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Molecular Pathogenesis

Identification of Genes Transcribed by Actinobacillus pleuropneumoniae in Necrotic Porcine Lung Tissue by Using Selective Capture of Transcribed Sequences

Nina Baltes, Gerald F. Gerlach
Nina Baltes
Department of Infectious Diseases, Institute for Microbiology, University of Veterinary Medicine Hannover, Hannover, Germany
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  • For correspondence: nbaltes@gmx.de
Gerald F. Gerlach
Department of Infectious Diseases, Institute for Microbiology, University of Veterinary Medicine Hannover, Hannover, Germany
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DOI: 10.1128/IAI.72.11.6711-6716.2004
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  • FIG. 1.
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    FIG. 1.

    Southern dot blot showing (a) SCOTS clones and (b) 1 μg (top) or 0.1 μg (bottom) of genomic A. pleuropneumoniae DNA as positive controls, hybridized to a radiolabeled probe generated from lung-derived (1) or culture-derived cDNA (2) after three rounds of normalization.

  • FIG. 2.
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    FIG. 2.

    PCR following reverse transcription of 5 μg of RNA derived from A. pleuropneumoniae-infected lung (lanes 1 to 5). Also shown is serial dilution of the cDNA template, ranging from undiluted (lane 1) to 1:10,000 (lane 5). Lanes a and b show positive controls using primer pairs oADH1/oADH2 (a) and oFLPD1/oFLPD2 (b), with chromosomal A. pleuropneumoniae DNA as template. −, No template.

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  • TABLE 1.

    Characteristics of bacterial strains and primers used in this study

    Strain, plasmid or primerCharacteristics and/or sequencesSource and/or reference
    Strains
        A. pleuropneumoniae C1569 A. pleuropneumoniae serotype 9, clinical isolateDiagnostic unit of the Department of Infectious Diseases
        Escherichia coli TOP10 F′F−mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 rec A1 deoR araD139 Δ(araleu)7697 galU galK rpsL (Strr) endA1 nupGTOPO TA Cloning (Invitrogen)
    Plasmid
        pCR 2.1-TOPOTopoisomerase I enhanced E. coli cloning vectorTOPO TA Cloning (Invitrogen) (35)
    Primers
        M13 forward5′ CAGGAAACAGCTATGAC 3′Amersham Biosciences
        M13 reverse5′ GTAAAACGACGGCCAG 3′Amersham Biosciences
        oSCOTS-N9-15′ GTGGTACCGCTCTCCGTCCGA-N9 3′ 8
        oSCOTS-N9-25′ CGGGATCCAGCTTCTGACGCA-N9 3′ 8
        oSCOTS15′ GTGGTACCGCTCTCCGTCCGA 3′ 8
        oSCOTS25′ CGGGATCCAGCTTCTGACGCA 3′ 8
        oRRN16-15′ TGGGTCGTAGGTTCAAATCC 3′This work
        oRRN16-25′ GCGTCAGTACATTCCCAAGG 3′; amplifies 5′ end of 16s rRNA sequence; product size, 990 bp
        oRRN16-35′ TGTAGCGGTGAAATGCGTAG 3′This work
        oRRN16-45′ ACTTGAACCACCGACCTCAC 3′; amplifies 3′ end of 16s rRNA sequence; product size, 1,000 bp
        oRRN23-15′ TTGGAAACAAGCTGAAAACTGA 3′This work
        oRRN23-25′ GGACAGGAACCCTTGGTCTT 3′; amplifies 5′ end of 23s rRNA sequence; product size, 1,480 bp
        oRRN23-35′ TCAGAAGTGCGAATGCTGAC 3′This work
        oRRN23-45′ CTGGCGAGACAACTGGTACA 3′; amplifies 3′ end of 23S rRNA sequence; product size, 1,466 bp
        oADH75′ ACACGTAATGACGGCGGTA 3′This work
        oADH85′ CGAGTGGATTCACCCAATTT 3′; amplifies a 158-bp product from an A. pleuropneumoniae autotransporter adhesin; acc. no. ZP 00204542
        oFLPD15′ TTTCTCTGTAGAGATGGTTTGTGC 3′This work
        oFLPD25′ AACCCAATTAACCCAAATGGT 3′; amplifies a 154-bp product from an A. pleuropneumoniae fimbria-like protein; acc. no. NP 873737
  • TABLE 2.

    Genes identified via SCOTS

    ClassClone(s)Gene and/or possible functiona% Identity/ % similaritybSpancReference(s)dGenBank accession no.
    SecretedH7ApxIVA var3 RTX toxin (A. pleuropneumoniae) 33 AAD01905
    Cell surfaceB5Autotransporter adhesin, similar to Hsf in H. influenzae (A. pleuropneumoniae) 29, 39, 41 ZP_00204542
    E4 flpD, fimbria-like protein (H. ducreyi); flp genes are essential for microcolony formation in H. ducreyi65/66188 12, 31, 37 NP_873737
    B9Outer membrane protein similar to immunogenic protein PomA of P. multocida (A. pleuropneumoniae) 13, 42 E47068
    F4Outer membrane protein of unknown function (A. pleuropneumoniae) ZP_00135264
    MetabolismA7 guaA GMP synthase (A. pleuropneumoniae), linked to virulence in various organisms, including P. multocida 12, 27, 32, 34 ZP_00134556
    A8Diadenosine tetraphosphatase similar to ApaH in S. enterica serovar Typhimurium (A. pleuropneumoniae); linked to virulence in H. parasuis and serovar Typhimurium 21, 24 ZP_00134055
    A9 fuc1, l-fucose isomerase (H. influenzae); in Bacteroides thetaiotaomicron, the l-fucose metabolic pathway is coordinated with the production of fucosylated glycans in enterocytes76/78222 22, 25 P44779
    C2 ptsB, sucrose-specific phosphotransferase EII component (P. multocida); linked to virulence in serovar Typhimurium, S. aureus, and H. influenzae83/85216 28 NP_246785
    H3Sugar phosphate permease (A. pleuropneumoniae) ZP_00135166
    C7Oxygen-sensitive ribonucleoside-triphosphate reductase (A. pleuropneumoniae) ZP_00134686
    D2Thiol:disulfide interchange protein, similar to DsbA in H. influenzae (A. pleuropneumoniae); essential for virulence factor expression in P. aeruginosa 17, 34 ZP_00134426
    D33-Polyprenyl-4-hydroxybenzoate decarboxylase (A. pleuropneumoniae) ZP_00134932
    G6Ribosomal protein RpS19 (H. influenzae)100/10090 P44385
    E12Biotin-(acetyl-coenzyme A carboxylase) ligase (A. pleuropneumoniae) ZP_00134488
    F12Dethiobiotin synthetase (A. pleuropneumoniae) ZP_00135280
    F11NADH ubiquinone oxidoreductase subunit 5 (A. pleuropneumoniae) 34 ZP_00135039
    G8SAM-dependent methyltransferase (A. pleuropneumoniae) 34 ZP_00134291
    A4Uncharacterized conserved protein involved in oxidation of intracellular sulfur (A. pleuropneumoniae) ZP_00133817
    A11Transposase (A. pleuropneumoniae) ZP_00134532
    D13-Oxoacyl-(acyl-carrier protein) reductase (H. influenzae)78/82238 P43713
    D4Predicted GTPase (A. pleuropneumoniae) ZP_00134954
    G11ATPase (A. pleuropneumoniae) ZP_00134371
    E9Restriction-modification system (ALXA and HSDM, M . haemolytica); M. haemolytica AlxA activates leukotoxin transcription in E. coli81/81156 20 P95510
    F8Molybdopterin biosynthesis protein (H. ducreyi)84/86174 34 AAP95073
    H6Leucyl-tRNA synthetase (A. pleuropneumoniae) ZP_00133841
    F9Predicted hydrolase of alkaline phosphatase superfamily (A. pleuropneumoniae) ZP_00134565
    RegulatoryA3 rpoB, RNA polymerase subunit (H. ducreyi)91/94207 AAP96608
    B2Cell shape-determining protein, similar to MreC (A. pleuropneumoniae) ZP_00134717
    A10Signal recognition particle protein (H. influenzae)88/93243 P44518
    H5Molecular chaperone (A. pleuropneumoniae) ZP_00134922
    H4Putative transcriptional regulator (A. pleuropneumoniae) ZP_00134590
    StressC9 tufA, elongation factor (EF) Tu (H. ducreyi); EF-Tu acts as a fibronectin binding protein in M. pneumoniae, linked to virulence in K. pneumoniae100/100135 9, 40 NP_872680
    TransportA5 apaA, putative ABC transporter, immunogenic (A. pleuropneumoniae) 30 AF109148
    H1 dmsA, dimethyl sulfoxide reductase, catalytic subunit (A. pleuropneumoniae); deletion mutants are attenuated 2 AY138463
    C4 hgbA, hemoglobin-binding protein (A. pleuropneumoniae); enables iron uptake from hemoglobin 5, 38 AAO33396
    G1Na+/H+ antiporter (A. pleuropneumoniae); essential for in vivo survival in S. agalactiae 27 ZP_00134319
    E1Predicted metal-binding protein (A. pleuropneumoniae) ZP_00135073
    E7Membrane components of Kef-type potassium transport systems (A. pleuropneumoniae) ZP_00134994
    F1Anaerobic C4-dicarboxylate transporter (A. pleuropneumoniae) 34 ZP_00134386
    F2 tbpA, transferrin receptor; small subunit (A. pleuropneumoniae); upregulated and essential in vivo 1 CAA90896
    F5ATPase; cation transport (A. pleuropneumoniae) ZP_00135075
    G10Di- and tricarboxylate transporters (A. pleuropneumoniae) ZP_00135062
    UnknownB8Hypothetical protein PA2218 (P. aeruginosa)76/83162 Q01609
    G7, F7e A. pleuropneumoniae; no homology to known genes
    • ↵ a Genes with highest similarity to SCOTS sequences, obtained from public databases, source organism in parentheses, genes names given where available. SAM, S-adenosylmethionine.

    • ↵ b Identity/similarity percentages apply to amino acids.

    • ↵ c Span means the number of nucleotides over which similarity was detected.

    • ↵ d Publications reporting the isolation of identical or similar sequences.

    • ↵ e Sequences have not been submitted as these are partial open reading frames only.

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Identification of Genes Transcribed by Actinobacillus pleuropneumoniae in Necrotic Porcine Lung Tissue by Using Selective Capture of Transcribed Sequences
Nina Baltes, Gerald F. Gerlach
Infection and Immunity Oct 2004, 72 (11) 6711-6716; DOI: 10.1128/IAI.72.11.6711-6716.2004

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Identification of Genes Transcribed by Actinobacillus pleuropneumoniae in Necrotic Porcine Lung Tissue by Using Selective Capture of Transcribed Sequences
Nina Baltes, Gerald F. Gerlach
Infection and Immunity Oct 2004, 72 (11) 6711-6716; DOI: 10.1128/IAI.72.11.6711-6716.2004
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KEYWORDS

Actinobacillus Infections
Actinobacillus pleuropneumoniae
Bacterial Proteins
Genes, Bacterial
lung
Transcription, Genetic

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