HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 48, 38111-38119, December 1, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/48/38111    most recent M006367200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roshick, C. Articles by McClarty, G. Search for Related Content PubMed PubMed Citation Articles by Roshick, C. Articles by McClarty, G. Social Bookmarking                      What's this?

Cloning and Characterization of Ribonucleotide Reductase from Chlamydia trachomatis^*

Christine Roshick, Emma R. Iliffe-Lee, and Grant McClarty

From the Department Of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada

Received for publication, July 18, 2000, and in revised form, September 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In all organisms the deoxyribonucleotide precursors required for DNA synthesis are synthesized from ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a previous study we showed that Chlamydia trachomatis growth was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase, and a mutant resistant to the cytotoxic effects of the drug was isolated. Here we report the cloning, expression, and purification of the R1 and R2 subunits of the C. trachomatis ribonucleotide reductase. In comparison with other ribonucleotide reductases, the primary sequence of protein R1 has an extended amino terminus, and the R2 protein has a phenylalanine where the essential tyrosine is normally located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus of the R1 protein is not required for enzyme activity, it is needed for allosteric inhibition mediated by dATP. Results with site-directed mutants of protein R2 suggest that the essential tyrosine is situated two amino acids downstream of its normal location. Finally, Western blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation just upstream of the ATG start codon of the R2 protein. The possibility that this mutation affects translational efficiency is discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chlamydiae are obligate eubacterial parasites that cause a variety of diseases in humans, birds, and numerous animals (1, 2). Of the four recognized chlamydial species, two, Chlamydia pneumoniae and Chlamydia trachomatis, are established pathogens of humans. Human chlamydial infections are a leading cause of sexually transmitted disease, blindness, and respiratory disease (3). One of the reasons for chlamydiae's success as a pathogen is no doubt linked to its highly specialized biphasic growth cycle (4). The elementary body (EB)¹ is the metabolically inert, osmotically stable extracellular form that is capable of initiating infection by attaching to and entering the host cell. Within a few hours, the internalized EB differentiates into the metabolically active, osmotically fragile reticulate body (RB), which divides by binary fission. About 20 h later, the process of RB differentiation back to EB begins.

Over time, chlamydiae have evolved an intimate metabolic relationship with their host. Early studies on chlamydial metabolism indicated that they were auxotrophic for amino acids, nucleotides, and many other components of intermediary metabolism that most free living bacteria are capable of synthesizing themselves (4, 5). Studies from our laboratory have shown that chlamydiae are capable of transporting NTPs but not dNTPs directly from the host cell (6, 7). In addition, we isolated a mutant C. trachomatis isolate resistant to the cytotoxic effects of hydroxyurea, a rather specific inhibitor of class I ribonucleotide reductases (RNRs) (8). Taken together, these two observations suggested that chlamydiae encode an RNR for the synthesis of deoxyribonucleotides from ribonucleotides.

Ribonucleotide reductase was the first enzyme shown to contain a protein free radical (9), and it is now known that the free radical is used to activate the ribonucleotide substrate (10). Currently there are three classes of RNRs, which are differentiated on the basis of the mechanism they use for radical generation and the enzymes' complicated and precise allosteric regulation (11). Two subgroups of class I enzymes exist. Both consist of two homodimers, the R1 protein (₂), which contains the active site and the binding site(s) for allosteric effectors, and the R2 protein (₂), which contains a dinuclear iron center and a tyrosyl free radical (11). Class Ia are found in almost all eucaryotes, large DNA viruses, and several procaryotes, which typically exist in an environment where there is a close association with eucaryotes. Class Ib enzymes are found exclusively in bacteria. Although they show limited overall sequence identity with class Ia enzymes, catalytically important residues are conserved (12). Class II enzymes, which occur in many procaryotes, have a simpler structure ( or ₂) and use adenosylcobalamin as a cofactor, which is cleaved to produce a 5'-deoxyadenosyl radical (13, 14). Class III enzymes only function under anaerobic conditions and are found in procaryotes. Like class I enzymes, class III enzymes have an ₂₂ structure; however, they have a glycyl radical on the  subunit, and the  subunit contains an iron sulfur cluster (15).

Chlamydiae have a relatively small genome, approximately 1-1.2 million base pairs. This has facilitated the complete genome sequencing of four isolates C. trachomatis serovar D (16), C. pneumoniae CWL 029 (17), C. pneumoniae AR 39, and C. trachomatis mouse pneumonitis (18). In addition, two more genomes, C. psittaci guinea pig inclusion conjunctivitis and C. trachomatis serovar L2, are being sequenced. As expected from the results of our metabolic studies (6, 7), chlamydial genome sequence annotation identified a ribonucleotide reductase homologue (16-18). In agreement with our hydroxyurea sensitivity studies (8), the amino acid sequences of the R1 and R2 protein homologues suggest that the chlamydial RNR belongs to class Ia. The chlamydial R1 and R2 protein sequences do show some unusual characteristics. The R1 protein has a calculated molecular mass of 119,000 Da, which is about 30 kDa larger than the prototype class Ia Escherichia coli R1, and the R2 protein has a phenylalanine at the position where the essential free radical tyrosine is located in all other class I R2 proteins.

To initiate more detailed studies on the chlamydial RNR and to define the molecular mechanism of hydroxyurea resistance in our mutant chlamydiae we cloned, expressed, purified, and raised antibody against both subunits of the enzyme. We demonstrate that despite its rather unusual primary sequence, the chlamydiae RNR is active. Deletion mutagenesis was employed to show that the 30-kDa amino-terminal extension of the chlamydial R1 protein is not essential for enzyme activity, but it is required for the allosteric inhibitory effects of dATP. Results from site-directed mutagenesis studies with the chlamydial R2 protein suggest that tyrosine 129 (chlamydiae numbering) is probably the location of the essential tyrosyl free radical. Finally, our C. trachomatis mutant is resistant to hydroxyurea because it overproduces both the R1 and R2 proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- [5-³H]CDP and the enhanced chemiluminescence kit used for Western blot analysis were obtained from Amersham Pharmacia Biotech. Hydroxyurea and various nucleotides were purchased from Sigma. DNA cycle sequencing kit, restriction endonucleases, and oligonucleotides were obtained from Life Technologies, Inc. All other chemicals were of the highest purity available.

Bacterial Strains and Plasmids-- C. trachomatis L2/434/Bu was originally obtained from C. C. Kuo (University of Washington, Seattle, WA) and has been maintained in our laboratory since that time. An initial description of the hydroxyurea-resistant mutant C. trachomatis L2 isolate (L2H^R-25) used in this study has been published (8). E. coli BL21 (DE3) (hsdS gal clts857 ind1 Sam7 nin5 lacUV5-T7 gene 1) was obtained from Novagen, Inc. DH5 (supE44 lacU169 (80 lacZM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) is routinely maintained in our laboratory. Expression vector pET3a was obtained from Novagen, Inc.

HeLa Cell Line and Culture Condition-- Wild type HeLa 229 cells are continuously maintained in our laboratory and are routinely cultured in minimal essential medium containing 10% heat-inactivated (56 °C for 30 min) fetal bovine serum at 37 °C in an atmosphere of 5% CO₂/95% humidified air. HeLa cells were infected with C. trachomatis L2 and the mutant L2H^R-25 as described previously (6, 8). Following infection, the chlamydiae inoculum was removed, and the cell monolayer was washed three times with sterile phosphate-buffered saline. The chlamydiae-infected cells were cultured in minimal essential medium containing 10% heat-inactivated fetal bovine serum and 1 µg/ml cycloheximide. Highly purified preparations of EBs and RBs were prepared by centrifugation through Renografin density gradients (19, 20). RBs and EBS were purified from 20-24 h and 40-42 h infected cultures, respectively.

Construction of C. trachomatis Ribonucleotide Reductase Expression Vectors-- The pET expression system (Novagen, Inc.) was used for inducible overexpression of the cloned chlamydial wild type R1, R11-248, wild type R2, L2H^R-25 R2, R2Y129F, R2F127Y, and R2F127Y/Y129F. Initial construction and identification of recombinant plasmids were completed in E. coli DH5. For protein production, plasmids were transferred to BL21 (DE3). BL21 (DE3) contains a lysogenic  phage derivative, DE3, carrying the gene for T7 RNA polymerase under the control of an inducible lacUV5 promoter. Expression of recombinant proteins cloned into pET expression vectors is from a T7 RNA polymerase promoter.

Oligonucleotide primers used for the different genes were designed based on the published sequence of the C. trachomatis serovar D or L2 genome (16). Plasmid pET3a-CTR1 and pET3a-CTR11-248 express wild type C. trachomatis full-length protein R1 and protein R1 with a deletion of 248 amino acids from the amino terminus, respectively. The DNA inserts of pET3a-CTR1 and pET3a-CTR11-248 were produced by PCR amplification of wild type C. trachomatis L2 genomic DNA. PCR primers 5'- CCCCTCTAGACATATGGTCGATCTACAAG-3' and 5'-CCCCTCTAGAGGATCCGCTTGCATAAGAACC-3' encompassed the initiation and termination codons of the full-length R1 gene encoding a product of 1047 amino acids. For the R1 protein with 248 amino acids deleted at the amino terminus of the protein, encoding a product of 799 amino acids (R11-248), the PCR primers were 5'-CCCCTCTAGACATATGACGCATTCGCAG and 5'-CCCCTCTAGAGGATCCGCTTGCATAAGAACC-3'. Underlined portions of the oligonucleotide primers indicate the XbaI-NdeI and XbaI-BamHI restriction sites included for cloning purposes. The DNA inserts of pET3a-CTR2 and pET3a-CTH^R-25R2 were produced from PCR amplification of wild type C. trachomatis L2 and hydroxyurea-resistant C. trachomatis L2H^R-25 genomic DNA, respectively. PCR primers 5'- CCCCGAATTCCATATGCAAGCAGATATTTTAG-3' and 5'-CCCCGGATCCCTACCAAGTTAAGCTTGC-3' encompassed the initiation and termination codons of the full-length wild type R2 and L2H^R-25 mutant R2 genes encoding products of 346 amino acids. Underlined portions of the oligonucleotide primers indicate the EcoRI-NdeI and BamHI restriction sites included for cloning purposes.

PCR, using paired complementary oligonucleotides containing the desired point mutations, was used to introduce specific mutations into wild type C. trachomatis R2 as described previously (21). In all cases, wild type C. trachomatis L2 genomic DNA was used as template. The insert for plasmid pET3aR2Y129F was created using paired complementary primers 5'-GCACACATTTTTGTTTATTTGTG-3' and 5'-CACAAATAAACAAAAATGTGTGC-3'. The insert for plasmid pET3aR2F127Y was created using paired complementary primers 5'-GCACACATATTTGTATATTTGTG-3' and 5'-CACAAATATACAAATATGTGTGC-3', and the insert for plasmid pET3aR2F127Y/Y129F was created using paired complementary primers 5'-GCACACATATTTGTTTATTTGTG-3' and 5'-CACAAATAAACAAATATGTGTGC-3'. The flanking primers used were those encompassing the initiation and termination codons of the R2 gene as described above. The insert in all plasmid constructs was verified by dideoxynucleotide sequencing.

Expression and Purification of Recombinant Wild Type and Mutant C. trachomatis R1 and R2 Proteins-- Recombinant chlamydial full-length wild type R1 protein, mutant R11-248, wild type R2 protein, mutant L2H^R-25 R2, R2Y129F, R2F127Y, and R2F127Y/Y129F were expressed and purified as described earlier (22-24). Briefly, E. coli BL21(DE3) was transformed with one of the pET3a expression plasmids described above. The bacterial culture was grown in 600 ml of TB medium in 6-liter flasks with 50 µg/ml carbenicillin at 30 °C with vigorous shaking to an OD of 1.0 at 600 nm. At this time the incubator temperature was reduced to 15 °C, and when the OD reached approximately 2, protein expression was induced with IPTG at a final concentration of 50 µM. The cultures were incubated for a further 15-20 h at 15 °C, by which time the OD had typically reached 12-16. All subsequent procedures were carried out at 4 °C. Bacteria were harvested by centrifugation, washed once in 50 mM Tris-HCl buffer (pH 7.6), and repelleted. The cell pellet was resuspended in 50 mM Hepes buffer (pH 7.3) and quick frozen at 80 °C. Recombinant chlamydial R1 and R11-248 were purified by chromatography on a dATP-Sepharose column as described previously for recombinant mouse R1 (22). For full-length protein R1, the column was washed with 0.5 mM ATP, and then protein R1 was eluted with 5 mM ATP. For R11-248, the column was washed with 0.15 mM ATP and was subsequently eluted with 5 mM ATP. Recombinant chlamydial wild type R2, mutant L2H^R-25 R2, R2Y129F, R2F127Y, and R2F127Y/Y129F were purified on a DE52 column as described previously for recombinant mouse R2 protein (23, 25).

Reverse Transcriptase-PCR (RT-PCR)-- Total RNA was isolated from C. trachomatis L2-infected HeLa cells (3.0 × 10⁷ cells/150-cm² flask) cultured in complete minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum at 24 h postinfection using the RNA extraction kit from Qiagen as described previously (26). RT-PCR was performed using Superscript^TM Reverse Transcriptase (Life Technologies, Inc.) according to the manufacturer's instructions. cDNA resulting from reverse transcription was ethanol-precipitated, resuspended in double distilled H₂O, and stored at 20 °C as template for PCR amplification. To detect any changes in the level of expression of the nrd genes, the PCR was maintained in the linear range by using 30 cycles. RT-PCR primers used were as follows: (a) for 23 S rRNA, 5'-GGGTTGTAGGATTGAGGA-3' and 5'-GTTTTAGGTGGTGCAGGA-3'; (b) within the coding sequence of nrdA, 5'-ATATACACCAGCTACGCCA-3' and 5-'AAGCATCGTGTAATCCCG-3'; (c) within the coding sequence of nrdB, 5'-TATGGAAGTCGGATCGTC-3' and 5'-AATCTCCGGGTTCTCTTC-3'; and (d) overlapping the coding sequence of nrdA and nrdB, 5'-CTTTATCTTGCCCAGCCA-3' and 5'-ATATTGTCTCGCTTCCGG-3'.

Polyclonal Antibody Production and Western Blot Analysis-- Mouse polyclonal antibodies against purified recombinant chlamydial R1 and R2 proteins were produced as described previously (27, 28). Cell extract proteins were analyzed on a 10% linear SDS-polyacrylamide gel and then transferred to nitrocellulose membrane for Western blot analysis as described previously (29, 30). Primary antibody binding was detected with a goat anti-mouse IgG conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories) and visualized by enhanced chemiluminescence as described in the manufacturer's instructions (Amersham Pharmacia Biotech).

Assay of Ribonucleotide Reductase Activity-- Ribonucleotide reductase activity was assayed by determining the reduction of CDP as described previously (30). Incubation was at 37 °C for 20 min in a final volume of 0.05 ml containing 25 mM Tris-Cl, 50 mM Hepes (pH 7.6), 6.4 mM MgCl₂, 20 µM FeCl₃, 10 mM dithiothreitol, 2 mM ATP, and 0.5 mM CDP (20 cpm/pmol). These conditions are referred to as standard assay conditions. One unit of enzyme activity corresponds to 1 nmol of dCDP formed per min.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequence Analysis-- Sequence data are available for both RNR subunits from each of the six chlamydial isolates that have had their genomes sequenced (16-18). Serovar L2 R1 protein contains 1047 amino acids, giving a calculated molecular mass of 119 kDa, about 30 kDa larger than prototype E. coli class Ia R1 protein (11). The deduced R1 protein sequences from the various chlamydiae are approximately the same molecular weight and show about 80% amino acid sequence identity to each other (data not shown). A comparison of the complete amino acid sequence of the chlamydial L2 R1 with that of other R1 proteins in the public data bases (Fig. 1A) provides some interesting information, which is summarized schematically in Fig. 1C. First, as previously noted by Jordan et al. (31), the chlamydial R1 shows the highest overall sequence identity, 47.7%, to the Pseudomonas aeruginosa R1. Second, as shown in Fig. 1B, the extended amino-terminal region of the chlamydial R1 protein (amino acids 1-221) appears to be a duplication of approximately 110 amino acids that show highest homology to the N-terminal region of various class II and class III enzymes. Finally, the remainder of the R1 protein (amino acids 220-1047) shows higher homology to full-length eucaryotic R1 proteins (37.1% amino acid identity to mouse R1) than to bacterial class Ia R1 proteins (25.7% amino acid identity to E. coli class Ia R1). Importantly, most of the residues that have been shown to be critical for R1 function (E. coli class Ia numbering is used for clarity), including the active site cysteines (Cys²²⁵, Cys⁴³⁹, Cys⁴⁶²), the proposed radical transfer pathway (Tyr⁷³⁰, Tyr⁷³¹), the cysteines that receive electrons from the external hydrogen donor (Cys⁷⁵⁴, Cys⁷⁵⁹), and the asparagine and glutamate residues of the active site (Asn⁴³⁷, Glu⁴⁴¹) (11, 32), are all conserved. Furthermore, key residues involved in effector nucleotide binding at the allosteric substrate specificity site are essentially conserved (32, 33). Since the chlamydial R1 has a duplication of approximately 110 amino acids at the N terminus, it contains three reasonable homologues of the signature sequence VXKRDG (Fig. 1A), which occurs at the N terminus of R1s that contain an allosteric activity site (11, 32).

View larger version (39K): [in this window] [in a new window]   Fig. 1.   A, comparison of the N-terminal region of the C. trachomatis L2 R1 protein with the R1 protein from P. aeruginosa (Pseudomonas Genome Project Online), E. coli (64), and mouse (55). The remainder of the C. trachomatis L2 sequence is shown by itself. Critical conserved residues identified in E. coli including the active site cysteines (Cys225, Cys439, Cys462), the C-terminal cysteines involved in shuttling electrons from glutaredoxin or thioredoxin (Cys754, Cys759), and the tyrosines in the radical transfer pathway (Tyr730, Tyr731) are shaded lightly. The three homologues of the signature sequence (VXKRDG) involved in effector binding at the N-terminal activity site are in boldface lettering. The start methionine in the deletion mutant R11-248 is marked with a plus sign. Identical amino acid residues are indicated by asterisks, strongly similar residues are shown by colons, and weakly similar residues are indicated by dots. Alignments were done using ClustalW version 1.8. B, comparison of the duplicated N-terminal region of the C. trachomatis L2 R1 protein with the N-terminal region of the class III E. coli nrdD (E.coliD) protein (46) and the class II Pyrococcus furiosus nrdJ protein (P.furJ) (65). The N-terminal 221 residues of the chlamydial R1 protein are split into two portions containing amino acids 1-113 (C.trachN) and 114-221 (C.trachC). The residues that are identical between the two chlamydial parts are indicated in boldface lettering. For the alignment of all four sequences, identical residues are indicated by asterisks, strongly similar residues by colons, and weakly similar residues by dots. Alignments were done using ClustalW version 1.8. c, schematic representation of the C. trachomatis R1 protein. The important structural features of the C. trachomatis R1 protein are summarized including the approximately 110-amino acid portion duplicated at the amino terminus (solid block arrows), the three homologues of the nucleotide effector binding activity site (solid blocks), the conserved cysteines, and the start site and size of the expressed recombinant full-length and truncated R1 proteins. See "Results" for details.

Serovar L2 R2 protein contains 346 amino acids, a size similar to the prototype E. coli class Ia R2 subunit (11). The deduced chlamydial R2 protein sequences show about 90% identity at the amino acid level to each other (data not shown). A comparison of the complete amino acid sequence of the chlamydial L2 R2 with that of several other R2 proteins in the public data bases is shown in Fig. 2. In general, the chlamydial R2 protein shows lower overall homology to procaryotic R2s (19.5% identity to E. coli R2) than to eucaryotic R2s (23.2% identity to mouse R2); however, as with the R1 protein, the chlamydial R2 shows highest overall homology (54.4% identity) to the P. aeruginosa R2. Most of the residues known to be important for R2 function, including the iron ligands (Glu¹¹⁵, His¹¹⁸, Glu²⁰⁴, Glu²³⁸, His²⁴¹), residues in the hydrophobic pocket surrounding the tyrosyl free radical (Phe²⁰⁸, Phe²¹², Ile²³⁴), and those that participate in the long range radical transport pathway (Trp⁴⁸, Asp²³⁷, Glu³⁵⁰, Tyr³⁵⁶) are conserved. Chlamydial R2 residues that are not conserved are the iron ligand Asp⁸⁴, which is conservatively substituted with glutamic acid, and a striking change, the critical tyrosyl free radical (Tyr¹²²), which is substituted with phenylalanine. Indeed, of all the R2 protein sequences deposited in the public data bases, only the chlamydial R2s (the Tyr to Phe change is conserved in all six chlamydial R2s) show this change.

View larger version (79K): [in this window] [in a new window]   Fig. 2.   Comparison of the C. trachomatis L2 R2 protein with the R2 protein from P. aeruginosa (Pseudomonas Genome Project Online), E. coli (64), and mouse (55). Critical conserved residues identified in E. coli including the iron ligands (Glu115, His118, Glu204, Glu238, His241), residues in the hydrophobic pocket surrounding the tyrosyl free radical (Phe208, Phe212, Ile234), and those that participate in the long range radical transport pathway (Trp48, Asp237, Glu350, Tyr356) are shaded lightly. Critical residues that are not conserved including the iron ligand (Asp84) and the tyrosyl free radical (Tyr122) are shown in boldface type. The tyrosine that probably carries the radical in the chlamydial R2 protein is marked with a plus sign. See "Results" for details.

Expression Analysis of Chlamydial nrdAB-- In E. coli, nrdAB are transcribed as a polycistronic mRNA (34, 35). Chlamydial genome sequence analysis indicates that there are 37 bases between the predicted TAA stop codon of nrdA and the ATG start codon of nrdB (16). This organization suggests that chlamydial nrdAB may be transcribed as a polycistronic mRNA. Since it has proven difficult to do Northern blots for chlamydial metabolic gene transcripts and the absence of consensus promoter sequences (36, 37) largely negates identification of potential transcripts by sequence analysis, this question was addressed using RT-PCR. Primer pairs were chosen within nrdA and nrdB coding sequences as well as a pair that spanned the two open reading frames. As shown in Fig. 3A, all primer pairs gave PCR products following RT-PCR, indicating that the nrdAB genes do constitute an operon in chlamydiae. We also used RT-PCR to analyze the expression of nrdAB throughout the chlamydial developmental cycle as described previously (26). Total RNA was isolated from C. trachomatis-infected HeLa cells at 2, 6, 16, 24, 36, 48 h postinfection and used as template for cDNA synthesis. The amount of cDNA used as template for each time point was then roughly equalized using primers specific to chlamydial 23 S rRNA so that the 23 S rRNA PCR products were of similar intensity when run on an agarose gel (Fig. 3B). This amount of cDNA was kept constant for subsequent reactions, and the primers employed were within the coding region of the nrdB gene. nrdAB expression is barely detected at our earliest time point of 2 h postinfection, increases until 16 h postinfection, and then remains essentially constant throughout the remainder of the life cycle (Fig. 3B).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   RT-PCR analysis of total RNA extracted from Chlamydia-infected cells. A, RT-PCR indicates that nrdAB are transcribed as a polycistronic mRNA. RNA was extracted at 24 h postinfection and treated with DNase. All lanes labeled with a plus sign represent samples subjected to RT-PCR. All lanes labeled with a minus sign represent RNA extracts subjected to PCR with no reverse transcriptase step. Primer pairs used to amplify nrdA, nrdB, and nrdAB are listed under "Experimental Procedures." B, RT-PCR analysis of total RNA extracted from chlamydiae-infected cells at different time points in the chlamydial developmental cycle. Each lane contains RNA samples subjected to RT-PCR analysis. Time points indicate the number of hours after infection at which the RNA sample was isolated. Primers used for 23 S rRNA and nrdAB are described under "Experimental Procedures." RT-PCR using 23 S rRNA primers and nrdAB primers are shown. C, semiquantitative RT-PCR analysis of total RNA extracted from wild type C. trachomatis L2 (L2)- and hydroxyurea-resistant L2HR-25 (HUR)-infected cells. Semiquantitative RT-PCR indicates that nrdAB mRNA is not elevated in the hydroxyurea-resistant C. trachomatis isolate. RNA was isolated from 24-h infected cells and subjected to RT-PCR as described for A. Primers used are the same as those for B.

Expression and Purification of Wild Type and Mutant C. trachomatis R1 and R2 Proteins-- In agreement with previous findings with cloned overexpressed mouse (22), viral (38, 39), and Arabidopsis thalania (40) R1 proteins using bacterial expression systems, we found that insoluble inclusion body formation decreased and yield of soluble R1 protein increased if lower growth temperature and reduced IPTG concentration were used (data not shown). Therefore, these were the conditions employed for expression of chlamydial protein R1. Recombinant full-length R1 (1047 amino acids) and a truncated R1 with 248 amino-terminal residues deleted, R11-248 (799 residues), were overexpressed in E. coli using a pET expression system and subsequently purified on a dATP-Sepharose affinity column. The effect the 248-amino acid segment had on RNR activity was investigated, because this region contains the three homologues of the activity site signature sequence (VXKRDG). Both R1 proteins remained in the soluble fraction following overexpression; however, the yield of the full-length R1 (0.5-1.0 mg/liter) was significantly lower than that of R11-248 (2-3 mg/liter). On SDS-polyacrylamide gel electrophoresis, the wild type recombinant R1 showed several bands with mobilities close to that of the molecular mass (119,421 Da) deduced from the cloned nrdA gene (Fig. 4). R11-248 gave two bands on gel analysis, a major band with mobility of approximately 90 kDa and a less prominent band running at approximately 85 kDa (Fig. 4). The molecular mass, as deduced from the truncated nrdA insert cloned into the pET vector, is 90,820 Da.

View larger version (116K): [in this window] [in a new window]   Fig. 4.   SDS/polyacryamide gel electrophoresis of full-length and truncated (1-248) C. trachomatis L2 R1 protein and wild type R2 protein. R1 proteins were purified by dATP-Sepharose chromatography and R2 protein by DEAE-cellulose chromatography. Purified samples were run on a 10% SDS-polyacrylamide gel. Sizes are indicated in kilodaltons.

The unusual primary structure of the chlamydial R2 in the vicinity of the critical free radical tyrosine prompted us to construct mutants in this area. Visual inspection of the chlamydial R2 sequences suggested that Tyr¹²⁹ might have assumed the role of the free radical tyrosine. Three mutant R2s, R2F127Y, R2Y129F, and R2F127Y/Y129F were constructed using a PCR mutagenesis approach as described under "Experimental Procedures." Following overexpression in E. coli, wild type R2 and two of the three R2 mutants, R2Y129F and R2F127Y, remained in the soluble fraction and after purification gave similar yields of 7-9 mg of protein R2 per liter. On SDS-polyacrylamide gel electrophoresis, the wild type recombinant R2 gave one major band with mobility of approximately 40 kDa (Fig. 4) in good agreement with the molecular mass deduced from the cloned nrdB gene (40,516 Da). Purified mutant R2 proteins were of similar purity and molecular mass on gel analysis (data not shown). Unfortunately, when overexpressed, R2F127Y/Y129F formed inclusion bodies of recombinant protein with little soluble protein being formed. We tried many different growth conditions (temperature, medium, IPTG inducer concentration) in an attempt to minimize inclusion body formation. As with R1 protein, optimal yield (0.5-1.0 mg) of soluble R2F127Y/Y129F was obtained by using a lower growth temperature (15 °C) and reduced IPTG concentration (50 µM).

Recombinant Chlamydial Ribonucleotide Reductase-- Initial characterization and optimization showed that CDP reductase activity depended on the presence of both purified chlamydial R1 and R2 proteins; neither protein showed any enzymatic activity on its own. Fig. 5A shows CDP reductase activity obtained with a set amount (1.5 µg) of R2 protein and increasing amounts of full-length R1 or truncated R11-248 protein. The enzyme activity of both increased with increasing amount of protein R1, a result indicating that the amino-terminal 248 amino acids of protein R1 are not essential for enzyme activity in vitro. Of CMP, CDP, or CTP, only CDP was used as a substrate. As with other class Ia RNRs (12, 30, 31, 41), activity was stimulated by the presence of Mg²⁺ and dithiothreitol (data not shown). The specific activities, in the presence of ATP as a positive effector, were 180, 35, and 48 nmol/min/mg for chlamydial protein R2, R1, and R11-248, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   CDP reduction by C. trachomatis L2 R1 in the presence of C. trachomatis L2 R2. A, CDP reduction as a function of increasing amounts of C. trachomatis L2 R1 in the presence of C. trachomatis R2. Assays were carried out under standard conditions with various amounts of full-length R1 () or truncated R11-248 () in the presence of 1.5 µg of R2. B, effect of ATP (2 mM) and/or dATP (100 µM) on CDP reduction by full-length R1 (2.0 µg) or truncated R11-248 (2.0 µg) in the presence of 5.0 µg of R2. Open bars, no effectors; vertical striped bars, with ATP-dATP; horizontal striped bars, without ATP plus dATP; solid bars, with ATP plus dATP. Each assay was run in triplicate, and the results shown are the mean ± S.D.

Numerous studies with RNR purified from both eucaryotes and procaryotes have shown that enzyme activity is regulated by nucleoside triphosphate effectors (ATP, dATP, dGTP, dTTP) that bind to the R1 subunit (11). Results of preliminary experiments assessing the effects of the allosteric modulators, ATP and dATP, on CDP reductase activity of full-length R1 or truncated R11-248 are shown in Fig. 5B. As has been found with many other RNRs (11), ATP (2 mM) stimulates CDP reductase activity of both chlamydial R1 proteins, although both R1 preparations showed low level basal activity in the absence of any nucleotide effector. With full-length R1, the addition of dATP (100 µM) alone or together with ATP (2 mM) inhibited CDP reductase activity. In contrast, when the truncated protein R11-248 was the source of the large subunit, CDP reductase activity was stimulated by dATP (100 µM) whether added alone or together with ATP (2 mM). These results suggest that the inhibitory effects caused by dATP depend on the presence of the amino-terminal 248 amino acids of the R1 protein and are consistent with current models that assign the allosteric dATP binding activity site to the amino terminus of R1 (11, 32).

CDP reductase activities of wild type chlamydial R2 and the mutant R2s, Y129F, F127Y, and F127Y/Y129F measured in the presence of purified recombinant full-length R1 (5 µg) and ATP (2 mM) as a positive effector are shown in Fig. 6A. Wild type R2 and the F127Y mutant R2 both displayed good activity, which increased with increasing amounts of R2 protein. The double mutant R2 protein, F127Y/Y129F, showed about 10-15% of the activity displayed by the wild type R2, and the Y129F R2 mutant showed no significant activity.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   CDP reduction by C. trachomatis wild type and mutant R2s in the presence of C. trachomatis R1. A, CDP reduction as a function of increasing amounts of wild type R2 (), mutant R2Y129F (), R2F127Y (), and R2F127Y/Y129F (*) in the presence of 5 µg of R1. Assays were carried out under standard conditions. B, hydroxyurea-dependent inhibition of CDP reduction. Aliquots of purified recombinant C. trachomatis wild type R2 () or hydroxyurea-resistant L2HR-25 R2 () were incubated at 4 °C with the indicated concentration of hydroxyurea, diluted into an assay mixture, and assayed under standard conditions. The final assay mix contained 1.5 µg of R2 and 5 µg of full-length R1. 100% activity corresponds to 34 milliunits.

A combination of full-length chlamydial R1 protein and wild type R2 were used for a CDP reductase assay to determine the hydroxyurea sensitivity of the enzyme activity. As can be seen in Fig. 6B, the enzyme activity was sensitive to hydroxyurea in a concentration-dependent fashion. Class I RNRs are typically sensitive to hydroxyurea because it scavenges the tyrosyl-free radical, which is required for enzyme activity (9).

Molecular Characterization of a C. trachomatis Hydroxyurea-resistant Mutant-- In a previous study, we reported the isolation and initial characterization of a mutant C. trachomatis L2 selected for resistance to the RNR inhibitor hydroxyurea (8). The mutant C. trachomatis L2 isolate, L2H^R-25, was approximately 65-fold more resistant to hydroxyurea than wild type C. trachomatis L2. We conducted a series of studies to define the molecular mechanism underlying drug resistance in this isolate.

Since the tyrosyl free radical of protein R2 is the target of hydroxyurea, we decided to test the hydroxyurea sensitivity of recombinant L2H^R-25 R2 protein in vitro. The L2H^R-25 nrdB gene was PCR-amplified, cloned into pET 3a, overexpressed in E. coli BL21(DE3), and subsequently purified as described above. CDP reductase activity resulting from a combination of L2H^R-25 R2 protein and full-length R1 displayed hydroxyurea sensitivity similar to that found with wild type R2 (Fig. 6B). This suggests that the L2H^R-25 R2 protein tyrosyl radical is just as sensitive to scavenging by hydroxyurea as it is in the wild type R2 protein.

A common mechanism accounting for hydroxyurea resistance in mammalian cells (29, 42) and viruses (43) is overexpression of the tyrosyl radical-containing protein R2. Whole cell proteins of wild type and L2H^R-25-purified EBs and RBs were separated by SDS-polyacrylamide gel electrophoresis and Western blotted to nitrocellulose membrane. The major outer membrane protein of C. trachomatis L2 was used as a standard to assess the amounts of protein loaded for each sample. The R1 and R2 proteins were immunodetected with mouse polyclonal antiserum raised against purified L2 R1 and R2, respectively. Major outer membrane protein was detected with a mouse monoclonal antibody (44). There was an obvious increase in the amount of R1 and R2 protein in L2H^R-25 cell extract prepared from either purified RBs or EBs compared with wild type L2 samples (Fig. 7). Interestingly, similar to purified recombinant full-length R1, which showed multiple bands on an SDS-polyacrylamide gel (Fig. 4), several bands in the 100-kDa molecular mass range were detected with the R1 antibody on the Western blot. In contrast, if crude whole cell lysate, prepared from an E. coli clone overexpressing recombinant full-length R1 or truncated R11-248, was used as the source of R1, a single band was observed. These results suggest that the R1 subunit is susceptible to proteolysis during the purification of chlamydiae EBs, RBs, or recombinant R1 protein.

View larger version (72K): [in this window] [in a new window]   Fig. 7.   Western blot analysis for ribonucleotide reductase subunits in wild type and hydroxyurea-resistant C. trachomatis EBs and RBs. Samples of purified wild type (L2) and hydroxyurea-resistant (L2HR-25) EBs and RBs or E. coli BL21 expressing recombinant chlamydial full-length R1, truncated R11-248, or wild type R2 were heated at 95 °C in the presence of SDS sample buffer prior to loading on the gel. Samples were blotted to nitrocellulose and then incubated with one of the following primary antibodies: mouse monoclonal anti-chlamydial major outer membrane protein, mouse polyclonal anti-chlamydial R1, or mouse polyclonal anti-chlamydial R2. Lane a, L2HR-25 EBs; lane b, L2 EBs; lane c, L2 RBs; lane d, L2HR-25 RBs; lane e, E. coli expressing chlamydial R11-248; lane f, E. coli expressing full-length chlamydial R1; lane g, E. coli expressing wild type chlamydial R2.

Southern blot analysis of wild type L2 and L2H^R-25 genomic DNA, digested with various restriction endonucleases, probed with a PCR product encompassing nrdAB showed no gross alterations in genomic organization or gene copy number (data not shown). Semiquantitative RT-PCR was employed to determine if there was an alteration in the level of nrdAB mRNA in L2H^R-25 compared with wild type L2. Total RNA was isolated from wild type L2 and L2H^R-25 mutant C. trachomatis-infected HeLa cells at 24 h postinfection and then used as template for cDNA synthesis. The results shown in Fig. 3C indicate that there was no major change in the level of nrdAB mRNA in L2H^R-25 compared with wild type L2.

We decided to investigate whether there were any changes in the DNA sequence in the L2H^R-25 nrdAB operon, compared with wild type L2, that could provide an explanation for the dramatic increases in the level of R1 and R2 protein in L2H^R-25. We used a PCR fragment encompassing part of the L2 nrdAB operon to screen an L2H^R-25 genomic pUC19 HindIII partial digest library. Two recombinant plasmids, pH^R25-1 and pH^R25-2, containing the entire nrdAB operon were isolated. A portion of the insert in pH^R25-1 encompassing a region from 300 bases upstream of the R1 protein ATG start codon to 100 bases downstream of the R2 protein TAG stop codon was sequenced from both directions using the dideoxynucleotide method. The wild type L2 and L2H^R-25 nrdAB operon sequences were identical except for a single point mutation (C to A transversion) at base 3510 downstream from the nrdA ATG start codon. This point mutation lies in the putative ribosome binding site region upstream of nrdB (Fig. 8). The mutation was confirmed by sequencing the same region of the insert in pH^R25-2, the second plasmid containing the nrdAB operon.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Point mutation identified in the C. trachomatis L2HR-25 nucleotide sequence upstream of the nrdB translational start site. The nucleotide sequence between the nrdA TAA stop codon and the nrdB ATG start codon is shown for wild type C. trachomatis (L2) and hydroxyurea-resistant C. trachomatis (L2HU-25). The start and stop codons are underlined. The putative ribosome binding site is shown in boldface type, and the C to A transversion mutation is lightly shaded. The nucleotide sequence of the 3'-end of the chlamydial 16 S rRNA is shown, and the region that is complementary to the putative Shine-Dalgarno sequence is highlighted in boldface type.

Since there was only a single base pair change between the wild type L2 and the L2H^R-25 nrdAB sequence, it was important to ensure that it was not a simple sequence polymorphism. This was especially true, since the wild type L2 sequence we were using for comparison was from the chlamydial genome sequencing project and not the wild type L2 isolate used in our own laboratory, from which the hydroxyurea-resistant mutant chlamydiae was initially isolated (8). To check this, we screened our wild type L2 genomic pUC19 HindIII partial digest library (45) with the nrdAB PCR fragment, isolated a plasmid clone, and then sequenced the region of the nrdAB insert where the mutation lies. The sequence was identical to the L2 sequence deposited in the public data base.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The data presented here indicate that the C. trachomatis L2 nrdAB operon is expressed, R1 and R2 proteins are produced, and recombinant enzyme is active at reducing CDP to dCDP. Protein R1 and R2 amino acid sequence homologies, nrdAB operon structure, substrate phosphorylation state, allosteric regulation characteristics, and sensitivity to hydroxyurea clearly indicate that the chlamydial RNR belongs to the class Ia enzymes.

The prototypical class Ia enzyme is encoded by the nrdAB operon in E. coli (11). Recently, it has been shown that E. coli also has a class Ib enzyme, encoded by nrdE (R1E) and nrdF (R2F) (12), and an anaerobic class III enzyme, encoded by nrdD (46). With the release of complete genome sequence data for so many organisms, it has become clear that it is not unusual for procaryotes to have more than one RNR. Indeed, some Pseudomonas species contain genetic information for all three classes of RNRs and simultaneously express active class I and class II enzymes (31). While being essentially universal in eucaryotes and large DNA viruses, class Ia reductases are not that common in procaryotes (11). It is intriguing that of the procaryotes that contain class Ia enzymes, most (Chlamydiae species, Treponema palidum, Helicobacter pylori, P. aeruginosa, E. coli, Salmonella typhimurium) have documented associations with eucaryotes, typically human or non-human mammals. This suggests the possibility of horizontal gene transfer of the class Ia enzyme from eucaryotes to procaryotes. In the case of chlamydiae, T. palidum, and H. pylori, the only RNR present is the class Ia enzyme. Presumably, if these organisms have acquired the enzyme from eucaryotes, the original reductase present in their genomes has been lost. This implies that there is a selective advantage to keeping the class Ia enzyme.

Class Ia R1 proteins typically have two separate binding sites for allosteric effectors, one controlling substrate specificity, located at the homodimer interface, and the other regulating overall activity, located at the N-terminal domain (11, 47). The amino acid sequence of the chlamydial R1 proteins, as deduced from genome sequence data, indicate that there is an extended amino terminus, which includes an obvious 110-amino acid duplication. This region contains the VXKRDG allosteric activity site signature motif, which is characteristic of class Ia as well as some class II and most class III reductases (11). This region shows highest homology to the amino terminus of the class II and class III enzymes that contain the allosteric activity site. Our results indicate that while this region is not required for CDP reductase activity, it is essential for the allosteric inhibitory effects of dATP. These characteristics are not surprising, since active class Ib (12) and several class II enzymes (33) naturally lack the N-terminal domain and as a result do not show dATP negative activity control. In contrast, most class Ia reductases have the N-terminal domain and are feedback-inhibited by dATP (11). There is not, however, a strict correlation between the presence of the N-terminal domain and negative allosteric control. The class Ia enzymes of bacteriophage T4 (48), Trypanosoma brucei (49, 50) and P. aeruginosa (31), contain the N-terminal domain, but it has lost its physiological function.

The N-terminal extension of the chlamydial R1 protein raises the question of the location of the functional activity site. There is substantial sequence variation between the three putative signature motifs, with the one closest to the N terminus (IVKRNG) being the most similar to the VXKRDG sequence found in other reductases with an activity site. Of note is the fact that this IVKRNG sequence is completely conserved in all six chlamydiae R1 protein sequences known, whereas the other putative signature motifs show significant sequence variation. In E. coli, the allosteric activity site is located within the first 100 residues, which form a four-helix bundle covered by a three-stranded  sheet (47). Compared with the E. coli R1 structure, our chlamydial R1 deletion mutant has the first 17 of these 100 activity site residues removed. Clearly, more work including selective deletion of just the first and the first and second putative chlamydial R1 activity sites, by deletion mutagenesis, is needed to determine which of the three sites is responsible for regulating overall enzyme activity in response to nucleotide effectors. In addition, nucleotide binding experiments and studies on the regulation of UDP, GDP, and ADP reduction are required to obtain a better understanding of the chlamydial reductase allosteric regulation.

The most unusual feature of the chlamydial R2 protein is that it has a phenylalanine (Phe¹²⁷ by chlamydiae numbering) in place of the highly conserved tyrosine (Tyr¹²² in E. coli), which harbors the stable free radical of the enzyme (51). Site-directed mutagenesis studies with both the E. coli (51) and mouse (52) R2 proteins have shown that a phenylalanine for tyrosine substitution results in an inactive enzyme. The three-dimensional structure of the R2 protein shows that the Tyr¹²² is buried deep inside the protein in a hydrophobic pocket close to the iron center (53). The spatial organization of this environment is critical for radical generation and stability (32).

Our results indicate that, despite its unusual primary sequence, wild type chlamydial R2 is enzymatically active when complexed with R1. Furthermore, results with site-directed R2 mutants suggest that Tyr¹²⁹ (chlamydiae numbering) is a likely candidate for the radical. The R2Y129F (double phenylalanine) mutant was inactive, and the R2F127Y (double tyrosine) mutant was active. Interestingly, unlike all other recombinant chlamydial R2s, the double mutant (R2F127Y/Y129F), which places the tyrosine in a position expected from sequence alignments and a phenylalanine where the tyrosine is naturally located, was difficult to express in soluble form. Perhaps the relocation of the critical radical carrying tyrosine was a necessity for this reason alone. The R2F127Y/Y129F mutant did show a low level of CDP reductase activity, suggesting that any of the protein that does fold correctly is active. Biophysical studies (light absorption, circular dichroism, electron paramagnetic resonance spectroscopy) on the chlamydial wild type and site-directed R2 mutants are needed to provide definitive information on the nature of the metal ion (iron) cluster and the tyrosyl radical.

Results from Western blot analyses establish the mechanism of resistance to hydroxyurea in our C. trachomatis mutant, as an overproduction of both the R1 and R2 proteins. Elevated expression of the R2 protein is a well established means of attaining hydroxyurea resistance in mammalian cells (29, 42) and viruses (43). Mammalian cells selected for high level hydroxyurea resistance overexpress both the R1 and the R2 subunits (54, 55). In mammalian cells, R2 gene amplification with concurrent increases in mRNA are often responsible for the elevated R2 protein levels (29, 55).

With our L2H^R-25 mutant we found no evidence of alterations in genomic organization or changes in nrdAB mRNA levels. Sequencing and subsequent comparison of the complete nrdAB operon, including the upstream promoter region, from both wild type and hydroxyurea-resistant chlamydiae uncovered only a single base difference between the two. The C to A transversion 5 bases upstream of the R2 protein ATG start codon. The mutation lies in a region where the Shine-Dalgarno ribosome binding site (RBS) is typically located (56). Numerous in vitro and in vivo studies have clearly shown that translational efficiency can be dramatically affected by single base changes in the region just upstream of the ATG start codon and that purine-rich sequences often favor enhanced translation (57-59). The mutation in L2H^R-25 makes the putative RBS entirely purines and increases the match to the 3'-end of the chlamydial 16 S rRNA (CCTCC) to three (AGG) from two nucleotides (GG). The chlamydial major outer membrane protein gene, which is highly expressed, has a four (GAGG) out of five match to the 16 S rRNA 3'-end (16). Interestingly, in E. coli one mechanism by which dihydrofolate reductase can be overexpressed in trimethoprim-resistant isolates is by mutations in the RBS (60). With E. coli, it has also been shown that simple mutations that change mRNA secondary structure stability or alter the accessibility of the AUG initiator can have a dramatic effect on translation (61, 62).

It is also intriguing that the single mutation 5' of nrdB can lead to elevated expression of protein R1, which lies upstream of protein R2. This result clearly suggests that the level of expression of the two subunits is linked, which may not be surprising given that the active RNR is a complex of the two homodimers. With ribosomal protein operons, it has been shown that the product of one gene in an operon can regulate the expression of other genes in the same operon post-transcriptionally by binding to cis-acting sequences (63). At the present time, we do not know exactly how nrdAB gene expression is regulated in chlamydiae.

In conclusion, we have shown that despite its unusual primary structure the chlamydial RNR is active. Further work is needed to obtain a complete understanding of the enzymes allosteric regulation. In addition, we have demonstrated that the molecular mechanism responsible for hydroxyurea resistance in the L2H^R-25 mutant is overexpression of both the R1 and R2 proteins as a result of a single base change in the putative RBS upstream of the nrdB ATG start site. This hydroxyurea-resistant mutant will be a valuable tool for studies on the process of translational regulation in chlamydiae.

	ACKNOWLEDGEMENTS

We thank Professor Lars Thelander for reading the manuscript and all of our Swedish RNR colleagues for helpful discussions.

	FOOTNOTES

^* This work was supported by Medical Research Council of Canada Grant GR-13301 (to G. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Medical Microbiology, University of Manitoba, 730 William Ave., Winnipeg, Manitoba R3E 0W3, Canada. Tel.: 204-789-3307; Fax: 204-789-3926; E-mail: mcclart@cc.umanitoba.ca.

Published, JBC Papers in Press, September 12, 2000, DOI 10.1074/jbc.M006367200

	ABBREVIATIONS

The abbreviations used are: EB, elementary body; RB, reticulate body; RNR, ribonucleotide reductase; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; IPTG, isopropyl-1-thio--D-galactopyranoside; RBS, ribosome binding site.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				C. S. Andersson and M. Hogbom A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold PNAS, April 7, 2009; 106(14): 5633 - 5638. [Abstract] [Full Text] [PDF]

				N. Voevodskaya, F. Lendzian, O. Sanganas, A. Grundmeier, A. Graslund, and M. Haumann Redox Intermediates of the Mn-Fe Site in Subunit R2 of Chlamydia trachomatis Ribonucleotide Reductase: AN X-RAY ABSORPTION AND EPR STUDY J. Biol. Chem., February 13, 2009; 284(7): 4555 - 4566. [Abstract] [Full Text] [PDF]

				R. DeMars and J. Weinfurter Interstrain Gene Transfer in Chlamydia trachomatis In Vitro: Mechanism and Significance J. Bacteriol., March 1, 2008; 190(5): 1605 - 1614. [Abstract] [Full Text] [PDF]

				W. Jiang, D. Yun, L. Saleh, E. W. Barr, G. Xing, L. M. Hoffart, M.-A. Maslak, C. Krebs, and J. M. Bollinger Jr. A Manganese(IV)/Iron(III) Cofactor in Chlamydia trachomatis Ribonucleotide Reductase Science, May 25, 2007; 316(5828): 1188 - 1191. [Abstract] [Full Text] [PDF]

				E. Torrents, M. Westman, M. Sahlin, and B.-M. Sjoberg Ribonucleotide Reductase Modularity: ATYPICAL DUPLICATION OF THE ATP-CONE DOMAIN IN PSEUDOMONAS AERUGINOSA J. Biol. Chem., September 1, 2006; 281(35): 25287 - 25296. [Abstract] [Full Text] [PDF]

				N. Voevodskaya, A.-J. Narvaez, V. Domkin, E. Torrents, L. Thelander, and A. Graslund Chlamydial ribonucleotide reductase: Tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster PNAS, June 27, 2006; 103(26): 9850 - 9854. [Abstract] [Full Text] [PDF]

				R. Binet and A. T. Maurelli Frequency of Spontaneous Mutations That Confer Antibiotic Resistance in Chlamydia spp. Antimicrob. Agents Chemother., July 1, 2005; 49(7): 2865 - 2873. [Abstract] [Full Text] [PDF]

				M. Kolberg, D. T. Logan, G. Bleifuss, S. Potsch, B.-M. Sjoberg, A. Graslund, W. Lubitz, G. Lassmann, and F. Lendzian A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H: COMBINED X-RAY DIFFRACTION AND EPR/ENDOR STUDIES J. Biol. Chem., March 25, 2005; 280(12): 11233 - 11246. [Abstract] [Full Text] [PDF]

				M. Hogbom, P. Stenmark, N. Voevodskaya, G. McClarty, A. Graslund, and P. Nordlund The Radical Site in Chlamydial Ribonucleotide Reductase Defines a New R2 Subclass Science, July 9, 2004; 305(5681): 245 - 248. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 275/48/38111    most recent M006367200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roshick, C. Articles by McClarty, G. Search for Related Content PubMed PubMed Citation Articles by Roshick, C. Articles by McClarty, G. Social Bookmarking                      What's this?