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Molecular Pathogenesis

Identification and Disruption of Two Discrete Loci Encoding Hyaluronic Acid Capsule Biosynthesis GeneshasA, hasB, and hasC inStreptococcus uberis

Philip N. Ward, Terence R. Field, William G. F. Ditcham, Emmanuelle Maguin, James A. Leigh
Philip N. Ward
Institute for Animal Health, Compton Laboratory, Compton, Berkshire RG20 7NN, United Kingdom,1and
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Terence R. Field
Institute for Animal Health, Compton Laboratory, Compton, Berkshire RG20 7NN, United Kingdom,1and
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William G. F. Ditcham
Institute for Animal Health, Compton Laboratory, Compton, Berkshire RG20 7NN, United Kingdom,1and
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Emmanuelle Maguin
Laboratoire de Genetique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France2
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James A. Leigh
Institute for Animal Health, Compton Laboratory, Compton, Berkshire RG20 7NN, United Kingdom,1and
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DOI: 10.1128/IAI.69.1.392-399.2001
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  • Fig. 1.
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    Fig. 1.

    Schematic representation of the has locus inS. pyogenes (A), and the homologous regions in S. uberis 0140J (B to E). Open arrows represent the orientations ofhas genes. Filled arrows indicate the locations and orientations of ISS1-encoded transposase (not shown to scale). Hatched bars represent the pGh9 vector sequence (also not shown to scale). Filled arrowheads indicate the orientation of putative open reading frames (orf). ISS1 insertions were mapped to the points indicated by vertical arrows in strains TRF0-6, TRF0-29, and TRF0-30. (B and D) Arrangements before plasmid excision. (C and E) Arrangements after plasmid excision. Partial and horizontal arrows denote key oligonucleotide primer locations (Table 1). H and E,HindIII and EcoRI restriction sites used in the cloning of flanking sequences, respectively. Erres., erythromycin resistant; Ersens., erythromycin sensitive.

  • Fig. 2.
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    Fig. 2.

    Southern analysis of integrants picked randomly from theS. uberis 0140J ISS1 mutant bank. Genomic DNAs from 12 mutant clones (lanes 1 to 12) and the parent strain 0140J (lane 13) were digested using HindIII and probed with DIG-labeled ISS1. Two clones (lanes 3 and 11) showed three bands, one of which corresponded to a 4.6-kb linearized pGh9::ISS1 fragment (arrow) characteristic of tandem pGh9::ISS1 integration.

  • Fig. 3.
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    Fig. 3.

    PCR amplification products from the 3′ end of theS. uberis homologue of hasA demonstrating band shifts due to the insertion of ISS1 in mutants TRF0-6 and TRF0-29, compared to the wild-type (WT) strain 0140J.

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    Fig. 4.

    Southern analysis of S. uberis 0140J mutants TRF0-6 and TRF0-29. Genomic DNA was digested withHindIII and hybridized with a DIG-labeled ISS1 probe at 65°C. Lane 1, wild type (WT); lane 2, wild-type 0140J transformed with pGh9::ISS1 and grown at 28°C (pG9); lanes 3 and 4, mutant TRF0-6 before (Err) and after (Ers) excision of the plasmid vector sequence, respectively; lanes 5 and 6, mutant TRF0-29 before (Err) and after (Ers) excision of the plasmid vector sequence, respectively. Lane 2 shows the linearized preintergrated free-plasmid form of pGh9::ISS1. Lanes 3 and 5 show that mutant TRF0-6 contained a tandem insertion, whereas mutant TRF0-29 had a single copy of pGh9::ISS1 inserted in the chromosome.

Tables

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  • Table 1.

    Oligonucleotide primer sequences and their applications

    DesignationSequenceApplicationTemplateAnnealing temp (°C)
    ISS1fora5′-GGAGAGAATGGGTTCTGTTGCISS1probepGh9::ISS160
    ISS1reva5′-GCTCTAGAGCATTCTCTGGTTC
    P0505′-GTTATCGTTCACCGTTCCCS. pyogenes hasA probeS. pyogenes genomic DNA47
    P0525′-TACGTGTTCCCCATTCCG
    P064a5′-AGAACCGAAGAATTCGAACGCTCAmplification of S. uberis DNA and sequencing primersWild-type and mutant S. uberis 0140J genomic DNAs50
    P067b5′-GGTGGAGCGTGCTGCTCA
    P0705′-CAAAACGAATAGATGTATCAATCC
    P082a5′-CCAACAGCGACAATAATCACATC
    P084c5′-CAGTCCCGACGGTGTAAAACG
    P085c5′-GCTAAAGAGGTCCCTAGACTCT
    • ↵a Primers complementary to ISS1.

    • ↵b A primer complementary to S. pyogenes hasA.

    • ↵c Primers complementary to the pGh9 vector.

  • Table 2.

    Putative translation products for mutant S. uberis hasA and hasC homologues

    StrainGenePredicted amino acid sequencea
    TRF0-6hasA… LVAFLVIILV LLQSFLISLF *
    TRF0-29hasA… LVAFLVIIFI VALCRNVHYM VKHPFAFLLS PFYGLIHRFC CKVF**
    TRF0-30hasC… DDLMDITNTV LLQSLKIKYK VYNPSCS*
    • ↵a ISS1-encoded amino acids are denoted by underlining. An asterisk denotes a stop codon.

  • Table 3.

    Resistance to phagocytosis and capsular status of mutants TRF0-6, TRF0-29, and TRF0-30 compared to wild-type S. uberis 0140J grown in CDM containing casein hydrolysate

    StrainaMean (SD)b
    % Survivalcμg of capsuled
    Wild-type 0140J103.02 (15.21)145.775 (27.89)
    TRF0-6 Err0.118 (0.06)0.325 (0.26)
    TRF0-6 Ers0.440 (0.12)0.625 (0.46)
    TRF0-29 Err0.250 (0.15)0.750 (0.31)
    TRF0-29 Ers0.135 (0.05)0.700 (0.37)
    Wild-type 0140J71.83 (33.67)122.35 (18.02)e
    TRF0-30 Err0.16 (0.06)1.29 (2.00)e
    TRF0-30 Ers0.22 (0.02)1.63 (1.54)e
    • ↵a Mutant strains were tested before (Err) and after (Ers) pGh plasmid excision.

    • ↵b Unless otherwise indicated, data from triplicate samples from two independently grown broth cultures were pooled (n = 6) .

    • ↵c Resistance to phagocytosis is shown as the percent survival of cells relative to cells at time zero.

    • ↵d Capsule status is shown as the amount of hyaluronidase-released N-acetylglucosamine per 1010 bacteria.

    • ↵e Data from quadruplicate samples from two independently grown cultures were pooled (n = 8) .

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Identification and Disruption of Two Discrete Loci Encoding Hyaluronic Acid Capsule Biosynthesis GeneshasA, hasB, and hasC inStreptococcus uberis
Philip N. Ward, Terence R. Field, William G. F. Ditcham, Emmanuelle Maguin, James A. Leigh
Infection and Immunity Jan 2001, 69 (1) 392-399; DOI: 10.1128/IAI.69.1.392-399.2001

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Identification and Disruption of Two Discrete Loci Encoding Hyaluronic Acid Capsule Biosynthesis GeneshasA, hasB, and hasC inStreptococcus uberis
Philip N. Ward, Terence R. Field, William G. F. Ditcham, Emmanuelle Maguin, James A. Leigh
Infection and Immunity Jan 2001, 69 (1) 392-399; DOI: 10.1128/IAI.69.1.392-399.2001
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KEYWORDS

Bacterial Capsules
Bacterial Proteins
Carrier Proteins
Chromosome Mapping
Genes, Bacterial
Hyaluronic Acid
membrane proteins
Streptococcus

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