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J Bacteriol. May 1995; 177(10): 2609–2614.
PMCID: PMC176928

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


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 sigma 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 sigma 70 subunit. This sigma 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 sigma 70 consensus promoter, and transcription by E sigma 70 from some of these promoters has been observed. Conversely, S. aureus RNAP does not transcribe from all E. coli sigma 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 sigma 70.

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Selected References

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  • Abbas-ali B, Coleman G. The characteristics of extracellular protein secretion by Staphylococcus aureus (Wood 46) and their relationship to the regulation of alpha-toxin formation. J Gen Microbiol. 1977 Apr;99(2):277–282. [PubMed]
  • Aboshkiwa M, al-Ani B, Coleman G, Rowland G. Cloning and physical mapping of the Staphylococcus aureus rplL, rpoB and rpoC genes, encoding ribosomal protein L7/L12 and RNA polymerase subunits beta and beta'. J Gen Microbiol. 1992 Sep;138(9):1875–1880. [PubMed]
  • Basheer R, Iordanescu S. The Staphylococcus aureus chromosomal gene plaC, identified by mutations amplifying plasmid pT181, encodes a sigma factor. Nucleic Acids Res. 1991 Sep 25;19(18):4921–4924. [PMC free article] [PubMed]
  • Betley MJ, Borst DW, Regassa LB. Staphylococcal enterotoxins, toxic shock syndrome toxin and streptococcal pyrogenic exotoxins: a comparative study of their molecular biology. Chem Immunol. 1992;55:1–35. [PubMed]
  • Borst DW, Betley MJ. Promoter analysis of the staphylococcal enterotoxin A gene. J Biol Chem. 1994 Jan 21;269(3):1883–1888. [PubMed]
  • Borukhov S, Sagitov V, Goldfarb A. Transcript cleavage factors from E. coli. Cell. 1993 Feb 12;72(3):459–466. [PubMed]
  • Burgess RR, Jendrisak JJ. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. [PubMed]
  • Cheung AL, Koomey JM, Butler CA, Projan SJ, Fischetti VA. Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6462–6466. [PMC free article] [PubMed]
  • Cheung AL, Ying P. Regulation of alpha- and beta-hemolysins by the sar locus of Staphylococcus aureus. J Bacteriol. 1994 Feb;176(3):580–585. [PMC free article] [PubMed]
  • Couch JL, Betley MJ. Nucleotide sequence of the type C3 staphylococcal enterotoxin gene suggests that intergenic recombination causes antigenic variation. J Bacteriol. 1989 Aug;171(8):4507–4510. [PMC free article] [PubMed]
  • Cowing DW, Bardwell JC, Craig EA, Woolford C, Hendrix RW, Gross CA. Consensus sequence for Escherichia coli heat shock gene promoters. Proc Natl Acad Sci U S A. 1985 May;82(9):2679–2683. [PMC free article] [PubMed]
  • Erickson JW, Gross CA. Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression. Genes Dev. 1989 Sep;3(9):1462–1471. [PubMed]
  • Gentry DR, Burgess RR. Overproduction and purification of the omega subunit of Escherichia coli RNA polymerase. Protein Expr Purif. 1990 Sep;1(1):81–86. [PubMed]
  • Hager DA, Jin DJ, Burgess RR. Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase. Biochemistry. 1990 Aug 28;29(34):7890–7894. [PubMed]
  • Harley CB, Reynolds RP. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. [PMC free article] [PubMed]
  • Hart ME, Smeltzer MS, Iandolo JJ. The extracellular protein regulator (xpr) affects exoprotein and agr mRNA levels in Staphylococcus aureus. J Bacteriol. 1993 Dec;175(24):7875–7879. [PMC free article] [PubMed]
  • Hawley DK, McClure WR. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. [PMC free article] [PubMed]
  • Hufnagle WO, Tremaine MT, Betley MJ. The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule. Infect Immun. 1991 Jun;59(6):2126–2134. [PMC free article] [PubMed]
  • Karls RK, Jin DJ, Donohue TJ. Transcription properties of RNA polymerase holoenzymes isolated from the purple nonsulfur bacterium Rhodobacter sphaeroides. J Bacteriol. 1993 Dec;175(23):7629–7638. [PMC free article] [PubMed]
  • Karls R, Schulz V, Jovanovich SB, Flynn S, Pak A, Reznikoff WS. Pseudorevertants of a lac promoter mutation reveal overlapping nascent promoters. Nucleic Acids Res. 1989 May 25;17(10):3927–3949. [PMC free article] [PubMed]
  • Kehoe M, Duncan J, Foster T, Fairweather N, Dougan G. Cloning, expression, and mapping of the Staphylococcus aureus alpha-hemolysin determinant in Escherichia coli K-12. Infect Immun. 1983 Sep;41(3):1105–1111. [PMC free article] [PubMed]
  • Lonetto M, Gribskov M, Gross CA. The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol. 1992 Jun;174(12):3843–3849. [PMC free article] [PubMed]
  • Lowe PA, Hager DA, Burgess RR. Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry. 1979 Apr 3;18(7):1344–1352. [PubMed]
  • Morfeldt E, Janzon L, Arvidson S, Löfdahl S. Cloning of a chromosomal locus (exp) which regulates the expression of several exoprotein genes in Staphylococcus aureus. Mol Gen Genet. 1988 Mar;211(3):435–440. [PubMed]
  • Newlands JT, Gaal T, Mecsas J, Gourse RL. Transcription of the Escherichia coli rrnB P1 promoter by the heat shock RNA polymerase (E sigma 32) in vitro. J Bacteriol. 1993 Feb;175(3):661–668. [PMC free article] [PubMed]
  • Novick RP, Brodsky R. Studies on plasmid replication. I. Plasmid incompatibility and establishment in Staphylococcus aureus. J Mol Biol. 1972 Jul 21;68(2):285–302. [PubMed]
  • Novick RP, Ross HF, Projan SJ, Kornblum J, Kreiswirth B, Moghazeh S. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J. 1993 Oct;12(10):3967–3975. [PMC free article] [PubMed]
  • Peng HL, Novick RP, Kreiswirth B, Kornblum J, Schlievert P. Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus. J Bacteriol. 1988 Sep;170(9):4365–4372. [PMC free article] [PubMed]
  • Regassa LB, Couch JL, Betley MJ. Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose. Infect Immun. 1991 Mar;59(3):955–962. [PMC free article] [PubMed]
  • Ross W, Thompson JF, Newlands JT, Gourse RL. E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 1990 Nov;9(11):3733–3742. [PMC free article] [PubMed]
  • Sandler P, Weisblum B. Erythromycin-induced stabilization of ermA messenger RNA in Staphylococcus aureus and Bacillus subtilis. J Mol Biol. 1988 Oct 20;203(4):905–915. [PubMed]
  • Schmidt MC, Chamberlin MJ. Amplification and isolation of Escherichia coli nusA protein and studies of its effects on in vitro RNA chain elongation. Biochemistry. 1984 Jan 17;23(2):197–203. [PubMed]
  • Smeltzer MS, Hart ME, Iandolo JJ. Phenotypic characterization of xpr, a global regulator of extracellular virulence factors in Staphylococcus aureus. Infect Immun. 1993 Mar;61(3):919–925. [PMC free article] [PubMed]
  • Strickland MS, Thompson NE, Burgess RR. Structure and function of the sigma-70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription. Biochemistry. 1988 Jul 26;27(15):5755–5762. [PubMed]
  • Tjian R, Losick R, Pero J, Hinnebush A. Purification and comparative properties of the delta and sigma subunits of RNA polymerase from Bacillus subtilis. Eur J Biochem. 1977 Mar 15;74(1):149–154. [PubMed]
  • Tremaine MT, Brockman DK, Betley MJ. Staphylococcal enterotoxin A gene (sea) expression is not affected by the accessory gene regulator (agr). Infect Immun. 1993 Jan;61(1):356–359. [PMC free article] [PubMed]

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