JOURNAL OF BACrERIOLOGY, May 1994, p. 3033-3039 Vol. 176, No. 100021-9193/94/$04.00+0Copyright X 1994, American Society for Microbiology

Enhancement of In Vitro Transcription by Addition of Cloned,Overexpressed Major Sigma Factor of Chlamydia psittaci 6BC

ANNEMARIE L. DOUGLAS, NIRMAL K. SAXENA,t AND THOMAS P. HATCH*

Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163

Received 27 December 1993/Accepted 10 March 1994

Obligate parasitic bacteria of the genus Chiamydia possess a developmental cycle that takes place entirelywithin eucaryotic host cells. Because standard methods of genetic analysis are not available for chlamydiae, anin vitro transcription system has been developed to elucidate the mechanisms by which chlamydiae regulategene expression. The in vitro system is specific for chlamydial promoters but is inefficient, presumably becausethe RNA polymerase is not saturated with sigma factor. Therefore, we prepared recombinant Chlamydia psittaci6BC major sigma factor to enhance transcription in the in vitro system. The gene encoding the major sigmafactor (sigA) was identified by using an rpoD box oligonucleotide and was subsequently cloned and sequenced.It was found to encode a potential 571-amino-acid protein (a6") that is greater than 90%o identical to thepreviously identified major sigma factors from the L2 and MoPn strains of Chlamydia trachomatis. sigA wasrecloned into a T7 RNA polymerase expression system to produce large quantities of co" in Escherichia coli.Overexpressed co" was identified by immunoblot by using monoclonal antibodies 2G10 (reactive) and 2F8(nonreactive) generated against E. coli Cr70. After purification by polyacrylamide gel electrophoresis, therecombinant protein was found to stimulate, by 10-fold or more, promoter-specific in vitro transcription by C.psittaci 6BC and C. trachomatis L2 RNA polymerases. Transcription was dependent on added chlamydial cr",rather than on potentially contaminating E. coli CF70 or other fortuitous activators, since the monoclonalantibody 2G10, and not 2F8, inhibited transcription initiation. Recombinant cr66 had no effect on transcriptionby E. coil core polymerase. The addition of recombinant r66 to the in vitro system should be useful fordistinguishing cu"-dependent transcription of developmentally regulated chlamydial genes from c66-indepen-dent transcription.

Chlamydia spp. are obligate intracellular parasites with aunique developmental cycle. The cycle is characterized by theinfection of eucaryotic host cells by elementary bodies, theconversion of elementary bodies to dividing reticulate bodies,the reorganization of reticulate bodies back to infectiouselementary bodies, and lysis of the host cell (24). The transi-tions between stages of the developmental cycle involve alter-ing the expression of chlamydial genes (26).The mechanisms that regulate gene expression in chlamyd-

iae have not been identified but are likely to occur at the levelof transcription initiation. RNA polymerase holoenzyme ini-tiates transcription by using the sigma subunit (ur) in a complexwith core enzyme, itself a complex of a2cP' subunits. Thesigma subunit confers the abilities to recognize specific pro-moters, to melt the DNA template at the site of initiation, andto incorporate the first few nucleotides before sigma is releasedfrom core enzyme (13, 20). Core enzyme is a DNA-bindingprotein complex that can initiate transcription at a low level atnonspecific sequences in vitro (13); its function in vivo, how-ever, is to continue elongating transcripts initiated by theholoenzyme. A cascade of alternate sigma factors, each withthe ability to recognize a specific cognate promoter, could beused by chlamydiae to express developmentally regulatedgenes in a manner similar to that of the sporulation process inBacillus subtilis (31). Alternatively, expression of stage-specificgenes could be dependent on the major sigma factor, with

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of Tennessee, 858 Madison Ave.,Memphis, TN 38163. Phone: (901) 448-4664. Fax: (901) 448-8462.Electronic mail address: THATCH@UTMEMI.UTMEM.edu.

t Present address: Department of Chemistry, Jabalpur University,Jabalpur MP482001, India.

regulation occurring by repression and activation of transcrip-tion by auxiliary proteins. At present only the major sigmafactor (r66) has been identified in Chlamydia spp. The pre-dicted peptide sequence of cr' bears strong homology to themajor sigma factor of Escherichia coli ((J70), particularly in theregions thought to bind to promoters (4, 16).

Several putative promoter regions of chlamydial genes havebeen identified by virtue of their positions from 5' ends ofmRNA (3, 8, 9, 18, 29). Some of the chlamydial promotersclosely resemble the consensus sequence recognized by themajor sigma factors of bacteria, whereas others are widelydivergent. For example, the promoters of omp2, a late-stage-specific gene, and the cryptic plasmid antisense gene, whichappears to be expressed throughout the cycle, vary from theconsensus by only one base at both the -35 and -10hexamers of their putative promoter regions (9, 18). In con-trast, the genes encoding the major outer membrane proteinand rRNA each possess two putative promoters, none of whichclosely resembles the consensus for major sigma factors (3, 29).However, the ability of major sigma factors to recognizedivergent sequences is not uncommon for genes that aresubject to positive activation (27). Thus, examination of thepromoter sequences of genes often fails to provide usefulinformation regarding the role of a given type of sigma factorin the expression of that gene. Definitive association of aspecific sigma factor with gene expression can be determinedonly by in vitro transcription studies or genetic analysis ofmutants.The inability to stably transform, transfect, conjugate, or

electroporate chlamydiae makes it impossible to determinemechanisms of gene expression by traditional genetic methods.For this reason, our laboratory has developed an in vitrotranscription system (23). The system consists of substrates,

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DNA templates, and chlamydial RNA polymerase-enrichedextracts (SS2), made by high-salt elution of the RNA poly-merase from pellets of reticulate body cell lysates. The systemhas been used, in conjunction with a monoclonal antibody(MAb) that specifically inhibits (r66-dependent transcription, todemonstrate that the plasmid antisense gene and the cysteine-rich protein operon require the major sigma factor for initia-tion of transcription (23). Other chlamydial genes have not yetbeen examined in the in vitro system.

Except for molecules engaged in initiation complexes, theRNA polymerase in chlamydiae, and therefore SS2 prepara-tions, is free of sigma factor and cannot initiate transcriptionfrom promoters. Consequently, the efficiency of transcriptionis low and also has proved to be variable from one SS2preparation to another. The purpose of the present study,therefore, was to improve the sensitivity and reliability of the invitro transcription system by more fully saturating SS2 prepa-rations of RNA polymerase with chlamydial sigma factor. Wecloned and sequenced the gene encoding the major sigmafactor of Chlamydia psittaci 6BC and demonstrated that thepurified recombinant sigma factor greatly enhances promoter-specific transcription in SS2 extracts prepared from C. psittaciand Chlamydia trachomatis.

MATERIALS AND METHODS

Growth of chlamydiae. C. psittaci 6BC and C. trachomatisLGV434 (serovar L2) were grown in L929 cells and HeLa cells,respectively, as previously described (12). For all experiments,reticulate bodies were harvested at 24 h postinfection and were

purified by Renografin density gradient centrifugation (2).Cloning and sequencing of sigA. The rpoD box oligonucle-

otide (5'-GCITTGICIIATCCACCAIGTIGCITAIGT-3') ofTanaka et al. (36) and oligonucleotide 101S (5'-GATCCTGTTCGTATGTAlTTTGAAAGAAATGGGAACC-3'), based on

the region 1 sequence of C. trachomatis (16), were used toidentify and clone sigA, the gene encoding the major sigmafactor (J66) of C. psittaci 6BC. The oligonucleotides, endlabelled with 125 ,uCi of [y-32P]ATP (NEN Research Products,Boston, Mass.) and polynucleotide kinase (New England Bio-Labs, Inc., Beverly, Mass.) (37), were used to probe Southernblots of restriction endonuclease-digested C. psittaci 6BCgenomic DNA. DNA was blotted onto MSI NitroPlus (MicroSeparations Inc., Westboro, Mass.) and was hybridized at lowstringency with 6 x SSC (1 x SSC = 0.15 M NaCl plus 0.015 Msodium citrate) without formamide at 42°C. Blots were washedin 6 x SSC-0.05% sodium pyrophosphate at 42°C. A 1.6-kbHindlIl fragment, which reacted with the rpoD box oligonu-cleotide, and a 1.8-kb PstI fragment, which reacted witholigonucleotide 101S, were isolated by using GeneClean (Bio101, La Jolla, Calif.) after agarose gel electrophoresis. TheHindIll and PstI fragments were ligated into pBluescript KS+(Stratagene, San Diego, Calif.), producing pALR100 andpALR101, respectively. The complete double-stranded se-

quence of sigA was determined by the dideoxy-chain termina-tion method (28) by using the Sequenase 2.0 kit (U.S. Bio-chemical Corp., Cleveland, Ohio). pALR100 was found, byhomology to other chlamydial major sigma factors, to encodethe 3' end of sigA, and pALR101 was found to encode the 5'end of the gene, overlapping pALR100 by 250 bp. All oligo-nucleotides were provided by the University of TennesseeMolecular Resource Center.The complete nucleotide sequence of sigA is available from

GenBank (accession no. U09442).Overexpression and purification of cr". The entire sigA

gene was cloned into pT7-5 by PCR amplification of C. psittaci

genomic DNA to overexpress "66 in E. coli. The forwardprimer, containing an EcoRI site, was 5'-CGGAATTCTlTTGTTGCAACCCAATAATTAA-3'; the reverse primer, con-taining a BamHI site, was 5'-CGGGATCCATGCGATTCTGCGGCGCGACAC-3'. Amplification was for 30 cycles eachof 1 min at 95°C, 2 min at 50°C, and 1 min at 72°C. The PCRproducts were digested with EcoRI and BamHI (New EnglandBioLabs), purified by GeneClean, and ligated into pT7-5,which contains a T7 RNA polymerase promoter (35). Ligationproducts were transformed into E. coli strain HMS174 (33),and clones containing inserts were identified by colony blotusing end-labelled oligonucleotide 101S (22). Three cloneswere selected and sequenced. The DNA sequence of one,pALR105, was identical to sigA as determined from clonespALR100 and pALR101.To overexpress r66, pALR105 was transformed into

BL21(DE3)pLysE (33). The transformant was grown at 37°Cto an A600 of 0.3 in 10 ml of L broth containing carbenicillin(100 ,ug/ml) and chloramphenicol (30 ,ug/ml). Isopropyl-P-D-thiogalactopyranoside (IPTG) was added to 400 ,uM to induceT7 RNA polymerase, and cultures were incubated for 30 min.Rifampin was added to 200 ,ug/ml to inhibit transcription fromnon-T7 promoters, and cultures were grown for an additional2 h. Cells were harvested by centrifugation, and the cell pelletwas suspended in lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mMEDTA, 0.3 mg of lysozyme per ml) and was incubated on icefor 20 min. Cells were lysed by sonication, and the lysate wascentrifuged at 10,000 x g for 10 min at 4°C. The pellet,containing insoluble inclusion bodies, was washed once (50mM Tris-HCl [pH 8.0], 10 mM EDTA, 0.1 M NaCl, 0.5%Triton X-100), was suspended in 300 ,ul of gel solubilizationbuffer (0.137 M Tris-HCl [pH 6.8], 2% glycerol, 0.006%bromophenol blue, 2% sodium dodecyl sulfate [SDS]), and wasloaded onto an SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel (gradient, 7.5 to 15%). After electrophoresis, a60-kDa band was visualized by incubating the gel in ice-cold0.25 M KCl-1 mM dithiothreitol for 5 min and was processedby a modification of the method of Hager and Burgess (10).The band was excised, and protein was eluted overnight atroom temperature with buffer (50 mM Tris-HCl [pH 7.9], 0.1%SDS, 0.15 M NaCl, 5 mM dithiothreitol, 0.1 mM EDTA, 0.1mg of bovine serum albumin per ml). Protein was precipitatedfrom the elution buffer by the addition of 4 vol of chilledacetone and by incubation for 30 min in an ethanol-dry icebath. The centrifugation pellet (10,000 x g for 10 min at 4°C)was dried, and protein was solubilized by suspension in 50 ,ul of6 M guanidinium-HCl in dilution buffer (50 mM Tris-HCl [pH8], 20% glycerol, 0.15 M NaCl, 1 mM dithiothreitol, 0.1 mMEDTA, 0.1 mg of bovine serum albumin per ml). Purified u66was renatured by the addition of 2.5 ml of dilution buffer andwas used directly in transcription reactions.

Immunoblots were done as described previously (7) by usingMAb 2G10 and 2F8 ascites at 1:1,000 dilution (kindly suppliedby Nancy Thompson, University of Wisconsin) made against E.coli o(J (15, 32).

In vitro assays. C. psittaci and C. trachomatis SS2 extracts ofRNA polymerase were prepared as described previously (23).Standard in vitro transcription reactions (100 pLI) were carriedout for 20 min at 37°C by using 10 RI of SS2, 2.5 ,ug of templateDNA, 25 ,uCi of [a-32P]UTP (NEN Research Products), 0.1nmol of UTP, and 33 nmol of ATP, GTP, and CTP asdescribed by Mathews et al. (23). Ten pul (approximately 40 ng)of purified (r66 preparation was added to the reaction mixturebefore substrates except when otherwise noted. For MAbinhibition studies, SS2, purified ur66, and 3.8 ,ug of 2G10 or 6 ,igof 2F8 were preincubated on ice for 30 min before substrates

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and templates were added. In some experiments, SS2 wassubstituted with 0.2 U of E. coli core RNA polymerase(Epicentre Technologies, Madison, Wis.) or 0.05 U of ou70-saturated E. coli holoenzyme (Epicentre), and the final NaClconcentration was adjusted to 150 mM. Templates werepALR202 (23), containing the promoter of the chlamydialcryptic plasmid antisense gene inserted into the transcriptionassay vector pUC19-spf' (5), and pUC19-spf'. Radiolabelledtranscripts were extracted by the hot-acid phenol method andassayed on 6% polyacrylamide-8 M urea DNA sequencing gels(23).

RESULTS

Identification of sigA. The gene encoding the major sigmafactor of C. psiuaci 6BC, sigA, was identified by Southern blotanalysis using a radiolabelled rpoD box oligonucleotide (36).The sequence of the oligonucleotide was based on a 14-amino-acid sequence within region 2 which is highly conserved amongthe major sigma factors of bacteria (20) and contains inosine atthe points of degeneracy. The oligonucleotide hybridized toonly one fragment in chromosomal Hindlll, BamHI, EcoRI,and PstI digests, suggesting that sigA4 is a single-copy gene (datanot shown). A 1.6-kb HindIII fragment was cloned into pBlue-script to form pALR100. On the basis of homology to otherchlamydial sigma factors, pALR100 was found to encode onlythe 3' end of sigA. The 5' end of the gene was identified bySouthern blot analysis of genomic DNA probed with anoligonucleotide (101S) based on region 1 of the C trachomatisL2 sigA sequence (data not shown). The 1.8-kb PstI fragment,which reacted with 101S, was cloned and found to encode the5' end of sigA, overlapping the pALR100 insert by 250 bp.

Sequence analysis. sigA encodes a potential 571-amino-acidprotein (molecular weight, 66,245) that is 91% identical to themajor sigma factor of C. trachomatis MoPn and 92% identicalto the major sigma factor of C. trachomatis L2 (Fig. 1). Allthree chlamydial sigma factors, which we will refer to as U66,contain the same number of amino acids. Differences amongthem were noted at the N terminus, between regions 1 and 2 asdefined for E. coli u70 and B. subtilis U(3 (20), and in the last 11amino acids of the C terminus. C. psittaci cu' is also almost44% identical to E. coli u70 over the entire length of theprotein. From amino acids 389 to 627 (Fig. 1), a region that isconserved among the major sigma factors of bacteria (20), theidentity is 66%; in the highly conserved region 2 of major sigmafactors, the identity is 92% over 89 amino acids. The predictedisoelectric point of each of the three chlamydial a' proteins isabout 9, whereas that of u70 is about 4.5. This difference can beaccounted for by a concentration of multiple acidic aminoacids (198 to 237) in c70 which is not found in the chlamydialsigma factors. The aberrant migration of u70 on SDS-PAGE(Mr = 90,000) has been attributed to this acidic region (13).The chlamydial major sigma factors migrate with an Mr ofabout 66,000 (4, 16; see also Fig. 2).

Overexpression of c66. sigA was cloned by PCR into pT7-5,overexpressed in E. coli, and purified as described in Materialsand Methods. When whole-cell extracts and the 10,000 x gcentrifugation pellet of the overexpressing clone were exam-ined by SDS-PAGE, a prominent Coomassie blue-stainingband of approximately 66 kDa was noted in both preparations(Fig. 2). The 66-kDa bands and others with lower molecularweights reacted on immunoblots with MAb 2G10 but not withMAb 2F8. Both MAbs were generated against u70 of E. coli(32); however, of the two, only 2G10 has been shown to reactwith u( of C. trachomatis (16). Therefore, the 2G10-reactivebands most likely represent intact C. psittaci 6BC r66 and

smaller peptides generated by degradation or anomalies asso-ciated with overexpression. The presence of (66 in the pelletfraction suggests that the overexpressed protein formed insol-uble inclusion bodies. A similar observation was made when E.coli u70 was overexpressed (14) in the same T7 phage system.The 90-kDa band in the whole-cell extract, which reacted withboth 2G10 and 2F8, is presumed to be E. coli C7u. Followingthe method of Hager and Burgess (10), a66 was purified byeluting the 66-kDa band of the pellet fraction from an SDS-polyacrylamide gel, solubilizing the protein in guanidinium-HCl, and renaturing the protein by dilution in buffer lacking achaotropic agent. A single major band of 66 kDa was noted onCoomassie blue-stained gels of the purified cru66 preparation.More significantly, the purified preparation failed to react withMAb 2F8, indicating that c66 was not contaminated with E. coli70 (Fig. 2).Effect of purified u6r on in vitro transcription by C. psittaci

RNA polymerase. The in vitro transcription system developedby our laboratory (23) makes use of the transcription assayvector pUC19-spf' (5). This vector contains the 3' end of thespf gene of E. coli, including its rho-independent terminator.Promoter regions of interest can be cloned upstream of the spfgene fragment, and transcripts initiated in vitro from thecloned promoters can be roughly quantified and identified onthe basis of their length on sequencing gels. pALR202, used asthe DNA template in the studies reported here, contains thechlamydial cryptic plasmid antisense gene promoter (-35:TTGCCA; - 10: TATATT), which closely resembles the con-sensus sequences recognized byE. coli u70. Multiple transcriptsare generated from the pALR202 template, including somefrom vector promoters and fortuitous promoter-like se-quences. Sequences initiated from the antisense promoterhave been shown to run on sequencing gels as a series of bandsof between 105 and 110 bases relative to a DNA sequencingladder (23). The multiplicity of bands reflects inexact termina-tion rather than imprecise initiation (23).As can be seen in Fig. 3, the addition of purified recombi-

nant cr66 to C. psittaci 6BC RNA polymerase preparations(SS2) significantly enhanced promoter-specific transcriptionfrom pALR202. Enhancement was dependent on the presenceof SS2, since no transcripts were noted when u66 was added tothe in vitro system lacking SS2. Maximum transcription oc-curred when 80 ng of purified (u66 was added; transcriptiondecreased when larger amounts were used, probably becauseguanidinium-HCl was present in the cr66 preparations. Wefound that the level of enhancement provided by u66 varieddepending on the basal level of activity of the RNA polymerasepreparation (i.e., the fold increase was greater for preparationswith low basal activity) but generally was at least 10-fold.Transcript length was not affected by the addition of u66,suggesting that the transcription start site was not altered bythe sigma preparation. Primer extension experiments con-firmed this supposition and also showed that the in vitro and invivo start sites were identical (data not shown). Transcriptionfrom the pUC19-spf' vector was also stimulated by purified66, confirming an earlier observation that the chlamydial

transcription apparatus is capable of recognizing some non-chlamydial promoter sequences (23).We examined the effects of MAbs 2G10 and 2F8 on

(F"-stimulated transcription to determine if enhancement wasdirectly related to the u6 in our preparations. We found that2F8, which does not react with u(66 in an immunoblot assay,failed to have an inhibitory effect on either the base level or thea66_stimulated transcription of pALR202 (Fig. 4). In contrast,we were able to demonstrate that 2F8 (reactive with u70 in animmunoblot assay) did inhibit u70-dependent in vitro transcrip-

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FIG. 1. Alignment of the deduced amino acid sequences of major sigma factors. The major sigma factors of C. psittaci 6BC (Cp66), C.trachomatis L2 (L266), MoPn (Mo66), and E. coli (Ec7O) were aligned by using the PileUp program of the University of Wisconsin GeneticsComputer Group (Biotechnology Center, University of Wisconsin, Madison, Wis.). The dots (.) indicate amino acid identity with the C. psittaci6BC sequence; gaps generated by the program are indicated by blank spaces. Also shown are regions of conservation among the major sigmafactors of eubacteria (20).

tion of pALR202 by E. coli RNA polymerase (data not shown).MAb 2G10, which reacts with C. psittaci u66, inhibited bothbasal transcription of pALR202 by SS2 and the stimulatoryeffects of added o66 (Fig. 4). These observations support theconclusion that o66, rather than E. coli u70 or a fortuitousactivator of transcription, is the active component in thepreparation of purified sigma factor.

Effect of C. psittaci O66 on transcription of C. trachomatis andE. coli RNA polymerases. We found that C. psittaci J66enhanced in vitro transcription by C. trachomatis L2 RNApolymerase SS2 preparations as effectively as it enhanced theC. psittaci preparations (Fig. 5). However, purified u66, whenpresent in concentrations sufficient to stimulate transcriptionby chlamydial RNA polymerases, did not augment promoter-specific transcription by E. coli core RNA polymerase. E. coliholoenzyme, in contrast, was found to effectively transcribepALR202 in vitro, an observation consistent with the previous

observation that the cryptic plasmid antisense promoter isexpressed well in E. coli (9). B. subtilis alternative sigma factorsK and E are synthesized as proforms which must be activatedby proteolysis (17, 21). It is possible, therefore, that the failureof u66 to enhance the activity of purified E. coli core poly-merase is due to the absence of an activating protease which ispresent in our SS2 preparations. However, we failed to noteany changes in the intensity or migration rate of the u66 bandon immunoblots following incubation of "66 with SS2 (data notshown).

DISCUSSION

The purpose of this study was to improve the chlamydial invitro transcription system that was previously developed in ourlaboratory (23). Although transcripts were shown to be initi-ated from the same start sites in vitro as in vivo, in vitro

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FIG. 2. Purification of ac6 expressed in E. coli. Samples producedat various stages of purification of recombinant ac66 were run intriplicate on a single SDS-PAGE gel. One set of samples was stainedwith Coomassie blue, and two sets were blotted to membrane filtersand reacted with either MAb 2G10 (reactive with C. psittaci) or MAb2F8 (nonreactive with C. psiffaci). Both MAbs were generated againstE. coli ac70 (15). The samples run on the gel were whole-cell lysates ofIPTG-induced BL21(DE3)pLysE transformed with pALR105(Whole), the centrifugation pellet of the whole-cell lysate (Pellet), andpurified a"66 obtained by elution of the 66-kDa gel band in the pelletfraction (Gel). Purification was carried out as described in Materialsand Methods except that bovine serum albumin was omitted in allsteps to permit visualization of the 66-kDa cr' band. The migrationdistances of E. coli c-70 (ca. 90 kDa) and C. psittaci a-66 (ca. 66 kDa) areindicated by the arrows on the right. Numbers on the left refer tostandard molecular weight markers (MW) and prestained molecularweight markers (PMW) in thousands.

transcription signals were often weak and occasionally difficultto detect. We hypothesized that inefficient transcription wasdue to a deficiency of sigma factor in our RNA polymerasepreparations. To rectify this potential deficiency, we suJpple-mented our preparations with purified recombinant cr , themajor sigma factor of C. psittaci 6BC. We consistently noted a10-fold or greater increase in transcription when preparationsof purified a-66 were added to the in vitro system. Although therecombinant sigma factor was not purified to homogeneity, E.coli ca70 was not detected in our preparations on immunoblots,and a MAb, which reacted with a66, abrogated the stimulatoryeffect of a-66. These results suggest that the stimulatory effect ofthe preparation was due to the chlamydial sigma factor and notfortuitous contaminating activators of transcription. Our spec-ulation that the RNA polymerase preparations consist largelyof core enzyme appears, therefore, to be correct. Because wehave no means of quantifying the subunits of chlamydial RNApolymerase, we were unable to determine the degree ofsaturation of polymerase in our preparations both before andafter the addition of recombinant a66.The amino acid sequence identity between the major sigma

factors of C. psittaci 6BC and C. trachomatis L2 is 92%. This isnotably higher than the identity between other proteins: 54%for the small cysteine-rich protein, 71% for the large cysteine-

p19 pALR202FIG. 3. Effect of a(66 on in vitro transcription. In vitro reaction

mixtures that contained C. psiftaci 6BC SS2 were incubated withvarious quantities (indicated above the lanes in nanograms) of purifieda66 or without added cr' (-); the DNA templates were pUC19-spf'(p19) or pALR202 (indicated below the appropriate lanes). A controlreaction without SS2 (-SS2) was also run. Radioactive transcriptswere detected on a sequencing gel as described in Materials andMethods. The dot (.) next to the DNA sequencing ladder indicates asequencing product of 110 nucleotides.

rich protein, and 67% for the major outer membrane protein(6, 7). Although all of these proteins are believed to playdistinct and critical roles in the life cycle of chlamydiae (1, 11,25, 34), the sigma factor is the only protein likely to be involved

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FIG. 4. Inhibition of a66-stimulated transcription by MAbs. SS2and MAbs (2G10 and 2F8) were incubated with or without a66 on icefor 30 min before the transcription reaction was initiated by theaddition of substrates and template. pALR202 was used as thetemplate except for a control reaction using vector pUC19-spf' (p19).The dot (*) indicates the DNA sequencing product that is 110nucleotides long.

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3038 DOUGLAS ET AL.

FIG. 5. Effect of recombinant C psittaci u' on in vitro transcrip-tion of pALR202 by C. trachomatis L2 and E. coli RNA polymerases.(A) SS2 preparations were made from purified C. psittaci 6BC (6BC)and C. trachomatis L2 (LGV) and were supplemented with recombi-nant U66 (40 ng) where indicated. (B) C. psittaci 6BC SS2 or E. coli coreRNA polymerase (Core) were supplemented with three differentpreparations of purified U66 (cl, u2, and o3). Reaction with E. coliholoenzyme (Holo) is also shown. The dot (.) indicates the DNAsequencing product that is 110 nucleotides long.

in the transcription and regulation of gene expression. Allchlamydiae are obligate intracellular parasites and in thiscontext have identical environments which may have restrictedevolutionary divergence of promoter sequences and regulatorymechanisms of gene expression. This speculation is supportedby the observation that DNA sequences upstream of the majorouter membrane protein and cysteine-rich protein genes are

more highly conserved than the structural genes (6, 7, 18, 30).Also supportive is our finding that the promoter of the C.trachomatis L2 antisense plasmid gene is efficiently transcribedin vitro by C. psittaci 6BC uo66-RNA polymerase.The level of identity between C. psittaci au66 and E. coli u70 iS

also high, especially in regions 1.2 and 2 and, to a lesser extent,in regions 3 and 4 (Fig. 1). These regions are typicallyconserved among the major sigma factors of eubacteria (13,20). The conserved regions include parts of region 2 (aminoacids 448 to 470) and region 4 (amino acids 595 to 617) whichare involved in sigma recognition of -10 and - 35 promoterregions. The observation that at least some chlamydial pro-moters are nearly identical to the E. coli u66 consensus isconsistent with the homology within these regions.Although purified C. psittaci ur66 was equally effective in

stimulating transcription by C. trachomatis and C. psittaci RNApolymerase preparations, it had no effect on transcription by E.coli core polymerase. E. coli U70_saturated holoenzyme, on theother hand, efficiently initiated transcription from the chlamyd-ial promoter in pALR202. Lesley and Burgess (19) reportedthat binding of Cr7° to core RNA polymerase is associated witha region somewhere between amino acids 374 and 404 (Fig.1).Although there is only one difference between u66 and ac°from amino acids 388 to 404, there are 15 differences, many ofthem radical, over the first 18 NH2-terminal amino acids in thepotential core binding region. These differences may account

for the failure of u66 to function with E. coli RNA polymerase;however, the NH2-terminal portion of the core binding regionis not highly conserved among sigma factors that are capable ofbinding to E. coli RNA polymerase (19).

or66 may function with core E. coli RNA polymerase in vitroat concentrations above those which we were able to attain.Such concentrations may also be unattainable in vivo, however,since cr66 appears to form insoluble inclusion bodies whenoverproduced in E. coli. The failure of u66 to interact produc-tively with E. coli RNA polymerase has negative implicationsregarding surrogate genetic studies. For example, the putativepromoters of several chlamydial genes studied to date divergesignificantly from the E. coli consensus and likely require acombination of a66 and transcriptional activators for expres-sion. One approach to identifying and cloning activators of agiven chlamydial gene would be to screen a chlamydialgenomic library for a clone that allows expression of that genein E. coli. Although promoter sequences recognized by E. coliu70 and chlamydial u66 appear to overlap, cr70 may fail torecognize divergent sequences, even in the presence of auxil-iary factors. Unfortunately, our findings predict that inclusionof u66 expression in the cloning system is unlikely to relieve thispotential problem.The addition of recombinant a66 to the in vitro transcription

system not only enhances transcription signals but also shouldfacilitate identification of transcriptional regulatory mecha-nisms. For example, developmental stage-specific genes can bepositively identified as u66 dependent if recombinant f66 aug-ments in vitro transcription from the same start site that is usedin vivo. By the same token, the effects of alternative sigmafactors on gene expression can be examined in vitro once thesefactors are identified and cloned.

ACKNOWLEDGMENTS

We thank Nancy Thompson and Richard Burgess for providingmonoclonal antibodies, Rebecca Crenshaw for her technical assis-tance, and Barbara Kuyper for her assistance in the writing of themanuscript.

This work was supported by Public Health Service grant A119570from the National Institutes of Health.

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ERRATUM

Enhancement of In Vitro Transcription by Addition of Cloned, OverexpressedMajor Sigma Factor of Chlamydia psittaci 6BCANNEMARIE L. DOUGLAS, NIRMAL K. SAXENA, AND THOMAS P. HATCH

Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163

Volume 176, no. 10, p. 3036: Figure 1 should appear as shown below. Sequence from amino acids 601 to 644 was omitted in theoriginal figure.

1Cp66 MLMNTQNGQA MEAAHEEEAQ KKLEELVSLAL266 .R.D.LDS.. A...Q.. QI. R. T..Mo66 .RIH.LDS.R AD. .Q.. .I. R. T..Ec7O M.QNP. SQ.KL ..TRG

51KDQGFITYEE INEILPMSFD TPEQIDQVLI................. ... ..........

.P...s..........E..YL..A. V.DH..EDIV DSDQIEDIIQ

FLTGMDIQVL NQ...... ... ..

...... ... ..

MINDMGIQVM EEAPDADDLM

100ADVERQKER KKEAKELEGL......... ..........A.....

L.ENTADEDA AEA.AQVLSS

101 151Cp66 AKRTEGTPDD PVRMYLKEMG TVPLLTREEE VEISKRIEKA QVQIERIILR FRYSSKEAISL266 ...S...... .......... .......... ......................T.. .V.Mo66 ...S...... .......... .......... ......................T.. .V.Ec7O VESEI.RTT. MR. E..E G. ID.A .... DG IN.VQCSVAE YPEAITYLLE

Region 1.2

201EEDAYLESLL FRLKQNNLSK EETAKLNDDL EK.......ER. LA ..DPA... PD... . .E.

...S...ER. LA..DPA... QDQ.K...E.Q..LDDDEDE DEEDGDDD.A DDDNSIDPE. AREKFAELRA

301L EQQINDLKVR.A..........

IMKLCVEQCK MPKKNFITLF TGNETSDTWF NAh.AMN.PW

IAQYLINGKE RFDKIISE K.V..K

...............VA KQYNRVEAEEA .LSDL.TGFV

251CRIRTQ AYLRCFHCRH NVTEDFGEV...... ......... .......... .... ....... ....

*.................. ............... ....

QYVVT.DTIK .KG.SHATAQ EEILKLS..F

351AERNKFAAAK LDAAKRRLYK REVAAGRTLE.......... . ...R.K.H.............A R.K.

S.KLHDVSEE VHRALQKLQQ I.EET.L.I.

VFKAY

.....KQFRL.P.QF

200EVEDKAHFLK LLPKLISLLK

.T...N..T...N.....T.N.

DPNAEEDLAP TATHVG.E.S

300DSFLQ

.YLVNSMRVM MDRVRTQERL

400EFKKDVRMLQ RWMDKSQEAK KEMVESNLRL... .... .. . .... .. .. .... ... .... .... . ......... .....

.......... ..........QQV.DIN.RMS 1GEA.ARR.....A..I-

401Cp66 VISIAKKYTN RGLSFLDLIQ EGNMGLMKAV EKFEYRRGYKL266 .......... .......... .......... ..........

Mo66 .......... .......... .......... ..........

Ec7O ............Q......... D....D

451 500FSTYATWWIR QAVTRAIADQ ARTIRIPVHM IETINKVLRG AKKLMMETGK EPTPEELAEE.......... .......... .......... ........................... ..

.......... .......... .......... ........................... ..

...............S..........LN..........LNI SRQMLQ.M.R ......... R1.

501Cp66 LGLTPDRVRE IYKIAQHPIS LQAEVGEGGE SSFGDFLEDTL266 ..F ................. ...... ... .......

Mo66 ..F....................... DS ..........Ec7O MLMPE.KI.K VL........ METPI.DDED .HI..

551 600

GVESPAEATG YSMLKDKMKE VLKTLTDRER FVLIHRFGLL DGKPKTLEEV GSAFNVTRER

A...........................................................A......... ........ K ................... .......... ..........

TL.L.LDSAT TES.RAATHD ..AG.....A K .RM.. .ID MNTDY KQ.D.

Region 4

601 644Cp66 IRQIEAKALR KMRHPIRSKQ LRAFLDLLEE EKIT GKAKN IKGKL266 .................... .......... ... GS. I.S Y.N

Mo66 .................... .......... .. TGS. I.S Y.N

Ec7O .......... ..EV .....D_ ~~~~~~~~~~~~~~~~~~~~~~~~I

FIG. 1

4196

Cp66L266Mo66Ec70

Cp66L266Mo66Ec7O