombinant antibody fragment on the surface E. acervulina merozoites. These resultant antibody fragments showed high specificity and binding capacity for soluble antigens and intact fixed merozoites which seems promising as diagnostic, therapeutic and/or vaccine tools against coccidiosis.     

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU. This mutant carries an R7L mutation on the α-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 Å PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lα cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lα cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lα cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the α-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.     

Isolation and characterization of oxygen-evolving thylakoid membranes and Photosystem II particles from a marine diatom Chaetoceros gracilis

Thylakoid membranes retaining high oxygen-evolving activity (about 250 μmol O₂/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 μmol O₂/mg Chl/h in the absence and presence of CaCl₂, respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.     

IS elements in Aliivibrio salmonicida LFI1238: Occurrence,variability and impact on adaptability

Insertion sequence (IS) elements are short, self-replicating DNA sequences that are capable of efficiently spreading over the host genome. Possessing varied integration specificity IS elements are capable of the irreversible inactivation of genes, which diversifies the pool of intact genetic determinants in host populations. In the current study, we performed a complex analysis of IS elements (Vsa IS) in the previously sequenced genome of Aliivibrio salmonicida LFI1238 and proposed a model of the spread of the Vsa IS elements over the genome of this microorganism. Along with the prediction of the integration sites for Vsa IS elements, the current study provides an overview of the properties of A. salmonicida IS elements, as well as information regarding their occurrence in different bacterial classes. An analysis of individual alleles of the IS elements has allowed us to depict a history of the accumulation of mutations and to describe distinctive microevolution lines for actively transposing Vsa IS elements in the genome of A. salmonicida LFI1238. Our results demonstrate the high importance of the dead end microevolution of actively transposing Vsa IS elements for the inactivation of genes in A. salmonicida LFI1238.     

Proliferation and copy number variation of BEL-like long terminal repeat retrotransposons within the Diabrotica virgifera virgifera genome

The proliferation of retrotransposons within a genome can contribute to increased size and affect the function of eukaryotic genes. BEL/Pao-like long-terminal repeat (LTR) retrotransposons were annotated from the highly adaptable insect species Diabrotica virgifera virgifera, the Western corn rootworm, using survey sequences from bacterial artificial chromosome (BAC) inserts and contigs derived from a low coverage next-generation genome sequence assembly. Eleven unique D. v. virgifera BEL elements were identified that contained full-length gag–pol coding sequences, whereas 88 different partial coding regions were characterized from partially assembled elements. Estimated genome copy number for full and partial BEL-like elements ranged from ~ 8 to 1582 among individual contigs using a normalized depth of coverage (DOC) among Illumina HiSeq reads (total genome copy number ~ 8821). BEL element copy number was correlated among different D. v. virgifera populations (R² = 0.9846), but individual element numbers varied ≤ 1.68-fold and the total number varied by ~ 527 copies. These data indicate that BEL element proliferation likely contributed to a large genome size, and suggest that differences in copy number are a source of genetic variability among D. v. virgifera.     

A novel t(4;16)(q25;q23.1) associated with EGF and ELOVL6 deregulation in acute myeloid leukemia

About 50% of acute myeloid leukemia (AML) patients show the occurrence of non-random chromosome rearrangements. Most of the recurrent karyotypic rearrangements in AML have been defined as distinct disease entities in the 2008 World Health Organization (WHO) classification. In this paper we report an AML case showing a novel t(4;16)(q25;q23.1) rearrangement causing the activation of epidermal growth factor (EGF) and elongation of long-chain fatty acids family member 6 (ELOVL6) genes, rather than the generation of a novel fusion gene.     

Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis

Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O₂ (mg Chl a)^− 1 h^− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl₂. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells.     

Gene expression and characterization of a stress-induced tyrosine decarboxylase from Arabidopsis thaliana

Full-length tyrosine decarboxylase cDNA (TyrDC) from Arabidopsis thaliana was identified by rapid amplification of cDNA ends-PCR and isolated by RT-PCR. The TyrDC mRNA was substantially induced by drought stress and wounding, and was considerably decreased by salt stress. By using TyrDC protein fusions with green fluorescent protein, an intracellular localization to the cytoplasm was shown. Recombinant (His)₆-TyrDC was expressed in Escherichia coli and enzymatically characterized: it exclusively catalyzed the conversion of l-tyrosine to tyramine, exhibited an optimum temperature of 50 °C, and an optimum pH at approximately 8.5-9. Recombinant TyrDC protein formed tetramers, as shown by blue native gel electrophoresis.

Structured summary

MINT-7040408:TyrDC (uniprotkb:Q8RY79) and TyrDC (uniprotkb:Q8RY79) bind (MI:0407) by blue native page (MI:0276)     

Occurrence and genetic diversity of Arcobacter spp. in a spinach-processing plant and evaluation of two Arcobacter-specific quantitative PCR assays

Some species of the genus Arcobacter are considered to be emerging food pathogens. With respect to recent vegetable-borne outbreaks, the aim of this work was to investigate the occurrence and diversity of Arcobacter within the production chain of a spinach-processing plant by a combination of cultivation and molecular methods. Samples including spinach, water, and surface biofilm were taken over a period of three years from the entire processing line. Ten 16S rRNA (rrs) gene clone libraries were constructed and analysed using amplified rRNA gene restriction analysis (ARDRA). Approximately 1200 clones were studied that resulted in 44 operational taxonomic units (OTUs). Sequences with high similarities to Arcobacter cryaerophilus (13% of clones, 3 OTUs), A. ellisii (4%, 6 OTUs), A. suis (15%, 3 OTUs), and the type strain of A. nitrofigilis (1%, 7 OTUs) were identified. This represents the first report of the detection of the recently described species A. ellisii, A. suis and, in addition, A. venerupis from alternative habitats. A total of 67% of the clones (22 OTUs) could not be assigned to a genus, which indicated the presence of uncharacterised Arcobacter species.