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Molecular Pathogenesis

Molecular Characterization of a NovelStaphylococcus aureus Serine Protease Operon

Samantha B. Reed, Carla A. Wesson, Linda E. Liou, William R. Trumble, Patrick M. Schlievert, Gregory A. Bohach, Kenneth W. Bayles
Samantha B. Reed
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052, and
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Carla A. Wesson
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052, and
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Linda E. Liou
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052, and
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William R. Trumble
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052, and
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Patrick M. Schlievert
Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
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Gregory A. Bohach
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052, and
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Kenneth W. Bayles
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052, and
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DOI: 10.1128/IAI.69.3.1521-1527.2001
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    Fig. 1.

    Purification of the Spl proteins. (A) The Spl proteins from S. aureus RN6390 grown to stationary phase were fractionated and then separated by SDS–12% PAGE. Lanes: 1, 10-kDa molecular size markers (Gibco-BRL); 2, exoproteins from an RN6390 culture; 3, partially purified exoproteins after IEF fractionation; 4, FPLC fraction containing purified SplA, SplB, and SplF separated in the presence of 2.0 M urea to enhance the resolution of these proteins; 5, purified SplC; 6, purified SplB. The molecular size standards used were the BenchMark Protein Ladder (Gibco-BRL). (B) Zymographic analysis of purified SplC (lane 2) and SplB (lane 3) using casein as the substrate. The molecular size markers used (lane 1) were the BenchMark Prestained Protein Ladder (Gibco-BRL).

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    Fig. 2.

    Schematic representation of the S. aureus sploperon (middle) and its disruption by plasmid insertion (top) and allele replacement (bottom). The PCR primers (splA1, splA2, splD3, and splD4) used to generate DNA fragments for allele replacement are indicated. The arrows 5′ and 3′ to the spl operon represent the putative position of the transcription start site and a putative factor-independent transcription terminator, respectively.

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    Fig. 3.

    Sequence alignment of SplA to F with staphylococcal V8 serine protease. High sequence conservation throughout the proteins is evident. Identical residues in all five sequences are boxed. The asterisks indicate residues comprising the catalytic triad.

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    Fig. 4.

    Sequence identities among the Spl proteins and V8 protease. Shown are the percentages of identical amino acids calculated using paired alignments of the proteins.

  • Fig. 5.
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    Fig. 5.

    Temporal regulation of the spl operon. Dot blot analysis of total cellular RNA isolated from RN6390 at 4, 6, 8, 10, and 12 h postinoculation and probed with ansplB-specific probe. The 8-h time point corresponds to the transition into stationary phase.

  • Fig. 6.
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    Fig. 6.

    Northern blot analysis of the spl operon. DNA probes specific for splA, splB, splC, and splF were hybridized with RNAs isolated from RN6390 (lanes 1) and KB601 (lanes 2). Transcript sizes were determined based on the migration of a 0.24 to 9.5-kb RNA ladder (Gibco-BRL).

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    Fig. 7.

    Transcriptional regulation of the spl operon. A DNA probe specific for splB was hybridized with RNAs isolated from RN6390 (lane 1), KB600 (lane 2), ALC136 (lane 3), ALC135 (lane 4), and RN6911 (lane 5). Transcript sizes were determined based on the migration of a 0.24 to 9.5-kb RNA ladder (Gibco-BRL).

Tables

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  • Table 1.

    Strains and plasmids used in this study

    Strain or plasmidRelevant phenotypeReference or source
    S. aureus
     RN6390Wild-type laboratory strain23
     RN4220Highly transformable strain23
     RN6911agr RN6390 strain18
     ALC135agr sar RN6390 strain8
     ALC136sar RN6390 strainAmbrose Cheung
     KB600RN6390 spl operon null mutantThis study
     KB601RN6390 splB mutantThis study
     BrittinghamTSSa isolateAmy Bryant
     Israel #1TSS isolateAmy Bryant
     Israel #2TSS isolateAmy Bryant
     NewmanProduces type 5 capsuleChia Y. Lee
     MProduces type 1 capsuleChia Y. Lee
     WrightProduces type 8 capsuleChia Y. Lee
     BeckerProduces type 8 capsuleChia Y. Lee
     ColMRSAbJohn J. Iandolo
     DU4916MRSAJohn J. Iandolo
     NovelBovine mastitis isolate33
     305Bovine mastitis isolate22
    E. coli DH5αHighly transformable strainBethesda Research Laboratories
    Plasmids
     pCL52.2Temperature-sensitive cloning vector30
     pER924Temperature-sensitive cloning vector5
     pRN5548High-copy gram-positive plasmid24
     pDG647Source of Emr cassette16
     pSR7pRN5548 derivative used in expression ofsplABCDThis study
    • ↵a TSS, toxic shock syndrome.

    • ↵b MRSA, methicillin-resistant S. aureus.

  • Table 2.

    Characteristics of mature Spl proteins

    ProteinNo. of amino acidsMolecular mass (kDa)Deduced pINo. of amino acids in signal sequence
    SplA20021.98.635
    SplB20422.49.136
    SplC20322.46.436
    SplD20322.08.936
    SplE20222.09.236
    SplF20321.98.936
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Molecular Characterization of a NovelStaphylococcus aureus Serine Protease Operon
Samantha B. Reed, Carla A. Wesson, Linda E. Liou, William R. Trumble, Patrick M. Schlievert, Gregory A. Bohach, Kenneth W. Bayles
Infection and Immunity Mar 2001, 69 (3) 1521-1527; DOI: 10.1128/IAI.69.3.1521-1527.2001

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Molecular Characterization of a NovelStaphylococcus aureus Serine Protease Operon
Samantha B. Reed, Carla A. Wesson, Linda E. Liou, William R. Trumble, Patrick M. Schlievert, Gregory A. Bohach, Kenneth W. Bayles
Infection and Immunity Mar 2001, 69 (3) 1521-1527; DOI: 10.1128/IAI.69.3.1521-1527.2001
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KEYWORDS

Operon
Serine Endopeptidases
Staphylococcus aureus

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