CHLAMYDIA-TRACHOMATIS CTP SYNTHETASE - MOLECULAR CHARACTERIZATION ANDDEVELOPMENTAL REGULATION OF EXPRESSION

Chlamydia trachomatis is a nucleotide parasite, being entirely depende nt on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chla mydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydi al CTP synthetase. This study addresses the developmental regulation o f CTP synthetase over the course of the C. trachomatis life cycle. Giv en the distinct life stages of C. trachomatis, analysis of temporal ch anges in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The r esults of immunodetection analysis indicate that CTP synthetase is pre sent in C. trachomatis elementary bodies and reticulate bodies and tha t it is widespread in other chlamydial strains. Reverse transcriptase- polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily duri ng the mid-and late stages of the chlamydial growth cycle. In addition , C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxyoctanoic acid synthetase (CMP-KDO synthetase ) as part of a polycistronic mRNA. The co-expression of these two enzy mes suggests a role for CTP synthetase in lipopolysaccharide biosynthe sis, potentially channelling CTP directly to CMP-KDO synthetase. The a bility of the intact operon to complement CTP synthetase and CMP-KDO d eficiencies in mutant Escherichia coli strains indicates that both enz ymes are efficiently translated from a single messenger RNA. Kinetic a nalysis revealed that the C. trachomatis CTP synthetase possessed co-o perativity patterns typical of both prokaryotic and eukaryotic CTP syn thetases. However, the K-m of the enzyme for UTP was lower than that o f E. coli CTP synthetase, presumably in response to the low intracellu lar concentration of this nucleotide in C. trachomatis.