SarA is a repressor of hla (alpha-hemolysin) transcription in Staphylococcus aureus: its apparent role as an activator of hla in the prototype strain NCTC 8325 depends on reduced expression of sarS

Abstract

In most Staphylococcus aureus strains, inactivation of sarA increases hla transcription, indicating that sarA is a repressor. However, in S. aureus NCTC 8325 and its derivatives, used for most studies of hla regulation, inactivation of sarA resulted in decreased hla transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its rsbU mutation, which leads to sigma(B) deficiency. This has now been verified by the demonstration that sarA repressed hla transcription in an rsbU+ derivative of strain 8325-4 (SH1000). That sarA could act as a repressor of hla in an 8325-4 background was confirmed by the observation that inactivation of sarA in an agr sarS rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sarA as an activator of hla in 8325-4 was not a result of the rsbU mutation alone, as inactivation of sarA in another rsbU mutant, strain V8, led to increased hla transcription. Northern blot analysis revealed much higher levels of sarS mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the sarS activator, tcaR, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in sarS transcription upon the inactivation of sarA was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of sarA in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of sarA means a net decrease in repressor activity and, therefore, enhanced hla transcription.

FIG. 1.

(A) Hemolysin production by strains 8325-4 (rsbU) and SH1000 (rsbU⁺) and their corresponding sarA (PC1839 and KT3001), sarS (WA501 and WA502), and sarA sarS (WA504 and WA507) mutant derivatives, grown on a rabbit blood agar plate. wt, wild type. (B) Hemolysin production by KT3001 (SH1000 sarA) and WA1215 (SH1000 sarA hla), grown on a rabbit blood agar plate. (C) Immunoblot analysis of α-hemolysin in culture supernatants of PC1839 (8325-4 sarA) (lane 1), 8325-4 (lane 2), SH1000 (lanes 3 and 4), KT3001 (SH1000 sarA) (lane 5), and WA502 (SH1000 sarS) (lane 6).

FIG. 2.

Northern blot analysis of sarS, sarA, RNAIII, 16S rRNA, and hla in strain SH1000 (rsbU⁺) and its derivatives WA502 (sarS) and KT3001 (sarA) and in strain 8325-4 (rsbU) and its derivative PC1839 (sarA). The main sarA transcript seen is the sigma B-dependent sarA P3 transcript, which is markedly enhanced in SH1000. The bottom panel (hla) shows a reduced exposure. Samples were taken at the specified time points during the growth of a representative culture. wt, wild type.

FIG. 3.

(A) Northern blot analysis of hla and RNAIII in strains V8 and WA845 (V8 sarA). (B) Northern blot analysis of levels of sarS and 16S rRNA in strains PC1839 (8325-4 sarA), 8325-4, WA845 (V8 sarA), and V8. Samples were taken at the specified time points during growth.

FIG. 4.

Northern blot analysis of hla, RNAIII, and 16S rRNA in WA1434 (8325-4 sarS, containing pKT601 with xylose-inducible sarA). Samples were taken at the indicated time points during growth in liquid media with or without 0.05% xylose (xyl; final concentration).

FIG. 5.

(A) Northern blot analysis of hla and 16S rRNA in WA1049 (agr sarS rot sarA), WA1029 (agr sarS rot), WA1430 (agr rot sarA), WA1428 (agr sarS sarA), and RN6390 (wild type [wt]), grown in liquid cultures with samples taken at the indicated time points. (B) Hemolysin production by strains, grown on a rabbit blood agar plate, from left to right as follows: row 1, 8325-4 (wt) and WA1217 (agr sarS); row 2, PM466 Δrot::tet (agr rot) and WA1049 (agr sarS rot sarA); and row 3, PM466 (agr) and WA1029 (agr sarS rot).

FIG. 6.

Northern blot analysis of RNAIII and hla in WA505 (8325-4 sarA sarS, containing pIK64 with xylose-inducible sigB). Samples were taken at the indicated time points during growth in liquid media with or without 0.05% xylose (xyl; final concentration).

FIG. 7.

Northern blot analysis of hla and 16S rRNA in WA900 (sarS sarA rot triple mutant of SH1000) and 8325-4. Samples were taken at the indicated time points during the growth of representative cultures.

FIG. 8.

Effect of complementation of the sarS mutant derived from SH1000. Northern blot analysis of RNAIII in WA519 (SH1000 sarS with pKT210 carrying sarS), WA502 (SH1000 sarS), and SH1000. Samples were taken at the indicated time points during the growth of representative cultures.

FIG. 9.

Schematic overview of regulatory interactions involving sarA, sarS, sarT, rot, agr (RNAIII), and σ^B in the control of hla transcription. The arrows indicate stimulation, and the bars indicate repression. The asterisks indicate genes known to be upregulated by σ^B. In the exponential phase of growth, hla transcription is suppressed by sarA, sarS, and rot (45, 67) (Fig. 5). The transcription of sarS is stimulated by rot (56, 59) and sarT (62) and is suppressed by sarA (15, 67). The transcription of sarT is repressed by sarA and agr (RNAIII) (63). RNAIII expression is enhanced by sarA (13, 17, 65). In the post-exponential phase, RNAIII is supposed to neutralize Rot activity (23, 45), whereas the transcription of sarA and sarS is σ^B dependent (4, 34, 67) (Fig. 2). In addition, σ^B seems to suppress hla transcription via mechanisms not involving sarA, sarS, and rot (referred to as X) (Fig. 7). For further explanation, see the text.