©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of the Chlamydia trachomatis Gene for CTP Synthetase (*)

(Received for publication, October 17, 1994; and in revised form, January 18, 1995)

Graham Tipples (§) Grant McClarty (¶)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A HindIII partial digest Chlamydia trachomatis L2 library in pUC19 was screened for the CTP synthetase gene by functional complementation in CTP synthetase-deficient Escherichia coli JF646. A complementing clone was isolated and contained a recombinant plasmid (pH-1) with a 2.7-kilobase C. trachomatis DNA insert. The entire insert was sequenced and found to encode two complete open reading frames (ORFs) that overlapped by 25 bases and the start of a third ORF that overlapped with ORF2 by 14 bases. The derived amino acid sequence of ORFs 1 and 2 shows 37% identity to kdsB, an E. coli gene that codes for CMP-2-keto-3-deoxyoctulosonic acid synthetase and 48% identity to pyrG, an E. coli gene that codes for CTP synthetase, respectively. To obtain downstream sequence data for ORF3, colony hybridization screening of the HindIII chlamydial DNA library was used to isolate a second recombinant plasmid (pH-11) that contained a 1.7-kilobase chlamydial DNA insert. The deduced amino acid sequence of ORF3 is not significantly homologous to any protein in the translated GenBank data base. Recombinant chlamydial CTP synthetase appears to be similar to the E. coli enzyme in that it is sensitive to inhibition by CTP, requires UTP, ATP, Mg, GTP, and glutamine for activity, and can also utilize ammonia as an amido-group donor.


INTRODUCTION

Chlamydiae are obligate intracellular Gram-negative bacteria that are capable of infecting a wide range of eucaryotic host cells. Chlamydia trachomatis is a leading cause of preventable blindness in developing countries, and in industrialized nations it is a prevalent cause of sexually transmitted infections(1) . Chlamydiae have evolved a unique biphasic life cycle to accommodate survival both intracellularly and extracellularly. The metabolically inert but infectious elementary body (EB) (^1)is the extracellular form, and the non-infectious, metabolically active reticulate body (RB) is the intracellular vegetative form that replicates by binary fission(2, 3) . The chlamydial life cycle takes place within the confines of a membrane-bound vacuole that avoids fusion with host cell lysosomes(2) .

It has been suggested that chlamydiae likely draw on the host cell cytoplasm for a wide variety of metabolites that other free-living bacteria must synthesize for themselves(2, 4) . Such a loss of biosynthetic capacity has been used as an explanation for the small size of the chlamydial genome, 1.0 times 10^6 bp(5) . For a variety of reasons it is difficult to conduct definitive studies on chlamydial metabolism(3) . Conditions for cell-free growth have not been established, host-free purified RBs are fragile and display minimal metabolic activity, limited expression of chlamydial genes occurs in heterologous hosts, and a system for gene transfer has not been developed.

For the past several years we have been employing an in situ approach, using eucaryotic host cell lines with well defined mutations in nucleotide metabolism, to dissect the nucleotide biosynthetic capabilities of chlamydiae(4) . These studies have shown that C. trachomatis L2 is incapable of de novo purine and pyrimidine nucleotide biosynthesis(6) , that RBs have a limited capacity for nucleotide salvage(7) , and that RBs draw on the host cell ribonucleoside triphosphate (NTP) pool as a source of all four NTPs (8) . Recently, we have shown that C. trachomatis L2 is auxotrophic for only three (ATP, GTP, UTP) of the four ribonucleotides because they can synthesize CTP from UTP, a reaction catalyzed by CTP synthetase(6) . Since C. trachomatis L2 can obtain CTP directly from the host cell, it is unclear why they would encode a CTP synthetase.

To initiate the study of the molecular mechanisms regulating CTP synthetase expression, the C. trachomatis L2 CTP synthetase gene was cloned by functional complementation, and the recombinant enzyme was partially characterized. Nucleotide sequence analysis indicated that the chlamydial CTP synthetase is likely encoded in an operon with CMP-KDO synthetase, an enzyme involved in lipopolysaccharide (LPS) biosynthesis(9, 10, 11) , and a third gene of unknown function. The intriguing possibility that chlamydiae encode a CTP synthetase to support LPS biosynthesis is discussed.


EXPERIMENTAL PROCEDURES

Chemicals

[5,6-^3H]Uridine triphosphate (38 Ci/mmol) was obtained from DuPont NEN. [-P]ATP (7500 Ci/mmol) and [alpha-P]dATP (3500 Ci/mmol) were obtained from ICN. [6-^3H]Uracil (30 Ci/mmol) was obtained from Moravek Biochemicals Inc. Cycloheximide was purchased from Sigma. The random primer labeling kit and the DNA cycle sequencing kit were bought from Life Technologies, Inc. Oligonucleotides were synthesized on a Beckman DNA synthesizer.

Bacterial Strains

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. Escherichia coli strain JF646 (relevant genotype pyrE pyrG cdd argE his4 proA thr1 thi1 recA) has been previously described(12, 13) , as has recombinant plasmid pMW5 containing E. coli pyrG, encoding CTP synthetase(14) .

Preparation of C. trachomatis Genomic DNA and HindIII Library

C. trachomatis L2 was grown in mouse L cells in suspension culture, and EBs were purified through Renografin density gradients as described previously(15) . Chlamydial genomic DNA was isolated from highly purified EBs by standard procedures(16) . To construct the C. trachomatis L2 library, the chlamydial genomic DNA was partially digested with HindIII, and 2-4 kb fragments were ligated into pUC19(16) .

Complementation Screening for Chlamydial CTP Synthetase

Forty µl of competent E. coli JF646 was transformed with 20 ng of recombinant chlamydial DNA library by electroporation. After a 90-min recovery period in SOC medium (16) the cells were washed 2 times in Hanks' balanced salt solution and then plated onto selective medium and incubated at 37 °C until colonies appeared. The selective medium for the growth of E. coli JF646 consisted of: 1 times minimal A salts (60.2 mM K(2)HPO(4), 33.1 mM KH(2)PO(4), 7.6 mM (NH(4))(2)SO(4), and 1.7 mM sodium citrate), 0.2 mg/ml MgSO(4), 5 mg/ml glucose, 0.2 mg/ml thiamine, 1 mg/ml casamino acid, 10 µg/ml uracil, and 50 µg/ml ampicillin.

Incorporation of [6-^3H]Uracil into Recombinant E. coli JF646 Nucleic Acid

Duplicate 5-ml overnight cultures of E. coli JF646 harboring pH-1 (plasmid containing the chlamydial CTP synthetase gene), pMW5 (positive control plasmid containing the E. coli CTP synthetase gene), or pUC19 (negative control plasmid containing no insert) were grown in LB broth containing 50 µg/ml ampicillin. The cells were centrifuged (3,000 times g for 10 min) and resuspended in minimal A selective medium. A 1-ml aliquot was diluted to 2 ml in minimal A selective medium (A 0.6), and 10 µl of [6-^3H]uracil was added. The culture was incubated at 37 °C for 3 h, and the cells were collected by centrifugation as above. Total nucleic acid was isolated from cultures, degraded to free nucleobases by boiling in acid, and neutralized as described previously (17) . Isotope incorporation into nucleobases was monitored by on-line flow detection (Beckman 171 flow detector) after separation of the nucleobases by high performance liquid chromatography (HPLC)(17) . The identity of the radioactive peaks was confirmed by simultaneously monitoring the As of known uracil and cytosine standards. Data analyses were done with an IBM PC50 using Beckman System Gold software.

Extract Preparation and Conditions for in Vitro CTP Synthetase Assay

E. coli JF646 were transformed by electroporation with pH-1, pMW5, or pUC19 and then incubated for 90 min at 37 °C in SOC. One liter of LB was then inoculated with the SOC culture and incubated a further 2 h at 37 °C. 50 µg/ml ampicillin was added, and the culture was incubated overnight at 37 °C. Cells were collected by centrifugation, and extract was prepared as described by Long and Koshland(18) . CTP synthetase assay conditions were adapted from Anderson(19) . The CTP synthetase assay was carried out in a total volume of 100 µl as follows. The prereaction mixture (2 mM glutamine, 0.5 mM ATP, 0.1 mM UTP, 0.1 mM GTP, 10 mM MgCl(2), 1 µCi of [5,6-^3H]UTP in 20 mM Tris acetate buffer, pH 7.2) was equilibrated to 37 °C on a sand heating block. The reaction was started with the addition of the appropriate amount of extract and terminated after the appropriate amount of time by adding 20 µl of 4 N perchloric acid and immediately placing on ice. The mixture was neutralized by extracting with 1.1 times volumes of 78.1:21.9 (v/v) Freon-tri-N-octylamine. Fifty µl of the top aqueous layer was injected for HPLC analysis. The nucleotides were separated using 0.44 M ammonium phosphate buffer, pH 2.4, containing 2.5% acetonitrile on a Whatman Partisil 5 SAX column at a flow rate of 1 ml/min. The identity of the radioactive peaks was confirmed by simultaneously monitoring the As of known UTP and CTP standards. Data analyses were done with an IBM PC50 using Beckman System Gold software.


RESULTS

Screening by Functional Complementation

C. trachomatis L2 DNA was partially digested with HindIII and ligated into the pUC19 cloning vector. The resulting C. trachomatis L2 HindIII partial digest library was then screened for CTP synthetase by functional complementation in E. coli JF646. JF646 is deficient in functional CTP synthetase activity (12, 13) and is therefore auxotrophic for cytidine. After transformation with the chlamydial library, two colonies were isolated that grew on selective medium (containing ampicillin and lacking cytidine) and thus complemented the CTP synthetase activity deficiency of the host E. coli JF646. The plasmids from these complementing recombinant E. coli JF646 colonies were isolated and designated pH-1 and pH-2. The two plasmids appeared identical by restriction analysis (data not shown); therefore only one, pH-1, was further studied in detail. To confirm the complementation activity, pH-1 was again used to transform E. coli JF646 and was found to complement the CTP synthetase deficiency, this time giving confluent growth of recombinant colonies on the selective medium. The plasmid pH-1 contained a 2.7-kb chlamydial DNA insert in the HindIII cloning site of pUC19.

In Vivo CTP Synthetase Activity of Recombinant E. coli JF646

To determine if the recombinant E. coli containing pH-1 was capable of converting UTP to CTP, in vivo CTP synthetase activity was examined. [6-^3H]Uracil was added to the selective growth medium of cultures of recombinant E. coli JF646. The recombinant E. coli JF646 contained either pH-1, pMW5, or pUC19. After 3 h of incubation the nucleic acid was isolated and acid hydrolyzed, and the resulting free bases were analyzed by HPLC. The HPLC eluent was simultaneously monitored for UV absorbance and radioactivity. Radiolabeled cytosine was found in nucleic acid-derived free bases of the recombinant E. coli cultures containing either pMW5 or pH-1 (Fig. 1). The pUC19 negative control culture showed no labeling of cytosine bases.


Figure 1: Incorporation of [6-^3H]uracil into nucleic acid of the CTP synthetase activity-deficient E. coli JF646. E. coli JF646 has been transformed with pUC19 (dashed line), pH-1 (plasmid contains chlamydial CTP synthetase) (solid line), or pMW5 (plasmid contains E. coli CTP synthetase) (dotted line). Logarithmically growing E. coli JF646 were cultured in the presence of radiolabel for 3 h in minimal selective medium lacking cytidine. Total nucleic acid was extracted and acid-hydrolyzed to free nucleobases. The identity of the radioactive peaks was confirmed by simultaneously monitoring the A of known cytosine (C), uracil (U), and thymine (T) standards as shown on the chromatograms.



Nucleotide Sequence of the Insert in pH-1 and pH-11

To identify the gene(s) coding for the complementing activity, the 2.7-kb fragment was sequenced. DNA sequencing was carried out with double-stranded cycle sequencing based on the dideoxy chain termination method. The complete nucleotide sequence of the 2.7-kb HindIII insert of pH-1 is shown in Fig. 2. All nucleotide sequences shown were confirmed by cycle sequencing both strands of the double-stranded DNA. Analysis of this sequence for protein coding regions indicated the presence of two large overlapping ORFs, designated ORF1 and ORF2, plus part of a potential third ORF (ORF3) that overlapped with ORF2 (Fig. 3). ORF1 and ORF2 were 765 (nucleotide 350-1114) and 1620 (nucleotide 1090-2709) bases long, respectively. To determine whether a third ORF existed, the C. trachomatis L2 HindIII partial digest library was screened, by colony hybridization, for the genomic DNA fragment downstream of ORF2. A recombinant plasmid, designated pH-11, containing the desired downstream fragment was isolated. Partial sequence analysis of the 1.7-kb insert of pH-11 established that a third ORF (ORF3) of 447 (nucleotide 2696-3142) bases exists downstream of ORF2 ( Fig. 2and Fig. 3). The G + C ratio of the entire sequence is 41%.


Figure 2: Nucleotide sequence of C. trachomatis L2 DNA inserts in pH-1 and pH-11 (partial sequence). The deduced amino acid sequences of the three open reading frames are also shown. ORF1 (4) codes for CMP-KDO synthetase, ORF2(1090-2709) codes for CTP synthetase, and ORF3 (2696-3142) codes for a protein of unknown function. Potential ribosome binding sites are overlined. Numbers to the right and left of the sequence correspond to nucleotides and amino acids, respectively.




Figure 3: Schematic outline of pH-1, pH-11, and overlapping open reading frames determined from the DNA sequence derived from the chlamydial DNA inserts in these recombinant plasmids. pH-1 is a plasmid isolated by functional complementation using a partially digested HindIII C. trachomatis L2 genomic DNA library to transform CTP synthetase-deficient E. coli JF646. pH-11 was isolated by using a PCR-generated probe for colony hybridization screening of a partially digested HindIII C. trachomatis L2 DNA library. The thickersolidline represents the chlamydial DNA insert, and the thinnerdashedline represents the pUC19 cloning vector. Selected restriction enzyme sites are marked: H, HindIII; X, XbaI; P, PstI; and E, EcoRI. Forward (F) and reverse (R) primers as well as the lacZ transcriptional promoter (P) of the pUC19 vector are also shown. The open reading frames are represented by openrectangles and correspond to the genes for CMP-KDO synthetase (ORF1), CTP synthetase (ORF2), and unknown (ORF3). The direction of ORFs is indicated (5` 3`). The expanded regions of the gene boundaries are shown to indicate relative positions of the putative ribosome binding sites, the translational start sites (ATG), and the translational stop sites (TGA, TAA, or TAG).



The deduced amino acid sequences encoded by ORF1, ORF2, and ORF3 are shown under the corresponding nucleotide sequence in Fig. 2. Results from a comparison of the predicted amino acid sequences of ORF1, ORF2, and ORF3 with the translated GenBank data base (release 84) are shown in Table 1. ORF1 codes for a 254-amino acid polypeptide that is 37.6% identical with the E. coli kdsB gene product (10) and 34.6% identical with the E. coli kpsU gene product (11) . Both of these E. coli genes code for CMP-KDO synthetase. Alignment of the amino acid sequences of the two E. coli and C. trachomatis CMP-KDO synthetases is shown in Fig. 4. ORF2 codes for a 539-amino acid polypeptide that is 48.6% identical with the E. coli pyrG gene product. pyrG codes for CTP synthetase. Alignment of the amino acid sequences of all known CTP synthetases is shown in Fig. 5. ORF3 codes for a 148-amino acid polypeptide that is not significantly homologous to any sequence in the translated GenBank data base. In addition, no nucleotide sequence homology was found to any sequence in GenBank.




Figure 4: Comparison of deduced amino acid sequence of CMP-KDO synthetases of C. trachomatis L2 (CHTKDSB), E. coli kdsB (ECOKDSB) (10) , and E. coli kpsU (ECOKPSU)(11) . Gaps are used to give the best alignment. Identical amino acid residues are indicated by an asterisk, and similarity between amino acids is shown by a dot. Alignments are done by the FASTP program(30) .




Figure 5: Comparison of deduced amino acid sequence of CTP synthetases of C. trachomatis L2 (CHTCTPS), B. subtilis (BSCTPS)(26) , E. coli (ECCTPS)(14) , Azospirillum brasilense (ABCTPS), (^2)Spiroplasma citri (SMECTPS), (^3)Saccharomyces cerevisiae URA7 (SC7CTPS)(28) , S. cerevisiae URA8 (SC8CTPS)(29) , and human (HUCTPS)(27) . Identical amino acid residues are indicated by an asterisk, and similarity between amino acids is shown by a dot. Alignments are done by the FASTP program(30) . Also indicated above the sequence alignment (plus sign) are the residues of the glutamine amidotransferase domain consensus sequence(28) : (NH(2) terminus). . . . G. . . . G-C-G-Q. . . . HPE. . . . (COOH terminus).



Analysis of the overlapping region between the ORFs indicates a 25-bp overlap between the ATG start codon of ORF2 and the TGA stop codon of ORF1 and a 14-base overlap between the ATG start codon of ORF3 and the TAA stop codon of ORF2 (Fig. 3). The arrangement of the cloned genes in an operon seemed likely considering the overlapping arrangement of the genes. It appears that the putative operon consists of only ORF1, ORF2, and ORF3 for two reasons: 1) no ATG start codon or any putative ribosome binding site is evident either upstream or downstream of the TAG stop codon of ORF3, and 2) the nucleotide sequence immediately upstream of ORF1 and immediately downstream of ORF3 indicate many stop codons are present in all reading frames.

The orientation of the 2.7-kb DNA insert in pH-1 places the lacZ transcriptional promoter upstream of the ORFs (Fig. 3). An experiment in which the orientation of the 2.7-kb insert in the pUC19 vector was reversed failed to show complementation activity after transformation of E. coli JF646. This suggests that transcription of the chlamydial DNA was most likely driven by the lacZ promoter of pUC19. No rho-independent transcriptional stop sequences are evident at the 3`-end of any of the three genes. Putative ribosome binding sites were identified upstream from the presumed initiation codon for each open reading frame ( Fig. 2and Fig. 3). Interestingly, each open reading frame terminates with a different stop codon; ORF1-TGA, ORF2-TAA, and ORF3-TAG.

A PCR product, corresponding to the entire CTP synthetase ORF, was random primer P-labeled and used to probe a Southern blot of genomic DNA, from several sources, completely digested with a number of restriction enzymes (data not shown). The results suggest that C. trachomatis L2 CTP synthetase is a single copy gene. Under the conditions used, there was no cross-hybridization with E. coli, Acholeplasma laidlawii, or Chlamydia psittaci DNA.

In Vitro CTP Synthetase Assay

Extracts were prepared from E. coli JF646 transformed with pH-1, pMW5, and pUC19 for in vitro CTP synthetase assays as outlined under ``Experimental Procedures.'' Negative control extracts derived from E. coli JF646 transformed with pUC19 showed no detectable activity under any conditions (data not shown). The results of the in vitro assays for extracts derived from recombinant E. coli JF646 expressing the gene for the C. trachomatis CTP synthetase are shown in Table 2. Using complete assay mix for the glutamine assay, CTP synthetase activity was found to be 11.6 ± 0.7 nmol of CTP produced per min per mg of protein. Eliminating GTP (activator for the glutamine assay) or ATP (energy source) from the reaction mixture reduced enzyme activity to below the sensitivity of the assay. Decreasing the concentration of MgCl(2) or glutamine reduced enzyme activity by 54 and 85%, respectively. The presence of CTP (feedback inhibitor) in the reaction mixture decreased CTP synthetase activity by 95%. The ammonia assay, which does not require the activator GTP, also showed enzyme activity. Results for pMW5, E. coli CTP synthetase, follow the same trends (i.e. requires ATP, GTP, MgCl(2), and glutamine for maximal activity and is feedback inhibited by CTP) as the C. trachomatis CTP synthetase activity but express relatively higher amounts of activity. For example, using complete assay conditions for the glutamine assay the activity was 24.9 ± 0.8 nmol of CTP produced per min per mg of protein.




DISCUSSION

Our previous work (6) showing that C. trachomatis L2 could convert UTP to CTP suggested that chlamydiae encode a CTP synthetase; an intriguing observation since CTP can also be obtained directly from the host cell cytoplasm(8) . In our present study, the existence of a C. trachomatis L2-specific CTP synthetase was confirmed by a number of experiments. (i) A C. trachomatis-specific DNA fragment cloned into pUC19 (pH-1) was capable of complementing the deficiency of CTP synthetase activity in E. coli JF646. (ii) E. coli JF646 transformed with pH-1 was capable of converting radiolabeled exogenous uracil to cytosine nucleotides. (iii) The derived amino acid sequence of a portion of the C. trachomatis-specific DNA fragment was shown to share high sequence identity (42-49% overall) with known CTP synthetases. The amidotransferase amino acid consensus sequence common to all CTP synthetases sequenced so far was completely conserved in the chlamydial enzyme (Fig. 5). (iv) Southern hybridizations with genomic DNA preparations indicated that the gene was C. trachomatis-specific and single copy. (v) Finally, in vitro CTP synthetase activity was detected in extracts prepared from E. coli JF646 transformed with pH-1.

CTP synthetase of E. coli has been thoroughly studied and is known to carry out the conversion of UTP to CTP using both glutamine (in the presence of GTP) and ammonia as the amino-group donor (18, 20) . In addition, the E. coli CTP synthetase also requires Mg and ATP for enzymatic activity(18, 20) . Although the in vitro C. trachomatis CTP synthetase assays were performed with only crude enzyme preparations, it was evident that there was an absolute requirement for ATP and GTP in the glutamine-donor assay. Sufficient concentrations of MgCl(2) and glutamine are also required for maximal activity. In addition, like CTP synthetases of E. coli and mammalian cells(18, 21) , the C. trachomatis CTP synthetase is inhibited by CTP. Like E. coli, the C. trachomatis CTP synthetase reaction can use ammonium sulfate (ammonia) as the amino donor in the absence of the allosteric effector, GTP. These results suggest that the CTP synthetase of C. trachomatis L2 shares similar properties with the well studied E. coli CTP synthetase. CTP synthetase activity was not detected in crude extracts prepared from purified RBs (data not shown). Possibly the combination of low CTP synthetase enzyme amounts, competition for UTP substrate, and/or the presence of inhibitors (such as CTP) may explain the lack of activity detected from the crude RB extract. Further enzymatic characterization of the CTP synthetase of C. trachomatis L2 will require the preparation of highly purified recombinant protein.

The overlapping arrangement of the chlamydial CTP synthetase gene with two other open reading frames suggests that these genes may be in an operon. Although several chlamydial operons have been characterized, this is the first report of an overlapping gene arrangement of this sort in chlamydiae. This overlapping arrangement is similar to that described for a number of E. coli and Bacillus subtilis operons and may be suggestive of translational coupling (22) . Translational coupling is a proposed mechanism of regulation whereby gene translation from a polycistronic mRNA is at least partially dependent on translation of an upstream gene. The function of translational coupling would be to allow the proportionate synthesis of functionally related proteins. The two requirements of translational coupling are slightly overlapping genes and the presence of a ribosome binding site in the vicinity of the ATG start codon for each gene of the operon(22) . Both of these conditions are present for the chlamydial operon. However, the translation stop-translation start overlaps (14- and 25-bp overlaps) are larger for this chlamydial operon than the 1-8-bp overlaps suggested by Zalkin and Ebbole (22) to favor translational coupling.

CMP-KDO synthetase is responsible for activating KDO, an 8-carbon sugar, for its subsequent incorporation into Gram-negative LPS(9, 23) . This reaction requires CTP and Mg. Thus, it appears that the CTP synthetase of C. trachomatis L2 is contained in an operon with a functionally related protein involved in LPS biosynthesis. CMP-KDO is the substrate for 3-deoxy-D-manno-octulosonic acid transferase (KDO transferase). KDO transferase transfers the KDO sugars onto the lipid A moiety during LPS biosynthesis(9) . The gene (gseA) for KDO transferase has recently been cloned from C. trachomatis(24) and C. psittaci(25) .

It may be that there is a certain period in the chlamydial life cycle when there is a high demand for LPS biosynthesis (for example during maximum RB replication). At this time of maximal LPS biosynthesis, there may be a higher than usual draw on the CTP pool in the RB such that the CTP taken directly from the host cell cytoplasm is not sufficient to meet the demand for both LPS biosynthesis and nucleic acid synthesis. Alternately, there may be a local draw on the CTP pool during LPS biosynthesis such that it is more efficient to channel the CTP directly to the CMP-KDO synthetase via the action of an associated CTP synthetase than to rely on the general intracellular CTP pool of the RB. Although there is no net use of cytidine nucleotides during LPS biosynthesis, there is a net loss of high energy phosphates. Being an energy parasite(2) , chlamydiae may not be able to resynthesize CTP via nucleotide kinases or may not be able to salvage cytidine nucleotides quickly enough back to the triphosphate level during LPS biosynthesis.

This is the first report of a CTP synthetase possibly being encoded as part of an operon, although E. coli pyrG (CTP synthetase gene) may be transcribed with the enolase gene (eno) as a pyrG eno polycistronic mRNA(14) . More definitive evidence, such as Northern blot analysis, reverse transcriptase-PCR, and/or S1 nuclease mapping, for the chlamydial CTP synthetase being encoded by a polycistronic mRNA is needed. Unfortunately, while chlamydial structural gene transcripts are readily detected by Northern blot analysis, it has proven difficult to detect transcripts for metabolic genes, most likely due to the low concentration and high instability of the particular mRNA species being studied.

Our finding that chlamydiae can obtain CTP in two ways, salvage directly from the host cell cytoplasm (8) and via de novo synthesis from host-supplied UTP(6) , is the first detailed description of alternate nutrient acquisition options for chlamydiae. The cloning of C. trachomatis L2 CTP synthetase will enable us to address a variety of questions about the regulation of chlamydial gene expression in response to altered nutrient (CTP) availability from the host cell. Studies of this type are currently ongoing.


FOOTNOTES

*
This work was supported by a grant from the Medical Research Council of Canada (to G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U15192[GenBank].

§
Supported by a studentship from the Manitoba Health Research Council.

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: 204-783-5255.

(^1)
The abbreviations used are: EB, elementary body; RB, reticulate body; bp, base pair(s); KDO, 2-keto-3-deoxyoctulosonic acid; LPS, lipopolysaccharide; kb, kilobase(s); HPLC, high pressure liquid chromatography; ORF, open reading frame; PCR, polymerase chain reaction.

(^2)
W. Zimmer and B. Hundeshagen, EMBL Data Library accession number S 25101.

(^3)
C. Citti, C. Saillard, and J. M. Bove, GenBank Data Library accession number L 22971.


ACKNOWLEDGEMENTS

We thank Dr. J. D. Friesen for supplying E. coli JF646 and Dr. H. Zalkin for recombinant plasmid pMW5.


REFERENCES

  1. Fraiz, J., and Jones, R. B. (1988) Annu. Rev. Med. 39, 357-370 [CrossRef][Medline] [Order article via Infotrieve]
  2. Moulder, J. W. (1991) Microbiol. Rev. 55, 143-190
  3. Stephens, R. S. (1993) Infect. Agents Dis. 1, 279-293
  4. McClarty, G. (1994) Trends Microbiol. 2, 157-163 [CrossRef][Medline] [Order article via Infotrieve]
  5. Birkelund, S., and Stephens, R. S. (1992) J. Bacteriol. 174, 2742-2747 [Abstract]
  6. Tipples, G., and McClarty, G. (1993) Mol. Microbiol. 8, 1105-1114 [Medline] [Order article via Infotrieve]
  7. Wang, L., Henson, E., and McClarty, G. (1994) Mol. Microbiol. 14, 271-281 [Medline] [Order article via Infotrieve]
  8. McClarty, G., and Tipples, G. (1991) J. Bacteriol. 173, 4922-4931 [Medline] [Order article via Infotrieve]
  9. Raetz, C. R. H. (1990) Annu. Rev. Biochem. 59, 129-170 [CrossRef][Medline] [Order article via Infotrieve]
  10. Goldman, R. C., Bolling, T. J., Kohlbrenner, W. E., Kim, Y., and Fox, J. L. (1986) J. Biol. Chem. 261, 15831-15835 [Abstract/Free Full Text]
  11. Pazzani, C., Rosenow, C., Boulnois, G. J., Bronner, D., Jann, K., and Roberts, I. S. (1993) J. Bacteriol. 175, 5978-5983 [Abstract]
  12. Friesen, J. D., An, G., and Fiil, N. P. (1978) Cell 15, 1187-1197 [Medline] [Order article via Infotrieve]
  13. Friesen, J. D., Parker, J., Watson, R. J., Fiil, N. P., Pedersen, S., and Pedersen, F. S. (1976) J. Bacteriol. 127, 917-922 [Medline] [Order article via Infotrieve]
  14. Weng, M., Makaroff, C. A., and Zalkin, H. (1986) J. Biol. Chem. 261, 5568-5574 [Abstract/Free Full Text]
  15. Fan, H., McClarty, G., and Brunham, R. C. (1991) J. Bacteriol. 173, 6670-6677 [Medline] [Order article via Infotrieve]
  16. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  17. McClarty, G., and Qin, B. (1993) J. Bacteriol. 175, 4652-4661 [Abstract]
  18. Long, C., and Koshland, D. E., Jr. (1978) Methods Enzymol. 51, 79-83 [Medline] [Order article via Infotrieve]
  19. Anderson, P. M. (1983) Biochemistry 22, 3285-3292 [Medline] [Order article via Infotrieve]
  20. Koshland, D. E., Jr., and Levitzki, A. (1994) Enzymes 10, 539-559
  21. McPartland, R. P., and Weinfeld, H. (1979) J. Biol. Chem. 254, 11394-11398 [Abstract]
  22. Zalkin, H., and Ebbole, D. J. (1988) J. Biol. Chem. 263, 1595-1598 [Free Full Text]
  23. Unger, F. M. (1981) Adv. Carbohydr. Chem. Biochem. 38, 23-38
  24. Belunis, C. J., Mdluli, K. E., Raetz, C. R. H., and Nano, F. E. (1992) J. Biol. Chem. 267, 18702-18707 [Abstract/Free Full Text]
  25. Mamat, U., Baumann, M., Schmidt, G., and Brade, H. (1993) Mol. Microbiol. 10, 935-941 [Medline] [Order article via Infotrieve]
  26. Trach, K., Chapman, J. W., Piggot, P., LeCoq, D., and Hoch, J. A. (1988) J. Bacteriol. 170, 4194-4208 [Medline] [Order article via Infotrieve]
  27. Yamauchi, M., Yamauchi, N., and Meuth, M. (1990) EMBO J. 9, 2095-2099 [Abstract]
  28. Ozier-Kalogeropoulos, O., Fasiolo, F., Adeline, M.-T., Collin, J., and Lacroute, F. (1991) Mol. & Gen. Genet. 231, 7-16
  29. Ozier-Kalogeropoulos, O., Adeline, M. T., Yang, W. L., Carman, G., and Lacroute, F. (1994) Mol. & Gen. Genet. 242, 431-439
  30. Lipman, D. J., and Pearson, W. R. (1985) Science 227, 1435-1441 [Medline] [Order article via Infotrieve]

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