In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus

Abstract

The RNA polymerase (RNAP) holoenzyme of Staphylococcus aureus was purified by DNA affinity, gel filtration, and ion-exchange chromatography. This RNAP contained four major subunits with apparent molecular masses of 165, 130, 60, and 47 kDa. All four subunits of the RNAP were serologically related to the subunits of Escherichia coli E 70 holoenzyme by Western immunoblot analysis. The 60-kDa subunit was subsequently isolated and found to react with a monoclonal antibody specific to the E. coli 70 subunit. This 70 -related protein allowed E. coli core RNAP promoter-specific initiation and increased transcription by S. aureus RNAP that is unsaturated with sigma. We therefore suggest that this 60-kDa protein is a sigma factor. Purified S. aureus RNAP transcribed from the promoters of several important S. aureus virulence genes (sea, sec, hla, and agr P2) in vitro. The in vitro transcription start sites of the sea, sec, and agr P2 promoters, mapped by primer extension, were similar to those identified in vivo. The putative promoter hexamers of these three genes showed strong sequence similarity to the E. coli 70 consensus promoter, and transcription by E 70 from some of these promoters has been observed. Conversely, S. aureus RNAP does not transcribe from all E. coli 70 -dependent promoters. Taken together, our results indicate that the promoter sequences recognized by purified S. aureus RNAP are similar but not identical to those recognized by E. coli E 70 .Staphylococcus aureus is an important human and animal pathogen. It causes a variety of human infections, from localized skin suppuration to life-threatening septicemia. This pathogen also causes mastitis among dairy cattle, sheep, and goats (3, 16). In addition, staphylococcal enterotoxins are the emetic toxins responsible for staphylococcal food poisoning (6). Therefore, studying virulence gene regulation in S. aureus is useful for understanding pathogenesis and medical treatment.Many virulence genes of S. aureus have been cloned and sequenced. Known virulence factors in S. aureus include ␣-hemolysin (encoded by hla), staphylococcal enterotoxin A (sea), and staphylococcal enterotoxin C (sec). The global accessory gene regulator (agr) regulates hla transcription and translation and sec transcription but does not regulate sea expression (4,27,33,36,43). In addition to agr, the exoprotein regulator (xpr) and staphylococcal accessory regulator (sar) genes were recently identified as two distinct global elements (12,13,20,40), but the mechanism by which these global regulators affect gene expression has not been clearly elucidated. The putative promoter regions of some virulence genes have been identified (8,30,35). Recent mutational analysis of sea suggests that S. aureus has promoter structures similar to those recognized by Escherichia coli E 70 (8). However, promoter recognition by S. aureus RNA polymerase (RNAP) is not well understood yet because of the lack of an in vitro transcription system.The genes encoding S. aureus RNAP subunits have been reported. The ge...

The molecular architecture of the sar locus in Staphylococcus aureus

“…Consequently, recognition by E. coli 70 of some of these S. aureus promoters has been observed. However, not all 70 -dependent promoters from E. coli are recognized by the RNA polymerase of S. aureus (27). The same promoter elements are also recognized by A -dependent RNA polymerase in Bacillus subtilis and probably resemble a conserved signal for eubacterial promoters (16).…”

Functional Characterization of the σ B -Dependent yabJ - spoVG Operon in Staphylococcus aureus : Role in Methicillin and Glycopeptide Resistance

“…2C) was identified. However, the sequence preceding the spoVG TSP candidate shared no homology with either a A consensus promoter (TTGACA-16/18 -TATAAT) (10,35) or a B consensus promoter (GTTTAA-12/15 -GGGTAT) (18), suggesting that the small spoVG transcript may possibly be a result of processing and not a product of a specific promoter.…”

Catabolite Control Protein E (CcpE) Is a LysR-type Transcriptional Regulator of Tricarboxylic Acid Cycle Activity in Staphylococcus aureus

“…2C). The transcriptional start point is preceded by nucleotide sequence ATGACA-17-TATAAT that strongly matched with the Ϫ35 and Ϫ10 hexamers identified in the promoters of genes shown to be transcribed by S. aureus A containing RNA polymerase (41,42). Because many LysR-type of regulators display autocrine regulation of their own transcription (43), we determined the capacity of CcpE to bind to its own promoter.…”