Since host cell-derived thymidine is not incorporated into Chlamydia trachomatis
DNA, we hypothesized that chlamydiae must synthesize dTMF de novo for DNA
replication. The only known enzyme performing de novo dTMP synthesis is thymidylate
synthase (TS). The goals of this thesis were to provide biochemical evidence for the
existence of TS in chlamydiae, to investigate the mechanism by which the parasite
obtains folate, a necessary cofactor for TS, and to provisionally characterize chlamydial
TS. Results of a series of in situ experiments using a mutant cell line as chlamydial host
which is incapable of de novo dTMP synthesis suggest that C. trachomatis converts
dUMP into dTXP. In vitro experiments conclusively establish these findings by the
demonstration of TS activity in extracts prepared from host-free chlamydial reticulate
bodies. Furthermore it was found that both sulfa-sensitive and sulfa-resistant chlamydial
strains can synthesize folates de novo; however strains vary significantly in their ability
to transport preformed folates from the host cell. A C. trachomatis gene which is capable
of complementing thymidine auxotrophy in Escherichia coli deficient in TS was cloned.
Auxotrophic E. coli containing the complementing chlamydial DNA sequence converts
dUMP to dTMP, using methylene tetrahydrofolate as the cofactor. The complementing
DNA fragment contains an open reading frame of 1587 bp. Surprisingly this open
reading frame shows absence of sequence homology to known TS. Unique in vitro
characteristics shared by the enzyme activities from both chlamydial extract and
recombinant E. coli extract suggest that C. trachomatis might encode a novel TS.
| Identifer | oai:union.ndltd.org:MANITOBA/oai:mspace.lib.umanitoba.ca:1993/22224 |
| Date | 02 October 2013 |
| Creators | Fan, Huizhou |
| Source Sets | University of Manitoba Canada |
| Detected Language | English |
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