[PMC free article] [PubMed] [Google Scholar] 15. skin illnesses to life-threatening deep infections, such as endocarditis, meningitis, arthritis, and toxic shock syndrome (26, 36, 39). The frequencies of both community- and hospital-acquired staphylococcal infections have increased steadily with little change in overall mortality (16). Treatment of these infections has become more difficult because of the emergence of multidrug-resistant strains (16, 37). The virulence of is dependent upon its ability to respond to a wide range of host conditions during the contamination process. Transcriptional regulators such as sigma factors are likely to play an important role in the bacterial adaptive responses needed for pathogenesis (29). Indeed, alternative sigma factors have been correlated with virulence in several pathogenic species (13, 17, 18). Recently, an alternative sigma factor gene known as was recognized within a four-gene operon (22, 40). With strong primary amino acid similarity to SigB of SigB has been Acebutolol HCl evaluated as a stress response and stationary-phase sigma factor and has been shown to be induced during stationary phase and upon warmth shock (22). Additionally, interruption of the gene causes increased sensitivity to hydrogen peroxide during the stationary growth phase (23). By in vitro transcription SigB has further been shown to participate in the transcription of the locus (12), which is usually itself a key regulator of virulence gene expression (7, 8, Acebutolol HCl 27). Furthermore, the expression of lipase and thermonuclease, which play important functions in abscess formation, have been associated with SigB control (23). Thus, SigB appears to participate directly and indirectly in the expression of virulence genes. While most sigma factors are themselves transcriptionally regulated, posttranslational control by other proteins known as anti-sigma factors also plays an important role in controlling their activity in some instances (4, 5, 31). Anti-sigma factor proteins bind and sequester a specific sigma factor, thus blocking transcription initiation (5, 19). The RsbW protein has been shown to function as an anti-sigma factor of the stress response regulator SigB (4, 14, 19). The gene is located immediately upstream of the gene, and the two are cotranscribed. RsbW and SigB demonstrate specific binding by column chromatography and coimmunoprecipitation with monoclonal antibodies to either protein (4). RsbW also efficiently blocked SigB-dependent transcription in vitro. Recent DNA sequence analyses of the operon revealed four complete open reading frames (in (22, 40). These similarities Acebutolol HCl suggest that RsbW of is an anti-sigma factor of SigB. In this report we demonstrate that RsbW binds to SigB and inhibits SigB-dependent transcription. We also identify two possible new members of the SigB regulon by demonstrating that the genes for alkaline shock protein 23 and coagulase show SigB and RsbW dependence in vitro. MATERIALS AND METHODS Strains and plasmids. The TA cloning vector pCRII was purchased from Invitrogen Corp. (Carlsbad, Calif.). BL21(DE3) and the vector pET32b, used for protein overexpression, were obtained from Novagen (Madison, Wis.). Isolation and purification of plasmids were performed by using the Qiagen system (Qiagen, Inc., Chatsworth, Calif.). strains ATCC 29213 and 8325-4 were used as sources of chromosomal DNA for the PCR amplifications. Construction of plasmids for overexpressing and (gene was amplified by PCR with primers SAF005 (5-ATCCATGGCGAAAGAGTCGAAATC-3) and SAR003 (5-CGGATCCTATTGATGTGCTGCTTCTTG-3); this PCR product was cloned into pCRII, and the resulting plasmid was designated pEM101. The Rabbit Polyclonal to GSPT1 (expression vector, pEM202. Purification of SigB and RsbW fusion proteins. pET32b-based, His6-thioredoxin (Trx) fusion proteins were expressed and purified according to the recommendations of the manufacturer (Clontech Laboratories, Inc., Palo Alto, Calif.) with some modifications. BL21(DE3) transformed with pEM102, pEM202, and pET32b (generating strains EMBL1, EMBL2, and EMBL3, respectively) was grown in 250 ml of Luria-Bertani medium containing ampicillin (100 g/ml) at 37C until the culture reached an optical density at 600 nm (OD600) of between 0.6 to 0.8. Induction with isopropyl–d-thiogalactopyranoside (IPTG) at 1 mM was conducted for 3 h, and then the cells were harvested, suspended in 10 ml of lysis buffer (50 mM NaH2PO4, 10 mM Tris-HCl, 100 mM NaCl; pH 8), and lysed by sonication. After centrifugation at 12,000 rpm for 30 min, the resulting supernatant was loaded onto a 2-ml column of metal affinity resin (Clontech) equilibrated with sonication buffer. After exposure to excess wash buffer (50 mM.
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