s activity depends on rsbu in staphylococcus aureus · general methods. all dna manipulations,...

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.6.1843–1852.2001

Mar. 2001, p. 1843–1852 Vol. 183, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

sB Activity Depends on RsbU in Staphylococcus aureusP. GIACHINO,1 S. ENGELMANN,2 AND M. BISCHOFF1*

Institute of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland,1 and Institute of Microbiologyand Molecular Biology, Ernst-Moritz-Arndt University, D-17487 Greifswald, Germany2

Received 6 September 2000/Accepted 14 December 2000

Derivatives of the widely used laboratory strain Staphylococcus aureus NCTC8325, which are natural rsbUmutants, were shown to be unable to produce RsbU, a positive regulator of the alternative sigma factor sB. Thelack of RsbU prevented the heat-dependent production of sB-controlled transcripts and resulted in reducedH2O2 and UV tolerance, enhanced alpha-hemolysin activity, and the inability to produce the alkaline shockprotein Asp23. After 48 h of growth, rsbU mutant strains failed to accumulate staphyloxanthin, the majorstationary-phase carotenoid. Transcription of Asp23 was found to be exclusively controlled by sB, making it anexcellent target for the study of sB activity in S. aureus. Reporter gene experiments, using the firefly luciferasegene (luc1) fused to the sB-dependent promoter(s) of asp23, revealed that sB is almost inactive in 8325derivatives. cis complementation of the 8325 derivative BB255 with the wild-type rsbU gene from strain COLproduced the rsbU1 derivative GP268, a strain possessing a sB activity profile comparable to that of the rsbU1

wild-type strain Newman. In GP268, the heat inducibility of sB-dependent genes, Asp23 production, alpha-hemolysin activity, pigmentation, and susceptibility to H2O2 were restored to the levels observed in strainNewman, clearly demonstrating that RsbU is needed for activation of sB in S. aureus.

Staphylococcus aureus is a major human pathogen causing awide spectrum of diseases and able to survive under a varietyof extreme conditions. In many bacteria, alternative sigma fac-tors have been shown to be important for survival under ex-treme conditions by regulating the coordinate expression ofstress response genes triggered by environmental as well asgrowth-dependent stimuli. As part of the RNA polymeraseholoenzyme, the sigma subunits are responsible for the bindingof the catalytic core to specific promoter regions and the ini-tiation of transcription of downstream genes. Thus, sigma fac-tors provide an elegant mechanism in eubacteria to ensuresimultaneous transcription of a variety of genetically unlinkedgenes, provided all these genes share the critical promoterelements. The alternative sigma factor sB of Bacillus subtilishas been shown to control the transcription of more than 100genes in response to different stimuli such as heat, ethanol, orsalt stress; acid shock; or glucose, oxygen, or phosphate star-vation (for reviews see references 23 and 46). In B. subtilis, sB

activity itself is controlled posttranslationally by a multicom-ponent signal transduction pathway comprising eight regula-tory proteins which—with the exception of Obg and RsbP—are coexpressed with the sigma factor as part of the sameoperon (3, 7, 24, 40, 44, 48, 50). One of these proteins, RsbU,a positive regulator of sB, is essential for the activation of sB

during exponential growth after environmental stress (45, 48,50). RsbU activity itself is controlled by the action of furtherRsb proteins encoded by the operon (1, 19, 50).

An operon encoding four proteins, sharing strong primaryamino acid similarity with RsbU, RsbV, RsbW, and sB of B.subtilis, has been identified in S. aureus (27, 49). The putativeS. aureus sB was shown to act as a sigma factor initiating the

transcription of sarC from the sar P3 promoter (17, 32). RsbW,on the other hand, was shown to be an anti-sigma factor,regulating sB activity posttranslationally (32). sB is activatedupon heat shock in S. aureus strain MA13 (20) and controls thetranscription of at least 30 genes encoding cytoplasmic proteins(21). Although sB was shown to be involved in the heat andacid shock response of strain MA13, it had no apparent func-tion in strain 8325-4, either in the heat shock response, star-vation survival, or pathogenicity, in a mouse abscess model (10,20).

A phenotypic comparison of genetically distinct wild-type S.aureus strains and their DrsbUVWsigB mutants revealed themutants to be almost unpigmented and to be unable to pro-duce the alkaline shock protein Asp23. Furthermore, the mu-tants showed increased alpha-hemolysin activity and weremore susceptible to hydrogen peroxide (28, 33). Remarkably,the 8325 derivative BB255 showed essentially the same phe-notype as DrsbUVWsigB mutants. This phenomenon was tracedback to an 11-bp deletion in the 59 part of the rsbU gene ofstrain BB255, generating a stop codon within a short distancedownstream. This 11-bp deletion was also found in the 8325derivatives 8325-4 and RN4220 (20, 28).

In this study, we demonstrate that 8325 derivatives are un-able to produce the positive regulator RsbU. The lack of thisprotein results in dramatic changes in sB activity compared tothat in rsbU1 strains. cis complementation of the 8325 deriv-ative BB255 with the rsbU1 allele from COL restored the sB

activity profile as well as the sB-dependent phenotypic prop-erties to the levels seen in the Newman strain.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. The bacterial strains andplasmids used in this study are listed in Table 1. S. aureus was routinely grown inLuria-Bertani (LB) medium at 37°C and 200 rpm. Antibiotics were used at thefollowing concentrations: chloramphenicol, 30 mg ml21; erythromycin and tet-racycline, 10 mg ml21; ampicillin and kanamycin, 50 mg ml21.

* Corresponding author. Mailing address: Institute of Medical Mi-crobiology, University of Zurich, Gloriastr. 32, Postfach, CH-8028Zurich, Switzerland. Phone: 41 1 634 26 70. Fax: 41 1 634 49 06. E-mail:[email protected].

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General methods. All DNA manipulations, basic molecular methods, andhandling of Escherichia coli were performed in accordance with standard proto-cols (39). Genetic manipulation of S. aureus was done as described earlier (27).S. aureus carotenoids were extracted and analyzed according to the methods ofMarshall and Wilmoth (31) or Raisig and Sandmann (37). The general trans-ducing phage 80a was used for transductions. Preliminary sequence data wereobtained from The Institute for Genomic Research (TIGR) through the website(http://www.tigr.org).

Northern blot analyses. For the heat shock experiments, isolation of totalRNA and analysis of transcription were performed as described by Gertz et al.(20). The specific RNA probes for sigB and crtM were prepared by in vitrotranslation with T7 polymerase and the appropriate PCR fragments as thetemplate. The PCR fragments were generated by using chromosomal DNA of S.aureus strain COL which was purified with the chromosomal DNA isolation kit(Promega) according to the protocol of the manufacturers and oligonucleotidesSasigB1 (59-AAATAATGGCGAAAGAGTCG-39) and SasigB(T7)2 (59-CTAATACGACTCACTATAGGGAGACATAATGGTCATCTTGTTGC-39) (cor-responding to nucleotides 2669 to 2688 and 3228 to 3248, respectively, of Gen-Bank accession no. Y07645) and SacrtM1 (59-CAGAAGATCAAAGAAAGCG-39) and SacrtM(T7)2 (59-CTAATACGACTCACTATAGGGAGCCTGTCTCAACTTCGTCC-39) (nucleotides 317 to 335 and 985 to 1002, respectively, of

accession no. X73889). Nucleotides corresponding to the T7 promoter consensusare underlined. The hybridizations specific for asp23 were conducted with digoxi-genin-labeled RNA as described previously (20).

For all other Northern blot analyses, total RNA was isolated as described byCheung et al. (13). Eight micrograms of total RNA of each sample was electro-phoresed through a 1.5% agarose–0.66 M formaldehyde gel in morpholinepro-panesulfonic acid (MOPS) running buffer (20 mM MOPS, 10 mM sodium ace-tate, 2 mM EDTA [pH 7]). RNA was blotted onto a positively charged nylonmembrane (Roche, Basel, Switzerland) with a vacuum blotter (Pharmacia, Upp-sala, Sweden). The intensities of the 23S and 16S rRNA bands stained withethidium bromide were verified to be equivalent in all the samples before trans-fer. Labeling and hybridization were done by use of the digoxigenin labeling anddetection kits according to the manufacturer’s instructions (Roche). The follow-ing specific primers were used to generate the digoxigenin-labeled DNA probesby PCR labeling: Saasp23A1 (59-ATGACTGTAGATAACAATAAAGC-39)and Saasp23A2 (59-TTGTAAACCTTGTCTTTCTTGG-39) (nucleotides 343 to365 and 828 to 849, respectively, of accession no. S76213) and luc int1 (59-GGAGAGCAACTGCATAAGGC-39) and luc int2 (59-GGCGAAGAAGGAGAATAGG-39) (nucleotides 111 to 130 and 914 to 932, respectively, of accession no.U47122).

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Relevant genotype or phenotypea Reference or source

StrainsE. coli

DH10B F2 f80dlacZDM15 recA1 Gibco, Gaithersburg, Md.BL21(DE3) F2 ompT gal [dcm] [lon] hsdSB (rB

2 mB2), with DE3 Novagen, Madison, Wis.

S. aureusRN4220 NCTC8325-4 r2 m1 (restriction minus, modification plus) 26BB255 Essentially the same as NCTC8325 58325-4 NCTC8325, cured of known prophages 34RN6390 Derivative of NCTC8325 that maintains its hemolytic pattern when

propagated on sheep erythrocytes35

COL mec, high-Mcr clinical isolate, Tcr 25Newman Clinical isolate, high level of clumping factor (ATCC 25904) 18IK181 BB255 DrsbUVWsigB Emr 28IK183 COL DrsbUVWsigB Emr 28IK184 Newman DrsbUVWsigB Emr 28GP268 BB255 (rsbUVWsigB)1-tetL Tcr This studyGP269 8325-4 (rsbUVWsigB)1-tetL Tcr This studyMB25 RN4220 asp231 (asp23P::luc1)-pEC-ermB Emr This studyMB32 Newman asp231 (asp23P::luc1)-pEC-ermB Emr This studyMB33 BB255 asp231 (asp23P::luc1)-pEC-ermB Emr This studyMB49 BB255 (rsbUVWsigB)1-tetL asp231 (asp23P::luc1)-pEC-ermB Tcr Emr This studyMB61 RN4220 asp231 (asp23P::luc1)-pBT-tetL Tcr This studyMB69 Newman DrsbUVWsigB asp231 (asp23P::luc1)-pBT-tetL Tcr Emr This studyMB90 BB255 DrsbUVWsigB asp231 (asp23P::luc1)-pBT-tetL Tcr Emr This study

PlasmidspET-24b(1) Kmr; expression vector NovagenpSP-luc1 Apr; firefly luciferase casette vector PromegapBC SK1 Cmr; cloning vector StratagenepAW8 Tcr; pAMa1 origin and tetL gene of pHY300PLK, ColE1 origin A. Wada, unpublished datapBT Tcr; 1.6-kb PCR fragment of tetL gene of pHY300PLK into Alw26I-

digested pBC SK(1)This study

pEC1 Apr; Emr; 1.45-kb ClaI ermB fragment of Tn551 in pUC18 9pIK6 Apr; 6.6-kb PstI-EcoRI sigB fragment from strain 8325 in pUC18 27pPG11 Apr; Tcr; 252-bp MluI-BstXI fragment of rsbU from strain COL

replacing the corresponding region of the 6.6-kb PstI-EcoRI sigBfragment from strain 8325 in pUC19

This study

pETasp23 510-bp PCR fragment of asp23 from strain 8325 in pET24b This studypETrsbUCOL 1-kb PCR fragment of rsbU from strain COL in pET24b This studypETsigB 770-bp PCR fragment of sigB from strain 8325 in pET24b This studypSPasp23P 1.1-kb PCR fragment of asp23 promoter from strain COL in pSP-luc1 This studypECasp23P::luc1 2.7-kb KpnI-EcoRI asp23P::luc1 fragment of pSPasp23P in pEC1 This studypBTasp23P::luc1 2.7-kb KpnI-EcoRI asp23P::luc1 fragment of pSPasp23P in pBT This study

a Abbreviations are as follows: Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Emr, erythromycin resistant; Kmr, kanamycin resistant; Mcr, methicillinresistant; Tcr, tetracyclin resistant.

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Construction of plasmid pPG11. A 6.6-kb PstI-EcoRI fragment of strainBB255, including the whole sigB operon (27), was subcloned into the MCS ofpUC19. The plasmid obtained was digested with MluI and BstXI, excising a252-bp fragment from the rsbU gene including the 11-bp deletion. The excisedfragment was replaced by the corresponding fragment of the rsbU1 allele fromCOL. In a next step, a 1.6-kb PCR fragment of the tetL gene of pAW8 was clonedinto a blunted Bsp119I site downstream of the sigB operon (corresponding topositions 3545 to 3550 of the sigB operon of strain 8325, accession no. Y07645).The resulting plasmid was electroporated into S. aureus RN4220 DrsbUVWsigB topromote a crossover upstream of rsbU, and screening for double-crossover trans-formants sensitive to erythromycin and resistant to tetracycline was carried out(Fig. 1). In a last step, the engineered chromosomal region of a positive trans-formant was transduced into different 8325 derivatives.

Construction of plasmids pECasp23P::luc1 and pBTasp23P::luc1. A DNAfragment carrying 1.1 kb of the asp23 gene, including its sB-dependent promot-ers, was generated by PCR with primers Saasp23P1 (59-GGGATCCTTTGAGTGAGGAGAAACC-39) including a KpnI linker (underlined), and Saasp23P2(59-CTACAGCCATGGTAGATTCTCCTTTTAC-39) including an NcoI linker(underlined). The PCR product was digested with KpnI and NcoI and cloned infront of the luciferase gene of plasmid pSP-luc1. The identity of the constructwas confirmed by sequence analysis and comparison to the respective COLsequence of the TIGR database. The 2.7-kb KpnI-EcoRI fragment, including theasp23 promoter region fused to the luciferase coding region, was then cloned intoplasmids pEC1 and pBT, respectively. The plasmids obtained were electropo-rated into RN4220 and subsequently transduced into strains BB255, Newman,and GP268 (pECasp23P::luc1) and their respective DrsbUVWsigB mutants(pBTasp23P::luc1) (Fig. 2C).

Construction of E. coli vectors for overexpression of His-tagged RsbU, sB, andAsp23. A DNA fragment carrying 999 bp of the rsbUCOL gene was amplified byPCR using primers SarsbU11 including an NdeI linker (underlined) (59-GGAG

ATATACATATGGAAGAATTTAAGCAAC-39 [the start methionine shown inboldface type]) and SarsbU12 including an XhoI linker (underlined) (59-GGTGGTGCTCATTTACTCTTTTTATAATC-39) (italics correspond to positions 785to 772 and 1764 to 1782, respectively, in accession no. Y09929). The PCRproduct was cloned into pET24b to obtain pETrsbUCOL. Similarly, the sigB geneand the asp23 gene were amplified by PCR using, respectively, primer SasigB11including an NdeI linker (underlined) (59-GGAGATATACATATGGCGAAAGAGTCGAAATCAGC-39) combined with primer SasigB12 including an XhoIlinker (underlined) (59-GTGGTGCTCGAGTTGATGTGCTGCTTCTTG-39)(italics correspond to positions 2674 to 2696 and 3424 to 3441, respectively, inaccession no. Y07645) and primer Saasp23231 (59-GGAGATATACATATGACTGTAGATAACAATAAAGC-39) combined with primer Saasp232 (59-GGTGGTGCTCGAGTTGTAAACCTTGTCTTTCTTGG-39) (italics correspond to posi-tions 343 to 365 and 828 to 849, respectively in accession no. S76213). The PCRproducts were cloned into pET24b to obtain pETsigB or pETasp23, respectively.The junction regions and the introduced PCR products were sequenced toensure proper ligation and fidelity of the PCR. E. coli strain BL21(DE3) wastransformed with the plasmids obtained. Overexpression and purification of theHis-tagged proteins were performed using Ni-nitriloacetic acid (NTA) columnsaccording to the recommendations of the manufacturer (Qiagen, Basel, Switzer-land). The purified proteins were separated using sodium dodecyl sulfate–12%polyacrylamide gel electrophoresis (SDS–12% PAGE), and bands containing theprotein were cut out of the gels. N-terminal sequencing confirmed the identitiesof the desired proteins. The gel slices containing the respective proteins wereinjected into rabbits to raise anti-RsbU, anti-SigB, and anti-Asp23 polyclonalantibodies (BioScience, Gottingen, Germany). The resulting antisera were pu-rified against the immobilized antigens.

Hydrogen peroxide experiments. The MICs and minimal bactericidal concen-trations (MBCs) of hydrogen peroxide were determined by broth microdilutionusing the National Committee for Clinical Laboratory Standards protocol with

FIG. 1. Genetic organization of the sigB operon. (A) Schematic representation of the sigB operon of S. aureus strain NCTC8325. Open readingframes, putative promoters (3), termination signals (E), and restriction sites used for construction of pPG11 are indicated. The 11-bp deletionwithin the rsbU gene of strain 8325, resulting in a truncated open reading frame for RsbU (solid area), is indicated by a triangle (‚). (B) Schematicrepresentation of the rsbU1 construct pPG11 and of the strategy for the integration of this construct into the chromosome of S. aureus BB255. Inplasmid pPG11, a 252-bp MluI-BstXI restriction fragment of the rsbU gene of strain COL including the 11 bp (shaded area) replaces thecorresponding fragment of the rsbU allele from strain BB255 harboring the 11-bp deletion, leading to an open reading frame that encodes afunctional RsbU protein. A tetL resistance gene was introduced as a selective marker downstream of the proposed termination signal of the sigBoperon, in order not to disrupt the transcriptional control of this locus. Strain RN4220 DrsbUVWsigB, in which the major part of the sigB operonis replaced by an ermB resistance cassette (28), was used for electroporation to promote a double crossover of the modified sigB operon of theintroduced pPG11 suicide plasmid upstream of the rsbU gene and downstream of the tetR gene. The chromosomal region of a positive transformantwas phage transduced into strain BB255 to obtain strain GP268.

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serial dilutions of hydrogen peroxide (2.2 M to 0.125 mM). Microtiter plateswere incubated for 24 and 48 h at 37°C.

Luciferase assay. Bacterial cells were harvested by centrifugation (at 11,000 3

g for 1 min. at room temperature), and the cell pellet was resuspended inphosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mMNa2HPO4, 1.4 mM KH2PO4 [pH 7.3]) to an optical density at 600 nm (OD600)of 10 and snap-frozen in liquid nitrogen. Luciferase activity was determined byrapidly mixing PBS-resuspended cells (10 ml) with an equal volume of luciferaseassay reagent (Promega, Madison, Wis.). Luminescence was measured on aTurner Designs TD-20/20 Luminometer (Promega) for a period of 10 s with adelay of 2 s.

UV-stress experiments. Bacterial cells were diluted to McFarland 0.5 andstreaked out on LB agar plates. After plating, cells were immediately exposed tofar-UV light (254 nm) or near-UV light (312 nm) for different time periods, usinga Stratalinker (Stratagene, La Jolla, Calif.) as the light source. The bacteria werethen incubated for 24 h at 37°C.

RESULTS

Occurrence of RsbU and sB in different S. aureus strains.The rsbU gene in S. aureus strain COL encodes a 323-amino-acid open reading frame, while a deletion in the 59 region ofrsbU in strain 8325 generates a premature stop codon, givingrise to an open reading frame of only 74 amino acids. The samedeletion was found in all 8325 derivatives tested (BB255,8325-4, RN4220, RN6390, and BB270) (20, 28). Western blotanalyses using antigen-purified polyclonal antibodies revealedthe presence of RsbU in the clinical isolates COL and Newmanbut not in the 8325 derivatives (Fig. 3B), while sB was detect-

FIG. 2. Genetic organization of the asp23 operon of S. aureus. (A)Schematic representation of the asp23 operon of S. aureus based on acomparison of the respective sequence region of strain COL, obtainedfrom the unfinished TIGR microbial database. The probes used forNorthern blot analyses, open reading frames, putative promoters, andthe transcripts detected are indicated. (B) Putative promoter se-quences of the asp23 locus. Nucleotides of the 235 and 210 regions ofthe putative promoters of the asp23 locus which are identical to thesB-dependent promoter consensus of B. subtilis are boldfaced. Spacerregions between the 235 and 210 hexameric nucleotide sequences,and between the promoter sequence and the proposed start codons ofthe closest open reading frames, are indicated. (C) Schematic repre-sentation of the integration of asp23P::luc1 fusion constructs into theS. aureus chromosome by single crossover. For construction of plas-mids pECasp23P::luc1 and pBTasp23P::luc1, and integration of theconstructs into the S. aureus chromosome, see Materials and Methods.

FIG. 3. Western blot analyses of different S. aureus strains. Cyto-plasmic protein fractions (10 mg/lane) of different S. aureus overnightcultures, grown in LB medium at 37°C and 200 rpm, were separatedusing SDS–10% PAGE and blotted onto nitrocellulose. The blottedproteins were either stained with amido black (A) or subjected toWestern blot analyses using antigen-purified anti-RsbU antibodies(B), anti-SigB antibodies (C), or anti-Asp23 antibodies (D). Thebroad-range molecular size marker (Gibco-BRL) was used. Relevantprotein signals are indicated.


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able in all strains analyzed except BB255 DrsbUVWsigB (Fig.3C).

Fifteen independent clinical isolates of S. aureus, selected atthe University Hospital of Zurich in 1999, were tested for thepresence of the deletion in rsbU by PCR using oligonucleotidesflanking the deletion site (28). None of the clinical isolatestested had such a deletion. The presence of RsbU in all clinicalisolates was demonstrated by Western blot analysis (data notshown). These results indicated (i) the general presence ofRsbU in clinical isolates and (ii) that 8325 derivatives areunable to produce the potential sB activator RsbU. Further-more, they excluded the possibility that an RsbU protein, trun-cated at its N terminus, is translated by use of a cryptic startcodon downstream of the deletion. Strain 8325 derivatives aretherefore natural rsbU mutants.

Complementation of strain BB255 with rsbUCOL. The find-ing that 8325 derivatives are devoid of RsbU and the fact thatmost studies on sB have been conducted with these strainsprompted us to investigate the effects of RsbU on the pheno-type of S. aureus in the 8325 isogenic background by replacingthe truncated rsbU gene of BB255 with the intact rsbU1 allelefrom strain COL. For this purpose, we constructed the suicideplasmid pPG11, harboring a 6.6-kb chromosomal region in-cluding the sigB operon of strain BB255 and the rsbU gene ofstrain COL. In order not to disrupt the transcriptional integrityof the sigB operon, the tetL gene was inserted as a selectivemarker downstream of the operon (Fig. 1). To promote cross-over events upstream of the rsbU region, we used RN4220DrsbUVWsigB mutants for electroporation and selected fortransformants that were resistant to tetracycline but sensitiveto erythromycin, the selective marker that replaced the sigBoperon in the RN4220 derivative (28). Transformants possess-ing these resistance characteristics should have undergone adouble crossover, thereby replacing the DrsbUVWsigB deletionregion through the sigB operon including the rsbU gene fromCOL (Fig. 1B). The corresponding chromosomal region ofsuch a transformant was then transduced into 8325 derivativesto obtain the respective tetracycline-resistant rsbU1 deriva-tives. Transductants were analyzed by Southern hybridizationfor correct integration and loss of the suicide vector (data notshown). Strain GP268 was thus generated and characterized(see below). As a final proof for correct construction, thechromosomal region of the sigB operon was further phagetransduced from GP268 into the natural rsbU1 strain Newman.The phenotypes of the resulting transductants, harboring thechromosomal region of the sigB operon of GP268, and that ofthe Newman strain were found to be identical (data notshown), confirming that all manipulations had occurred asintended.

Growth of S. aureus strains. Different S. aureus strains and theirrespective DrsbUVWsigB mutants were analyzed for their station-ary-phase cell densities, measured as the OD600. The wild-typestrains COL and Newman were found to reach significantlyhigher OD600 values than their respective DrsbUVWsigBmutants, while strain BB255 reached a cell density that wasindistinguishable from that of its DrsbUVWsigB mutant (Table2). In contrast, the rsbU1 derivative GP268 reached a celldensity that was clearly higher than that of the correspondingstrain BB255 or the respective DrsbUVWsigB mutant. The ratiobetween the cell densities of GP268 and BB255 was compara-

ble to those found for the other two rsbU1 strains and theirDrsbUVWsigB mutants.

Increased H2O2 tolerance conferred by RsbU. Kullik et al.(28) reported the MBC of H2O2 to be four times higher thanthe MIC in strains COL and Newman, whereas for theirDrsbUVWsigB mutants as well as for BB255, the MICs andMBCs were found to be identical. Consistent with the data forthe rsbU1 strain Newman, we demonstrate here that the MBCof H2O2 for GP268 is four times higher than the MIC (Table3).

Alpha-hemolysin activity is negatively correlated to sB ac-tivity. It has been shown previously that DrsbUVWsigB mutantspossess higher alpha-hemolysin activities than their respectivewild-type mutants (15, 33). Alpha-hemolysin activities wereanalyzed here by examining the lysed zones around spottedcolonies grown on horse blood agar (Fig. 4). The DrsbUVWsigBmutants as well as strain BB255 produced clearly visible zonesof hemolysis. In contrast, the rsbU1 strains Newman andGP268 showed almost no lytic zones. The lytic zones of BB255and its respective DrsbUVWsigB mutant were indistinguishable.

Production of Asp23. The alkaline shock protein Asp23, a169-amino-acid polypeptide of still unknown function, isknown to be highly inducible in S. aureus strains 912 and MA13by a pH upshift to pH 10 (20, 29). It was, however, neitherdetectable nor inducible in strain 8325-4 (20). Asp23 was foundto be highly abundant in the cytoplasmic fraction of stationary-phase protein extracts of strains COL and Newman, while itwas missing in their respective DrsbUVWsigB mutants as well asin the 8325 derivatives (20, 28). A sB-dependent promotermotif has been proposed (20, 28) and recently confirmed (32)upstream of the asp23 open reading frame. Northern blotanalysis suggested asp23 expression to be highly dependent onthe alternative stress sigma factor sB (20). Here we presentfurther evidence for asp23 being under the sole control of sB

in S. aureus.Kuroda et al. (29) reported 0.7- and 1.5-kb asp23 transcripts.

TABLE 2. Cell densities of different S. aureus strainsa

Strain OD600b

GP268 ......................................................................................7.97 6 0.235BB255 ......................................................................................6.33 6 0.225BB255 DrsbUVWsigB .............................................................6.18 6 0.19Newman ..................................................................................8.89 6 0.16Newman DrsbUVWsigB..........................................................6.52 6 0.135COL .........................................................................................8.65 6 0.25COL DrsbUVWsigB ................................................................6.99 6 0.235

a Strains were grown for 48 h at 37°C and 200 rpm. Cell densities weremeasured photometrically as OD600.

b The OD600 values of rsbU1 strains are boldfaced. Values shown are resultsof four independent assays.

TABLE 3. Susceptibilities of different S. aureus strains tohydrogen peroxide

Strain MIC (mM) MBC (mM)

GP268 0.5 2BB255 0.5 0.5BB255 DrsbUVWsigB 0.5 0.5Newman 0.5 2Newman DrsbUVWsigB 0.5 0.5


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Our Northern blot experiments demonstrated that sequenceshybridizing to the asp23 probes can be detected on three dif-ferent RNAs, including a 3.3-kb transcript that was not previ-ously detected. This longer RNA includes an open readingframe with strong homology to OpuD of B. subtilis. Transcrip-tion of the asp23 locus (Fig. 5) was analyzed by use of threedifferent DNA probes (as indicated in Fig. 2A; data for probe2 and 3 not shown). The 0.7- and 1.5-kb transcripts were foundto be highly abundant, and all three transcripts were heatinducible in strains Newman and GP268, while they were onlyweakly expressed and not heat inducible in strain BB255 andwere not detectable at all in the DrsbUVWsigB mutants ofBB255 and Newman (Fig. 5 and 8A). Western blot analysiswith anti-Asp23 antibodies confirmed that Asp23 was highlyabundant in strains COL, Newman, and GP268, less abundantin strain BB255, and undetectable in the DrsbUVWsigB mutant(Fig. 3D).

The abundance of asp23 transcripts and their sole depen-dence on sB makes the asp23 promoter(s) an ideal candidatefor studying sB activity in S. aureus. We therefore fused thepromoter region of asp23 to the firefly luciferase gene (luc1)and integrated the construct into the chromosome (Fig. 2C).Except for the missing 3.3-kb transcript due to the chromo-somal integration of asp23P::luc1, transcription was found tobe similar to that of the original chromosomal region as dem-onstrated by Northern blotting (Fig. 5). The sB activity deter-mined indirectly by the use of the luciferase reporter systemconfirmed that sB was almost inactive in strain BB255, while instrain GP268 the sB activity profile was comparable to thatfound in strain Newman (Fig. 6). The sB activity profiles of theabove five strains were confirmed by the use of further lucif-erase fusions to the promoter of csb7, another sB-controlledgene (21). While luciferase activities derived from csb7P::luc1strains were found to be 10-fold lower compared to theasp23P::luc1 data, relative intensities in the different strainswere essentially identical (data not shown).

Pigmentation. A characteristic feature of many S. aureusstrains is the increase in pigmentation, with cells turning brightorange from pale yellow when incubated for 48 h at 37°C. Thisphenomenon has also been observed in COL, Newman, andMA13 but did not occur in 8325 derivatives, which kept theirpale-yellow pigmentation even after 96 h of incubation at 37°C.Although pigment production by S. aureus has been describedas a rather unstable characteristic (47), it has clearly been

demonstrated by Kullik et al. (28) that the orange pigmenta-tion of S. aureus is influenced by sB. They showed that sigBdeletion mutants of strains COL and Newman were unable toproduce the orange pigment, while a sigB-complemented strainof the 8325 derivative BB255 did. Corroborating these findings,we observed that GP268, the BB255 derivative complementedwith rsbU1, accumulated staphyloxanthin, the orange endproduct of S. aureus carotenoid biosynthesis (31), as its majorstationary-phase pigment after 48 h of growth (data notshown). In contrast, BB255 produced only trace amounts ofthe staphyloxanthin precursors 4,49-diapophytoene (colorless)and 4,49-diaponeurosporene (yellow), the products of the di-apophytoene synthase (CrtM) and diapophytoene desaturase(CrtN), respectively (37, 47). Consistent with its increased pig-mentation, GP268 was found to be more tolerant to UV radi-ation, especially to near-UV light (312 nm), than its unpig-mented donor, BB255 (Fig. 7). In the 8325-4 background, thetolerance to UV light was even more pronounced. GP269, thersbU-complemented 8325-4 derivative, was significantly more

FIG. 4. Alpha-hemolysin activities of different S. aureus strains.Cells of different S. aureus strains (3 ml of McFarland 0.5 dilutions)were spotted on horse blood agar plates and incubated for 24 h at37°C. The resulting colonies were scanned and analyzed for theirsurrounding lytic zones.

FIG. 5. Northern blot analyses of the asp23 operon. (A) Growthcurves of the S. aureus strains investigated. Solid squares, BB255; solidcircles, IK181 (BB255 DrsbUVWsigB); solid triangles, GP268 (BB255rsbU1); open squares, MB33 (BB255 asp23P::luc1); open circles,MB90 (BB255 DrsbUVWsigB asp23P::luc1); open triangles, MB49(BB255 rsbU1 asp23P::luc1). Time points of sampling are indicated.(B) Total RNAs (8 mg/lane) of S. aureus strains BB255 (lanes 1 to 3),IK181 (BB255 DrsbUVWsigB) (lanes 7 to 9), GP268 (BB255 rsbU1)(lanes 13 to 15), MB33 (BB255 asp23P::luc1) (lanes 4 to 6), MB90(BB255 DrsbUVWsigB asp23P::luc1) (lanes 10 to 12), and MB49(BB255 rsbU1 asp23P::luc1) (lanes 16 to 18), obtained from cellsgrown in LB medium at 37°C and harvested 1 h (lanes 1, 4, 7, 10, 13,and 16), 3 h (lanes 2, 5, 8, 11, 14, and 17), and 5 h (lanes 3, 6, 9, 12, 15,and 18) after inoculation of the medium with log-phase cells, wereblotted onto a positively charged nylon membrane and subjected toNorthern blot analysis. The blotted membranes were hybridized usinga digoxigenin-labeled DNA probe specific for asp23 (for construction,see Materials and Methods). The RNA molecular size marker I(Roche) was used. Positions of the 16S and 23S rRNAs are indicatedby diamonds (}) on the left, and relevant transcript signals are indi-cated on the right.


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tolerant to UV light than its donor (Fig. 7). The differences inUV tolerance observed between the BB255 and the 8325-4strains are probably due to the fact that the BB255 strains, incontrast to 8325-4 strains (34), still harbor temperate bacterio-phages which are known to be excised by UV radiation (41).The marked differences in near-UV-light tolerance betweenthe rsbU1 strains and their unpigmented relatives (Fig. 7B), asopposed to the marginal differences in far-UV-light tolerance(Fig. 7A), reflect the findings of Tuveson et al. (43). Theseauthors, using different light qualities, investigated the UV-

protective capacity of pigmentation in an E. coli strain that wastransformed with the carotenoid biosynthesis cluster of Erwiniaherbicola. They showed that carotenoids protected the trans-formed E. coli strain against high fluences of near-UV light(320 to 400 nm) but not against far-UV light (200 to 300 nm).

The observation that strain BB255 did not efficiently accu-mulate the staphyloxanthin precursors 4,49-diapophytoene and4,49-diaponeurosporene argues for an influence of sB on ca-rotenoid biosynthesis, either on gene products governing theformation of 4,49-diaponeurosporene or 4,49-diapophytoene oron a prior synthetic step. Consistent with this assumption thatformation of 4,49-diapophytoene may be affected, we coulddetect an influence of sB on the transcription of crtMN byNorthern blot analysis (Fig. 8B). Both the transcript levels ofGP268 compared with those of BB255 and the heat inducibilityof the detected transcripts argue for a sB dependence ofcrtMN. However, sB dependence of crtMN alone is not suffi-cient to explain the inability of BB255 to produce staphyl-oxanthin, as overproduction of crtMN under the control of axylose-inducible promoter resulted, after 24 h of growth, in astrong accumulation of 4,49-diaponeurosporene, which was notfurther converted to staphyloxanthin even after 96 h of growth.Overproduction of crtMN in the rsbU1 strain Newman resultedin an equal accumulation of 4,49-diaponeurosporene after 24 hof growth, but in contrast to the situation in BB255, almost allthe 4,49-diaponeurosporene was converted to staphyloxanthinafter 96 h of growth (data not shown). Thus, sB is likely tocontrol more than one of the intermediate steps of carotenoidbiosynthesis in S. aureus.

Induction of sB-dependent transcripts after heat shock. InB. subtilis, sB directs transcription of its own gene when acti-vated through a variety of stress stimuli (4, 7, 8, 22, 45, 50).Transcription starts within the sigB operon upstream of thersbV gene. A similar situation has been proposed for S. aureus,as a promoter sequence highly similar to the sB consensus ofB. subtilis is found upstream of the rsbV gene (27, 49). Tran-scription from this promoter would lead to an mRNA of ap-proximately 1.6 kb. In agreement with this prediction, Gertz et

FIG. 6. sB activity during growth of S. aureus. The expression of asp23P::luc1 during growth of S. aureus strain BB255 (A) and strain Newman(B), grown in LB medium at 37°C, is shown. Bacterial growth was measured as the OD600 (solid symbols). sB transcriptional activity wasdetermined by measuring the luciferase activity of Luc1 (open symbols), the product of the luc1 reporter gene fused to the sB-dependentpromoters of asp23 (asp23p). (A) Squares, S. aureus strain MB33 (BB255 asp23P::luc1); circles, strain MB90 (BB255 DrsbUVWsigB asp23P::luc1);triangles, strain MB49 (BB255 rsbU1 asp23P::luc1). (B) Squares, S. aureus strain MB32 (Newman asp23P::luc1); circles, strain MB69 (NewmanDrsbUVWsigB asp23P::luc1).

FIG. 7. UV tolerances of different S. aureus strains. McFarland 0.5dilutions of different S. aureus strains were streaked out onto LB agarplates and exposed to far-UV light (254 nm) (A) or near-UV light (312nm) (B) for different time periods. After light exposure, plates wereincubated for 24 h at 37°C.


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al. (20) detected a 1.6-kb transcript that was heat inducible instrain MA13 but was not detectable in strain 8325-4. Here wedemonstrate that BB255 cells expressed the 1.6-kb transcriptwhen complemented with rsbU (Fig. 8) and that the transcriptwas heat inducible as in MA13 (20). In addition to sigB andasp23, we found transcription of crtM to be heat inducible anddependent on sB (Fig. 8). The time course of transcript induc-tion after heat stress resembled that of the 1.6-kb sigB tran-scripts in strain MA13 (20), with a maximum induction withinthe first 3 to 6 min and a decrease thereafter to or below theuninduced level after 12 min.

DISCUSSION

In the gram-positive bacterium B. subtilis, RsbU has beenshown to be essential for activation of sB in response to dif-ferent environmental stress stimuli such as heat shock, saltstress, or ethanol stress (45, 48, 50). A similar function hasbeen proposed for the RsbU homologue of S. aureus (27, 49).

In this study, we clearly demonstrate that RsbU of S. aureus isindeed an essential factor for sB activity, as strains lacking thisprotein were unable to render activity from this sigma factor(Fig. 6). Furthermore, the lack of RsbU in 8325 derivativesresulted in phenotypes comparable to those of DrsbUVWsigBmutants of rsbU1 strains such as COL or Newman (28).Complementation of strain BB255 with the rsbU1 allele fromCOL resulted in the rsbU1 derivative GP268. This strain ex-hibited a sB activity profile comparable to that of the rsbU1

wild-type strain Newman (Fig. 6) and restored the sB-depen-dent phenotypic traits to the levels seen in Newman. Overex-pression of RsbU in BB255 altered the phenotype to thatfound for GP268, while overexpression in the correspondingDrsbUVWsigB mutant had no apparent influence, suggestingthat RsbU acts primarily through sB (unpublished data). Theobservations that sB is produced by 8325 derivatives (Fig. 3C)and that BB255 was phenotypically indistinguishable from itssigB derivative under the conditions that we tested indicatethat although sB is detectable in 8325 derivatives, it cannot beactivated to relevant levels due to the absence of RsbU in thisgenetic background. However, the presence of detectableamounts of Asp23 and sB activity at a low level in BB255suggests that RsbU is not the sole determinant of sB activity inS. aureus. Significant amounts of Asp23 were detectable in the8325 isogenic background only in BB255, which harbors atleast four prophages, and not in any of the 8325-4 derivatives(i.e., 8325-4, RN4220, and RN6390), which have been curedfrom the respective prophages, implying that the loss of theprophages from 8325 may influence such residual RsbU-inde-pendent sB activity.

The finding that 8325 derivatives are almost unable to acti-vate sB is of particular interest, as 8325 derivatives are thelaboratory strains most frequently used in S. aureus research.Most studies on starvation survival, pathogenicity, and the reg-ulation of the two global regulators agr (accessory gene regu-lator) and sar (staphylococcal accessory gene regulator) and, inparticular, the influence of sB in these processes, have beencarried out in this genetic background (2, 6, 10, 11, 12, 14, 15,16, 17, 30, 42). The observed lack of sB activity in the 8325isogenic background revives the question if, and to what extent,sB is involved in these processes. The findings in rsbU1 strainssuch as COL, Newman, and GP268 of the inducibility of tran-scription of sB-dependent genes, of staphyloxanthin accumu-lation, of reduced susceptibility to hydrogen peroxide (Table2), and of higher cell densities in overnight cultures comparedto those for their respective DrsbUVWsigB mutants (Table 3)argue for an influence of sB on the survival capacity of S.aureus.

sB has been shown to be a major player in the general stressresponse of B. subtilis, by controlling the transcription of morethan 100 genes under a variety of stress conditions (23, 46). Sofar, more than 30 genes in S. aureus have been determined tobe controlled by sB (21). These proteins are likely to be in-volved in the general stress response of S. aureus. In addition,pigmentation of S. aureus by the carotenoid staphyloxanthin,the biosynthesis of which is clearly influenced by sB, is alsolikely to be a protective measure against various environmentalstress factors, such as UV radiation (Fig. 7) or free radicals. Asbiological antioxidants, carotenoid pigments have been shownto protect many bacteria against the harmful effects of light, in

FIG. 8. Heat shock induction of sB-dependent transcripts in S.aureus. Total RNA was isolated from S. aureus GP268 (BB255 rsbU1)(lanes 1 to 7) and S. aureus BB255 (lanes 9 to 15) grown at 37°C (lanes1, 2, 9, and 10) and 1 min (lanes 3 and 11), 3 min (lanes 4 and 12), 6min (lanes 5 and 13), 9 min (lanes 6 and 14), and 12 min (lanes 7 and15) after shifting the cultures to 48°C. The RNAs (15 mg/lane) wereblotted onto positively charged nylon membranes and subjected toNorthern blot analyses. The blotted membranes were hybridized usingdigoxigenin-labeled RNA probes specific for asp23 (A), crtM (B), andsigB (C) (for construction see Materials and Methods). A digoxigenin-labeled RNA size marker (lane 8) (Roche) was used as a standard.Relevant transcript signals are given on the left.


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particular against high fluences of near-UV light (320 to 400nm). They act as scavengers of reactive molecules that aregenerated within cells and that can induce oxidative damage,e.g., singlet molecular oxygen (1O2) (36, 43). Thus, pigmentedS. aureus cells are very likely to survive longer periods ofdaylight exposure than their unpigmented relatives.

The lower susceptibility of rsbU1 strains to hydrogen perox-ide may also be due to the pigmentation, as carotenoids havebeen shown to protect efficiently against oxygen radicals (36).Alternatively, the increased resistance of rsbU1 strains to hy-drogen peroxide may be due to a sB-dependent transcriptionalcontrol of enzymes directly involved in the degradation ofreactive oxygen species, such as catalase or superoxide dis-mutase. Transcriptional control of the katA gene, coding forthe sole catalase thus far identified in S. aureus, however, wasfound to be independent of sB in Northern blot analysis (datanot shown). Notwithstanding, the higher tolerance of rsbU1

strains to hydrogen peroxide is likely to provide considerablebenefit for S. aureus strains invading a host, enabling them totolerate higher concentrations of oxygen radicals that are pro-duced by the host defense system (38).

We note that rsbU mutants were unable to accumulate thepigment staphyloxanthin, even after 72 h of growth. This find-ing indicates that—unlike the situation in B. subtilis—sB isinactive even during the stationary-growth phase, providedthat RsbU is absent. Since all analyzed clinical isolates of S.aureus were found to be rsbU1, we consider it important toreinvestigate these processes. Most importantly, the regulationof the two global regulators agr and sar will have to be studiedin a genetic background representative for the majority ofclinical isolates. Preliminary data suggest a strong impact of sB

on sar expression in rsbU1 strains (M. Bischoff, unpublisheddata). Strain GP268, which has sB activity, provides the pos-sibility of studying these processes in the well-characterized8325 isogenic background.

ACKNOWLEDGMENTS

We thank B. Berger-Bachi, A. Schaller, and M. Hecker for criticalreading of, and comments on, the manuscript. We are very grateful toA. Wada for providing plasmid pAW8 and to A. Raisig for HPLCanalysis of carotenoids. Preliminary sequence data were obtained fromThe Institute for Genomic Research (TIGR) through the website athttp://www.tigr.org. Sequencing of S. aureus COL was accomplishedwith support from National Institute of Allergy and Infectious Dis-eases (NIAID) and the Merck Genome Research Institute (MGRI).

This work was supported by Swiss National Science Foundationgrant NF 31-46762.96 to F. H. Kayser.

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