Article Figures & Data
Figures
- 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).
- 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.
- 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.
- 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.
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.
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).
- 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
- Table 1.
Strains and plasmids used in this study
Strain or plasmid Relevant phenotype Reference or source S. aureus RN6390 Wild-type laboratory strain 23 RN4220 Highly transformable strain 23 RN6911 agr RN6390 strain 18 ALC135 agr sar RN6390 strain 8 ALC136 sar RN6390 strain Ambrose Cheung KB600 RN6390 spl operon null mutant This study KB601 RN6390 splB mutant This study Brittingham TSSa isolate Amy Bryant Israel #1 TSS isolate Amy Bryant Israel #2 TSS isolate Amy Bryant Newman Produces type 5 capsule Chia Y. Lee M Produces type 1 capsule Chia Y. Lee Wright Produces type 8 capsule Chia Y. Lee Becker Produces type 8 capsule Chia Y. Lee Col MRSAb John J. Iandolo DU4916 MRSA John J. Iandolo Novel Bovine mastitis isolate 33 305 Bovine mastitis isolate 22 E. coli DH5α Highly transformable strain Bethesda Research Laboratories Plasmids pCL52.2 Temperature-sensitive cloning vector 30 pER924 Temperature-sensitive cloning vector 5 pRN5548 High-copy gram-positive plasmid 24 pDG647 Source of Emr cassette 16 pSR7 pRN5548 derivative used in expression ofsplABCD This study - Table 2.
Characteristics of mature Spl proteins
Protein No. of amino acids Molecular mass (kDa) Deduced pI No. of amino acids in signal sequence SplA 200 21.9 8.6 35 SplB 204 22.4 9.1 36 SplC 203 22.4 6.4 36 SplD 203 22.0 8.9 36 SplE 202 22.0 9.2 36 SplF 203 21.9 8.9 36
