TcaR, a putative MarR-like regulator of sarS expression

Abstract

TcaR, which shares sequence homology with MarR-like transcriptional regulators, has been identified as a novel Staphylococcus aureus regulator affecting the expression of the global regulatory element SarS (SarH1), as well as that of the cell surface-associated protein SasF (N315-SA2439). Microarray analysis, confirmatory Northern blots, and genetic complementation experiments showed that TcaR upregulates sarS and thus spa transcription. In addition, it attenuates whole-length transcription of sasF, thereby producing a truncated transcript lacking the 3' terminus, which codes for the cell wall anchor motif. Hence, in strains containing an intact tcaR gene, TcaR is likely to decrease the amount of the surface-associated protein SasF and to increase that of the surface-associated protein A. The widely used laboratory strains derived from NCTC8325 were found to be natural, truncated mutants of tcaR, harboring an inactive TcaR and therefore expressing very low levels of sarS. The data presented here identified TcaR as a further activator of sarS, and a modulator of sasF expression that has to be taken into account in studies of virulence gene expression in S. aureus.

FIG. 1.

Schematic representation of the tcaRAB locus. (A) Map of the chromosomal region surrounding the tcaRAB operon. The nomenclature of the ORFs corresponds to the published S. aureus N315 genome sequence. (B) Depiction of the Tn917 induced deletion that occurred in BB1372.

FIG. 2.

Transcription of sarS, spa, and sasF during growth. (A) Growth curve of S. aureus strain COL. The arrows indicate the OD₆₀₀ points at which RNA was isolated. (B) Northern blot analyses of sarS and spa. Samples 1 to 5 correspond to RNA samples isolated at the OD₆₀₀ values indicated in panel A. (C) Transcription of sasF. Specific transcripts are indicated on the right; two additional hybridizing bands, which corresponded to the sizes of the 23S rRNA and 16S rRNA, are indicated on the left. (D) As an indication of RNA loading, the 16S rRNA band from the ethidium bromide-stained gel is shown.

FIG. 3.

Complementation of the COL ΔtcaRAB phenotype by tcaR. Northern blots of BB1372 complemented with the empty plasmid vector pMGS100, ptcaA, or ptcaR_COL against the sarS, spa, and sasF probes. Total RNA was harvested at growth stages corresponding to the OD₆₀₀ values indicated above the lanes. As an indication of RNA loading, the 16S rRNA band from the ethidium bromide stained gel is shown.

FIG. 4.

Amino acid sequence alignment of TcaR against MarR from E. coli, SarA, and identified SarA homologues from S. aureus and other homologous protein sequences identified from the S. aureus N315 genome by BLASTP analysis. The sequences of SarS (SarH1) and SarH2, which both contain two domains with homology to SarA, were truncated so that only the N-terminal domain of each was included in the alignment. The region containing the predicted helix-turn-helix motif of TcaR (Network Protein Sequence analysis [9]) and several of the other homologues is highlighted in gray. Strongly conserved residues are indicated in the consensus line and universally conserved residues are in bold type. The arrowhead represents the position (amino acid 79) at which the TcaR protein in NCTC8325-4 is truncated.

FIG. 5.

Transcription of sarS and sasF in BB255 and BB255 complemented with either the empty plasmid vector pMGS100, the tcaR gene from B255 (ptcaR_BB255), or the tcaR gene from COL (ptcaR_COL). Total RNA was harvested at growth stages corresponding to the OD₆₀₀ values shown. As an indication of RNA loading the 16S rRNA band from the ethidium bromide-stained gel is shown.

FIG. 6.

Effect of sarS mutation on the expression of spa and sasF. Northern analysis of COL and BB1372 and their corresponding sarS mutants, COLsarS::pAD21 and BB1372sarS::pAD21. Total RNA was harvested at growth stages corresponding to the OD₆₀₀ values shown. As an indication of RNA loading the 16S rRNA band from the ethidium bromide-stained gel is shown.