(Received for publication, October 17, 1994; and in revised form, January 18, 1995)
From the
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.
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) ()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 10
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.
Figure 1:
Incorporation of
[6-H]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.
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), ()Spiroplasma citri (SMECTPS), (
)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
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.
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
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U15192[GenBank].