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, Mg2+, GTP, and glutamine for activity, and can also utilize ammonia as an amidogroup donor.
Highlights
§ To whom correspondence should be addressed: Dept. of Medical Microbiology, University of Manitoba, 730 William Ave., Winnipeg, Manitoba R3E OW3, Canada
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)
Screening by Functional Complementation-C. trachomatis L2 DNA was partially digested with HindIII and ligated into the pUC19 cloning vector
Summary
Chemicals-[5,6-3HlUridine triphosphate (38 Ci/mmol)was obtained from DuPont NEN. [y_32PIATP (7500 Ci/mmol) and [a_3 2PldATP (3500 Ci/mmol) were obtained from ICN. [6-3HIUracii (30 Ci/mmol) was obtained from Moravek Biochemicals Inc. Genomic DNAwas isolated from highly purified EBs by standard procedures (16).To construct the C. trachomatis L2. 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).Chlamydia! C. trachomatis CTP Synthetase library, the chlamydial genomic DNA was partially digested with HindIII, and 2-4 kb fragments were ligated into pUC19 (16). Extract Preparation and Conditions for in Vitro CTP Synthetase Assay-E. coli JF646 were transformed by electroporation with pH-I, pMW5, or pUC19 and incubated for 90 min at 37°C in SOC. The identity of the radioactive peaks was confirmed by simultaneously monitoring the A25.s of known UTP and CTP standards. Data analyses were done with an IBM PC 50 using Beckman System Gold software
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