RscA, a member of the MDR1 family of transporters, is repressed by CovR and required for growth of Streptococcus pyogenes under heat stress

Abstract

The ability of Streptococcus pyogenes (group A streptococcus [GAS]) to respond to changes in environmental conditions is essential for this gram-positive organism to successfully cause disease in its human host. The two-component system CovRS controls expression of about 15% of the GAS genome either directly or indirectly. In most operons studied, CovR acts as a repressor. We previously linked CovRS to the GAS stress response by showing that the sensor kinase CovS is required to inactivate the response regulator CovR so that GAS can grow under conditions of heat, acid, and salt stress. Here, we sought to identify CovR-repressed genes that are required for growth under stress. To do this, global transcription profiles were analyzed by microarrays following exposure to increased temperature (40 degrees C) and decreased pH (pH 6.0). The CovR regulon in an M type 6 strain of GAS was also examined by global transcriptional analysis. We identified a gene, rscA (regulated by stress and Cov), whose transcription was confirmed to be repressed by CovR and activated by heat and acid. RscA is a member of the MDR1 family of ABC transporters, and we found that it is required for growth of GAS at 40 degrees C but not at pH 6.0. Thus, for GAS to grow at 40 degrees C, CovR repression must be alleviated so that rscA can be transcribed to allow the production of this potential exporter. Possible explanations for the thermoprotective role of RscA in this pathogen are discussed.

FIG. 1.

RNase protection assays confirm temperature stress microarray analysis. Fifty micrograms of RNA from strain JRS4 that was grown to early log phase at 37°C (lane 1) and then shifted to 40°C for 5 min (lane 2), 10 min (lane 3), or 20 min (lane 4) was hybridized to [α-³²P]UTP-labeled groEL (A), ctsR (B), clpC (C), purC (D), and ska (E) probes. Lane 5 contains a size marker. Arrows indicate the band of the size expected for the hybridization product with each probe.

FIG. 2.

RNase protection assays confirm acid stress microarrays. Fifty micrograms of RNA from strain JRS4 that was grown to early log phase at pH 7.5 (lane 1) and then shifted to pH 6.0 for 5 min (lane 2), 10 min (lane 3), or 20 min (lane 4) was hybridized to [α-³²P]UTP-labeled atpB (A) and ska (B) probes. Lane 5 contains a size marker. Arrows indicate the band of the size expected for the hybridization product with each probe.

FIG. 3.

Transcription of SPy0229 is increased at pH 6.0 and at 40°C. RNase protection assays in which 50 μg of RNA from strain JRS4 was hybridized to the [α-³²P]UTP-labeled SPy0229 probe are depicted. (A) RNA from JRS4 was grown to early log phase at pH 7.5 (lane 1) and then shifted to pH 6.0 for 5 min (lane 2), 10 min (lane 3), or 20 min (lane 4). (B) RNA was harvested from JRS4 grown to early log phase at 37°C (lane 1) and then shifted to 40°C for 5 min (lane 2), 10 min (lane 3), or 20 min (lane 4). Lane 5 contains a size marker in both panels. Arrows indicate the band of the size expected for the hybridization product with the SPy0229 probe.

FIG. 4.

CovR represses transcription of SPy0229. For the RNase protection assay, 50 μg of RNA from strain JRS4 or JRS948 harvested at the late exponential phase was hybridized in duplicate to an [α-³²P]UTP-labeled SPy0229 probe. Arrows indicate the band of the size expected for the hybridization product. WT, wild type.

FIG. 5.

Acid-dependent increase in rscA transcript levels is largely CovR dependent. For the RNase protection assay, 50 μg of RNA from strain JRS4 or JRS948 was hybridized in duplicate to an [α-³²P]UTP-labeled rscA (SPy0229) probe. Each panel contains RNA from JRS4 (wild type [WT]) grown to early log phase at pH 7.5 (lanes labeled 1) and shifted to pH 6.0 for 5 min (lanes labeled 2) and RNA from JRS948 (ΔcovR) grown to early log phase at pH 7.5 (lanes labeled 3) and shifted to pH 6.0 for 5 min (lanes labeled 4). Lane 5 contains a size marker in both panels. Arrows indicate the band of the size expected for the hybridization product.

FIG. 6.

Growth of JRS4 and JRS1819 cultures shifted from 37°C to 40°C. Cultures grown in THY broth at 37°C (closed circles) were diluted and shifted to 40°C at early log phase (open circles). (A) JRS4 (wild type); (B) JRS1819 (rscA mutant). Growth curves were repeated at least twice for each strain with similar results.

FIG. 7.

Efficiency of plating (e.o.p) (rscA). Cultures grown overnight at 37°C in THY broth were plated at 37°C and 40°C and incubated for up to 48 h. Numbers are reported as CFU/milliliter at 40°C divided by CFU/milliliter at 37°C and are the means of at least two experiments, with error bars indicating the standard deviations.