Dual control of streptokinase and streptolysin S production by the covRS and fasCAX two-component regulators in Streptococcus dysgalactiae subsp. equisimilis

Abstract

Synthesis of the plasminogen activator streptokinase (SK) by group A streptococci (GAS) has recently been shown to be subject to control by two two-component regulators, covRS (or csrRS) and fasBCA. In independent studies, response regulator CovR proved to act as the repressor, whereas FasA was found to act indirectly as the activator by controlling the expression of a stimulatory RNA, fasX. In an attempt at understanding the regulation of SK production in the human group C streptococcal (GCS) strain H46A, the strongest SK producer known yet, we provide here physical and functional evidence for the presence of the cov and fas systems in GCS as well and, using a mutational approach, compare the balance between their opposing actions in H46A and GAS strain NZ131. Sequence analysis combined with Southern hybridization revealed that the covRS and fasCAX operons are preserved at high levels of primary structure identity between the corresponding GAS and GCS genes, with the exception of fasB, encoding a second sensor kinase that is not a member of the GCS fas operon. This analysis also showed that wild-type H46A is actually a derepressed mutant for SK and streptolysin S (SLS) synthesis, carrying a K102 amber mutation in covR. Using cov and fas mutations in various combinations together with strain constructs allowing complementation in trans, we found that, in H46A, cov and fas contribute to approximately equal negative and positive extents, respectively, to constitutive SK and SLS activity. The amounts of SK paralleled the level of skc(H46A) transcription. The most profound difference between H46A and NZ131 regarding the relative activities of the cov and fas systems consisted in significantly higher activity of a functional CovR repressor in NZ131 than in H46A. In NZ131, CovR decreased SK activity in a Fas(+) background about sevenfold, compared to a 1.9-fold reduction of SK activity in H46A. Combined with the very short-lived nature of covR mRNA (decay rate, 1.39/min), such differences may contribute to strain-specific peculiarities of the expression of two prominent streptococcal virulence factors in response to environmental changes.

FIG. 1.

Detection of covRS mRNA in H46A and NZ131 incubated in the indicated media. Shown is a Northern blot of total RNA probed with covR (boxed).

FIG. 2.

Determination of mRNA half-lives of the indicated NZ131 genes under conditions in which new mRNA synthesis is inhibited by rifampin. Estimation of decay kinetics of the mRNAs (left) is based on Northern blots, representative results of which are shown on the right.

FIG. 3.

Detection of the fasCAX operon by Southern hybridization of H46A and SF370 chromosomal DNAs restricted with the indicated enzymes and probed with fasX_H46A (boxed).

FIG. 4.

SK and SLS activities in cell-free supernatant fluids of BHI cultures of the specified H46A wild-type and mutant strains containing comparable numbers of CFU. For SK determination, cultures were sampled after 16 h of growth, whereas log-phase cultures grown to an OD₆₀₀ of 0.30 were used for SLS determination. Relative activities of 100% correspond to 80 U of SK per ml or 0.75 OD₅₄₀ hemolytic units produced by 200 μl of a 1:40 diluted supernatant fluid obtained from an H46A culture. (For comparison, an OD₅₄₀ value of 1.15 corresponds to complete hemolysis produced by the same volume of water.)

FIG. 5.

CovR-regulated SK production by wild-type NZ131, the covR mutant, and complemented covR mutant strains. SK activities of culture supernatant fluids were compared by measuring plasminogen activation with a plasmin-specific chromogenic substrate as a function of time. The inset shows a slot blot hybridization of total RNA extracted from the indicated strains and probed with ska.

FIG. 6.

Transcriptional regulation of skc expression by the cov and fas regulatory systems demonstrated by Northern hybridization of total RNA extracted from the indicated strains and probed with skc (boxed). The ethidium bromide (EtBr)-stained gel on the left demonstrates equal loading of the slots and integrity of the 23S and 16S rRNAs.

FIG. 7.

Cotranscription of fasAX and dependence of separate transcription of fasX from its own promoter on the presence of FasA, demonstrated by Northern hybridization of total RNA extracted from the indicated strains and probed with fasX (boxed). The ethidium bromide (EtBr)-stained gel on the left demonstrates equal loading of the slots and integrity of the 23S and 16S rRNAs.