JOURNAL OF BACTERIOLOGY, Feb. 1996, p. 994–1002 Vol. 178, No. 40021-9193/96/$04.0010Copyright q 1996, American Society for Microbiology

The 18-Kilodalton Chlamydia trachomatis Histone H1-LikeProtein (Hc1) Contains a Potential N-Terminal Dimerization

Site and a C-Terminal Nucleic Acid-Binding DomainLOTTE BANG PEDERSEN,1 SVEND BIRKELUND,1 ARNE HOLM,2 SØREN ØSTERGAARD,2 AND

GUNNA CHRISTIANSEN1*

Department of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C,1 and ChemistryDepartment, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C,2 Denmark

Received 13 June 1995/Accepted 28 November 1995

The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the meta-bolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation inEscherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due toHc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptidesHc12–57 and Hc153–125, corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. Bychemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombi-nant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc12–57).Moreover, circular dichroism measurements indicated an overall a-helical structure of this region. By usinglimited proteolysis, Southwestern blotting, and gel retardation assays, Hc153–125 was shown to contain adomain capable of binding both DNA and RNA. Under the same conditions, Hc12–57 had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc153–125-DNA complexes revealed differences suggest-ing that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1.

The life cycle of the obligate intracellular bacterium Chla-mydia trachomatis is unique among eubacteria and is charac-terized by two alternating developmental forms: elementarybodies (EBs) and reticulate bodies (RBs). Infection of a eu-karyotic host is mediated by small (0.3 mm in diameter), ex-tracellular, metabolically inactive EBs, which adhere to thehost cells and induce their own uptake. At 4 to 6 h afterinfection, the EBs start transforming to the larger (1 mm indiameter), osmotically fragile, and metabolically active RBs.The RBs multiply by binary fission and remain within thephagosome during the entire intracellular stage of the lifecycle. At 24 to 48 h postinfection, when the inclusion is filledwith RBs, they start reorganizing into EBs. The EBs are finallyliberated into the surroundings by bursting of the inclusion,and a new infectious cycle can be initiated.During the morphological transformation of RBs to EBs,

the chlamydial cell undergoes a dramatic reduction in size. Toaccommodate the DNA within the much smaller EB, the RB-to-EB transition involves condensation of the chlamydial chro-mosome, which results in a nucleoid appearing as a dark,electron-dense body positioned in the center of the EB (10).Two very basic EB-specific DNA-binding proteins (41) arethought to be responsible for compaction of the chlamydialchromosome. Although no direct evidence of the involvementof these proteins in chlamydial chromosome condensation isavailable, a potential role for Hc1 in the condensation of DNAis suggested by the finding that expression of Hc1 in recombi-nant Escherichia coli causes condensation of the E. coli chro-mosome (4). Furthermore, Hc1 purified from recombinant E.coli forms complexes with double-stranded DNA in vitro,which appear as condensed spherical bodies when examined by

electron microscopy. The formation of these complexes seemslargely independent of DNA concentration but highly depen-dent on protein concentration, indicating cooperative bindingof Hc1 to DNA (8). In addition to a structural role in com-paction of DNA, evidence has been provided that Hc1 expres-sion in E. coli is self-limiting and results in down regulation oftranscription, translation, and replication at concentrationssimilar to those observed in chlamydial EBs. These eventscoincide with an overall decrease in the superhelical density ofE. coli chromosomal DNA (3). In a recent report, we describedthe binding of purified, recombinant Hc1 to RNA and single-stranded DNA and showed that Hc1 strongly represses tran-scription and translation in vitro (26). These results suggestthat Hc1 may act as a general repressor of transcription andtranslation, which correlates well with the apparent lack ofmetabolic activity of chlamydial EBs.The 18-kDa protein Hc1 is conserved among serovars,

whereas the other, Hc2, varies between 23 and 32 kDa inapparent molecular mass among different serovars (14, 16).Cloning and sequencing of the genes encoding these proteinsrevealed that both proteins are homologous to eukaryotic hi-stone H1 but not to any other known histone-like prokaryoticproteins (15, 27, 37).Despite the highly basic nature of Hc1 (calculated pI of 11.4)

and homology to eukaryotic histone H1 (15), the structuralbasis for the functional properties of Hc1 are unknown. Tolocate the putative functional domains within Hc1, we usedrecombinant DNA techniques and proteolytic digestion to gen-erate two polypeptides, Hc12–57 and Hc153–125, correspondingto the N- and C-terminal parts of Hc1, respectively (numbersrefer to amino acids of the Hc1 primary sequence [15]). Bychemical cross-linking we found that a potential dimerizationsite is located within the N-terminal part of Hc1, coveringamino acids 2 to 57. Circular dichroism (CD) measurements ofHc12–57 revealed a high a-helix content in Hc12–57, and com-puter analysis predicted that Hc1 contains an N-terminal am-

* Corresponding author. Mailing address: Department of MedicalMicrobiology and Immunology, The Bartholin Building, University ofAarhus, DK-8000 Aarhus C, Denmark. Phone: 4589421749. Fax:4586196128. Electronic mail address: chlam@biobase.dk.

994

phipathic a-helix with a putative hydrophobic interface forprotein-protein interaction. In addition, by limited proteolysis,Southwestern blotting, and gel retardation assays, we foundthat Hc153–125 contains a domain capable of binding bothDNA and RNA, whereas under the same conditions, the N-terminal part (Hc12–57) displays no significant nucleic acid-binding activity. Electron microscopy of Hc153–125-DNA andHc1-DNA complexes revealed differences suggesting that theN-terminal part of Hc1 is important for the formation of highlycondensed Hc1-DNA complexes.

MATERIALS AND METHODS

Cultivation of C. trachomatis. C. trachomatis serovar L2 LGV-II/434/Bu wascultivated in monolayers of McCoy cells (obtained from the American TypeCulture Collection, Rockville, Md.) and purified as previously described (5).Production and purification of recombinant Hc1 protein. Recombinant puri-

fied Hc1 protein was obtained as previously described (26). The final concen-tration of Hc1 was 5.65 mg/ml as determined by Bradford analysis using theBio-Rad Protein Assay Reagents (Bio-Rad Laboratories, Richmond, Calif.).Cloning of the gene encoding 6xHis-Hc12–57 in pET9d. Synthetic oligonucle-

otides for PCR amplification of the gene encoding 6xHis-Hc12–57 were obtainedfrom DNA Technology Aps, Aarhus, Denmark. The 59 oligonucleotide (primer11386) contained an NcoI restriction endonuclease site, the nucleotide sequenceencoding six adjacent histidine residues, a factor Xa cleavage site, and theamino-terminal region of hctA (59-GCC ATG GCT CAC CAC CAT CAC CACCAT ATC GAA GGT CGT GCG CTA AAA GAT ACG GC-39). The 39oligonucleotide (primer 11024) contained the sequence encoding amino acids 50to 57 of Hc1, followed by a stop codon and a BamHI restriction endonucleasesite (59-GGA TCC CTA TTC TGC TTT AAT GGA CTC TTT ACG-39). PCRwas done with a Perkin-Elmer Cetus thermal cycler (948C for 1 min, 458C for 1min, and 728C for 1 min for 35 cycles) by using the above-described primers,purified C. trachomatis L2 DNA (23), and Thermus aquaticus DNA polymerase(Boehringer GmbH, Mannheim, Germany). The PCR product 6xhis-hc12–57 wasgel purified and ligated into the TA Cloning Vector (pCRII) and introduced intoINVaF9-competent E. coli (Invitrogen Corp., San Diego, Calif.). The amplifiedgene product was control sequenced, digested with NcoI and BamHI, and ligatedinto expression vector pET9d (Novagen, Madison, Wis.). pET9d with 6xhis-hc12–57 was multiplied in E. coli XL1-Blue and subsequently electrotransformedinto E. coli K-12 BL21(DE3)/pLysS (35). This transformant clone, designatedBL21(DE3)/pLysS pET6xHis-Hc12–57-9d, was used for expression of 6xHis-Hc12–57.Induction and purification of 6xHis-Hc12–57. Recombinant BL21(DE3)/pLysS

pET6xHis-Hc12–57-9d was grown in Luria-Bertani (LB) medium with kanamycin(40 mg/ml) and chloramphenicol (34 mg/ml). Synthesis of 6xHis-Hc12–57 wasinduced with isopropyl-b-D-thiogalactopyranoside (IPTG) as previously de-scribed (8). At 2 h after induction, the cells were harvested, washed with coldphosphate-buffered saline (PBS), and stored at 2708C. Recombinant 6xHis-Hc12–57 was purified under denaturing but nonreducing conditions with a 1-mlNi-activated HiTrap Chelating column (Pharmacia, Uppsala, Sweden). The pu-rified protein (.95% pure by sodium dodecyl sulfate [SDS]-polyacrylamide gelelectrophoresis [PAGE]) was dialyzed against PBS, concentrated in Centricon 3tubes (GIBCO BRL), and stored at 2708C in PBS–30% (vol/vol) glycerol at aconcentration of 1.06 mg/ml.Enzymatic hydrolysis. Enzymatic hydrolysis of purified, recombinant Hc1

(1.38 mg/ml) with Staphylococcus aureus V8 protease (0.19 mg/ml; Boehringer)was carried out in PBS for 1 h at 378C. The Hc153–125 product was analyzed bySDS–15% PAGE and N-terminal sequencing. As a control, V8 protease withoutHc1 was analyzed by SDS–15% PAGE and Coomassie staining or immunoblot-ting with K101 anti-Hc1 serum (8). Hc1 (0.5 mg/ml) was digested with endopro-teinase Pro-C (9.1 U/ml; United States Biochemical Corporation, Cleveland,Ohio) in PBS for 1 h at 378C and analyzed by SDS–15% PAGE. To remove theN-terminal histidine tag, purified 6xHis-Hc12–57 (1 mg/ml) was cleaved for 24 hat 48C with 10 mg of restriction protease factor Xa (Boehringer) per ml in PBScontaining 30% (vol/vol) glycerol and 2 mM CaCl2. Removal of the histidine tagwas verified by SDS–15% PAGE and Coomassie blue staining. The cleavedprotein fragment was designated Hc12–57. Hc12–57 and Hc153–125 were used forcross-linking and nucleic acid-binding assays without further purification.N-terminal sequencing. For N-terminal sequencing, proteins were dissolved in

SDS sample buffer without b-mercaptoethanol (0.125 mM Tris [pH 6.8], 2.4%[wt/vol] SDS, 10% [wt/vol] glycerol, 5% [vol/vol] bromphenol blue solution),boiled for 5 min, subjected to SDS–15% PAGE, and electroblotted onto apolyvinylidene difluoride membrane (Immobilon; Millipore Corp., Bedford,Mass.) as previously described (44). Protein bands were cut out and sequencedby Edman degradation on an ABI Procise sequencer (Applied Biosystems, Per-kin Elmer).Chemical cross-linking. For chemical cross-linking, 1 or 10 mM ethylene

glycol-bis (succinic acid N-hydroxysuccinimide ester) (EGS; Sigma ChemicalCo., St. Louis, Mo.) in dimethylformamide was prepared and used at a 1:10

(vol/vol) dilution. All incubations with EGS were carried out for 10 min on ice.After cross-linking, the proteins were acetone precipitated, solubilized in 20 ml ofSDS sample buffer without b-mercaptoethanol, and boiled for 5 min beforeanalysis by SDS–14 or 15% PAGE and Coomassie blue staining.For cross-linking as a function of protein concentration, 500, 250, 62.5, 31.3, or

15.6 mg of purified Hc1 protein per ml in PBS (500 mg/ml corresponds to 36.5mM Hc1) was reacted with 1 mM EGS. To analyze the stability of Hc1 cross-linking, Hc1 (250 mg/ml) was chemically cross-linked with 1 mM EGS in PBScontaining 0, 1, or 10% (vol/vol) Triton X-100 or Tween 20 or 0.15, 0.5, 0.75, or1 M NaCl. To study the effect of DNA on cross-linking of Hc1, Hc1 (250 mg/ml)was heated for 5 min at 378C in PBS containing 0, 50, 100, or 150 mg ofpBluescript KS1 DNA (containing predominantly monomeric supercoiled,nicked circular, and linear forms, as determined by electron microscopy; Strat-agene, La Jolla, Calif.) per ml before addition of 1 mM EGS. For analysis fordimerization of Hc12–57 and Hc153–125, the polypeptides were cross-linked with0.1 mM EGS.For cross-linking with dimethyl suberimidate (DMS; Sigma), Hc1 (250 mg/ml)

in PBS was incubated on ice for 10 min with 1.0, 0.5, 0.25, or 0.125 mM DMS.SDS-PAGE, immunoblotting, and DNA-binding assay. SDS-PAGE was car-

ried out by standard procedures (21). For determination of relative proteinamounts, Coomassie blue-stained gels were scanned on a Hewlett-Packard Scan-Jet 3C/T with CREAM software (Kem-En-Tec, A/S, Copenhagen, Denmark).Immunoblotting with anti-Hc1 rabbit serum (K101) was done as previouslydescribed (8). DNA-binding assay with proteins immobilized on nitrocellulosemembranes was performed as previously described (8). After visualization ofbound, 32P-labelled DNA by autoradiography and staining of the membraneswith Ponceau S (17), protein bands were cut out and counted in a scintillationcounter.Peptide synthesis. The peptide H-ALKDTAKKMTDLLESI-OH was synthe-

sized by solid-phase peptide synthesis with fluorenylmethoxycarbonyl amino ac-ids in threefold excess with tert-butyl as a side-protecting group for Asp, Thr, andSer, except for the Asp-Thr sequence, where fluorenylmethoxycarbonyl-Asp (1-adamantyl)-OH was used, with tert-butyloxycarbonyl as a side-protecting groupfor Lys (Calbiochem-Novabiochem). O-(1H-benzotriazol-1-yl)-N,N,N9,N9-tetra-methyluroniumtetrafluoroborate (Millipore) was used as the coupling agent inthe presence of diisopropylethylamine (Aldrich-Chemie, GmbH, Steinheim,Germany), and all couplings were carried out in dimethylformamide with anacid-labile PepSyn resin with the first amino acid attached (substitution, 0.1meq/g; Milligen/Biosearch).The peptide was cleaved from the resin with trifluoroacetic acid-H2O-

ethanedithiol-thioanisol (90:5:3:2 [vol/vol/vol/vol]) for 2 h at room temperature,washed with trifluoroacetic acid-H2O (95:5 [vol/vol]), and then purified by high-pressure liquid chromatography to give one single component. The peptide wasanalyzed by mass spectroscopy with a JEOL AX 505W instrument (JEOL, Ltd.,Tokyo, Japan) in the fast atom bombardment positive-ion mode and a glycerolmatrix. The calculated and measured molecular masses (monoisotonic) were1,775.97, and 1,777 g/mol, respectively. The product was designated peptide A1.Computer analyses. Calculation of the mean residue weights and molecular

weights of proteins and peptide A1; prediction of the enzymatic cleavage sites,secondary structure, and helical-wheel structure of Hc12–19; and calculation ofthe sizes of DNA fragments of HaeIII-digested pBluescript KS1 DNA wereperformed with GCG software (Program Manual for the Wisconsin Package,Version 8, September 1994, Genetics Computer Group, Madison, Wis.).CD. For CD measurement, Hc12–57 was centrifuged in Centricon 3 tubes

(GIBCO BRL) and washed with PBS until the content of the N-terminal histi-dine tag was reduced to less than 5% (mol/mol) of Hc12–57. Factor Xa proteasewas removed by gel filtration in 0.2% (vol/vol) acetic acid in H2O on a SephadexG50 column (Pharmacia), and purified Hc12–57 was lyophilized and solubilized inPBS to a concentration of 0.15 mg/ml as determined by amino acid analysis witha Waters AccQTag system. For CD measurement, lyophilized peptide A1 wasdissolved in PBS containing 0, 20, or 40% (vol/vol) trifluoroethanol to give aprotein concentration of 0.21, 0.168, or 0.126 mg/ml, respectively. The CD spec-tra were recorded on a JASCO J-710 spectropolarimeter with a cuvette with a1.0-mm path length.Gel retardation assays. Interaction between Hc1 or Hc153–125 and nucleic

acids was analyzed by agarose gel electrophoresis as previously described (26),with TAE electrode buffer (31) containing 0.5 mg of ethidium bromide per ml.The amounts of agarose used for the gels were 0.7% (wt/vol) for undigestedpBluescript KS1 DNA and 2% (wt/vol) for HaeIII-digested DNA. Analysis ofthe interaction between Hc12–57 and undigested pBluescript KS1 DNA wasdone by using twofold dilutions of 20.8 to 0.65 mM purified Hc12–57.Electron microscopy. Purified Hc1 or Hc153–125 at 7.5, 3.87, 1.94, 0.97, 0.48, or

0.24 mM was incubated for 5 min at 378C with 6.5 or 8 mg of circular (containingsupercoiled, nicked circular, and linear forms) or HaeIII-digested pBluescriptKS1 DNA (Stratagene) per ml. The samples were analyzed by electron micros-copy as previously described with a JEM 1010 electron microscope (8).

RESULTS

Generation of Hc153–125 and Hc12–57. A domain is the small-est protein unit having a defined, independently folded struc-

VOL. 178, 1996 DIMERIZATION AND NUCLEIC ACID BINDING OF Hc1 995

ture and is typically 50 to 150 amino acids long (6, 18). Theclassical method used to define domain organization withinproteins is limited proteolysis since proteases tend not to cutwithin domains because cleavage sites within domains are pro-tected (43). To analyze the location of putative functionaldomains within Hc1, we generated two overlapping polypep-tides corresponding to the N- and C-terminal parts of the Hc1 pri-mary sequence, respectively (Fig. 1).The C-terminal fragment, Hc153–125, was generated by en-

zymatic hydrolysis of purified, recombinant Hc1 with V8 pro-tease. Since this protease cleaves at glutamine (E) residues, weexpected a product corresponding to Hc153–125 because ofglutamines present at positions 15, 25, 35, 43, 52, and 57 in theHc1 primary sequence (Fig. 1). However, N-terminal aminoacid sequencing of the product revealed that the first six aminoacids were 53SIKAEK58; therefore, no cleavage had occurredat E-57, indicating that this residue is located within a foldeddomain. To estimate its purity, Hc153–125 was analyzed bySDS–15% PAGE and staining with Coomassie blue or immu-noblotting with rabbit anti-Hc1 serum. As seen in Fig. 2A, onlyone electrophoretically resolvable band of approximately 9.5kDa was observed (lane 3; the lower-molecular-weight bandsresulted from cleavage of Hc1 degradation products present in

the Hc1 preparation [lane 1]). Immunoblotting of the gel withrabbit anti-Hc1 serum gave a similar result (Fig. 2B, lane 3),indicating that the N-terminal portion of Hc1 was completelydigested by V8 protease.To obtain the N-terminal part of Hc1, purified, recombinant

Hc1 was subjected to enzymatic cleavage with endoproteinasePro-C. Since this protease cleaves at the C termini of proline(P) residues, we expected cleavage to occur at positions 79,104, and 110 of the Hc1 primary sequence (Fig. 1). However,analysis of the cleavage product by SDS–15% PAGE revealeda band with a molecular weight only slightly lower than that offull-length Hc1 but larger than that expected if cleavage hadoccurred at P-79.Because of the lack of suitable enzymatic cleavage sites in

the C-terminal part of Hc1 for the generation of an N-terminalproteolytic fragment, recombinant DNA techniques were usedto generate Hc12–57 (in C. trachomatis L2, the formylmethi-onine of Hc1 is cleaved off [15]). The purity and antigenicity ofHc12–57 were confirmed by SDS–15% PAGE and Coomassieblue staining (Fig. 2A, lane 2) and immunoblotting with rabbitanti-Hc1 serum (Fig. 2B, lane 2).Chemical cross-linking of Hc1. Purified Hc1 was subjected

to cross-linking with the bifunctional cross-linker DMS or EGS(span lengths of 12 and 16 Å, respectively [9]), and the prod-ucts were analyzed by SDS-PAGE. Cross-linking with DMSdid not produce any significant cross-linking products. Cross-linking with 0.1 mM EGS revealed the presence of Hc1 mul-timers. Without addition of EGS a faint band migrating as Hc1dimers (2.5%) was observed (Fig. 3, lane 1). After cross-link-ing, 37% of Hc1 was present as dimers and 7% was present astrimers (Fig. 3, lane 2). Chemical cross-linking of Hc153–125

showed 4% dimers (Fig. 3, lane 4), while Hc12–57 showed thepresence of 14% dimers and 15% trimers (Fig. 3, lane 6).These results indicate that a substantial amount of Hc1 can beobserved as dimers after cross-linking with EGS and that thesite most likely to be responsible for dimerization probably islocalized within the N-terminal part of Hc1. To analyze thestability of Hc1 dimers and trimers, cross-linking was per-formed at different protein concentrations (15.6 to 500 mg[1.14 to 36.5 mM] of purified, recombinant Hc1 per ml), in thepresence of nonionic detergents (1 or 10% Triton X-100 orTween 20), at different sodium chloride concentrations (0.15 to1 M), and after addition of DNA. Addition of nonionic deter-gents, sodium chloride, and DNA did not influence the relativeamount of Hc1 dimers or trimers (monomers, 66% 6 6%;dimers, 28% 6 4%; trimers, 6% 6 3% [mean 6 standarddeviation]). At different concentrations of Hc1, there was morevariation in the observed amounts of dimers and trimers (48%6 6% for monomers, 38% 6 12% for dimers, and 14% 6 6%for trimers).

FIG. 1. Amino acid sequences of Hc1 (15), peptide A1, Hc12–57, and Hc153–125. The calculated molecular masses are 13,567, 1,777, 7,798, and 6,316 g/mol,respectively. Peptide A1 was synthesized by standard solid-phase peptide synthesis. The DNA sequence encoding 6xHis-Hc12–57 (described in Materials and Methods)was amplified by PCR and cloned into expression vector pET9d. Expression of the cloned gene was induced with IPTG in E. coli K-12 BL21(DE3)/pLysS (35), andthe target protein was purified on a nickel column. After dialysis and cleavage with factor Xa protease, purified Hc12–57 was obtained. Recombinant, purified Hc1 wasobtained as previously described (26), and Hc153–125 was generated by enzymatic hydrolysis of Hc1 with V8 protease. The N-terminal sequences of Hc1 and Hc153–125

were confirmed experimentally by amino acid sequencing. The putative sites of cleavage by V8 protease and endoproteinase Pro-C are indicated.

FIG. 2. (A) Coomassie blue-stained SDS–15% polyacrylamide gel showingpurified, recombinant Hc1 protein (lane 1), Hc12–57 (lane 2) obtained by factorXa cleavage of affinity-purified, recombinant 6xHis-Hc12–57, and Hc153–125 (lane3) obtained by enzymatic hydrolysis of Hc1 with V8 protease. The samples wereanalyzed under nonreducing conditions. (B) Immunoblotting of an SDS–15%polyacrylamide gel run in parallel with the gel in panel A. Rabbit anti-Hc1 serum(K101 [8]) was used for the reaction. Molecular mass markers are indicated inkilodaltons on the left.

996 PEDERSEN ET AL. J. BACTERIOL.

When Hc1 (250 mg/ml) was cross-linked with various con-centrations of DMS (0.125 to 1 mM) and the products wereanalyzed, the only electrophoretically resolved band detectedwas monomeric Hc1. However, a significant amount of large,insoluble aggregates of Hc1 was detected in the stacking gel,indicating that DMS reacted very strongly with Hc1 (resultsnot shown). DMS was therefore not used for further analyses.Secondary structure of Hc12–57. Computer prediction

showed that Hc12–57 is mainly a-helical. The secondary-struc-ture predictions of amino acids 2 to 19 of Hc1 showed a strongtendency for this region to form an a-helix (Fig. 4) with acalculated hydrophobic moment of 0.42 (13, 30). To examinethis prediction experimentally, purified Hc12–57 and syntheticpeptide A1 (Fig. 1), covering amino acids 2 to 17, were ana-lyzed for secondary-structure content by CD measurement (1).As seen in Fig. 5, the CD spectrum of Hc12–57 in PBS revealeda minimal peak at 209 nm and a shoulder at 222 nm, suggestiveof an overall a-helical structure (7). Calculation of the a-heli-cal content of Hc12–57 in PBS by using the value of the meanresidue ellipticity (u) at 222 nm and assuming a maximumvalue of 2u222 of 33,0008 cm

2/dmol (25) indicates approxi-mately 46% a-helical content (Fig. 5). The CD spectrum ofpeptide A1 in PBS revealed a mostly random coil conforma-tion, which may be due to the short length and consequentconformational flexibility of peptide A1. In the presence oftrifluoroethanol (20 or 40%), which is known to stabilize onlypreexisting secondary structures of peptides (12), the CD spec-trum of peptide A1 was indicative of an a-helical structure(results not shown). These results demonstrate that Hc12–57

has a defined secondary structure which is approximately 46%a-helical.DNA and RNA binding of Hc153–125. Since both Hc12–57 and

Hc153–125 were found to exist in a folded conformation, wetested whether the nucleic acid-binding activity is confined to

both or only one of these fragments. Purified Hc1, Hc12–57, andHc153–125 immobilized on nitrocellulose membranes were an-alyzed for nucleic acid-binding activity. Bound DNA was de-tected by autoradiography and quantified by scintillationcounting of the pieces of the nitrocellulose membrane corre-sponding to the locations of Hc1, Hc12–57, and Hc153–125, re-spectively. As seen in Fig. 6, Hc153–125 bound approximately78% 32P-labelled DNA relative to Hc1, whereas no DNA wasbound by Hc12–57. These results show that under the experi-mental conditions used in this study, Hc12–57 displays no DNA-

FIG. 3. Chemical cross-linking of Hc1, Hc153–125, and Hc12–57. Purified pro-tein (50 mM) was incubated for 10 min on ice with the cross-linker EGS, and theproducts were analyzed by SDS–15% PAGE and staining with Coomassie blue.Lanes: 1, Hc1 with no cross-linker added; 2, Hc1 cross-linked with 100 mM EGS;3, Hc153–125 without EGS; 4, Hc153–125 with 100 mM EGS (the double bandaround 34 kDa in lanes containing Hc153–125 is V8 protease [double arrows] asdetermined by SDS–15% PAGE of V8 protease alone and immunoblotting withK101 anti-Hc1 serum [8; the band was not recognized by the antiserum]); 5,Hc12–57 without EGS; 6, Hc12–57 with 100 mM EGS. Arrows: Hc1 monomer anddimer. Arrowheads, dimer and trimer bands of Hc12–57.

FIG. 4. Helical-wheel prediction of amino acids 2 to 19 from the Hc1 primarysequence. Hydrophobic amino acids are boxed. The wheel was predicted by usingGCG software.

FIG. 5. CD spectrum of Hc12–57 in PBS. The mean residue ellipticity (u orul) was calculated from the formula ul 5 uobs 3 MRW/10 3 d 3 c0 (1), whereuobs is the observed ellipticity, MRW is the mean residue weight for Hc12–57

(112.79 g/mol), d is the path length of the cuvette (0.1 cm), and c0 is theconcentration of Hc12–57 (0.15 mg/ml). Assuming a maximum 2u222 value of33,0008 cm2/dmol (25), the a-helix content of Hc12–57 is 46% [(15,038/33,000) 3100%].

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binding activity whereas Hc153–125 displays DNA-binding ac-tivity comparable to that of full-length Hc1.The interaction between Hc1 or Hc153–125 and DNA was

analyzed by gel retardation assays with agarose gels. As seen inFig. 7A, Hc153–125 caused retention of plasmid DNA in theslots at molar concentrations comparable to those of Hc1,where similar retention of DNA occurred although Hc1, butnot Hc153–125, showed a preference for binding of supercoiledDNA over nicked circular and linear plasmid DNAs (lanes 6and 7). Furthermore, for Hc1, complete retention of DNA inthe slots occurred at and above 2.5 mM Hc1 whereas forHc153–125, the DNA was partially released at 2.5 mM. Whensimilar assays were performed with Hc12–57, no retention ofDNA occurred even at relatively high molar concentrations(0.65 to 20.8 mM) of protein (results not shown).When HaeIII-digested pBluescript KS1 DNA (fragment

sizes, 11 to 605 bp) was used, the gel retardation profile ofHc153–125-DNA complexes was similar to that of Hc1-DNAcomplexes (Fig. 7B), although the smeared appearance of theDNA observed in the presence of 1.25 mM Hc153–125 was notseen in the corresponding Hc1 lanes (Fig. 7B).To determine whether the DNA- and RNA-binding activi-

ties of Hc1 are located in the same domain or separate do-mains, purified Hc1, Hc12–57 and Hc153–125 were incubated for5 min at 378C with bacteriophage MS2 RNA and the com-plexes were analyzed by electrophoresis in agarose gels. By thisanalysis, Hc153–125 displayed RNA-binding activity similar tothat of full-length Hc1 (Fig. 7C), whereas RNA binding ofHc12–57 was not observed. Thus, the DNA- and RNA-bindingactivities of Hc1 are located within Hc153–125. Since domainstypically are 50 to 150 amino acids long (6), and since Hc1 wasresistant to Pro-C cleavage at P-79, it is unlikely that Hc153–125

contains more than one domain. We conclude that the DNA-and RNA-binding activities of Hc1 are located in the samedomain within Hc153–125. The results thus suggest that theforces involved in DNA binding of Hc1 are different fromthose involved in protein-protein interaction.Interaction between Hc1 or Hc153–125 and DNA was exam-

ined by electron microscopy. Different molar amounts of pu-rified Hc1 or Hc153–125 were incubated for 5 min at 378C with

FIG. 6. Southwestern blotting of purified, recombinant Hc1 protein (lane 1),Hc12–57 (lane 2), and Hc153–125 (lane 3). The proteins were solubilized in non-reducing SDS sample buffer, subjected to SDS–15% PAGE, and electrotrans-ferred to a nitrocellulose membrane. The membrane was incubated with nick-translated pBluescript KS1 DNA, and bound 32P-labelled DNA was revealed byautoradiography. Scintillation counting of the cut out protein bands revealed thatHc153–125 bound 78% 32P-labelled DNA relative to Hc1, whereas no DNA wasbound by Hc12–57. Open arrowhead, DNA bound by Hc1; closed arrowhead,DNA bound by Hc153–125. Molecular mass markers are shown in kilodaltons onthe left.

FIG. 7. (A) Gel retardation of DNA-Hc1 and DNA-Hc153–125 complexesformed in vitro. pBluescript KS1 DNA (45.5 nM), containing supercoiled DNA,as well as nicked open circles and linear forms, was mixed with twofold dilutionsof 10 to 0.078 mM (as indicated above the lanes) purified Hc1 (lanes 3 to 10) orHc153–125 (lanes 13 to 20), incubated for 10 min at 378C, and subjected toelectrophoresis in a 0.7% agarose gel containing ethidium bromide. Lane Stcontained HindIII-cleaved l DNA, and lane V8 contained DNA with V8 pro-tease (83.3 mg/ml). (B) Same as panel A, except that HaeIII-digested pBluescriptKS1 DNA (30.5 nM) was used (Hc1-DNA in lanes 2 to 8; Hc153–125-DNA inlanes 9 to 15) and the samples were analyzed in a 2.0% agarose gel. Lane V8contained DNA with V8 protease (83.3 mg/ml). (C) Gel retardation analysis withMS2 RNA (83.3 nM). The RNA was mixed with twofold dilutions of 4.8 to 0.15mM Hc1 (lanes 4 to 9) or Hc153–125 (lanes 10 to 15) and electrophoresed in a0.7% agarose gel containing ethidium bromide. Lane St contained HindIII-cleaved l DNA, lane 2 contained RNA without protein, and lane V8 containedRNA with V8 protease (7 mg/ml).

998 PEDERSEN ET AL. J. BACTERIOL.

uncut or HaeIII-cleaved pBluescript KS1 DNA and analyzedby electron microscopy (Fig. 8). At high concentrations ofprotein (7.5 mM), both Hc1 and Hc153–125 formed highly con-densed complexes when incubated with pBluescript KS1DNA(Fig. 8A and B). No free DNA was detected corresponding tothe gel retardation assay shown in Fig. 7A (lanes 10; 5 mM). Atan intermediate concentration of protein (1.94 mM), Hc1 con-densed pBluescript KS1DNA into spherical aggregates with ahighly condensed center surrounded by a halo of DNA coils(Fig. 8C). A few unpacked open circular DNA molecules weredetected outside the condensed particles. A different conden-sation was seen when Hc153–125 was used. A branched networkof condensed fibers was surrounded by a meshwork of DNAcoils (Fig. 8D). Outside these networks, free supercoiled andopen circular DNA molecules were detected. These resultsconfirm the results shown by the gel retardation assay (Fig. 7A,lanes 3 and 4). At a lower concentration of protein, the com-plexes formed became smaller and less condensed and morefree DNA molecules were seen to be unassociated with thecomplexes. Similarly, HaeIII-cleaved pBluescript KS1 DNAbecame highly condensed when incubated with a high concen-tration (7.5 mM) of both Hc1 and Hc153–125. At an intermedi-ate concentration (1.94 mM), Hc1 formed spherical bodies witha condensed center containing a high number of DNA frag-ments surrounded by a lesser condensed halo (Fig. 8E). At thisconcentration, not all DNAmolecules were associated with thecomplexes. As seen in Fig. 8G, most of these fragments wereshort, indicating preferential association of the longest linearmolecules with the complexes also seen by gel retardation (Fig.7B, lanes 4 and 5). In addition, many of the short linear frag-ments were kinked, looped, or branched. Hc153–125 formedsimilar complexes with HaeIII-cleaved pBluescript KS1 DNA;however, a much less condensed halo of DNA was seen (Fig.8F). As judged from the size of the uncomplexed DNA (Fig.8H), there was no preferential association of the longest linearmolecules with the complexes and the uncomplexed DNA didnot appear to be kinked. These data thus suggest that theN-terminal part of Hc1 affects the DNA-binding property ofHc1 by promoting better condensation of the DNA and en-abling Hc1 to induce bends in the DNA.

DISCUSSION

Prokaryotic histone-like proteins often exist in homo- orheterodimeric form (11). One of the best-characterized exam-ples of these is the HU protein from E. coli, which is a het-erotypic dimer composed of two closely related subunits, HUaand HUb, displaying amino acid homology to eukaryotic his-tone H2B (11, 29). The HU protein from Bacillus stearother-mophilus (HBs) has been crystallized, and its three-dimen-sional structure has been determined. It appears that thefunctional unit is a wedge-shaped dimer of two identical HBsmonomers with a hydrophobic interior and two antiparallelb-sheet arms responsible for binding DNA and that hydropho-bic interactions between several such HBs molecules inducebending of the DNA, thus promoting the formation of HBsnucleosome-like particles in which the DNA is negatively su-percoiled (36, 42).Another well-studied example of prokaryotic proteins with

histone-like properties is DNA-binding protein HMf from thehyperthermophilic archaeon Methanothermus fervidus. HMf isa mixture of two very similar polypeptides, HMfA (HMf-1) andHMfB (HMf-2), which display more than 30% homology tothe consensus sequences derived from eukaryotic core histonesH2A, H2B, H3, and H4 (33). Electron microscopy and topo-logical studies of HMf-DNA complexes have revealed that the

DNA is wrapped around a core of HMf molecules in positivetoroidal coils forming characteristic nucleosome-like particles(24, 33). The formation of these complexes is mediated bysequential and reversible interactions between individual HMfmolecules, and since the relative amounts of HMfA and HMfBare growth phase dependent, the proportion and compositionof the protein complexes may vary substantially during the lifecycle, resulting in different biological properties of these com-plexes (32).In eukaryotes, the core histones are associated to form an

octamer which, when DNA is wrapped around it, makes up anucleosome core (20). Non-core histone H1 interacts with thelinker DNA between adjacent nucleosome cores and stabilizesthe coiling or folding of nucleosome chains into the 30-nmchromatin fiber (38). At low ionic strength, the fiber unfoldsand the characteristic bead-like repeating units of nucleosomesbecome visible. This unfolding occurs within a narrow range ofsalt concentrations and appears to be a consequence of achange from cooperative binding between H1 and DNA tononcooperative binding (28). In contrast to HU and HMf,which display similarity to one or more of the eukaryotic corehistones (11, 33), Hc1 shares 34.9% sequence identity in a106-amino-acid overlap with the C-terminal part of histone H1from Lytechinus pictus but displays no significant homology toeukaryotic core histones or to any other known histone-likeproteins (15). This may explain the different effects of DNA onoligomerization of HU and Hc1. Moreover, previous studies byelectron microscopy revealed that Hc1-DNA complexes werelargely spherical, consisting of a highly condensed center witha tight meshwork of DNA looping out at the periphery of thecomplex (8), but the characteristic beads-on-a-string-like ap-pearance observed for HU-DNA (11) or HMf-DNA (33) com-plexes was not observed.The data presented in this report are the first experimental

evidence for dimerization and trimerization of Hc1 in vitro. Inthis respect, Hc1 shows some similarity to other prokaryotichistone-like proteins, such as HU and HMF. For E. coli HU,formation of large protein complexes (trimers, tetramers, etc.)apparently requires the presence of DNA (11, 22), whereas forHc1, interaction between individual Hc1 molecules apparentlyis unaffected by DNA. Since Hc1 dimers were detected even inthe absence of a cross-linker, the molecular interactions areapparently very strong, suggesting that dimeric Hc1 representsa stable and functionally important form of the protein. Sincethe Hc1 concentration within chlamydial EBs is approximately46 mg/ml (8), this is theoretically sufficient for Hc1-Hc1 com-plex formation in vivo. The in vitro experiments presented inthis study were all performed with purified recombinant Hc1.A concern when using such proteins overproduced in E. coli isthat they may be misfolded and thus produce unspecific aggre-gates. We found, however, that upon V8 digestion, only asingle polypeptide band (Hc153–125) was produced, leavingE-57 protected. Similarly, digestion with endoproteinase Pro-Cleft P-79 protected. From these results, it thus seems that theDNA-binding C-terminal part of Hc1 is uniformly folded. Fur-thermore, the recombinant Hc12–57 showed, by CD measure-ment, approximately 46% a-helical content, indicative of awell-defined structure in this part of the molecule. The highlysine content of Hc1 makes this molecule highly reactive forcross-linking. Lack of oligomerization of Hc153–125 upon EGStreatment indicates that in solution the molecules are presentin the monomeric form, which is indicative of electrostaticrepulsion between the amino groups. From these in vitro ex-periments, Hc1 thus seems to contain a dimerization site in theN-terminal part of the molecule and a DNA-binding domain inthe C-terminal part.

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FIG. 8. Electron microscopy of complexes formed between Hc1 and DNA (A, C, E, and G) and between Hc153–125 and DNA (B, D, F, and H). Proteins wereincubated with DNA for 5 min at 378C prior to being mounted for electron microscopy. After dehydration in increasing concentrations of ethanol, the samples wererotary shadowed with tungsten. In A and B, 7.5 mM protein was incubated with pBluescript KS1 DNA (6.25 mg/ml); in C to H, 1.94 mM protein was incubated withpBluescript KS1 DNA (8 mg/ml). A to D, uncleaved DNA; E to H, HaeIII-digested DNA. Bar, 0.2 mm.

1000

Gel shift assays and electron microscopy of Hc1-DNA andHc153–125-DNAcomplexes revealedmarked differences between theDNA-binding modes of full-length Hc1 and Hc153–125. First, Hc1exhibited a preference for binding of supercoiled DNA overnicked circular and linear DNAs and for binding of the longestover the shortest linear DNA fragments, whereas Hc153–125 didnot display such binding preferences. Second, for both undi-gested and HaeIII-digested pBluescript KS1 DNAs, the com-plexes formed between Hc153–125 and the DNA appeared to beless condensed by electron microscopy than those formed be-tween Hc1 and the DNA. Third, HaeIII-digested plasmidDNA had a more kinked or bent appearance in the presence ofHc1 than in the presence of Hc153–125.Since by CD spectroscopy, Hc12–57 was found to be approx-

imately 46% a-helical, we propose that this region indirectlyaffects the DNA-binding property of Hc1 by virtue of a proteininteraction site. Interestingly, Barry et al. (3) reported thatlow-level expression of Hc1 in E. coli resulted in differentialeffects on gene expression, probably through Hc1-mediatedalterations of DNA topology, whereas high concentrations ofHc1 resulted in complete condensation of the E. coli chromo-some and cessation of macromolecular synthesis (3). Consid-ering the data presented here and those of Barry et al. (3), it ispossible that at low, substructural Hc1 concentrations, Hc1preferentially binds to supercoiled DNA because the relativelyhigh flexibility of supercoiled DNA would promote the inter-action between individual Hc1 molecules bound to separatedsites on the DNA (for a review of DNA topology and protein-DNA interactions, see reference 40). A consequence of inter-action between Hc1 molecules bound to distant sites on theDNA would be the formation of loops or bends in the DNA,which is fully compatible with electron microscopic observa-tions of Hc1-DNA complexes (this report; 8). The preferentialbinding of eukaryotic histone H1 to superhelical DNA hasbeen shown to involve the central globular domain of H1 (34),which binds highly cooperatively to DNA (39) and affects theability of the C-terminal domain to induce condensation of thenucleofilament (2). The high primary sequence homology ofHc1 to eukaryotic H1 may thus be reflected at the functionallevel. Although a detailed topological analysis is required toconfirm the proposed explanation for the apparent bindingpreferences of Hc1, the evidence recently provided by Kaul etal. (19) suggests that the N-terminal part of Hc1 may be func-tionally very important. Those investigators reported that theinterspecies diversity among chlamydial Hc1 genes was largestfor the C-terminal half of Hc1 (25% deduced amino acididentity between Hc1 from C. psittaci Mn and C. trachomatisL2), whereas there was a high level of identity (87%) amongthe first 66 amino acid residues (19). This pronounced conser-vation of the N-terminal part of Hc1 also includes the potentialfor a-helix formation and may reflect the functional impor-tance of a protein interaction site. The N-terminal 2 to 19amino acids of Hc1 were predicted to form an amphipathica-helix with a putative hydrophobic interface for dimerization.Such helices are often involved in dimerization of DNA-bind-ing proteins (reviewed in reference 40) and have been pre-dicted by a database search to be present in a large number ofdifferent nucleic acid-binding proteins (30).Although we have shown that Hc1 forms homodimers in

solution, our data do not exclude possible interactions betweenHc1 and other chlamydial proteins, such as the variable histoneH1 homolog Hc2. Identification of chlamydial proteins thatpotentially interact with Hc1 may shed light not only on theultrastructural arrangements within the chlamydial nucleoidbut also on the mechanisms involved in the transition between

the transcriptionally inactive and active stages of the uniquechlamydial life cycle.

ACKNOWLEDGMENTS

We are grateful to Charlotte Holm, Karin Skovgaard, and IngerAndersen for excellent technical assistance. Special thanks are due toKaren Jørgensen, H. C. Ørsted Instituttet, University of Copenhagen,Copenhagen, Denmark, for performing the CD spectroscopy.This work was supported by grants from the Danish Veterinary and

Agricultural Research Council and the Danish Health Research Coun-cil (12-0150-1 and 20-3503-1) and by the Aarhus University ResearchFoundation, the Carlsberg Foundation, the John and Birthe MeyerFoundation, the Marie and M. B. Richters Foundation, and the Fac-ulty of Science, University of Aarhus, Aarhus, Denmark.

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