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(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 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) ( 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 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-
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
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), (
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
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 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 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].
Volume 270,
Number 14,
Issue of April 7, 1995 pp. 7908-7914
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, GTP, and glutamine for activity, and can also
utilize ammonia as an amido-group donor.
)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) .
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.
Chemicals
[5,6-
H]Uridine
triphosphate (38 Ci/mmol) was obtained from DuPont NEN.
[
-
P]ATP (7500 Ci/mmol) and
[
-P]dATP (3500 Ci/mmol) were obtained from
ICN. [6-H]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 minimal A salts (60.2
mM K
HPO
, 33.1 mM KHPO, 7.6 mM (NH)SO, and 1.7 mM sodium citrate), 0.2 mg/ml MgSO, 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-
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
H]Uracil
into Recombinant E. coli JF646 Nucleic Acid 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-H]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 A
s 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, 1 µCi of [5,6-H]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 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.
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-H]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.
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.
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%.
) 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).
)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).
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 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, 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.
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.. 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) .
)))
We thank Dr. J. D. Friesen for supplying E. coli JF646 and Dr. H. Zalkin for recombinant plasmid pMW5.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 S. Gronow and H. Brade Invited review: Lipopolysaccharide biosynthesis: which steps do bacteria need to survive? Innate Immunity, February 1, 2001; 7(1): 3 - 23. [Abstract] [PDF]
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 G. Leclerc, S. P. Wang, and B. Ely A New Class of Caulobacter crescentus Flagellar Genes J. Bacteriol., October 1, 1998; 180(19): 5010 - 5019. [Abstract] [Full Text]
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D. B. Ostrander, D. J. O'Brien, J. A. Gorman, and G. M. Carman Effect of CTP Synthetase Regulation by CTP on Phospholipid Synthesis in Saccharomyces cerevisiae J. Biol. Chem., July 24, 1998; 273(30): 18992 - 19001. [Abstract] [Full Text] [PDF]
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J. L. Wylie, L. L. Wang, G. Tipples, and G. McClarty A Single Point Mutation in CTP Synthetase of Chlamydia trachomatis Confers Resistance to Cyclopentenyl Cytosine J. Biol. Chem., June 28, 1996; 271(26): 15393 - 15400. [Abstract] [Full Text] [PDF]
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W.-L. Yang, M. E. C. Bruno, and G. M. Carman Regulation of Yeast CTP Synthetase Activity by Protein Kinase C J. Biol. Chem., May 10, 1996; 271(19): 11113 - 11119. [Abstract] [Full Text] [PDF]
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C. Roshick, E. R. Iliffe-Lee, and G. McClarty Cloning and Characterization of Ribonucleotide Reductase from Chlamydia trachomatis J. Biol. Chem., November 22, 2000; 275(48): 38111 - 38119. [Abstract] [Full Text] [PDF]
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