Cloning and expression of the Chlamydia trachomatis gene for CTP synthetase

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.