Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression.
Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that 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 during 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-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA. The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the K(m) of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.
Wylie
J L
JL
Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada.
Berry
J D
JD
McClarty
G
G
D000818:Animals; D016466:CHO Cells; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D002692:Chlamydia trachomatis / Q000201:enzymology*; D006224:Cricetinae; D003570:Cytidine Triphosphate / Q000378:metabolism; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D014544:Uridine Triphosphate / Q000378:metabolism
D012333:RNA, Messenger; D003570:Cytidine Triphosphate; D012194:RNA-Directed DNA Polymerase; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D014544:Uridine Triphosphate