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Journal of Bacteriology, January 2009, p. 651-660, Vol. 191, No. 2
0021-9193/09/$08.00+0     doi:10.1128/JB.01083-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutagenesis of Region 4 of Sigma 28 from Chlamydia trachomatis Defines Determinants for Protein-Protein and Protein-DNA Interactions

Ziyu Hua,^1,2, Xiancai Rao,^1,2 Xiaogeng Feng,³ Xudong Luo,¹ Yanmei Liang,¹ and Li Shen^1,2*

Division of Infectious Diseases, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118,¹ Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112,² Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112³

Received 4 August 2008/ Accepted 24 October 2008

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Transcription factor ²⁸ in Chlamydia trachomatis (²⁸_Ct) plays a role in the regulation of genes that are important for late-stage morphological differentiation. In vitro mutational and genetic screening in Salmonella enterica serovar Typhimurium was performed in order to identify mutants with mutations in region 4 of ²⁸_Ct that were defective in ²⁸-specific transcription. Specially, the previously undefined but important interactions between ²⁸_Ct region 4 and the flap domain of the RNA polymerase β subunit (β-flap) or the –35 element of the chlamydial hctB promoter were examined. Our results indicate that amino acid residues E206, Y214, and E222 of ²⁸_Ct contribute to an interaction with the β-flap when ²⁸_Ct associates with the core RNA polymerase. These residues function in contacts with the β-flap similarly to their counterpart residues in Escherichia coli ⁷⁰. Conversely, residue Q236 of ²⁸_Ct directly binds the chlamydial hctB –35 element. The conserved counterpart residue in E. coli ⁷⁰ has not been reported to interact with the –35 element of the ⁷⁰ promoter. Observed functional disparity between ²⁸_Ct and ⁷⁰ region 4 is consistent with their divergent properties in promoter recognition. This work provides new insight into understanding the molecular basis of gene regulation controlled by ²⁸_Ct in C. trachomatis.

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Chlamydia trachomatis is a leading causative pathogen of bacterial sexually transmitted diseases and ocular infections (trachoma) in humans (36). The obligate intracellular parasitic feature of Chlamydia has hindered genetic and biochemical studies. As a result, little is known about how gene expression is regulated in Chlamydia. Recent studies indicate that gene expression takes place coordinately at the level of transcription throughout the developmental cycle of Chlamydia (2, 33, 37). Bacterial RNA polymerase (RNAP) is a key enzyme that controls transcription (15). Chlamydial RNAP is identical to the well-studied Escherichia coli RNAP in containing subunits ₂ββ' and a factor, despite the lack of a recognizable subunit (43). The factors associate with the RNAP core to form an RNAP holoenzyme that initiates promoter-specific transcription (15, 19, 46). Three known factors, ⁶⁶, ⁵⁴, and ²⁸, and some transcription regulators have been revealed in the C. trachomatis genome (43). Both chlamydial ⁶⁶ and C. trachomatis ²⁸ (²⁸_Ct), but not ⁵⁴, belong to members of the E. coli ⁷⁰ family that share up to four conserved amino acid regions (regions 1 to 4) (26). Chlamydial ⁶⁶ serves as the primary factor that directs transcription of housekeeping genes or most constitutively expressed genes (2, 7, 23, 27, 33), whereas ²⁸_Ct directs transcription of several late-stage genes that are required for differentiation from the noninfectious but metabolically active reticulate bodies to the infectious but metabolically inactive elementary bodies (3, 39, 48). ²⁸_Ct also performs its task of transcription by responding to stressful conditions (39, 50). It is not known how the ²⁸_Ct function is regulated in Chlamydia. C. trachomatis does not appear to encode an identifiable anti-²⁸ homologue, which is known as FlgM in Salmonella (18, 22). A putative chlamydial RsbW, encoded by the rsbW gene, was shown to bind ²⁸_Ct in a glutathione S-transferase pull-down assay (A. L. Douglas and T. P. Hatch, personnel communication). However, there is no direct evidence to show an inhibitory effect of RsbW on chlamydial ²⁸_Ct-specific transcription (17, 22). Even less is known about the function of chlamydial ⁵⁴, and only two putative ⁵⁴-like promoters have been reported (28). Defining the molecular basis of factor action is central for understanding the mechanism by which Chlamydia completes its unique intracellular developmental cycle (16) and adapts to environmental cues.

Several lines of evidence demonstrate that the DNA- and -core interactions are essential for the process of transcription initiation and elongation (5, 11, 30, 31, 35). Such molecular interactions are well characterized in E. coli ⁷⁰-mediated transcription regulation (5, 11, 25). Regions 2 and 4 of E. coli ⁷⁰ recognize the promoter –10 and –35 elements, respectively (4, 11, 34). Region 2 of ⁷⁰ also interacts with the β' coiled coil in core RNAP. This interplay is essential for holoenzyme formation (1, 5, 12, 34). Also, the ⁷⁰ region 4 interacts with the flexible flap structure in the β subunit (β-flap). This interaction is required to properly position ⁷⁰ regions 2 and 4, allowing for simultaneous contact with the –35/–10 promoter elements. Furthermore, the ⁷⁰ region 4/β-flap interaction can be a target of transcription factors, such as the anti-⁷⁰ factor T4 AsiA (13, 21). By sequence analogy with E. coli ⁷⁰, conserved region 4 in an alternative factor is likely to interact with the β-flap for recognition of the –35/–10 promoters. Indeed, direct interactions of region 4 of Helicobacter pylori ²⁸ with the β-flap (6), as well as those of region 4 of E. coli ³⁸ with the β-flap (27, 36) have been identified. An apparent difference in the strengths of the E. coli ³⁸/β-flap and E. coli ⁷⁰/β-flap interactions was found, although E. coli ³⁸ and ⁷⁰ recognize similar promoter consensus sequences (19). The question is raised of whether the variability of region 4 and the β-flap interaction contributes to promoter recognition in a -specific manner.

Previously, we have characterized the range of promoter elements that are recognized by chlamydial ²⁸ (39, 40). This study was performed in order to explore the details of ²⁸_Ct action in the process of transcription. Specifically, we report that certain ²⁸_Ct determinants are required for contact with the chlamydial β-flap of RNAP or the –35 promoter element. Because ²⁸_Ct plays a role in regulation of genes important for late-stage morphological differentiation and the stress response, our data can shed light on molecular mechanisms underlying these processes. Importantly, given the uncertainty about whether a transcription regulator would affect the ²⁸_Ct activity, our ability to define the molecular interactions of the transcription machinery provides us with a useful genetic tool and information to facilitate the discovery and characterization of potential regulatory factors for ²⁸_Ct in Chlamydia, a genetically intractable pathogen.

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Bacterial strains and growth. The bacterial strains used are listed in Table 1. C. trachomatis was propagated and purified as previously described (38). DH5 or XL1-Blue was used as the host for cloning. Salmonella enterica serovar Typhimurium fliA, encoding Salmonella ²⁸, mutant strains TH5504 and TH7034 were kindly provided by Kelly T. Hughes (University of Utah). Reporter strains BN317 (34) and KS1 (8) were kindly provided by Ann Hochschild (Harvard University). Reporter strains ZY101 containing the test promoter Plac_hctB-35 linked to lacZ on an F' episome were constructed as described previously (45) (see Fig. 6). Briefly, the EcoRI-HindIII-digested DNA fragment carrying the synthetic promoter Plac_hctB- 35 was cloned into pFW11 via EcoRI and HindIII sites. The resultant plasmid was introduced into strain CSH100, allowing homologous recombination of Plac_hctB- 35 and lacZ onto an F' episome. Further mating with strain FW102 was performed in order to finalize construction of strain ZY101. In this test promoter, an ectopic chlamydial hctB promoter –35 element with the sequence TAAAGTTT was centered at bp –45.5 relative to the transcription initiation site of lac. Using a similar strategy, the reporter strain ZY102 was constructed. ZY102 is identical to ZY101, except that it contains a mutated ectopic chlamydial hctB promoter –35 element (gAAAGTTT). E. coli and S. enterica serovar Typhimurium strains were grown in Luria-Bertani (LB) medium at 37°C. MacConkey agar supplemented with 1% lactose (Difco) plus 0.02% arabinose (MacConkey-lactose-arabinose) was used in indicator plates for β-galactosidase activity. When required, medium was supplemented with 25 µg/ml chloramphenicol, 50 µg/ml kanamycin, 10 µg/ml tetracycline, and/or 100 µg/ml carbencillin.

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TABLE 1. Strains and plasmids used in this study

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FIG. 6. Bacterial one-hybrid assay: characterization of the 28Ct substitutions that affect interaction with the hctB –35 DNA element. (A) Cartoon illustrating the test promoter with wild-type or mutant ectopic hctB –35. The ectopic hctB –35 element serves as a binding site for the tethered 28Ct region 4 moiety. When they interact with each other, transcription from the test promoter is activated, resulting in an increase of reporter lacZ (β-galactosidase). (B) Effects of substitution in the moiety of the -28Ct chimera on transcription from the test promoter. Strain ZY101 harboring plasmids directing the synthesis of the -28Ct or derivatives was grown in the presence of different concentrations of IPTG as indicated. β-Galactosidase activity was measured in duplicate on at least three independent occasions. Values shown are the averages from one experiment. (C) Change (n-fold) in the β-galactosidase activity before and after IPTG (100 µM) induction. Shown are changes of transcription from the test promoter with wild-type hctB –35 (TAAAGTTT) (in ZY101, black bars) or mutant hctB –35 (gAAAGTTT) (in ZY102, light gray bars). ZY101 and ZY102 harboring plasmids that directed the synthesis of -28Ct or derivatives were grown in the absence or presence of IPTG and assayed for β-galactosidase activity. β-Galactosidase activity was measured in duplicate on at least three independent occasions.

Plasmid construction. The plasmids used in this study are listed in Table 1. The hybrid ²⁸ genes (Fig. 1) were generated by overlapping PCR amplification and recloned into expression vectors pS28H and pES28H (39), creating pH²⁸_Ct-Ec4 and pH²⁸_Ec-Ct4. The hybrid ²⁸ genes are under the control of an arabinose-inducible P_ara promoter. Plasmids pACcI, pBR, pBRLN and pACcI-β-flap (8-10, 25) were kindly provided by Ann Hochschild (Harvard University). pACcI-β-flap_Ct directs synthesis of cI fused to chlamydial β-flap under the control of a lacUV5 promoter. pBR-²⁸ encodes the E. coli N-terminal domain of the subunit of RNAP (NTD) fused to the chlamydial ²⁸ region 4 under the control of tandem lpp and IPTG (isopropyl-β-D-thiogalactopyranoside)-inducible lacUV5 promoters. pBR-⁶⁶ encodes NTD fused to region 4 of chlamydial ⁶⁶. Plasmid pGHR, carrying promoters of chlamydial hctB and the rRNA, was created by inserting the hctB promoter region into XbaI-EcoRV-digested pMT504 (44). pGHR together with pGS₉, pGP_hctB, pGS₁₄, pGP_hctBup, pGP_tar, or pGP_tar+ (40) was used as a supercoiled template in the in vitro transcription assay. Inserts in all constructs were confirmed by restriction mapping and DNA sequencing. Details of primers and procedures for these constructs are available upon request.

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FIG. 1. In vitro promoter selectivity of holoenzymes containing hybrid 28 proteins. (A) Construction of hybrid 28, showing the organization of 28Ct, 28Ec, 28Ec-Ct4 (28Ec residues 1 to 165 fused to region 4 of the 28Ct [residues 179-253]), and 28Ct-Ec4 (28Ct residues 1 to 178 fused to region 4 of 28Ec [residues 166 to 239]). (B) Gel analysis of in vitro transcription products using the RNAP holoenzyme containing 28Ct or hybrid 28Ec-Ct4 protein. (C) Gel analysis of in vitro transcription products using the RNAP holoenzyme containing 28Ec or hybrid 28Ct-Ec4 protein. RNAP is designated by the 28 proteins as indicated on the left. Above each lane is shown the test promoter used in the transcription assay. On the right side of each panel are shown transcripts from different test promoters. PfliC serves as an internal control in the same reaction.

Mutant ²⁸_Ct library and genetic screening. For convenience in cloning, a silent mutation (T to A) was introduced at position 723 of the ²⁸_Ct coding region using PCR-based site-directed mutagenesis in order to remove a natural HindIII site in pLF28 (40). The resultant plasmid was named pLF28a. Error-prone PCR was performed with Taq DNA polymerase (New England Biolabs) for introducing random mutations of the ²⁸_Ct gene into appropriate fragments of pLF28a. The mutagenized PCR fragments were digested with HindIII-SphI, and a 187-bp DNA fragment containing region 4 of ²⁸_Ct was ligated with the HindIII-SphI-digested fragment of pLF28a. DH5 was transformed with the ligation mixture in order to generate a mutant library of ²⁸_Ct region 4. This mutant library was then subjected to a genetic screen in S. enterica serovar Typhimurium fliA mutant strain TH7034. The selection strains formed white or pink colonies on MacConkey-lactose-arabinose indicator plates when transformed with mutant ²⁸_Ct (the wild type was red). The plasmids carrying potential mutations were analyzed by restriction enzyme digestion, and the full-length ²⁸_Ct gene was sequenced in order to confirm the presence of mutations.

Expression and purification of wild-type ²⁸_Ct and derivatives. For the production of N-terminally His₆-tagged ²⁸_Ct and derivatives, pS28H and derived plasmids were introduced into S. enterica serovar Typhimurium strain TH7034. Cells were grown in LB containing carbenicillin. The fusion proteins were induced by treatment with 0.02% arabinose for 2 h. Proteins were purified under denaturing conditions on an Ni-nitrilotriacetic column (Qiagen), followed by a HiTrap HP column (GE Healthcare), as described previously (42). Purified proteins were renatured by serial dialysis. The final dialysis buffer contained 50% glycerol, 50 mM Tris-HCl (pH 7.9), 0.01% (vol/vol) Triton X-100, 0.1 mM EDTA, 150 mM NaCl, and 0.1 mM dithiothreitol. Protein was stored at –20°C for future use. Protein concentrations were determined using a protein assay kit (USB Corporation) and confirmed by Western blot analysis.

In vivo assay of chlamydial hctB promoter activity in Salmonella: relative β-galactosidase activity. To examine the effect of mutant ²⁸_Ct proteins on transcription from the chlamydial hctB promoter, pS28H or derived plasmids were introduced into Salmonella fliA mutant strain TH5504 cells harboring pRV_hctB, which carries the reporter hctB::lacZ. Cells were grown in LB containing carbencillin and chloramphenicol to ensure selection of both plasmids. The protein was induced by the addition of 0.02% (wt/vol) arabinose starting from an optical density at 600 nm of 0.3 for 1 hour. Aliquots of cells were collected. Cell lysates in sodium dodecyl sulfate (SDS) loading buffer were electrophoresed on a 10% glycine-SDS-polyacrylamide gel, followed by Western blot analysis. Levels of cellular ²⁸_Ct and RNAP β subunit were measured by Western blot analysis with polyclonal anti-²⁸_Ct antibody (a gift from Thomas P. Hatch, University of Tennessee) and monoclonal antibody 8RB13 against the β subunit of RNAP (NeoClone) as probes, respectively. The β-subunit band was used as an internal standard for correcting the amount of protein loading. The activity of β-galactosidase was measured as described previously (29) and then normalized to the abundance of cellular ²⁸_Ct protein. The resultant relative β-galactosidase activity was used to evaluate ²⁸_Ct function.

Core-binding assays: coimmunoprecipitation. To test the in vivo core- binding, cellular lysates of Salmonella fliA mutant TH7034 expressing ²⁸_Ct (from pLF28 or its derivatives) in buffer (10 mM Tris [pH 7.5], 150 mM NaCl) were used for coimmunoprecipitation. To test core- binding in vitro, a reaction mixture (15 µl) containing purified ²⁸ proteins (4 pmol) and E. coli core RNAP (1 pmol) (Epicenter) in buffer A (10 mM Tris-Cl [pH 7.5], 1 mM β-mercaptoethanol, 0.3 M NaCl, 10 mM MgCl₂, 0.1 mM EDTA, 0.01% Triton 100, 250 µg/ml bovine serum albumin) was used. Monoclonal antibody B8R13 (1 µl) against the β subunit was incubated with cell lysate or a mixture of ²⁸ and core at 4°C overnight, and then complexes of core and were purified using protein A-agarose (Sigma). After three time washes with buffer A to remove unbound factor, the bound proteins were separated on a 10% SDS-polyacrylamide gel, followed by Western blot analysis. Bands of the bound ²⁸_Ct and β subunit were probed with polyclonal ²⁸_Ct antibody and β antibody (8RB13), respectively. Protein bands were quantified with Quantity One software (Bio-Rad).

In vitro transcription assays. The ²⁸RNAP holoenzyme was formed by incubation of a threefold excess of purified His₆-tagged ²⁸ protein or derivatives with E. coli core RNAP enzyme (Epicenter) on ice for 15 min. In some cases, the ratio of mutated ²⁸ to core enzyme was increased in order to make ²⁸-saturated RNAP. ⁷⁰RNAP holoenzyme was purchased from Epicenter. The in vitro transcription system contained RNAP holoenzyme, supercoiled plasmids (1 µg), 10 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 5 mM dithiothreitol, and transcription was initiated by adding 400 µM ATP, 400 µM UTP, 1.2 µM CTP, 0.20 µM [-³²P]CTP (3,000 µCi/mmol), and 100 µM 3'-O-methylguanosine 5'-triphosphate (GE Healthcare). The reaction was performed as described previously (39). mRNA made by the RNAP holoenzyme was separated and detected on a 6% (wt/vol) polyacrylamide-8 M urea polyacrylamide gel electrophoresis. Signal intensities from autoradiographs were determined with Quantity One software (Bio-Rad).

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Replacement of ²⁸_Ct region 4 with ²⁸_Ec region 4 changes the behavior of RNAP holoenzyme in vitro. Previously, we found that ²⁸_CtRNAP is permissive in recognizing promoters with altered length between the promoter –35 and –10 elements, and preferentially activates promoters with upstream AT-rich sequences; in contrast the ²⁸RNAP of E. coli (²⁸_EcRNAP) does not have such a preference (40). In order to test whether differences in region 4 of ²⁸ were related to the observed disparity, we assessed the complementary functionality of ²⁸_Ct region 4 by making a reciprocal pair of hybrid ²⁸ proteins. Specifically, region 4 of ²⁸_Ec was exchanged with the relevant region 4 of ²⁸_Ct (Fig. 1A). S. enterica serovar Typhimurium fliA mutant strain TH7034 carrying the constructs directing the synthesis of the hybrid ²⁸ (²⁸_Ec-Ct4 and ²⁸_Ct-Ec4) appeared as red colonies on MacConkey-lactose-arabinose indicator plates, suggesting that both ²⁸_Ec-Ct4 and ²⁸_Ct-Ec4 transcribe from the same fliC promoter that was recognized by wild-type ²⁸_Ct (23, 40).

We next tested the in vitro activities of purified ²⁸_Ec-Ct4 or ²⁸_Ct-Ec4 in the context of a holoenzyme with several defined ²⁸ promoters. We chose the chlamydial hctB promoter (P_hctB) (native AT-rich upstream sequence plus core promoter of hctB), chlamydial hctB promoters with spacer lengths of 9 or 14 bp between the –35 and –10 element (P_S9 and P_S14), the core promoter of hctB (P_hctBup), and the E. coli tar promoter (P_tar) (native GC-rich upstream sequence plus core promoter of tar) and its derivative P_tar+ (AT-rich upstream sequence of hctB plus core promoter of tar) (40). In the reaction mixture containing plasmid templates and reconstituted RNAP holoenzymes, the resultant transcripts were dependent on the addition of RNAP holoenzyme. RNAP holoenzyme made from hybrid ²⁸_Ec-Ct4, which contains ²⁸_Ct region 4, exhibited higher activities with P_S9, P_hctB, P_S14, and P_tar+ (P_test/P_fliC transcript ratio of 1) but was weakly active with P_hctBup and P_tar (P_test/P_fliC ration of <1) (Fig. 1B). Such behaviors of ²⁸_Ec-Ct4 RNAP are comparable to those of wild-type ²⁸_CtRNAP. In contrast, reconstituted ²⁸_Ct-Ec4RNAP, which contains ²⁸_Ec region 4, conferred strong activities for P_hctB, P_hctBup, P_tar, and P_tar+, but it was weakly active with P_S9 and P_S14 (Fig. 1C). These actions are in agreement with results from using wild-type ²⁸_EcRNAP. Taken together, the results obtained with the hybrid ²⁸ proteins indicate that the observed differences in promoter selectivity between ²⁸_Ct and ²⁸_Ec are, at least in part, specified by region 4.

Identification of mutants defective in ²⁸_Ct-dependent transcription in Salmonella. We next sought to identify residues in region 4 of ²⁸_Ct that are important for ²⁸_Ct-dependent transcription. A plasmid library carrying mutagenized ²⁸_Ct genes driven from an arabinose-inducible P_araB promoter was transformed into Salmonella fliA mutant TH7034, which allowed us to screen mutants defective in transcription from ²⁸-dependent fliC::lacZ. In this background, only ²⁸_Ct-dependent transcription can be detected, because no functional Salmonella ²⁸ exists. Cells were selected for a lower level of ²⁸-dependent transcription of lacZ (white or pink colonies) than for the wild type (red colony) by plating on MacConkey-lactose-arabinose agar. The candidates were further confirmed by measurement of β-galactosidase activity. Approximately 7,000 clones were screened, and four with single-residue substitutions in ²⁸_Ct (E206G, Y214C, E222G, and Q236L) were isolated (Fig. 2). These mutants were further characterized as described below.

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FIG. 2. Mutagenesis of 28Ct region 4. (A) Alignment of the amino acid sequences of region 4 from E. coli 70, Thermus aquaticus A, E. coli 32 (32Ec), Salmonella 28 (28St), 28Ec, Aquifex aeolicus 28 (28Aa), and 28Ct using the CLUSTAL W program. The amino acid sequence of 28Sa is identical to that of 28Ec. Regions were defined based on previous studies (26, 41). The number for each amino acid position is relative to that from the start of each protein sequence. Identical or similar residues are shown in black or gray shadow, respectively. Asterisks at the bottom indicate residues undergoing mutation. (B) Isolation of 28Ct mutants. Mutated nucleotides and the deduced amino acid residues are indicated based on the position of 28Ct and corresponding residue positions in different factors.

Mutations in region 4 of ²⁸_Ct decrease transcription from the chlamydial hctB promoter. We compared the effects of ²⁸_Ct mutants on transcription from the ²⁸_Ct-dependent hctB promoter in Salmonella. Plasmid-encoded wild-type ²⁸_Ct or derivatives were expressed in Salmonella fliA mutant strain TH5504 carrying pRV_hctB, and ²⁸_Ct-driven transcription from P_hctB::lacZ was assessed by measurement of β-galactosidase activity as described in Materials and Methods. Relative β-galactosidase activities from strains carrying ²⁸_Ct substitutions Y214C, E222G, and Q236L were significantly lower than that of wild-type ²⁸_Ct (i.e., 7.9%, 25.1%, and 1.0%, respectively) (Fig. 3A). ²⁸_Ct E206G also decreased levels of β-galactosidase activity, to about 41.9% relative to wild-type ²⁸_Ct.

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FIG. 3. Effect of single-residue substitution on 28-dependent transcription from chlamydial hctB promoter. (A) Activity of mutated 28Ct in vivo using a lacZ reporter assay. Cellular β-galactosidase activity was normalized to levels of 28Ct determined by Western blot analysis using specific anti-28 antibody. We previously found that mutation in region 4 of 28Ct did not impair its reactivity with the antibody to 28Ct. The β-galactosidase activity from each strain containing the indicated mutated proteins is represented as a percentage relative to the β-galactosidase activity of the strain with wild-type 28Ct. Asterisks above the bars indicated significantly reduced activities compared with that of the wild type (P < 0.05). (B) Transcripts from single-round transcription assay in vitro. Plasmid pGHR containing two chlamydial promoters (PhctB and P1CtrRNA) was used as a template in the presence of 70RNAP or 28CtRNAP as indicated on the top. The transcripts generated from promoters are indicated on the left. The intensity of each transcript band was quantified using Quantity One. The amount of transcript produced by each mutant RNAP is reported as a percentage of the transcription of wild-type 28CtRNAP.

Next, the direct effect of mutant ²⁸_CtRNAP holoenzyme on transcription from P_hctB was examined in vitro. In the presence of both ²⁸_CtRNAP and ^70_CtRNAP holoenzymes, plasmid template pGHR produced two divergent transcripts: a 152-bp transcript from the ²⁸-dependent P_hctB and a 130-bp transcript from the ⁶⁶-dependent rRNA P1 (P1_CtrRNA) (data not shown). The RNAP containing ²⁸_Ct Y214C or Q236L was severely defective in generating transcripts from hctB, showing a yield of less than 10% of the wild-type level (Fig. 3B). The RNAP containing ²⁸_Ct E206G or E222G reduced transcription activity to 79.1% and 49.0% of that of wild-type ²⁸_CtRANP, respectively (Fig. 3B). None of the mutant ²⁸_CtRNAPs was able to transcribe from the P1_CtrRNA. We also noted that the RNAP saturated with mutated ²⁸_Ct did not mount transcription activity in vitro. Moreover, mutated ²⁸_CtRNAPs did not change their behavior for transcription from P_S9, P_hctB, P_S14, P_hctBUP, P_tar, and P_tar+ in vitro (data not shown).

Taken together, these transcription studies indicate that ²⁸_Ct substitutions Y214C and Q236L severely decreased transcription from hctB, whereas substitutions E206G and E222G moderately affected hctB transcription.

Effect of ²⁸_Ct substitutions on core binding. The abilities of wild-type and mutant ²⁸_Ct to bind core RNAP were compared using coimmunoprecipitations. The levels of mutant proteins that bound to Salmonella core RNAP were similar to or slightly higher than the wild-type level. (Fig. 4). As validation, we also examined potential core-binding differences among these proteins using the E. coli core and purified recombinant ²⁸_Ct or derivatives in vitro. We found that each of the ²⁸_Ct mutants effectively bound E. coli core RNAP by coimmunopreciptation relative to wild-type ²⁸_Ct (data not shown). This confirms that none of the ²⁸_Ct substitutions severely reduced affinity for the core RNAP under our test conditions.

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FIG. 4. Binding of 28 and derivatives to core RNAP in Salmonella. (A) Immunoblot showing the results of a coimmunoprecipitation assay from Salmonella fliA mutant TH7034, which expresses 28 or derivatives as indicated above each lane. A strain expressing E. coli 28, which is identical to Salmonella 28, was the negative control. Cell lysates were subjected to immunoprecipitation using anti-β monoclonal antibody as described in Materials and Methods. Precipitated proteins were separated by PAGE and immunoblotted with anti-28 antibody or anti-β monoclonal antibody. (B) Relative binding affinity of 28 or derivatives for core. The amount of precipitated 28 protein is reported as a percentage of the level of β in core RNAP.

Influence of ²⁸_Ct substitutions on the interaction of ²⁸_Ct region 4 with the β-flap. We examined whether substitutions in the ²⁸_Ct region 4 would disrupt protein-protein interaction of ²⁸_Ct region 4 with the β-flap in a bacterial two-hybrid assay (8, 10). This assay involves the use of (i) a reporter strain, KS1, which carries a chromosomal copy of the test promoter plac O_R2-62 linked to lacZ; (ii) a plasmid encoding the cI fused to the β-flap from Chlamydia (cI-β-flap_Ct) or E. coli (cI-β-flap); and (iii) a second plasmid encoding the N-terminal domain of NTD fused to ²⁸_Ct region 4 or its derivatives. Interaction of the β-flap with ²⁸_Ct region 4 stabilizes RNAP binding to the test promoter, thus, mediating transcription activation from the test promoter plac O_R2-62::lacZ. This can be monitored by measuring β-galactosidase activity (Fig. 5A).

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FIG. 5. Bacterial two-hybrid assay: characterization of 28Ct substitution that affects its interaction with the β-flap. (A) Diagram showing how interaction of the 28Ct region 4 (in pBR-28Ct) with the β-flap (in pACcI-β-flapCt or pACcI-β-flap) activates transcription from the test promoter plac OR2-62 located on the chromosome in KS1. (B) Effects of substitution in the moiety of the -28Ct chimera on transcription from plac OR2-62 in the presence of the chimera cI-β-flapCt. (C) Effects of substitutions in the moiety of the -28Ct chimera on transcription from plac OR2-62 in the presence of the chimera cI-β-flap. KS1 cells harboring two compatible plasmids, which direct the synthesis of -28Ct or derivatives and cI-β-flapCt or pACcI-β-flap as indicated, were grown in the presence of carbencillin, chloramphenicol, and kanamycin. Protein expression was induced by treatment with different concentrations of IPTG for 1 hour when cultures reached an optical density at 600 nm of 0.3. β-Galactosidase activity was measured in duplicate on at least three independent occasions. Values shown are the averages from one experiment.

As shown in Fig. 5B, in the presence of the chlamydial β-flap fusion (cI-β-flap_Ct), the chimera -²⁸_Ct activates transcription from plac O_R2-62 up to 14.0-fold relative to that of the negative control expressed from pBR-. Such an increased magnitude compared to no obvious increase in the paired -⁷⁰/cI-β-flap did not surprise us, because ²⁸_Ct contains a residue at G228 corresponding to ⁷⁰D581 (25, 34). Substitution at ⁷⁰D581G stabilizes the folded structure of the tethered ⁷⁰ region 4 moiety and enhances interaction of ⁷⁰ region 4 with the DNA –35 element (25, 34). In strains coexpressing chimera cI-β-flap_Ct and chimera -²⁸E206G, -²⁸Y214C, or -²⁸E222G, the magnitude of transcription activation from plac O_R2-62 decreased about 8.3-, 4.5-, and 6.9-fold, respectively. The decrease of transcription activation observed is not due to instability of chimera proteins, as the chimeras were able to interact with the hctB –35 element in a one-hybrid assay (Fig. 6). In contrast, chimera -²⁸Q236L strengthened the interaction between the β-flap and ²⁸ in the presence of cI-β-flap_Ct; transcription from plac O_R2-62 was increased up to 23.4-fold (Fig. 5B) compared to the wild-type ²⁸_Ct (14.0-fold increase).

In the presence of an E. coli β-flap fusion (cI-β-flap), the -²⁸_Ct chimera increased transcription from plac O_R2-62 up to 8.5-fold, compared to 14-fold in the presence of the cI-β-flap_Ct chimera. Substitutions E206G, Y214C, and E222G in ²⁸_Ct reduced transcription from plac O_R2-62 to 3.4-, 3.4-, and 5.4-fold, respectively. However, the -²⁸_Ct Q236L chimera stimulates transcription from plac O_R2-62 14.4-fold, compared to a change of 23.4-fold in the presence of the cI-β-flap_Ct chimera (Fig. 5C).

These results indicate that substitution E206G, Y214C, or E222G in ²⁸_Ct weakens interactions between ²⁸_Ct and the β-flap from both E. coli and Chlamydia. Thus, these residues are involved in interaction of the cI-β-flap with ²⁸ region 4, whereas ²⁸_Ct residue Q236 seems not to be important for β-flap binding. E. coli and chlamydial β-flap share 62.2% amino acid identity and 73.8% similarity. The finding that ²⁸_Ct can bind the E. coli β-flap could be relevant to its ability to transcribe from both ²⁸_Ec- and ²⁸_Ct-dependent promoters when it associates with the E. coli core enzyme in a heterologous genetic system or in vitro.

Effect of ²⁸_Ct mutants on binding of the –35 element from the hctB promoter. We next examined whether residue substitutions in ²⁸_Ct region 4 would affect interaction of region 4 and the –35 element of the promoter, using a one-hybrid assay (34). This in vivo DNA-binding assay is designed to use a test promoter, which contains an ectopic hctB –35 element upstream of the lac promoter in strain ZY101, and a plasmid-encoded -²⁸ fusion. The direct interaction of ²⁸ region 4 with the ectopic hctB –35 element recruits and stabilizes RNAP to the test promoter, causing an increase of reporter gene (lacZ) expression. This can be monitored by measuring β-galactosidase activity (Fig. 6A).

We introduced a plasmid encoding chimera -²⁸ or its derivatives into the reporter strain ZY101. We chose chimera -⁶⁶ as a negative control, as previous report showed that chlamydial ⁶⁶ did not recognize the ²⁸-dependent promoter (48). The ²⁸L243K mutant bears enhanced transcriptional activity from the hctB promoter (Z. Hua et al., unpublished data) and was the positive control. In the presence of -²⁸, transcription activation from the test promoter was observed, and the increase of stimulation occurred in an IPTG dose-dependent fashion (Fig. 6B). Similarly, expression of -²⁸E206G, -²⁸E222G, or -²⁸Y214C was able to activate transcription from the test promoter. Although the stimulations are small (<1.5-fold), these increases are reproducible in all experiments. Chimera -²⁸L243K increased transcription up to 1.8-fold, indicating that this system functioned. In contrast, chimera -²⁸Q236L failed to stimulate transcription from the test promoter (Fig. 6B). Because -²⁸Q236L interacts with chimera cI-β-flap (Fig. 5), the inability of -²⁸Q236L to interact with the ectopic promoter is unlikely to be related to the poor protein expression. The substitution ²⁸_CtQ236L disrupted -²⁸/hctB –35 interactions, which may be an explanation for our findings. As expected, chimera -⁶⁶ was unable to activate transcription from test promoter in ZY101 (Fig. 6B and C); however, -⁶⁶ stimulated transcription from placCons-35C in BN317, which contains a ⁷⁰-specific ectopic –35 element (data not shown).

In ZY102, which bears a mutant ectopic hctB –35 element (gAAAGTTT), none of the wild-type and mutant -²⁸ chimeras stimulated transcription from the test promoter (Fig. 6C). The first base pair T of the hctB –35 element has been shown to be the major determinant for ²⁸_Ct recognition (49). This result confirmed that the observed stimulatory effects of -²⁸_Ct, -²⁸E206G, -²⁸Y214C, and -²⁸E222G in ZY101 are a result of the specific interaction of the chimeras with the ectopic hctB –35 element. Substitution ²⁸_CtQ236L interrupts interaction between ²⁸ and the hctB –35 element, indicating that ²⁸_Ct Q236 is essential to be in contact with the –35 element.

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We focused on characterization of several ²⁸_Ct mutants with mutations in region 4 that are defective in ²⁸-dependent transcription. Since there is no successful genetic system available to manipulate genes in Chlamydia, our strategy has been to study ²⁸-dependent transcription using complementary approaches. By switching ²⁸_Ct region 4 with ²⁸_Ec region 4, we found that amino acids in ²⁸_Ct region 4 might specify the function of the ²⁸_CtRNAP holoenzyme for use of an altered spacer length between promoter –35 and –10 elements, as well as preference for the UP-like sequences in vitro (Fig. 1). We also screened for ²⁸_Ct mutants defective in transcription from the ²⁸-dependent fliC promoter in Salmonella (Fig. 2). We then examined the effects of four single-residue substitutions in ²⁸_Ct region 4 on transcription from the chlamydial hctB promoter both in vitro and in a fliA mutant Salmonella strain. We found that substitutions ²⁸_Ct, Y214C and Q236L, significantly decreased transcription from the hctB promoter, while ²⁸_CtE206G and -E222G moderately reduced hctB transcription (Fig. 3); the promoter specificity was unchanged, and the effect was stronger in vivo than in vitro. A possible explanation for this is that the existence of additional host cell regulatory factors in vivo might lead to a bias for a diminished in vitro effect seen with ²⁸_Ct mutations. It is unlikely that the difference observed is due to the inhibition of FlgM encoded by the surrogate strain of S. enterica serovar Typhimurium, because FlgM specifically negatively regulates Salmonella ²⁸ but not ²⁸_Ct (22).

The apparent transcriptional deficiency observed might result from ²⁸_Ct mutations that have a reduced affinity of with the core RNAP, a disrupted /β-flap interaction, and/or an occluded /promoter interaction. Our data do not support a direct strong effect of mutant ²⁸_Ct on core affinity, since RNAP saturated by mutant ²⁸ did not increase transcription. Moreover, the mutant ²⁸_Ct effectively bound core RNAP in a coimmunoprecipitation experiment (Fig. 4). However, modest indirect effects cannot be excluded. By taking advantage of the bacterial two-hybrid system, we found that the three substitutions in ²⁸_Ct, E206G, Y214C, and E222G, impaired interaction of ²⁸_Ct with the β-flap from Chlamydia or from E. coli (Fig. 5). Of these residues, only the corresponding residue ²⁸_CtY214 has been mapped and directly contacts the β-flap in the X-ray structure of the Thermus aquaticus ^A holoenzyme-DNA complex (34) (Fig. 7A and B). The corresponding residues of ²⁸_CtE206 and -E222 may indirectly involve such /β-flap interactions. The X-ray structure of Aquifex aeolicus ²⁸, free or in complex with FlgM, the anti-²⁸ factor, does not explain our observations, as both core and DNA-binding domains are buried in the folded compact structure (Fig. 7C) (41, 42). We speculate that ²⁸_Ct is mostly in an active conformation when it associates with the core RNAP in Salmonella or in vitro, consistent with their transcription activities. Perhaps substitution E206G, Y214C, or E222G in ²⁸_Ct caused a deficiency of ²⁸_Ct in binding the β-flap, resulting in an unstable or mispositioned region 4 in the ²⁸_CtRNAP holoenzyme and an inability to simultaneously bind promoter –10 and –35 elements, leading to poor transcription activity. Our results further add support to the idea that RNAP remodeling of ²⁸ region 4 is required to expose the recognition domains of both the –35 binding site and β-flap on ²⁸ (41, 42). Previous studies indicated that the ⁷⁰ residues E555, F563, and E575 (corresponding to ²⁸_Ct E206, Y214, and E222, respectively) (Fig. 3) have been implicated in interaction with the E. coli β-flap (12, 13).

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FIG. 7. Positions of residues in substitutions that affect the interaction of 28Ct region 4 with the β-flap. (A) Locations of residues based on the crystal structures of Thermus aquaticus A holoenzyme and DNA (PDB ID no. 1I9Z) (32). Shown are the surfaces of (green), β-flap (gray), β' (cyan), (orange), and factor (blue). The DNA promoter (magenta) is shown as a cartoon. (B) Enlarged illustration of region 4 of T. aquaticus A (labeled with corresponding amino acid numbers in 28Ct) (see amino acid alignments in Fig. 2) and the β-flap contact. The β-flap (gray) is shown as the cartoon. The DNA promoter is hidden. (C) Locations of residues based on the crystal structures of the 28Aa/FlgM complex (labeled with corresponding amino acid numbers in chlamydial 28Ct) (PDB ID no. 1SC5 and 1RP3) (41). Blue, 28Aa; yellow-brown, FlgM. Residues corresponding to the T aquaticus A or 28Aa were identified and mutated to the naturally occurring residues in chlamydial 28Ct as indicated. This figure was generated using Pymol 0.99rc6 (http://pymol.sourceforge.net).

Our data suggest a role of residue Q236 of ²⁸_Ct in binding the –35 element of the hctB promoter because (i) substitution Q236L in ²⁸_Ct disrupted the interaction of ²⁸_Ct with the hctB –35 sequence as determined by the one-hybrid assay (Fig. 6), and the same protein interacted with the β-flap as determined by the two-hybrid assay (Fig. 5); (ii) Q236 is located in the DNA-binding helix-turn-helix motif in of ²⁸_Ct; and (iii) in T. aquaticus ^A, the corresponding residue Q414 is structurally positioned on the DNA-binding interface (4). Consistent with our results, Kourennaia et al. (24) reported that the substitution Q269A in E. coli ³² (corresponding to ²⁸_Ct Q236) impeded recognition of the –35 element of the E. coli groE promoter but did not reduce its ability to bind to the core enzyme. The corresponding residue in ⁷⁰ (Q589) has not been implicated in contact with a specific DNA base. Instead, the adjacent residue R588 of ⁷⁰ can help position residue 585 for direct contact with bacteriophage P_RM DNA and mediate binding to the DNA activator (20). Presumably, the ²⁸_Ct substitution Q236L, which changes a polar residue to an aliphatic hydrophobic residue, interrupts the side chain contacting the –35 element sequence and/or indirectly affects its ability to bind DNA by distorting or destabilizing the structure of ²⁸_Ct region 4.

Given the favorable effect of the presence of AT-rich sequences upstream of the promoter on ²⁸_Ct-dependent transcription (40), we wondered whether a possible positive influence is induced by the C-terminal domain of the subunit (CTD). The interaction of region 4 with the CTD can stimulate transcription from a subset of UP element-dependent promoters by tightening RNAP-promoter associations (14). We failed to detect direct interaction of wild-type or mutant ²⁸_Ct with the CTD in a two-hybrid assay (Hua et al., unpublished data). We still cannot rule out the possibility that this association, if any, might be transient and/or weak, thus making it difficult to detect in vivo.

These studies have allowed us to begin defining the determinants in ²⁸_Ct that are essential for contact with the β-flap and with the –35 element; both are required for recognition of the –35/–10 promoter. Because there is no overlapping promoter recognition specificity between ²⁸_Ct and the primary ⁶⁶ (⁷⁰) (39, 40), it is fair to assume that there might be some differences regarding the /core interaction as well as /promoter interaction. Supporting this, ²⁸_Ct Q236 has been shown to be important for the ²⁸_Ct-specific –35 element binding. This function has not been reported for the counterpart residue of E. coli ⁷⁰. While this study does not directly address whether ²⁸ activity is inhibited by a regulator, observations with several ²⁸_Ct region 4 mutants defective in contact with the β-flap indicates that inactivation may play a role in chlamydial ²⁸ activity control. In the future, we will use a stabilized or deficient protein-binding mutant of chlamydial ²⁸ in order to facilitate identification and characterization of potential ²⁸ regulators in a two-hybrid assay.

 
We gratefully acknowledge Ann Hochschild and Padraig G. Deighan for their suggestions and help with design of the one-hybrid and two-hybrid assays used in this study, as well as for comments on the manuscript. We thank You-xun Zhang and Sean J. Garrity for helpful discussions, Kelly T. Hughes for S. enterica serovar Typhimurium strains, Tom P. Hatch for chlamydial ²⁸ antibody, Pieter L. deHaseth and Kathleen Mathews for plasmids, and Ronald Luftig and Timothy P. Foster for critical reading of the manuscript.

This work was supported by grants from the National Institutes of Health (AI055869) and the Louisiana State University Health Sciences Center Fund to L.S.

 
* Corresponding author. Mailing address: Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112. Phone: (504) 568-4076. Fax: (504) 568-2918. E-mail: lshen{at}lsuhsc.edu

Published ahead of print on 31 October 2008.

Present address: Children's Hospital, Chongqing University of Medical Sciences, Chongqing, China.

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Journal of Bacteriology, January 2009, p. 651-660, Vol. 191, No. 2
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