¹ Department of Pediatrics and ² Department of Biological Chemistry, University of California, Davis , CA 95616, USA and ³ Life Sciences Division, Los Alamos National Laboratories, Los Alamos , NM 87545, USA

Received March 29, 1996; Revised and Accepted June 20, 1996

ABSTRACT

Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear homology to eukaryotic histone H1. Changes in macromolecular conformation of DNA mediated by the histone H1-like protein (Hc1) appear to regulate stage specific differentiation. We have developed a cross-linking immunoprecipitation protocol to examine in vivo protein-DNA interaction by immune precipitating chlamydial Hc1 cross linked to DNA. Our results strongly support the presence of sequence specific binding sites on the chlamydial plasmid and hc1 gene upstream of its open reading frame. The preferential binding sites were mapped to 520 bp Bam HI- Xho I and 547 bp Bam HI- Dra I DNA fragments on the plasmid and hc1 respectively. Comparison of these two DNA sequences using Bestfit program has identified a 24 bp region with >75% identity that is unique to the chlamydial genome. Double-stranded DNA prepared by annealing complementary oligonucleotides corresponding to the conserved 24 bp region bind Hc1, in contrast to control sequences with similar A+T ratios. Further, Hc1 binds to DNA in a strand specific fashion, with preferential binding for only one strand. The site specific affinity to plasmid DNA was also demonstrated by atomic force microscopy data images. Binding was always followed by coiling, shrinking and aggregation of the affected DNA. Very low protein-DNA ratio was required if incubations were carried out in solution. However, if DNA was partially immobilized on mica substrate individual strands with dark foci were still visible even after the addition of excess Hc1.

INTRODUCTION

Chlamydia trachomatis is an obligate intracellular bacterium with a unique developmental cycle. Infection of eukaryotic host cells is mediated by adherence of extracellular, metabolically inert elementary bodies (EBs) that induce their own uptake. Once inside host cells, chlamydial EBs transform into metabolically active reticulate bodies (RBs) and reside there during the entire intracellular stage of the life cycle. The non-infectious RBs divide by binary fission within cytoplasmic inclusions until they re-differentiate into condensed EBs which are released, capable of infecting other host cells. During transformation of RBs to EBs, the chlamydial chromosome becomes condensed, leading to the appearance of an electron-dense body in the center of the cell, whereas RB chromatin appears more pleomorphic, as in most bacteria ( 1 ).

Recently, two lysine-rich proteins of 18 and 32 kDa with homology to eukaryotic histone H1 like proteins have been described in C.trachomatis . They are thought to play a key role in the chromosome condensation during RB to EB transition ( 2 - 5 ). Both proteins are expressed late in the life cycle at a time when chlamydial DNA is undergoing compaction into its dense nucleoid form. The common 18 kDa protein, designated Hc1, is conserved among all C.trachomatis serovars ( 6 , 7 ) while the second protein, designated Hc2, exhibits variable molecular weights of 25-32 kDa depending upon the serovar ( 8 ). It has been suggested that the range of molecular weights observed in different serovars is due to truncated forms of a common protein, since they all share amino acid sequence in the amino terminus. Both Hc1 and Hc2 are expressed late in the life cycle, at a time when chlamydial DNA is undergoing compaction into its dense nucleoid form, accompanied by down regulation of transcription and metabolic processes ( 9 - 11 ). Whereas expression of the Hc1 gene in Escherichia coli was found to induce compaction of the E.coli chromosome in vivo, purified recombinant protein bound cooperatively to double-stranded DNA in vitro, forming condensed spherical Hc1-DNA complexes ( 9 - 11 ). This DNA binding ability of Hc1 was found to be restricted to its carboxyl portion-a function similar to its eukaryotic counterpart ( 12 ). The interaction appears complex and dependent upon the ionic conditions and the protein-DNA ratio. Evidence that Hc1 down regulates transcription and translational processes through modulation of DNA macromolecular conformations and/or by interacting directly with DNA and RNA supports its role in gene regulation ( 10 , 11 ). Recently, Solbrig et al. ( 13 ) reported that highly supercoiled DNA is associated with the EB stage of the developmental cycle.

Evidence is mounting that Hc1 may serve as a non specific yet carefully regulated transcription repressor in a manner similar to eukaryotic histone H1 and prokaryotic histone-like protein H-NS ( 10 , 11 , 14 - 16 ). Although it seems certain that all kinds of DNA will bind Hc1 there are reports that some sequences bind Hc1 better than others ( 10 ). It is also possible that chlamydial Hc1 may prefer AT-rich over GC-rich sequences, similar to eukaryotic histone HI which also shows preferential binding to AT-rich sequences ( 17 , 18 ). Other possibilities include interactions between H1 and sequence-dependent DNA conformations. Recently, Yaneva et al. ( 19 ) reported the existence of high affinity sites for chicken erythrocyte histone H1 in plasmid pBR322. Although this system has little physiological significance, these authors have shown that the preferred sequence specific DNA fragments possess intrinsic curvature. In an effort to unravel the mechanism of Hc1 interactions, we have identified a region of the cryptic chlamydial plasmid and hc1 gene upstream of its open reading frame (ORF) that binds preferentially to Hc1 in vivo . The preferential binding sites on chlamydial plasmid and hc1 were mapped to 520 bp Bam HI- Xho I and 547 bp Bam HI- Dra I DNA fragments, respectively, following immunoprecipitation of cross linked Hc1-DNA. Further comparisons between these two DNA sequences have identified a conserved 24 bp region that exhibits preferential binding for Hc1 in vitro . In addition, binding of Hc1 to these unique 24 bp fragments appears strand specific. We have also presented evidence that high affinity binding of Hc1 to DNA is not due to any intrinsic bends within this 24 bp fragment. We hypothesize, therefore, that site-specific binding of Hc1 to plasmid DNA is a sequence specific phenomenon.

MATERIALS AND METHODS

Chlamydia trachomatis L ₂ /434/Bu was grown in HeLa cells and EB purified as described previously ( 20 ). Histone H1-like protein (Hc1) was isolated from recombinant E.coli as described and checked for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of Hc1 was determined colorimetrically using BioRad protein assay kits (Hercules, CA). Total DNA from chlamydial EB was prepared as described, while plasmid DNA was isolated by alkaline lysis method using Qiagen purification kits (Chatsworth, CA). Restriction endonuclease generated DNA fragments were extracted from agarose gels using BIO 101 GeneClean kit (La Jolla, CA).

Southern blot analysis

Genomic DNA from C.trachomatis serovar L ₂ or cesium chloride purified recombinant plasmids pCTP1 (containing chlamydial plasmid cloned into pBR322; 12 ), pCT40-218 (encoding chlamydial MOMP; 21 ), pCTJS8 (encoding r-protein L6 gene; 22 ), pCTLS1 (encoding r-protein L6; 22 ), pCTH1 (encoding 18 kDa histone H1-like protein; 3 ) was single, double or triple digested with various restriction endonucleases and restriction fragments were resolved on 1% agarose gel. Gel was soaked in water containing 0.5 [mu]g/ml ethidium bromide, destained in water and visualized under UV light. Following visualization, agarose gels were Southern blotted overnight onto nitrocellulose membrane and the filters baked at 80^oC for 2 h, then hybridized to radiolabeled oligonucleotides or double-stranded DNA probes. Probe DNA was generated from Chlamydia -infected HeLa cells. Essentially, Chlamydia -infected HeLa cells at 30 h post infection were fixed with 1% formaldehyde for 20 min at 37^oC, then solubilized in RIPA buffer (containing 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% sodium dodecyl sulfate, 1% Triton X-100; 1% sodium deoxycholate; 1 [mu]g/ml aprotinin, 1 mM PMSF and 1 mM leupeptin). The cell suspension was sonicated briefly, spun at 2500 r.p.m. and the supernatant incubated with rabbit anti-C-terminal Hc1 antibodies overnight at 4^oC. Protein A Sepharose was added to the mixture next morning and incubation continued for another 3 h followed by precipitation and extensive washing of the pellet with RIPA buffer. The complex was suspended in TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA), heated at 70^oC for 3 h to dissociate Hc1-DNA complex, followed by phenol/chloroform extraction and ethanol precipitation. The purified DNA thus generated was radiolabeled using random primer technique while oligonucleotides were labeled by 5'-end labeling technique as described ( 3 ).

Sequence analysis and synthetic oligonucleotides

Optimal alignment between 520 bp Bam HI- Xho I fragment of chlamydial plasmid (EMBL accession number X7574; 23 ) and 545 bp Bam HI- Dra I fragment of chlamydial hc1 (EMBL accession number X 57311; 3 ) was carried out using Bestfit program (GCG, Madison, WI). Synthetic oligonucleotides representing regions of highest DNA homology between these two sequences, along with their complementary strands, were custom made by Gibco-BRL. Oligonucleotides used for studying protein-DNA interactions were:

(i) 5'-AATAGGGATTCCTGTAACAACAAG-3',

(ii) 5'-CTTGTTGTTACAGGAATCCCTATT-3',

(iii) 5'-AATAGGGTTTCTTTTAATAGAAAG-3',

(iv) 5'-CTTTCTATTAAAAGAAACCCTATT-3',

(v) 5'-ATTTATCGGAAACCTTGATAAAGG-3'

and (vi) 5'-CCTTTATCAAGGTTTCCGATAAAT-3'.

Binding reactions and gel shift assays

Equimolar amounts of unlabeled or radiolabeled complementary oligonucleotides were mixed, fully denatured by heating at 95^oC for 3 min in 10 mM Tris-HCl pH 7.5, 10 mM MgCl _2, 1 mM DTT and 50 mM NaCl and allowed to cool gradually to room temperature. Formation of double-stranded DNA was monitored by running the annealed samples on 20% polyacrylamide gel in 0.6% TBE buffer. Gel shift assays were performed by incubating radiolabeled annealed double-stranded DNA or individual oligonucleotides with purified Hc1 under binding conditions (20 mM Tris-HCl, pH 7.5, 70 mM KCl, 1.5 mM MgCl ₂ , 2.5 mM DTT, 10% glycerol) for 20 min at room temperature. Protein-DNA complexes were subsequently electrophoresed on a 10% pre-equilibrated polyacrylamide gels at 75 V followed by fluorography and examination of autoradiographs.

Atomic force microscopy

Changes in macromolecular conformation of DNA were examined by imaging the Hc1-DNA complexes in vitro using atomic force microscopy (AFM). Hc1 was extracted from recombinant E.coli carrying plasmid pCTH1 as described. Pst I digests of purified chlamydial plasmid DNA (cloned into the Pst I site of pBR322) were resolved by agarose gel and the 7.5 kb fragment extracted from agarose gel. The extracted DNA was further purified by ethanol precipitation and used for AFM studies. DNA samples were adsorbed onto freshly cleaved mica substrate in normal air environment for 60 s, rinsed briefly with a gentle blow of deionized water to remove excess unbound material and incubated with purified protein for an additional 15-60 s. Samples were rinsed with water, wicked dry and immediately scanned at low humidity using the Nanoscope III AFM (Digital Instruments, Santa Barbara, CA). All images were obtained using 450 [mu]m single beam, etched silicon probes at minimal constant force.

Hc1 induced inhibition of DNA circularization

A 500 bp Bam HI- Bam HI fragment comprising the Bam HI- Xho I fragment of plasmid pCTP1 was generated using PCR technology. Essentially, an internal 19mer forward oligonucleotide primer corresponding to sequence 7093-7111 and a 20mer reverse oligonucleotide complementary to sequence 103-84 were constructed at the DNA synthesis facility (Davis, UC). A two base change in the reverse sequence was introduced to generate an internal Bam HI site. Following amplification, the amplified product was cleaved with Bam HI and subcloned into the Bam HI site of plasmid SK ⁺ . Subsequently, recombinant plasmid pRK44 (carrying 493 bp insert in plasmid pSK ⁺ ) was purified, sized and analyzed by restriction endonuclease digestion. As a control we generated an internal 410 bp Eco RI fragment of plasmid pCTP1 corresponding to nucleotide sequence 5733-6149 ( 24 ). Both 493 bp Bam HI and 410 bp Eco RI fragments were dephosphorylated with calf alkaline phosphatase and end labeled with [[gamma]- ³² P]ATP and polynucleotide kinase. The reactions were chased with 1 mM unlabeled ATP and additional kinase to ensure that all ends were phosphorylated. The labeled DNA was incubated in the presence and absence of Hc1 (in varying DNA-Hc1 molar ratios) for 20 min at 22^oC in a volume of 25 [mu]l. The samples were then diluted to 300 [mu]l with 50 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 1 mM ATP and T4 DNA ligase to a final concentration of 1.0 U/ml. Aliquots were withdrawn at the indicated times and added to an equal volume of 0.5% SDS, 50 mM EDTA, 200 [mu]g/ml proteinase K as described by Schroth et al . ( 25 ). After 30 min at 37^oC the samples were extracted with phenol/chloroform, ethanol precipitated and subjected to electrophoresis in a 6% polyacrylamide gel. Resolved fractions were subsequently autoradiographed and the extent of DNA circularization examined. In some cases ligated DNA was subjected to restriction endonuclease digestion prior to phenol/chloroform extraction.

RESULTS

Selective binding of Hc1 to chlamydial plasmid and upstream region of hc1 gene

We have previously observed that Hc1 interacts with DNA through its carboxyl end while its amino terminus does not associate with DNA at physiological levels. The purpose of this investigation was to examine the interaction of Hc1 with chlamydial DNA and identify any sequences that associate with high specificity. As a part of this strategy we developed a protocol to immune precipitate Hc1 that is associated in vivo with chlamydial DNA. Essentially, in vivo DNA-Hc1 complexes were preserved by fixing with 1% formaldehyde and DNA sonicated to generate ~1-2 kb sized fragments (data not shown) followed by immune precipitation of protein-DNA complexes using polyclonal antibodies directed against the C-terminal domain of chlamydial Hc1. Purified DNA-Hc1-antibody complexes were uncross linked by heating at 70^oC for 3 h and extracted with phenol/chloroform. While protein complexes were retained in the phenol interphase, the aqueous layer comprised extracted DNA. The DNA thus extracted was labeled with ³² P and used as a probe. Figure 1 shows the Southern blot analysis of chlamydial DNA digested with various restriction endonucleases. One strong and one moderately hybridizing band of ~4.5 and 2.6 kb were observed among Eco RI digests (Fig. 1 B, lane 1). Bam HI digests (Fig. 1 B, lane 3) revealed a strongly hybridizing band of ~7.5 kb and a moderately hybridizing band of ~9 kb, while Pst I digests revealed a single band of ~7.5 kb. Other bands hybridized very weakly and presumably represent non-specific interaction of Hc1 with chlamydial DNA. The sizes of strong and moderately hybridizing DNA bands agreed closely with restriction fragment analysis of the chlamydial plasmid (Fig. 4 A) and chlamydial histone hc1 (Fig. 4 B). Similar hybridization pattern was consistently obtained irrespective of whether Hc1-DNA complexes were immune precipitated from Chlamydia infected HeLa cells at 24, 30, 36 or 48 h post infected samples (data not shown). Additional evidence that Hc1 is associated specifically with DNA sequences from the chlamydial plasmid and its own gene was obtained by performing Southern blot analysis on various recombinant clones that were available in our laboratory. The probe DNA strongly hybridized to a 7.5 kb Pst I fragment of plasmid pCTP1 and to a 2.6 kb Eco RI fragment of plasmid pCTH1(Fig. 2 B; lanes 2 and 6 respectively). Significantly weaker signals were observed with other cloned genes under similar conditions, even when autoradiographs were exposed for extended periods of time.

Figure 1 . Southern blot analysis of chlamydial genomic digests. ( A ) Ethidium bromide stained 0.8% agarose gel of various DNA digests from Chlamydia trachomatis serovar L ₂ . Genomic DNA was digested with restriction endonucleases Eco RI (lane 1); Pst I (lane 2) and Bam HI (lane 3). Mobility of [lambda] DNA digested with Hin dIII markers is shown on the left. ( B ) Autoradiograph of Southern hybridization of (A) hybridized to DNA that was associated with Hc1.

Figure 2 . Southern blot analysis of various recombinant clone digests. ( A ) Ethidium bromide stained agarose gel of [lambda] DNA digested with Hin dIII (lane 1); plasmid pCTP1 (containing chlamydial plasmid cloned into pBR322; 12) digested with Pst I (lane 2); pCt 40-218 (encoding chlamydial MOMP gene; 21) digested with Bam HI and Kpn I (lane 3); pCTJS8 (encoding r-protein L6 gene; 22) cleaved with Pst I and Hin dIII (lane 4); pCTLS1 (encoding r-protein L6; 22) cleaved with Sac I (lane 5); and pCTH1 (encoding histone Hc1; 3) cleaved with Eco RI (lane 6). ( B ) Autoradiograph of Southern hybridization of (A) hybridized to DNA that was associated with Hc1. Note only pCTP1 and pCTH1 hybridize to the said probe.

Identification of a DNA fragment that selectively binds Hc1 in vivo

In a search for DNA sequences on plasmid pCTH1 harboring chlamydial hc1 and chlamydial plasmid pCTP1 that bind preferentially to Hc1, we double or triple digested chlamydial plasmid pCTP1 and compared these electrophoretic patterns with corresponding Southern blots (compare Fig. 3 A with B). Autoradiographs of Southern blots identified only one strong hybridizing band irrespective of double or triple digested plasmid DNA. Also visible were other weak bands, corresponding to various pCTP1 restriction fragments. However, in each case fragments encompassing the 520 bp Bam HI- Xho I region revealed strong hybridization. Figure 4 A shows the localization of a high affinity Hc1 binding site on the chlamydial plasmid which lies ~500 bp upstream of the origin of replication and one third the distance of the plasmid from the Pst I cloning site. There are clearly larger as well as smaller DNA fragments that fail to hybridize under identical conditions. Similarly plasmid pCTH1 encoding chlamydial hc1 was double or triple digested and its electrophoretic pattern compared to corresponding Southern blots. One strong hybridizing band was observed in each case encompassing 545 bp Bam HI- Dra I fragment (data not shown). Figure 4 B shows the localization of this high affinity Hc1 binding site on hc1 upstream of its ORF.

Identification and characterization of Hc1 binding site(s) in vitro

Figure 3 . Southern blot analysis of chlamydial plasmid digests. ( A ) Ethidium bromide stained agarose gel of double and triple digested chlamydial plasmid pCTP1. Plasmid pCTP1 was digested with Eco RI and Pst I (lane 1); Eco RI, Pst I and Eco RV (lane 2); Eco RI, Pst I and Sma I (lane 3); Eco RI, Pst I and Xho I (lane 4); Eco RI, Pst I and Bam HI (lane 5); Eco RI, Pst I and Sac I (lane 6) and [lambda] DNA digested with Hin dIII (lane 7). ( B ) Autoradiograph of Southern hybridization of (A) hybridized to DNA that was associated with Hc1. Note the presence of one strong autoradiographic signal in lanes 1-6 of (B). We used Bestfit program to align the 520 bp Bam HI- Xho I fragment of plasmid pCTP1 ( 12 ) and 545 bp Bam HI- Dra I of plasmid pCTH1 ( 3 ) with an aim to identify a common region that might potentially exhibit Hc1 binding characteristics. Optimum alignment identified a 24 bp fragment with ~75% homology between these two sequences with a high A+T ratio (Fig. 5 ). To examine whether these conserved sequences possess Hc1 binding ability, we synthesized oligonucleotides corresponding to these sequences along with their complementary strands. As a control we synthesized a 24 bp fragment with similar A+T ratio corresponding to nucleotide sequence 4665-4688 that is located just outside of Bam HI- Xho I high affinity binding site on plasmid pCTA1. Double-stranded DNA generated by annealing 5'-end labeled complementary strands was mixed with Hc1 and protein-DNA complexes identified by autoradiography. At a molar ratio of 1:1, DNA complexes formed between 24 bp fragments (that map within plasmid pCTA1 and hc1 gene) and Hc1 were retained in polyacrylamide gel wells (Fig. 6 A; lanes 1 and 2) while control DNA failed to bind Hc1 as revealed by its inability to retard DNA mobility under similar conditions (Fig. 6 A; lane 3). These results strongly support the nomination of 24 bp fragments that map within chlamydial plasmid and upstream of hc1 as primary binding sites for Hc1. Earlier results have shown that Hc1 binds to RNA as well as to double- and single-stranded DNA. To examine whether Hc1 exhibits any preferential binding for one strand over the other we incubated radiolabeled oligonucleotides (representing primary Hc1 binding sites) with Hc1 and analyzed protein-oligonucleotide complexes by polyacrylamide gel electrophoresis. Surprisingly, only one strand out of each pair associated with Hc1 under our assay conditions. The protein-oligonucleotide complexes were retained in polyacrylamide gel wells while corresponding complementary strands exhibited either insignificant mobility shift or no shift at all under similar conditions (Fig. 6 B; compare lanes 1 with 2 and lanes 3 with 4). Additional evidence that Hc1 does not associate with control oligonucleotides was obtained by incubating these oligonucleotides with Hc1. Again, no shift in DNA mobility was observed (Fig. 6 B; lanes 5 and 6).

Figure 4 . Restriction map and localization of high affinity Hc1 binding site on chlamydial plasmid ( A ) and hc1 ( B ). Arrow indicates the direction of initiation of translation (B). Abbreviations for restriction endonucleases are: B, Bam HI; D, Dra I; E, Eco RI; H, Hin dIII; P, Pst I; RV, Eco RV; Sa, Sac I and S, Sma I.

Figure 5 . Conserved regions of 520 bp Bam HI- Xho I fragment of chlamydial plasmid and 545 bp Bam HI- Dra I fragment of hc1 (3). Optimum alignment was performed by using GCG Bestfit program. The numbers refer to nucleotide positions from unique Bam HI sites. The top string represents sequence that maps upstream of hc1 while the bottom string maps within the chlamydial plasmid.

Figure 6 . Gel mobility shift assays for analysis of DNA-Hc1 interaction in vitro . ( A ) Hc1 was incubated with double-stranded DNA generated by annealing complementary 24mer oligonucleotides 1 and 2 representing the primary binding site on hc1 (lane 1); 3 and 4 representing binding sites on the chlamydial plasmid (lane 2) and control oligonucleotides 5 and 6 with similar A+T ratio (lane 3). Note the retention of DNA-protein complexes in lanes 1 and 2 as compared to lane 3. ( B ) Lanes 1-6 represent interaction of Hc1 with individual oligonucleotides that were used above. Oligonucleotide-protein complexes were retained in lanes 1 and 3 only supporting the strand specific binding of Hc1.

Identification of chlamydial genomic sequences with homology to primary binding sites

The inability of Hc1 to associate specifically with genomic sequences other than two 24 bp fragments representing primary Hc1 binding sites that map within the chlamydial plasmid and upstream of hc1 is indicative of their uniqueness. To explore whether the chlamydial genome contains similar sequences, genomic DNA was cleaved with restriction endonucleases Bam HI, Eco RI and Hin dIII and hybridized to radiolabeled 24mer oligonucleotide that mapped upstream of hc1 . Interestingly, only two hybridizable bands were observed in each lane (Fig. 7 ). While 9 and 7.5 kb fragments were visible among Bam HI digests, Eco RI digests revealed fragments that measured 4.5 and 2.6 kb in size. The mobility of these bands agreed closely with restriction fragment analysis of the chlamydial plasmid and hc1 . These results are similar to Southern blot analysis performed with DNA that is crosslinked to Hc1 in vivo (Fig. 1 ). No other bands were visualized despite the fact that hybridization was performed under less stringent conditions, supporting the uniqueness of these sequences. These results may also help to explain why chlamydial histone Hc1 associates specifically with these two gene sequences in vivo .

In vitro imaging of DNA-Hc1 complexes

We examined the images of DNA-Hc1 complexes in vitro by atomic force microscopy to confirm localization of high affinity Hc1 binding site(s) on plasmid pCTP1. AFM can image both uncoated and DNA-Hc1 complexed molecules on a flat surface at submolecular resolution by raster scanning a sharp tip back and forth across the surface. Pst I linearized and gel purified 7.5 kb plasmid DNA was incubated with Hc1 either before or immediately following DNA spreading onto freshly cleaved mica. In the absence of any divalent cation, DNA bound very poorly to mica; however, pretreatment with 5 mM MgCl ₂ significantly enhanced spreading and binding capabilities of DNA for mica (data not shown). Figure 8 A shows AFM images of uncomplexed plasmid. Most of the DNA appeared completely linear. The average contour length of plasmid DNA was estimated to be 2.36 +- 0.083 [mu]M. Mixing of DNA and Hc1 at a ratio of 1:0.1-1:1.0 (w/w) in solution prior to spreading led to adsorption of multi-strand single-foci complexes to mica. The thickness of these bright foci was ~50 Å (Fig. 8 D). Formation of multi-stranded complex formation is probably mediated through protein-protein interaction and appears cooperative because of the fact that complex formation is highly dependent upon protein concentration. Some multi-stranded complexes revealed one or two small foci in addition to bright large foci. These large foci always originated around a region of DNA that was approximately one third the distance from the Pst I end. Coincidentally, the Bam HI- Xho I fragment of plasmid pCTP1 also lies approximately the same distance from the Pst I cloning site. However, at a DNA-Hc1 ratio of 1:2-1:20, very large complexes were observed that failed to reveal any molecular details (data not shown). In order to visualize single DNA-Hc1 complexes more clearly, we applied free DNA to the mica substrate 15-30 s prior to the addition of 10-20-fold excess Hc1. This DNA-Hc1 ratio is a reflection of the amount applied and not the amount deposited onto mica substrate because samples were rinsed with water before and after addition of Hc1. Despite its excess, in this sequential addition experiment Hc1 associated with adhered linear DNA around the same region (one third the distance from one end) as observed with preformed DNA:Hc1 complexes in solution (Fig. 8 B and C). Also, DNA started to form loops and coils around these bright foci, leading to shrinkage of contour length. However, exact contour length could not be measured accurately.


Circularization of DNA-Hc1 complexes

We examined whether DNA has intrinsic curvature, or if bending was induced by the high affinity binding of Hc1 to plasmid DNA, by following the rate of circularization of the Bam HI- Xho I fragment of plasmid pCTP1 in the presence or absence of Hc1. The assumption is that if Hc1 induces a bend in the DNA, it will decrease the distance between the two ends of the molecule and hence increase the probability of ring closure. For our experiment, we used a 500 bp Bam HI- Bam HI fragment encompassing the Bam HI- Xho I high affinity DNA binding site and an internal 400 bp Eco RI fragment of plasmid pCTP1 that acted as a control. Figure 9 shows that a 10-20-fold molar excess of Hc1 completely inhibits recircularization of both DNA fragments, irrespective of their affinity and size (lanes 6-10). Under these conditions, the rate of circularization for both DNA fragments in the absence of Hc1 protein was similar (lanes 1-5). Surprisingly, no circularization was observed at 1-3-fold molar excess of Hc1 either. Rather, it induced DNA dimerization and polymerization not seen among corresponding controls without Hc1 (lanes 11-13). Based on the gel mobility of these dimers and multimers, as compared to circular DNA, it is reasonable to conclude that they represent linear fragments.

Further evidence, that high affinity Hc1 binding is not due to any intrinsic bends was obtained by running free double-stranded oligonucleotides (specifying either control or primary DNA binding sites) on a 20% polyacrylamide gel. No unusual migration of 24 bp free double- stranded oligonucleotide fragments representing either control (Fig. 10 ; lane 1) or the two primary Hc1 binding sites was observed (Fig. 10 ; lanes 2 and 3).

Figure 7 . Southern blot analysis of chlamydial genomic digests. Genomic DNA from C.trachomatis serovar L ₂ was digested with restriction endonuclease Bam HI (lane 1), Eco RI (lane 2) and Hin dIII (lane 3) and hybridized to a 24mer oligonucleotide 3 probe that maps upstream of hc1 ORF and represents one of the two Hc1 binding sites. Mobilities of [lambda]/ Hin dIII fragment markers is shown on the left.

Figure 8 . AFM data-images of Pst I digested chlamydial plasmid DNA in the absence ( A ) and presence ( B - D ) of Hc1. Note the formation of single DNA molecule complexes showing condensation at unique sites when DNA was applied to mica substrate prior to addition of excess Hc1 (B and C). Mixing of DNA-Hc1 in solution resulted in the formation of multi-strand single-foci complexes (D). Bright foci in (D) are ~50 Å in thickness. All images are top-view height plots.

Figure 9 . Hc1 induced inhibition of DNA circularization. Autoradiogram of gels showing time course of circularization of a 500 bp Bam HI fragment ( A ) and a 400 bp Eco RI fragment ( B ). Circularization experiments were performed in the absence of Hc1 (lanes 1-5; A and B); in the presence of Hc1 containing molar excess ratio of 1:10 (lanes 6-10) or 1:2 (lanes 11-13) respectively. Rate of circularization was monitored at 0 min (lanes 1 and 6), 5 min (lanes 2, 7 and 11), 10 min (lanes 3, 8 and 12), 20 min (lanes 4, 9 and 13) and 30 min (lanes 5 and 10). Note the absence of circularized DNA among lanes 6-10 and presence of DNA dimers and multimers among lanes 11-13.

DISCUSSION

The rate and specificity of gene transcription are primarily regulated through sequence specific as well as sequence independent interactions between trans acting protein factors and cis acting DNA. Histone H1 is a eukaryotic repressor that plays a major role in the formation of chromatin structure (reviewed in 26 ). Recently, eukaryotic like histones have been described in Pseudomonas aeruginosa, Bordetella pertussis and C.trachomatis ( 2 - 5 , 27 , 28 ). The chlamydial DNA binding proteins are two lysine rich proteins of 18 and 32 kDa with sequence homology to eukaryotic histone H1. The common 18 kDa protein designated Hc1 is conserved among all C.trachomatis serovars and is expressed late in the life cycle at a time when chlamydial DNA is undergoing compaction into dense nucleoid form. Several groups, including ours, have shown that expression of Hc1 in E.coli is sufficient to induce nucleoid compaction, an observation that lends support to its role in DNA condensation ( 9 - 12 ). The mechanism by which the deposition of Hc1 is regulated is not understood, but it is believed that the affinity of different sequences for Hc1 may play a role in such processes. The identification of Hc1 specific DNA sequences may help to unravel their regulatory role during the chlamydial life cycle. Although many assays for DNA-binding proteins can be used to detect DNA-protein interactions in vivo , these assays are not very sensitive, particularly when proteins display both sequence specific and non-specific interactions. We have devised a more sensitive method based on immunoprecipitation of Hc1-DNA complexes followed by dissociation of these complexes and subsequent extraction of associated DNA and Southern blot analysis. Using this assay we have confirmed that Hc1 binds preferentially to its own gene upstream of the translational start site as well as to chlamydial plasmid DNA both in vitro and in vivo , in addition to its generalized non-specific interaction mediated through charge neutralization of DNA. We were able to identify one strong plasmid band and one moderately hybridizing band that include the hc1 gene itself. We questioned whether hybridization to chlamydial plasmid was a reflection of its multicopy number rather than any sequence specific interaction. However, hybridization of Hc1-associated DNA to plasmid pCTP1 as well as to hc1 gene sequences indicates otherwise. There are documented reports of site-selective binding by H1 in eukaryotes also ( 29 - 32 ). Further proof that Hc1-associated DNA hybridizes in a site-selective manner was obtained by examining Southern hybridization from double and triple digests of the recombinant chlamydial plasmids pCTP1 ( 12 ) and pCTH1 ( 3 ). One fragment encompassing the Bam HI- Xho I fragment of pCTP1 hybridized strongly in comparison to other restricted fragments (see Figs 3 and 4 ). Similar observations were made for plasmid pCTH1 in which case the Bam HI- Dra I fragment hybridized strongly compared to other restriction fragments (data not shown). The calculated A+T content of these fragments varied from 62.5% ( Bam HI- Dra I fragment) to 64.6% ( Bam HI- Xho I fragment) which reflects overall A+T content of the C.trachomatis genome ( 33 ). It has been shown that H1 binds preferentially to AT-rich over GC-rich sequences ( 17 , 18 ). However, that possibility was excluded because of the failure of other restriction fragments, including the 400 bp Eco RI fragment (with A+T content of 65%) and Eco RI- Sac I fragment (that encompasses AT rich tandem repeats), to hybridize to Hc1-associated DNA. The site-selective affinity of Hc1 was further documented by AFM data images. Binding always initiated from a unique point approximately one third from one end. Since these studies were performed on Pst I linearized 7.5 kb plasmid, the high affinity Bam HI- Xho I fragment is localized at one third the distance from one end. The estimated contour length of 2.36 +- 0.083 [mu]m is compatible with the expected value of 2.325 [mu]m for DNA in B-form. Unfortunately, we were not able to precisely localize the affinity site due largely to coiling and shrinking of affected DNA following initial interaction with Hc1. Coiling was always observed around the condensed center and was not seen at the other end of the DNA strand. Christiansen et al . ( 5 ) also noticed condensed centers following DNA-Hc1 interaction but these foci were localized more towards one end of the DNA strand and represented non-specific interaction. Visualization of individual foci by AFM was observed irrespective of the condition employed. Very low protein-DNA ratio was required if incubations were carried out in solution. However, if DNA was partially immobilized on mica substrate before addition of large amounts of Hc1, individual strands with dark foci were still visible. Multi-stranded aggregates observed in solution appear to be mediated through protein-protein interaction between Hc1 molecules. Similar observations have been made by other workers ( 5 ).

To precisely map principal Hc1 binding sites, we performed optimal alignment on two sequences that associated with Hc1 in vivo and in vitro with the hope of identifying common determinants that might exhibit similar characteristics. These searches led to the identification of common A+T-rich 24 bp fragments within both sequences that share 75% homology. Gel mobility shift experiments using double-stranded DNA (generated by annealing complementary oligonucleotides comprising conserved sequences) confirmed their Hc1 binding characteristics. In this study DNA-protein complexes were retained in polyacrylamide gel wells which is similar to what has previously been reported by us and others ( 10 - 12 ). In order to re-examine whether A+T-rich sequences are responsible for this characteristic we selected sequences with characteristically high A+T ratios that flanked the primary binding site on the chlamydial plasmid and used them to study protein-DNA interaction. No complexes were retained in polyacrylamide gel wells. These results strongly suggest that sequence rather than its base composition is responsible for protein interaction. Further, individual oligonucleotides specifying either control or conserved sequences were used to study protein-oligonucleotide interactions. Surprisingly, only one complementary strand out of each conserved pair associated with Hc1. Our results support strand specific interaction of Hc1.

DNA curvature has been implicated to play a role in transcription ( 25 , 34 , 35 ), replication ( 36 , 37 ), the excision of damaged nucleotides from DNA ( 37 ) and packaging of DNA into nucleosomes ( 39 , 40 ). To examine whether binding of Hc1 to a high affinity site is mediated through this phenomenon we examined the effects of Hc1 on DNA circularization. We used a Bam HI- Bam HI fragment which encompasses the Bam HI- Xho I high affinity site and a control Eco RI- Eco RI fragment. Both fragments were end labeled following dephosphorylation and used in circularization experiments. No difference in the rate of circularization between the 500 bp Bam HI fragment and 400 bp Eco RI fragment was observed in the absence of Hc1. These results argue convincingly against an intrinsic curvature in the 500 bp Bam HI fragment. Additional evidence that the Hc1 binding site is not curved was obtained by analyzing the migration of 24 bp free double-stranded oligonucleotides (representing both control and primary Hc1 binding sites) to the same distance in a polyacrylamide gel matrix. The hallmark of bent DNA, its anomalous migration in polyacrylamide gels, was not the case with these three double-stranded oligonucleotides. However, we failed to induce any circularization in the presence of Hc1. Our circularization results strongly suggest that the binding of Hc1 to the DNA reduces the ability of the intramolecular termini to approach each other. At the same time, Hc1 mediated multi-strand association as visualized from AFM data-image and the linear multimers formed in the DNA ligation experiment favors inter- rather than intramolecular ligation. We ruled out the possibility that Hc1-DNA interaction blocks accessibility to DNA ligase since linear dimers and multimers were formed under similar conditions at low protein-DNA molar ratios. The possibility that higher protein-DNA ratio blocks accessibility of DNA ligase may explain its inability to circularize or dimerize, although we can not exclude the possibility that intramolecular termini fail to approach under a compacted situation.

The significance of such site specific binding is unknown. It seems unlikely that the entire Chlamydia genome is compacted by binding of Hc1 to unique sites. Binding of Hc1 to its own gene upstream of the translational start site may reflect autoregulation of hc1 transcription and may be functionally distinct from a proposed compaction role. Regulation of Hc1 appears very important for cell survival. Overexpression of Hc1 in E.coli has been shown to repress transcription, translation and replication, including its effect on viability of these cells ( 10 , 11 ).

Figure 10 . Polyacrylamide gel electrophoresis of free double-stranded oligonucleotides generated by annealing 24mer oligonucleotides 1 and 2 representing the primary binding site on hc1 (lane 1); 3 and 4 representing binding sites on the chlamydial plasmid (lane 2) and control oligonucleotides 5 and 6 with similar A+T ratio (lane 3). Note the similar migration pattern of all three 24 bp fragments through 20% gel matrix.

The potential for stage specific transcriptional regulation by site-specific binding of Hc1 to plasmid DNA is intriguing. All C.trachomatis strains, with the exception of a single isolate, contain a 7.5 kb cryptic plasmid that exhibits development specific supercoiling with extraordinarily supercoiled (hypercoiled) plasmid DNA in EBs, and relaxed form in RBs. Modulation of gene expression by stage specific changes in superhelicity was observed by Barry et al. ( 10 ). These authors reported differential expression of E.coli outer membrane proteins OmpC and OmpF at substructural levels of Hc1 and down regulation of transcription, translation and replication during high level expression of Hc1 in E.coli . They concluded that net relaxation of chromosomal DNA was responsible for differential expression whereas superhelicity led to down regulation. It is, therefore, conceivable that deposition of Hc1 at the Bam HI- Xho I site of plasmid DNA at a structural level induces superhelicity. Recently, Mathews and Sriprakash ( 41 ) identified a strand specific endonucleolytic activity in high salt extracts of C.trachomatis . Coincidentally, this activity was specific for a DNA region encompassing the Bam HI- Xho I fragment of plasmid DNA and lies adjacent to the primary Hc1 binding site on the chlamydial plasmid. It is tempting to hypothesize an interplay between endonucleolytic activity and Hc1 induced superhelicity. One may envisage that preferential binding of Hc1 to the Bam HI- Xho I fragment at low levels allows access to endonucleolytic activity leading to site specific nicks, while high levels of Hc1 induce superhelicity and subsequently, deny accessibility to endonucleolytic activity. The formation of DNA dimers and polymers in the presence of low levels of Hc1 and failure of DNA fragments to circularize in the presence of high levels of Hc1 are supportive of a similar mechanism. These studies may eventually facilitate the identification of other factors involved with chlamydial gene regulation and may lead to the development of more detailed models of interaction.

ACKNOWLEDGEMENTS

We thank M. Remacha for help during the initial phase of this work. This work was supported in part by start up funds from the School of Medicine, University of California, Davis.

REFERENCES

1 Costerton,J.W., Pffenroth,L., Wilt,J.C. and Kordova,N. (1976) Can. J. Microbiol., 22, 16-28. MEDLINE Abstract

2 Hackstadt,T., Baehr,W. and Ying,Y. (1991) Proc. Natl. Acad. Sci. USA, 88, 3937-3941. MEDLINE Abstract

3 Tao,S., Kaul,R. and Wenman,W.M. (1991) J. Bacteriol., 173, 2818-2822. MEDLINE Abstract

4 Perara,E., Ganem,D. and Engel,J.N. (1992) Proc. Natl. Acad. Sci. USA, 89, 2125-2129. MEDLINE Abstract

5 Christiansen,G., Pedersen,L.B., Koehler,J.E., Lundemose,A.G. and Birkelund,S. (1993) J. Bacteriol., 175, 1785-1795. MEDLINE Abstract

6 Hackstadt,T. (1991) J. Bacteriol., 173, 7046-7049. MEDLINE Abstract

7 Kaul,R., Tao,S. and Wenman,W.M. (1992) Gene, 112, 129-132.

8 Hackstadt,T., Brickman,T.J., Barry,C.E. and Sager,J. (1993) Gene, 132, 137-141. MEDLINE Abstract

9 Barry,C.E., Hayes,S.F. and Hackstadt,T. (1992) Science, 256, 377-379. MEDLINE Abstract

10 Barry,C.E. 3d., Brickman,T. J. and Hackstadt,T. (1993) Mol. Microbiol., 9, 273-283. MEDLINE Abstract

11 Pedersen,L.B., Birkelund,S. and Christiansen,G. (1994) Mol. Microbiol., 11, 1085-1098. MEDLINE Abstract

12 Remacha,M., Kaul,R., Sherburne,R. and Wenman,W.M. (1996) Biochem. J., 315, 481-486. MEDLINE Abstract

13 Solbrig,M.V., Wong,M.L. and Stephens,R.S. (1990) Mol. Microbiol., 4, 1535-1541. MEDLINE Abstract

14 Croston,G.E., Kerrigan,L.A., Lira,L.M., Marshak,D.R. and Kadonaga,J.T. (1991) Science, 251, 643-649. MEDLINE Abstract

15 Zlatanova,J. (1990) Trends Biochem. Sci., 15, 273-276. MEDLINE Abstract

16 Hulton,C.S., Seirafi,A., Hinton,J.C., Sidebotham,J.M., Waddell,L., Pavitt,G.D., Owen-Hughes,T., Spassky,A., Buc,H. and Higgins,C.F. (1990) Cell, 63, 631-642. MEDLINE Abstract

17 Sponar,J. and Sromova,Z. (1972) Eur. J. Biochem., 29, 99-103.

18 Renz,M. and Day,L.A. (1976) Biochemistry, 15, 3220-3226. MEDLINE Abstract

19 Yaneva,J., Schroth,G.P., van Holde,K.E. and Zlatanova,J. (1995) Proc. Natl. Acad. Sci. USA, 92, 7060-7064. MEDLINE Abstract

20 Wenman,W.M. and Meuser,R.M. (1986) J. Bacteriol., 165, 602-607. MEDLINE Abstract

21 Kaul,R., Duncan,M.J., Guest,J. and Wenman,W.M. (1990) Gene, 87, 97-103. MEDLINE Abstract

22 Kaul,R., Gray,G.J., Koehncke,N.R. and Gu,L. (1992) J. Bacteriol., 174, 1205-1212. MEDLINE Abstract

23 Comanducci,M., Ricci,S. and Ratti,G. (1988) Mol. Microbiol., 2, 531-538. MEDLINE Abstract

24 Sriprakash,K.S. and MacAvoy,E.S. (1987) Plasmid, 18, 205-214. MEDLINE Abstract

25 Schroth,G.P., Cook,G.R., Bradbury,E.M. and Gottesfeld,J.M. (1989) Nature, 340, 487-488. MEDLINE Abstract

26 van Holde,K.E. (1988) Chromatin. Springer, New York.

27 Kato,J., Misra,T.K. and Chakrabarty,A.M. (1990) Proc. Natl. Acad. Sci. USA, 87, 2887-2891. MEDLINE Abstract

28 Scarlato,V., Arico,B., Goyard,S., Ricci,S., Manetti,R., Prugnola,A., Manetti,R., Polverino-De-Laureto,P., Ullman,A. and Rappuoli,R. (1995) Mol. Microbiol., 15, 871-881. MEDLINE Abstract

29 Sevall,J.S. (1988) Biochemistry, 27, 5038-5044. MEDLINE Abstract

30 Ristiniemi,J. and Oikarinen,J. (1989) J. Biol. Chem., 264, 2164-2174. MEDLINE Abstract

31 Izaurralde,E., Kas,E. and Laemmli,U.K. (1989) J. Mol. Biol., 210, 573-585. MEDLINE Abstract

32 Yaneva,J. and Zlatanova,J. (1992) DNA Cell Biol., 11, 91-99. MEDLINE Abstract

33 Hatt,C., Ward,M.E. and Clarke,I.N. (1988) Nucleic Acids Res., 16, 4053-4067. MEDLINE Abstract

34 Rees,W.A., Keller,R.W., Vesenka,J.P., Yang,C. and Bustamante,C. (1993) Science, 260, 1646-1649. MEDLINE Abstract

35 Perez-Martin,J. and Espinosa,M. (1993) Science, 260, 805-807.

36 Caddle,M.S., Dailey,L. and Heintz,N.H. (1990) Mol. Cell. Biol., 10, 6236-6243. MEDLINE Abstract

37 Gille,H., Egan,J.B., Roth,A. and Messer,W. (1991) Nucleic Acids Res., 19, 4167-4172. MEDLINE Abstract

38 Shi,Q., Thresher,R., Sancar,A. and Griffith,J. (1992) J. Mol. Biol., 226, 425-432. MEDLINE Abstract

39 Allen,M.J., Dong,X.-F., O'Neill,T.E., Yau,P., Kowalczykowski,S.C., Gatewood,J., Balhorn,R. and Bradbury,E.M. (1993) Biochemistry, 32, 8390-8396. MEDLINE Abstract

40 Griffith,J.D. (1975) Science, 187, 1202-1203.

41 Mathews,S.A. and Sriprakash,K.S. (1994) J. Bacteriol., 176, 4774-4778. MEDLINE Abstract

---

Return

^* To whom correspondence should be addressed at: Division of Infectious Diseases, Department of Pediatrics, 403 Neuroscience Building, School of Medicine, 1515 Newton Court, University of California, Davis, CA 95616, USA
CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				N. A. Grieshaber, E. R. Fischer, D. J. Mead, C. A. Dooley, and T. Hackstadt From The Cover: Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis PNAS, May 11, 2004; 101(19): 7451 - 7456. [Abstract] [Full Text] [PDF]

				L. A. Sitailo, A. M. Zagariya, P. J. Arnold, G. Vedantam, and D. W. Hecht The Bacteroides fragilis BtgA Mobilization Protein Binds to the oriT Region of pBFTM10 J. Bacteriol., September 15, 1998; 180(18): 4922 - 4928. [Abstract] [Full Text]

				L. Zhang, A. L. Douglas, and T. P. Hatch Characterization of a Chlamydia psittaci DNA Binding Protein (EUO) Synthesized during the Early and Middle Phases of the Developmental Cycle Infect. Immun., March 1, 1998; 66(3): 1167 - 1173. [Abstract] [Full Text] [PDF]

This Article Abstract Print PDF (174K) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (6) Request Permissions Commercial Re-use Guidelines for Open Access NAR Content Google Scholar Articles by Kaul, R Articles by Wenman, W. Search for Related Content PubMed PubMed Citation Articles by Kaul, R Articles by Wenman, W. Social Bookmarking          What's this?