Abundance of the {delta} Subunit of RNA Polymerase Is Linked to the Virulence of Streptococcus agalactiae

Group B streptococcus (GBS) remains a major cause of morbidityand mortality among newborn children. The bacterium is a commensalorganism colonizing the rectum and the gastrointestinal andurogenital tracts of adults, but it can be transmitted to neonatesby an ascending infection of the maternal genital tract or duringparturition. We previously reported that a transposon insertiondisrupting rpoE resulted in the decreased survival of the mutantin the neonatal rat sepsis model of GBS infection. rpoE encodesthe ${delta}$ protein, a subunit of RNA polymerase (RNAP) that has beencharacterized in Bacillus species. In this study, we confirmthe association of the ${delta}$ protein with purified GBS RNAP and showthat it is expressed in strains representing all nine serotypes.Flow cytometric analysis of a reporter strain containing a transcriptionalfusion of the rpoE promoter to gfp revealed that, in vitro,this gene is continuously expressed. Analysis of ${delta}$ expressionin the transposon mutant by quantitative Western blotting revealeda 10-fold reduction in relative abundance (which was linkedto the attenuation in virulence that was observed for this mutant)compared to that for the wild-type strain. These data suggestthat a minimum intracellular concentration of ${delta}$ is necessaryfor this organism to cause disease.

INTRODUCTION

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Introduction

Streptococcus agalactiae, or group B streptococcus (GBS), remainsa leading cause of pneumonia, sepsis, and meningitis in newbornswithin the developed world (37). GBS infection can be separatedinto two distinct syndromes, one occurring among neonates ofup to several days old (early onset disease) and the other amonginfants that are several weeks or months older (late-onset disease).Although the screening of at-risk obstetric patients, followedby the administration of intrapartum antibiotics, has drasticallyreduced the incidence of early onset disease (41), invasiveGBS disease still accounts for significant morbidity and mortalityin newborns (27, 43). Increasing numbers of reports of invasivedisease among the elderly and in immunocompromised adults havealso classified GBS as an important pathogen in these two populations(3). Recognition of the prevalence and severity of human neonataland adult disease underscores the need for investigating themechanisms that are important to the pathogenesis of GBS infections.

The GBS bacterium is a commensal organism in adults that colonizesthe rectum and the urogenital and gastrointestinal tracts (13).To establish early onset disease, the bacterium must ascendfrom the acidic environment of the mother's vaginal mucosa tothe lung epithelia of the neonate during birth and access thehighly oxygenated environment of the blood (for a review, seereference 2). Thus, an important feature that is exhibited bythis pathogen is an ability to survive and grow in diverse hostenvironments that differ with respect to the physiological conditionsand immune defenses. This ability to adapt likely involves analteration in the pattern of gene expression. The regulationof gene expression in bacteria primarily occurs at the levelof transcription and is controlled through the activity of thetranscribing enzyme, RNA polymerase (RNAP). Typically, bacterialRNAP can coexist in two distinct forms: (i) the core enzymecomprised of ß and ß' and two identical ${alpha}$ subunits and (ii) as a holoenzyme which, in addition to thecore, contains a dissociable ${sigma}$ subunit. Functionally, the coreenzyme has a low binding affinity for any DNA sequence but issufficient for transcriptional elongation and termination. Theassociation of a ${sigma}$ factor with the core enzyme drastically increasesthe affinity for ${sigma}$ -specific promoter sequences, allowing forthe initiation of transcription. The constitutively expressedsigma factors ${sigma}$ ⁷⁰ and ${sigma}$ ^A are responsible for initiating the transcriptionof the majority of bacterial genes in gram-negative and gram-positivebacteria, respectively. However, the induction of competence,adaptation to temperature change, control of sporulation, andsurvival response to oxidative stress have all been attributed,in part, to the association of specific secondary ${sigma}$ factors withRNAP (14, 15, 28, 31).

A number of accessory proteins are also found in associationwith RNAP during various stages in transcription. In additionto the core subunits and ${sigma}$ , RNAP purified from Bacillus subtilishas also been shown to contain a novel protein designated ${delta}$ ,which is encoded by the rpoE gene (1, 24, 26). An interrogationof the published sequence databases suggests that rpoE is ubiquitousamong gram-positive bacteria.

Activity for ${delta}$ has thus far been characterized in only Bacillusspp. by using in vitro assays. ${delta}$ has been shown to bind to thecore of Bacillus subtilis RNAP (25). It is thought to play arole in maintaining transcriptional specificity. Associationof ${delta}$ with RNAP reduces binding to DNA templates containing nonbiologicallyrelevant promoter sites (39) and inhibits transcription fromweak promoters. In addition, depending on the template, ${delta}$ hasbeen implicated in enhanced promoter melting (20) and may beinvolved in RNAP recycling (24). These data suggest that ${delta}$ actsas an allosteric modulator of RNAP conformation and lacks enzymaticactivity.

The function of ${delta}$ in vivo has not been established. We previouslyreported that a GBS mutant with a transposon insertion adjacentto rpoE exhibited decreased survival in a neonatal rat sepsismodel (18), suggesting a role for ${delta}$ in virulence. In this study,we confirm the association of the ${delta}$ protein with purified GBSRNAP and show that it is widely expressed among the serotypes.Using a reporter assay, we also demonstrate that rpoE expressionin vitro is growth-phase dependent. An analysis of ${delta}$ expressionin the transposon mutant by quantitative Western blotting revealeda 10-fold reduction in the relative abundance, which was linkedto the attenuation in virulence that was observed for this mutant.These data suggest that the abundance of ${delta}$ may be an importantaspect of GBS virulence.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and growth conditions. Bacterial strains and plasmids used in this study are listedin Table 1. GBS strains were grown in Todd-Hewitt broth (DifcoLaboratories) in 5% CO₂ at 37°C. Escherichia coli strainMC1061 served as a host strain for cloning purposes. E. coliOrigami was used as a host strain for the production of recombinantprotein. E. coli was grown at 37°C under aeration in Luriabroth (LB). When required, antibiotics were added to the mediumas follows. For E. coli, ampicillin (Ap), 50 µg ml^–1,spectinomycin (Sp), 100 µg ml^–1, and erythromycin(Em), 500 µg ml^–1, were used, and for GBS, 5 µgml^–1 Em or 100 µg ml^–1 Sp was added.

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TABLE 1. Bacterial strains and plasmids used in this study

Purification of RNAP. RNAP was purified from GBS strains A909 and AJ200 and B. subtilisstrain MH5636. For the isolation of RNAP from GBS, the strainswere grown to mid-logarithmic phase (optical density at 600nm [OD₆₀₀] was 0.4 to 0.6) and harvested by centrifugation.Approximately 5 g of cells (wet weight) were resuspended inprotoplast preparation buffer (0.3 M potassium phosphate buffer[pH 7], 40% sucrose) containing endo-N-acetylmuramidase (mutanolysin,0.5 U/µl) (Sigma) and incubated at 37°C for 90 min.Protoplasts were collected by centrifugation, resuspended inlysis buffer (50 mM Tris-HCl [pH 8], 10 mM MgCl₂, 0.1 mM dithiothreitol,0.1 mM EDTA [pH 8], 10% glycerol, 1 mM phenylmethylsulfonylfluoride), and lysed by sonication (Vibra Cell VC 750 sonicator;Sonics). Lysates were clarified by centrifugation at 23,000x g for 20 min, and the undiluted supernatant was applied toa heparin-agarose column (10 ml bed volume) (Sigma). Heparinis a polyanion that mimics DNA in its overall binding properties;thus, it is commonly used as a ligand for the purification ofvarious DNA binding proteins, including DNA-dependent RNA polymerase.The column was washed with 3 volumes of re-equilibration buffer(10 mM Tris-HCl [pH 8], 10 mM MgCl₂, 0.1 mM dithiothreitol,0.1 mM EDTA [pH 8], 10% glycerol), and proteins were elutedwith a linear salt gradient of 0.1 to 1.0 M NaCl in the samebuffer (20 ml total volume). Fractions that contained RNAP (basedon the presence of the ß/ß' subunit doublet)were pooled and concentrated, and the salt concentration wasadjusted to 0.1 M NaCl by buffer exchange using an Amicon-Ultrafilter device (Millipore). The concentrate was loaded onto ahigh-pressure liquid chromatography UNO Q1 column (Bio-Rad)that was equilibrated with pre-equilibration buffer, and proteinswere eluted as described above by using an NaCl step gradient(50 ml total volume). Holoenzyme-containing fractions were identifiedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and finally loaded on a high-pressure liquid chromatographyS-300 column (Amersham Biosciences) in re-equilibration buffercontaining 200 mM NaCl. Peak fractions (based on absorbanceat 280 nm) were pooled, concentrated, reconstituted in storagebuffer (10 mM Tris-HCl [pH 8], 10 mM MgCl₂, 100 mM NaCl, 0.1mM EDTA, 50% glycerol), and stored at –20°C. RNAPwas isolated from B. subtilis strain MH5636 as described previously(32).

DNA manipulations. Routine molecular biology techniques for cloning, sequencing,and PCR amplification were performed as previously described(36). Chromosomal DNA was isolated from GBS strains by usingthe method of Madoff et al. (30). Plasmid DNA was isolated fromGBS by using a modified QIAGEN plasmid mini-prep procedure (19).DNA restriction and modification enzymes were used accordingto the manufacturer's recommendations (New England Biolabs).GBS was transformed by electroporation as described previously(10).

Generation of recombinant RNAP subunits. Recombinant ${delta}$ was expressed in E. coli by using the pET32a expressionvector system (Novagen). A DNA fragment containing the entirecoding sequence of rpoE was PCR amplified by using high-fidelitypolymerase and the primers 5'-CATGCCATGGTATATGGATTAGAAAGAGAGGAATCand 5'-TTGCGGCCGCTTTTCTTGCTCGTTTTCC (underlining indicates eitheran EcoRI or an NotI restriction enzyme site) and A909 genomicDNA as a template. These primers were designed to include anNcoI or NotI restriction enzyme site in order to facilitatecloning into the pET32a plasmid vector. The resulting PCR productwas digested with NcoI and NotI, ligated to similarly digestedpET32a, and transformed directly into E. coli Origami (Novagen).The generation of the expected plasmid pET32rpoE was verifiedby restriction digest analysis and DNA sequencing of plasmids.To induce the expression of the thioredoxin (TRX) hexa-His-tagged- ${delta}$ fusion protein (TRX- ${delta}$ ), E. coli Origami containing pET32rpoEwas grown to an OD₆₀₀ of 0.6, and isopropyl-ß-D-thiogalactopyranoside(IPTG) was added at a final concentration of 1 mM for 3 h. TheTRX- ${delta}$ fusion protein was purified under native conditions byusing Ni-CAM HC agarose (Sigma) as the His tag affinity resin.TRX (containing the internal His tag) was cleaved from purifiedTRX- ${delta}$ with enterokinase (Stratagene), which was used accordingto the manufacturer's instructions. Cleaved TRX and uncleavedTRX- ${delta}$ were removed by further incubation with the Ni-CAM HC resin.The flowthrough containing pure ${delta}$ was reconstituted in a phosphate-bufferedsaline solution (PBS; pH 7.0) containing 10% glycerol and storedin aliquots at –80°C until required for use.

Recombinant ${sigma}$ ^A and ß were generated as His-tagged fusionproteins. For the cloning of rpoD ( ${sigma}$ ^A) and rpoB (ß),the trx coding sequence was first removed from pET32a by PCR,generating pET32ck. rpoD was amplified by using the primers5'-CATGCCATGGCAGAGAAAAAAGGAAATAC and 5'-TTGCGGCCGCATCTTCCATGAAATCTTTAAGTTG(underlining indicates either an NcoI or an NotI restrictionenzyme site), while rpoB was amplified by using the primers5'-CGGAATTCTTGGCAGGACATGAAGTTCAG and 5'-TTGCGGCCGCATCTTCTTGAACGACTTCAGA(underlining indicates either an EcoRI or an NotI restrictionenzyme site). Amplified products were digested with the appropriaterestriction enzymes and ligated to similarly digested pET32ck.E. coli clones containing the correct vector were grown andinduced as described above. Recombinant ${alpha}$ ^A and ß withcarboxyl-terminal His tags were purified under native conditionsthrough incubation with Ni-CAM HC agarose. Eluted protein fractionswere reconstituted in PBS and stored as described above.

Preparation of antisera. Antisera against recombinant ${delta}$ , ${alpha}$ ^A, and ß were generatedin female New Zealand White rabbits as a service from LampireBiological Laboratories (Pipersville, PA). In brief, rabbitswere immunized subcutaneously with 500 µg of recombinantprotein that was emulsified in complete Freund's adjuvant forthe first dose and in incomplete Freund's adjuvant for subsequentdoses (days 21 and 42 after initial immunization). Serum wasprepared from blood collected approximately 2 weeks after thelast dose was given.

Preparation of GBS lysates and Western blot analysis. GBS strains were grown to a designated OD₆₀₀, pelleted, washedin PBS, and resuspended in lysis buffer (60 mM Tris-HCl [pH6.8], 10% glycerol). Whole-cell lysates were prepared throughthe use of a FastPrep FP101 bead beater (Bio 101) by using 2x45-s bursts at 4°C at full speed, followed by the additionof SDS (final concentration, 3.3% wt/vol). The lysates wereboiled for 5 min, and insoluble material was pelleted by centrifugation.The protein content in the supernatant was quantified by usinga bicinchoninic acid assay (Pierce). Samples were normalizedfor protein concentration, mixed with 2x SDS gel loading buffer(36), and heated at 95°C for 5 min prior to SDS-PAGE analysis(36). The proteins were transferred to a nitrocellulose membraneby using a semidry transfer chamber (Bio-Rad) for 25 min at15 V. The membrane was blocked overnight at 4°C in a 5%(wt/vol) solution of nonfat dehydrated milk in PBS. The blotswere incubated at room temperature with a 1:1,000 dilution ofrabbit antisera (raised against the designated protein) for2 h, followed by a 90-min incubation with a 1:3,000 dilutionof Alexa Fluor-680 anti-rabbit immunoglobulin G (IgG) (MolecularProbes). Immunoreactive bands were visualized at 700 nm by usinga LiCor infrared imager (LI COR Biosciences).

RNA isolation. Total RNA was isolated from strains grown to an OD₆₀₀ of 0.3by using QIAzol (QIAGEN) according to the manufacturer's instructions,except that GBS cells were lysed through the use of a bead beateras described above. RNA samples were treated with DNase I (Promega)for 60 min at 37°C to remove any contaminating DNA and thenpurified by using an RNeasy mini kit (QIAGEN). RNA concentrationwas adjusted to 1 µg/ml, and samples were stored at –80°Cuntil required for use.

Promoter mapping. Rapid amplification of cDNA ends (RACE) was used to identifythe 5' end of the rpoE transcript in the wild-type strain A909.First-strand cDNA synthesis was carried out according to theprotocol of the 5'-RACE system (version 2.0; Invitrogen) byusing 2 µg of total RNA and the rpoE-specific primer 5'-CTAAACCTCTTCTTCCTC(GSP1), followed by dCTP tailing of the 5' end of the cDNA.Tailed cDNA was PCR amplified by using the 5' RACE-abridgedanchor primer (AAP; Invitrogen) and rpoE-specific primer 2 5'-CTAAACCTCTTCTTCCTCTTCTTCC(GSP2) with high-fidelity polymerase (Bioline). The productthat was generated served as a template for a second PCR byusing the nested primers' abridged universal amplification primer(AUAP, Invitrogen) and rpoE-specific primer 3 5'-AAAGCATTGACACGTTTCTTCTTACG(GSP3). The final PCR product was sequenced and aligned to thepublished genome sequence by using Sequencher version 3.1 software(Gene Codes Corporation) to identify the transcription initiationstart site, untranslated region, and promoter elements.

Construction of a complemented strain. To confirm that we had identified the correct promoter, we constructeda complemented strain for testing in our animal infection model.rpoE and the promoter were amplified by high-fidelity PCR byusing Bio-X-Act DNA polymerase (BioLine) and A909 chromosomalDNA as a template. Restriction enzyme sites for EcoR I wereincorporated into the primers to facilitate cloning into pLZ12,a low-copy streptococcal shuttle vector that replicates at 6to 9 copies per cell in GBS (7). The ligation mixture was transformedinto E. coli XL1-Blue, and clones containing the correct constructwere identified by PCR. Plasmid DNA was isolated from a positiveclone and designated pRN021. Electrocompetent AJ200 was transformedwith pRN021 (or pLZ12 as a control) and plated on medium containingSp to select for the plasmid. The complemented strain, AJ200containing pRN021, was designated RN114, and AJ200, containingthe vector pLZ12, was designated RN115.

Animal infection studies. Time-mated, barrier-sustained, female Sprague-Dawley rats wereobtained from Charles River Laboratories. Fifty-percent-lethal-dose(LD₅₀) assays were performed by using the neonatal rat sepsismodel as previously described (18). All procedures were performedin accordance with the guidelines provided by the Children'sHospital and Regional Medical Center Institutional Animal Careand Use Committee.

Construction of a reporter plasmid. To analyze rpoE expression, the native rpoE promoter was clonedupstream of the gfpmut3 allele to allow for monitoring of promoteractivity by flow cytometric analysis of green fluorescent protein(GFP) expression. The streptococcus-E. coli shuttle vector,pDC125 (4), was used to construct a reporter plasmid. The originalphoZ and cat reporter genes were removed from pDC125 by PCR,and ClaI and NotI sites were added to facilitate the cloningof inserts. A streptococcal ribosomal binding site (GGAGG) (9)was inserted into the vector 7 bp upstream of an ATG start codonthat was contained within the cloned ClaI site to optimize theexpression of GFP. The gfpmut3 allele, which has been optimizedfor bacterial codon usage (6), was PCR amplified by using pBL26as a template. ClaI and NotI sites were incorporated into the3' and 5' ends of gfpmut3, respectively. The resulting PCR productand plasmid were digested with ClaI and NotI and ligated togetherto generate pRSgfpmut3. A BamHI site was introduced into thegfp sequence of pRSgfpmut3 to facilitate the insertion of therpoE promoter fragment. An EcoR I-BamHI fragment, containingthe promoter and the first four codons of rpoE, was ligatedto pRSgfpmut3 cut with the same enzymes, generating pRSP_rpoE-gfp.This reporter construct contains an in-frame fusion of the rpoEpromoter to gfp.

Flow cytometry. Flow cytometric analysis was performed with a BD FACSCalibur(Becton Dickinson) equipped with a 488-nm argon laser and FlowJosoftware version 4.6.2. Overnight cultures of strains containingthe reporter construct and the control vector were subculturedinto fresh medium and grown to the indicated growth phases.Prior to analysis, cells were fixed at 4°C overnight in2% (wt/vol) paraformaldehyde in PBS, washed, and finally resuspendedin PBS. The PBS used in this study was filtered through a 0.22-µm-pore-sizefilter (Millipore) to minimize fluorescence. A total of 100,000events were analyzed for each sample at constant parameter settingsfor each experiment.

Northern hybridization. Total RNA was separated on a 0.8% agarose gel and transferredto a positively charged nylon membrane (Hybond N+, Roche) byalkaline transfer as described previously (36). A probe thatencompassed the complete rpoE open reading frame was generatedby incorporating digoxigenin-11-dUTP into a PCR product by usingthe protocol provided by the manufacturer (Roche Diagnostics)and the following primer pair: 5'-ATGACAAAAAAACATCTTAAAACG and5'-TTGCGGCCGCTTTTCTTGCTCGTTTTCC. The hybridization and washconditions used were as specified by Roche Diagnostics. Gene-specificbands were detected by chemiluminescence using CPSD (Roche Diagnostics)as the alkaline phosphatase substrate.

RESULTS

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Results

${delta}$ is a subunit of RNA polymerase in GBS. To identify the subunits of RNAP in GBS, native RNAP was purifiedfrom the wild-type strain A909 by using a modified affinityand size exclusion chromatography procedure that takes advantageof specific substrate binding and the size of the enzyme. Chromatographicsteps included heparin affinity, anion exchange (UNO Q1), andsize exclusion (S-300) chromatography. RNAP was isolated fromB. subtilis MH5636 for comparison. As seen in Fig. 1A, SDS-PAGEanalysis of the RNAP preparation from A909 revealed that itconsists of subunits that are consistent with the expected sizesfor ß' (135 kDa), ß (135 kDa), ${alpha}$ (34 kDa), ${sigma}$ ^A (42 kDa), and ${delta}$ (21 kDa). This is similar to what we observedfor B. subtilis MH5636. These results are in agreement withthe expected sizes for the gene products of rpoC, rpoB, rpoA,rpoD, and rpoE, respectively, based on the available GBS genomesequences (12, 40). As a control, we also isolated RNAP fromAJ200, a mutant in which rpoE has been replaced with a kanamycincassette by allelic exchange (19). As expected, while we detectedsubunits corresponding to ß', ß, ${alpha}$ , and ${sigma}$ ,the ${delta}$ protein was absent.

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FIG. 1. RNAP purification and subunit analysis. A total of 1 to 2 µg of purified RNAP from GBS strains A909 (1) and AJ200 (2) and B. subtilis strain MH5636 (3) were separated on a 12% SDS-polyacrylamide gel and stained with Coomassie blue (A). Subunit identity is shown on the left. Western blot analysis was performed on the RNAP preparations by using anti- ${delta}$ (B), anti- ${sigma}$ ^A (C), or anti-ß (D) antisera. Recombinant ${delta}$ (r ${delta}$ ), ${sigma}$ ^A (r ${sigma}$ ^A), and ß (rß) served as positive controls. Immunoreactive bands were detected by using an infrared imager after incubation with a rabbit anti-IgG infrared-labeled secondary antibody.

Western blot analysis was used to further confirm the associationof ${delta}$ with RNAP in GBS. Antiserum raised against purified recombinantGBS ${delta}$ was used to probe the purified RNAP preparations from A909,AJ200, and B. subtilis MH5636. The antiserum reacted with a21-kDa band corresponding to ${delta}$ in the RNAP that was isolatedfrom A909 (Fig. 1B). Although there was an approximately 21-kDaprotein visible in the Coomassie-stained gel of the RNAP preparationfrom AJ200 (Fig. 1A), as expected, ${delta}$ was not detected by Westernblot analysis in this preparation (Fig. 1B). The antiserum didnot recognize ${delta}$ in the bacillus RNAP. This observation can beexplained by the fact that there is a significant differencein the amino acid composition of ${delta}$ between GBS and B. subtilis(33% identity and 61% similarity). Alternatively, there maybe species-specific differences in the tertiary structure ofthis protein. Although the antisera cross-reacted with a 40-kDaprotein in this RNAP preparation, antisera raised against bacillus ${delta}$ also identified a similarly sized bacillus protein in additionto ${delta}$ (data not shown). To confirm that we had correctly identifiedthe other subunits of RNAP in GBS, Western blot analysis usingantisera directed against ${sigma}$ ^A (Fig. 1C) and the ß subunit(Fig. 1D) was performed. Both subunits were detected in theRNAP preparations from the GBS strains that we examined. Thesedata suggest that under these conditions, the sigma factor thatis associated with RNAP is ${sigma}$ ^A. The antisera also reacted withthe ${sigma}$ ^A and ß subunits in RNAP from B. subtilis MH5636,which is consistent with the high degree of amino acid sequencehomology that the B. subtilis ${sigma}$ ^A (65% identity and 78% similarity)and ß (68% identity and 82% similarity) subunits sharewith the GBS homologs.

${delta}$ is expressed among all serotypes of GBS. Western blot analysis was used to examine whether the expressionof ${delta}$ is limited to strains in serotype 1a. Whole-cell lysatesof strains representing all nine serotypes were prepared. Theanti- ${delta}$ antiserum detected an approximately 21-kDa band correspondingto ${delta}$ in all of the strains that were tested (Fig. 2). These dataindicate that the expression of ${delta}$ is not limited to A909.

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FIG. 2. Analysis of ${delta}$ expression across the serotypes. Western blot analysis was performed on lysates of strains representing all nine clinically relevant serotypes by using antiserum raised against recombinant ${delta}$ . The serotype of the strain is indicated above each lane. Ia, A909; Ib, DK14; II, DK23; III, COH1; IV, CNCTC 1/82; V, CNCTC 10/84; VI, NT6; VII, 87-603; VIII, M9; (- ${delta}$ ), AJ200; r ${delta}$ , recombinant delta protein.

rpoE is expressed from a ${sigma}$ ^A-dependent promoter. To identify the transcriptional start site and the promoterconsensus elements, we mapped the 5' end of the rpoE transcriptby 5'-RACE PCR. The positions of the primers and predicted sizesof the products are shown in Fig. 3A. Sequencing of the PCRproduct that was generated by using the gene-specific GSP3 andAUAP anchor primers identified the transcriptional initiationsite, which was located $~$ 109 bp upstream of the TTG start codon(Fig. 3C). Comparison of this sequence to the A909 genome sequenceidentified a putative –10 and –35 promoter regionthat was located 9 bp upstream of the 5' untranscribed region.The predicted –35 (TTGACG) and –10 (TAAAAT) elementsare consistent with what has been reported for ${sigma}$ ^A-dependent promotersin B. subtilis (16), suggesting that rpoE is transcribed bythe RNAP that is associated with ${sigma}$ ^A in GBS.

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FIG. 3. Identification of the rpoE promoter. (A) Schematic diagram of the 5'-RACE PCR strategy, primers, and product sizes. (B) Amplicons were obtained in primary PCR by using primers AAP and GPSP2 (lane 1) and were obtained in nested PCR by using primers AUAP and GSP3 (lane 4). Lanes 2 and 5 contain the respective negative controls for these PCRs, and lane 3 contains a DNA marker. (C) Sequence of rpoE promoter and 5'-untranslated region. The predicted –35 and –10 consensus elements are boxed. The 5'-untranslated region is underlined. The methionine start codon (TTG) is also indicated.

Complementation of virulence in neonatal rat sepsis model. The rpoE deletion mutant AJ200 was transformed with a low-copyvector containing the rpoE coding sequence and promoter elementsthat we identified by using the 5'-RACE technique. We comparedthe virulence of the complemented strain (RN114) to the wild-typestrain in the neonatal rat sepsis model. AJ200 containing emptyvector (RN115) was used as a control. In two separate experiments,providing rpoE expressed from the promoter that we identifiedon a low-copy vector restored virulence to wild-type levels(Table 2). The log of the LD₅₀ for RN114 was similar to thatfor the wild-type strain A909, while the log of the LD₅₀ forRN115 was 1.5 to 2 log units higher than that for the wild type.These data confirmed that we had correctly identified the nativerpoE promoter. In addition, the virulence of the original transposonmutant AJ8D3 was compared to those of A909 and the complementedstrain. The LD₅₀ for AJ8D3 was 2 log units higher than thosefor A909 and RN114 (Table 2), confirming that rpoE expressionis required for survival in the host.

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TABLE 2. Lethal dose values for complemented strains in neonatal rat sepsis model of infection

Analysis of rpoE expression. Once we confirmed that we had identified the native promoterfor rpoE, we generated a reporter construct to characterizeexpression during growth in vitro. The expression of rpoE wasanalyzed by measuring the fluorescence of strains containingtranscriptional fusions to gfpmut3. The promoter region forrpoE was cloned upstream of a promoterless gfpmut3 allele, generatingpRSPrpoE-gfp. A909 was transformed with the reporter plasmid,generating strain RS021, or empty vector as a control, generatingstrain RS020. We first confirmed that the expression of GFPfrom the rpoE promoter did not affect growth of the strain.As shown in Fig. 4A, the expression of GFP did not significantlyaffect the growth of RS021 in laboratory medium relative tothe control strain RS020. Flow cytometric analysis of GFP expressionin RS021 indicated that rpoE was expressed during all stagesof growth. GFP fluorescence increased from lag to early logarithmicphase, reached a maximum at the late logarithmic stage, anddropped off during stationary phase (Fig. 4B). These data areconsistent with what we have observed by using microarray analysisof rpoE expression in A909 during growth in vitro (unpublisheddata), confirming that the pattern of expression that we observedwith the plasmid construct is representative of gene expressionoccurring from the chromosomal locus.

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FIG. 4. Analysis of rpoE promoter activity using a reporter construct. (A) Growth of RS021 containing the rpoE reporter plasmid ( ${blacksquare}$ ) and the RS020 control strain ( ${square}$ ). (B) Flow cytometric analysis of RS021 and RS020. GFP fluorescence is shown as the mean fluorescence intensity (MFI) of the bacterial population, which was calculated by using the FlowJo software version 4.6.2. The data presented are means ± standard error of the means and are representative of three experiments.

rpoE is transcribed in AJ8D3. rpoE was first identified as being required for virulence ofGBS during a signature-tagged transposon mutagenesis (STM) screenutilizing a modified Tn917 (Tn917stm) (18). The transposon mutantAJ8D3 was severely attenuated for virulence and had a singleTn917stm insertion adjacent to rpoE. Sequencing of genomic DNAisolated from AJ8D3 subsequently revealed that the transposonhad inserted between the –10 promoter element and thetranscription initiation site (data not shown). To examine whetherrpoE was expressed in AJ8D3, Northern blot analysis was conductedon total RNA isolated from this mutant and from A909 for comparison.Using a probe that was specific for rpoE, we detected a singletranscript in RNA from A909 that we estimated to be 550 bp insize, indicating that rpoE is transcribed as a monocistronicmessage (Fig. 5A). In contrast, multiple transcripts were detectedin the AJ8D3 RNA. Three primary transcripts were detected (Fig.5A). However, none were large enough to represent a transcriptthat was being initiated from the native promoter now located8.6 kb upstream of the coding sequence for rpoE. These dataindicate that transcription of rpoE in AJ8D3 is being initiatedfrom promoters within the transposon. In addition, the totalband intensity of the rpoE-specific transcripts detected inAJ8D3 was considerably reduced compared to that of the singletranscript detected in A909, suggesting that the strength ofthe nonnative promoters may be weaker than the native one. Controlexperiments using a probe for rpsL and the same RNA samplesdemonstrated that equal amounts of total RNA from the isogenicstrains were used in the analysis (data not shown).

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FIG. 5. Transcriptional analysis of rpoE in isogenic strains. (A) Northern blot of RNA prepared from A909 (1), AJ8D3(2), and AJ200 (3) using a digoxigenin-labeled DNA probe that was specific for rpoE. The positions of size markers (in kilobases) and the 23S and 16S rRNAs are indicated. (B) Schematic diagram of the rpoE locus in the isogenic strains. (C) Promoter location within Tn917stm. The location of promoter regions within Tn917stm are indicated. Predicted transcript sizes that were initiated from these three promoters sites are given in kilobases. Shaded box, pBR322 ori/rop/tet region; gray arrows, erythromycin gene (erm), resolvase (tnpR), and transposase (tnpR); black arrow, rpoE gene. LTR, left terminal repeat; RTR, right terminal repeat. Promoter regions within are represented as P1, P2, and P3. Numbers on the right are reference numbers.

The 5'-RACE approach was used to identify the transcriptioninitiation sites for rpoE in AJ8D3. Consistent with our Northernanalysis, we obtained several amplification products from the5'-RACE PCR (data not shown). Previous sequencing of Tn917,together with transcription studies in Enterococcus faecalistransposon mutants, identified three functional promoters (P1,P2, and P3) (Fig. 5C) that were responsible for initiating thetranscription of Tn917 genes (38). The promoter P1 (–35[TTGATA] and –10 [TATAAT]) is located upstream of theerm gene, promoter P2 (–35 [TTAATG] and –10 [TATAAT])is immediately upstream of the resolvase gene tnpR and promoterP3 (–35 [ATGCCA] and –10 [TATAAA]) is located withinthe transposase (tnpA). Based on the size of the transcriptsand the sequence of the 5'-RACE PCR products that we obtainedfrom AJ8D3, it is likely that the transcription of rpoE is beinginitiated from these previously identified promoters in Tn917.

Abundance of ${delta}$ in AJ8D3 is reduced compared to that of the wild-type strain. Since we detected transcription of rpoE from nonnative promotersin AJ8D3, we sought to determine whether these messages weretranslated into protein. We compared the expression of ${delta}$ in whole-celllysates from A909 and AJ8D3 during growth in vitro by Westernblot analysis using the antiserum directed against ${delta}$ . In orderto estimate the relative quantity of ${delta}$ , a standard curve of seriallydiluted recombinant protein was prepared and analyzed in parallelwith the lysates. As shown in Fig. 6A, the amount of ${delta}$ in A909appeared constant throughout all phases of growth, indicatingcontinual expression. Interestingly, we also detected ${delta}$ in AJ8D3at all growth phases tested. However, the amount of ${delta}$ proteinin AJ8D3 was significantly reduced. Comparison of the band intensityto the standard curve (Fig. 6D) indicated that at all stagesof growth, there is at least 10-fold less ${delta}$ present in AJ8D3compared to that in A909. As expected, no ${delta}$ was detected in AJ200.As a control to demonstrate equal protein loading between samples,we repeated the analysis by using antisera directed against ${sigma}$ ^A (Fig. 6B) and the ß subunit (Fig. 6C). Western blottingusing anti- ${sigma}$ ^A and anti-ß antisera indicated that thelevels of these two subunits remained constant in the threestrains throughout growth. These data demonstrated that thetranscription of rpoE from the nonnative promoters in Tn917results in a reduction in ${delta}$ protein levels in AJ8D3. This findingprovides a likely explanation for the attenuated virulence thatwas observed for this mutant and suggests that a specific levelof ${delta}$ is required for virulence of GBS.

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FIG. 6. Immunoblot analysis of ${delta}$ , ${sigma}$ ^A, and ß subunits during growth. A909, AJ200, and AJ8D3 were grown to lag (LA) (OD₆₀₀ = 0.1), early log (EL) (OD₆₀₀ = 0.3), late-log (LL) (OD₆₀₀ = 0.3) and stationary (SA) phases (OD₆₀₀ = 1.0). Lysates were normalized for total protein (10 µg) and subjected to SDS-PAGE. Western blots were performed by using antisera directed against ${delta}$ (A), ${sigma}$ ^A (B), and ß (C). Protein levels were probed by using polyclonal sera raised against the recombinant protein. (D) Estimation of ${delta}$ levels. A total of 10 µg of A909 (1), AJ200 (2), and AJ8D3 (3) lysates were loaded together with 100 ng, 50 ng, 25 ng, 10 ng, and 5 ng of purified ${delta}$ . Blots were probed with antisera directed against ${delta}$ . Immunoreactive bands were detected by using an infrared imager after incubation with a rabbit anti-IgG infrared-labeled secondary antibody, and band intensity was compared between strains. r ${delta}$ , recombinant delta protein.

DISCUSSION

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Discussion

Bacterial RNAP is considered to play a pivotal role in globalchanges in gene expression. Although the core enzyme is a highlyconserved heteromeric structure (ßß' ${alpha}$ ₂),the composition of the holoenzyme can differ considerably amongbacterial species and with the environmental conditions. A significantamount of information regarding RNAP composition and the mechanicsof transcription has been generated. However, most of this informationis based on the analysis of only a few model organisms, includingE. coli and B. subtilis. Our knowledge of RNAP composition instreptococci, particularly in GBS, remains in its infancy. Yet,the elucidation of the composition of the transcriptional machineryis of fundamental importance for understanding the mechanismsthat this microbe utilizes to control the transcription process.

In this study, we used a well-documented multistage purificationprocedure to isolate RNAP from the cytosol of GBS. To our knowledge,this represents the first detailed description of RNAP isolationand subunit composition in GBS. The RNAP that we isolated consistedof subunits corresponding to ${alpha}$ , ß, ß', and ${delta}$ , which are consistent with RNAP composition analyses reportedfor other gram-positive bacterial species (7, 17, 28, 39). Thepreparation also contained a subunit corresponding to ${sigma}$ ^A, theprimary sigma factor in gram-positive bacteria. Interestingly,following size exclusion chromatography, the final GBS RNAPpreparation also contained a small number of protein bands inaddition to the ${alpha}$ , ß, ß', ${sigma}$ ^A, and ${delta}$ subunits,indicating that other potential RNAP-associated proteins mayhave been isolated. Analyses of the available genome sequenceshave identified genes encoding two alternative sigma factorsin addition to ${sigma}$ ^A (12, 40). Thus, these additional protein bandsmay represent alternative sigma factors. Alternatively, proteinssharing a similar binding affinities to the various columnsused in the chromatography steps may have also copurified, afeature that is indicative of isolating a multisubunit enzyme(17, 39). However, without further analysis, it is impossibleto determine whether these proteins represent additional sigmafactors, breakdown products of the core, or copurifying contaminatingproteins.

The ${delta}$ protein is a subunit of RNAP found in only gram-positivebacteria. This $~$ 21 kDa protein was originally identified in B.subtilis (26) but has also been demonstrated to copurify withRNAP in Staphylococcus aureus and Streptococcus pneumoniae (7,28). Here we provide evidence that the ${delta}$ homolog in GBS alsoassociates with RNAP following purification.

GBS strains are grouped into serotypes on the basis of capsularpolysaccharide on the bacterial surface. A number of virulencetraits, particularly surface proteins, have been reported tobe serotype specific (for a review, see reference 23). Westernblot analysis of a panel of strains representing all of thenine serotypes confirmed that ${delta}$ was expressed in all of the strainstested and not limited to one serotype. Taken together withthe observation that an rpoE homolog is present in every gram-positivebacterial genome currently sequenced (19), it appears that ${delta}$ is highly conserved among gram-positive bacteria.

The putative promoter for rpoE was identified by using a 5'-RACEPCR approach. The expression of rpoE from this promoter on alow-copy plasmid restored the virulence of the deletion mutantto wild-type levels, confirming that we had correctly identifiedthe promoter. Reporter constructs were then used to monitorrpoE expression from this promoter during growth in vitro. Theexpression of rpoE was continual throughout growth, but it appearedto reach a maximal level during exponential growth. A similarpattern of expression has been reported for rpoE in Bacillussubtilus (25), suggesting that this pattern may be conservedamong gram-positive bacteria.

${delta}$ was initially linked to the virulence of GBS when a mutantwith a transposon insertion adjacent to rpoE was identifiedin an STM screen (18). In the current study, we demonstratedthat rpoE is transcribed in the transposon mutant AJ8D3 butthat transcription is initiated from multiple promoters withinthe transposon and not from the native promoter. Additionally,the overall amount of rpoE transcript was substantially reducedcompared to that of the wild-type strain. The modified transposonused in the STM screen (Tn917stm) contains at least five knownpromoters. Three promoters were originally reported during thesequencing and analysis of Tn917 (38), and modification of thetransposon for STM introduced two additional promoters (18).The results presented here indicate that the transcription ofrpoE can occur from these promoters, although this appears tobe less efficient than from the native promoter. Western blotanalysis demonstrated that this message is translated into ${delta}$ protein. However, ${delta}$ is present in reduced abundance of $~$ 10-foldrelative to the wild-type strain. We have previously reportedthat AJ8D3 is as attenuated in the neonatal rat sepsis modelas it is in the rpoE deletion mutant that does not express any ${delta}$ (19). Based on these collective observations, we conclude thata critical amount of ${delta}$ is required within the cell for virulencebut that the levels of the protein in AJ8D3 do not reach thisthreshold. The decreased amount of ${delta}$ in AJ8D3 is likely a resultof the low abundance of the rpoE transcript. It is also possiblethat transcripts that were initiated from the promoters in thetransposon are less stable or are translated less efficiently.

It is not yet known what role ${delta}$ plays in the virulence of GBS.Functional activity for ${delta}$ has been demonstrated in Bacillus subtilisby using in vitro assays (20, 24, 39). The generation of rpoEmutants of Bacillus subtilis has been reported (21, 25), thoughthey lack a well-defined phenotype. More recently, it has beensuggested that rpoE may have a direct or indirect role in Bacillussubtilis sporulation by affecting the transcription of genesthat are required at specific stages (11). A mutation in rpoEhas also been reported to affect the ability of S. aureus torecover from nutrient starvation (42).

If the activities demonstrated for bacillus ${delta}$ can be extrapolatedto GBS, then maintaining the transcriptional specificity andefficient recycling of RNAP would appear to have a profoundeffect on virulence. These functions may allow this organismto adapt to environmental change more efficiently, making ita more successful pathogen. It seems unlikely that ${delta}$ has globaleffects on transcription since rpoE is not required for viabilityand mutants have only a limited number of phenotypic changes(11, 19, 25, 42). In GBS, ${delta}$ may impact the expression of a subsetof genes that are required at a critical stage for survivalin our animal model. Our data suggest that the relative abundanceof ${delta}$ is of critical importance for virulence of this organism.Further study is needed to determine how ${delta}$ influences RNAP activityand whether it affects gene expression in vivo.

ACKNOWLEDGMENTS

We thank J. D. Helmann for generously providing B. subtilisstrain MH5636 and antiserum directed against bacillus ${delta}$ as wellas Donald Chaffin for technical assistance with the HPLC.

This work was funded by the Streptococcal Initiative of theNational Institutes of Health, grant no. BWH 811501/N01-AI-75326and R01AI52299-01.

FOOTNOTES

* Corresponding author. Mailing address: Children's Hospital and University of Washington, Division of Infectious Disease, Department of Pediatrics, 307 Westlake Avenue N, Suite 300, Seattle, Washington, 98109. Phone: (206) 987-3573. Fax: (206) 987-7311. E-mail: amanda.jones{at}seattlechildrens.org.

Present address: Rosetta Inpharmatics, 401 Terry Avenue N, Seattle,WA 98109.

REFERENCES

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