Expression of staphylocoagulase by agr+ Staphylococcus aureus depends on the growth phase, being maximal during exponential growth and decreasing sharply postexponentially, while an agr-deleted strain continuously expresses an intermediate level of coagulase. Therefore, coagulase expression appears to be both positively and negatively modulated by an agr-dependent mechanism.Staphylocoagulase is an extracellular protein produced by Staphylococcus aureus which promotes fibrin formation in human plasma by nonenzymatic activation of prothrombin (14). It, like the cell wall-associated protein A, is mainly synthesized during exponential growth (3, 7). In contrast, other extracellular proteins and toxins (e.g., serine protease; nuclease; lipase; fibrinolysin; oa-, P-, and 8-hemolysin; toxic shock syndrome toxin 1; and enterotoxin B) are produced after the end of exponential growth (1). The expression of these products is controlled by a complex regulatory locus, agr (6, 9, 10), whose unique feature is control of the expression of its target genes by means of an RNA molecule (RNAIII) (8). agr RNAIII acts as a positive regulator on genes which are preferentially expressed postexponentially, whereas protein A and coagulase are inhibited. The kinetics of protein A gene (spa) transcription has been investigated with agr+ and agr mutant strains (12) and shown to be inversely related to the level of RNAIII. The proposed mechanism was that RNAIII had a strict inhibitory effect on spa expression. To our knowledge, no similar study of agr regulation of the coagulase gene (coa) has been conducted. Our results indicate that the regulation of coa expression is different from that of spa, in that coagulase expression may be modulated both positively and negatively by the agr system according to the growth stage.Kinetics of coagulase activity. The coagulase activity of strain RN6390 (9), an agr+ derivative of S. aureus 8325-4, was compared with that of RN6911, which was constructed by allelic replacement of the agr locus in RN6390 (8). Brain heart infusion broth (50 ml) in 500-ml Erlenmeyer flasks was inoculated with 10 colonies of overnight cultures on blood agar plates and incubated at 37°C with agitation at 190 rpm. Growth was monitored spectrophotometrically. Titration of coagulase activity was performed hourly for supernatants as described by McDevitt et al. (5) The two strains had similar latency times and growth rates under the cultivation conditions used in this study (Fig. 1). The agr+ strain RN6390 displayed a maximum clotting activity of 18.6 after 2 to 3 h of growth (Fig. 1A), which decreased rapidly to become undetectable after 7 h. The agr mutant strain RN6911 had a relatively constant coagulase activity of 8 (8.57 ± 1.68) over the same period (Fig. 1B).Kinetics of coa mRNA. The kinetics of coa mRNA expression was studied in the two strains by Northern (RNA) blot hybridization with a coa-specific RNA probe synthesized by in vitro transcription of pLUG12 (13) described previously (12). Hybridization and detection of l...

Coagulase deficiency in clinical isolates of Staph y o co ccus a ure us i n vo I ves bo t h t r a n sc r i p t i o n a I a n d post-transcriptional defects