In vivo and in vitro studies of Chlamydia trachomatis TrpR:DNA interactions.
We previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase (trpBA) and the tryptophan repressor (trpR). Here we employ primer extension analysis to identify the transcriptional origins of both trpR and trpBA, allowing for the identification of the putative operator sequences for both trpR and trpBA. Moreover we demonstrate that native recombinant chlamydial TrpR binds to the predicted operator sequence upstream of trpR. A restriction endonuclease protection assay was designed and used to demonstrate that 5-fluorotryptophan was the only tryptophan analogue capable of activating binding of native recombinant chlamydial TrpR to its operator. Additionally, 5-fluorotryptophan was the only analogue that repressed expression of trpBA at a level analogous to L-tryptophan itself. Based on these findings, a mutant selection protocol was designed and a C. trachomatis isolate containing a frameshift mutation in trpR was isolated. This chlamydial mutant synthesizes a truncated TrpR protein that cannot regulate expression of trpBA and trpR in response to changes in tryptophan levels. These findings provide the first genetic proof that TrpR acts as a negative regulator of transcription in C. trachomatis.
Carlson
John H
JH
Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, MT 59840, USA.
Wood
Heidi
H
Roshick
Christine
C
Caldwell
Harlan D
HD
McClarty
Grant
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009875:Operator Regions, Genetic; D018085:Regulon / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000235:genetics; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; D014364:Tryptophan; D014367:Tryptophan Synthase; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C026927:5-fluorotryptophan