Molecular characterization of a novel Staphylococcus aureus serine protease operon

Abstract

The present study identified and characterized a unique operon (spl) encoding six serine protease-like proteins. In addition, native Spl proteins were isolated and characterized. Typical of most exoproteins, the spl gene products contain putative 35- or 36-amino-acid signal peptides. The Spl proteins share 44 to 95% amino acid sequence identity with each other and 33 to 36% sequence identity with V8 protease. They also contain amino acids found in catalytic triads of enzymes in the trypsin-like serine protease family, and SplB and SplC were shown to degrade casein. The spl operon is transcribed on a 5.5-kb transcript, but several nonrandom degradation products of this transcript were also identified. Similar to other S. aureus exoprotein genes, the spl operon is maximally expressed during the transition into stationary phase and is positively controlled by the Agr virulence factor regulator. The Sar regulatory system did not affect spl operon expression. PCR analysis revealed the presence of the spl operon in 64% of the S. aureus isolates tested, although one spl operon-negative isolate was shown to contain at least two of the spl genes. Finally, intraperitoneal injection of an spl operon deletion mutant revealed no major differences in virulence compared to the parental strain.

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 spl operon (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 an splB-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).