Two glycine riboswitches activate the glycine cleavage system essential for glycine detoxification in Streptomyces griseus.
The glycine cleavage (GCV) system catalyzes the oxidative cleavage of glycine into CO2, NH4(+), and a methylene group, which is accepted by tetrahydrofolate (THF) to form N(5),N(10)-methylene-THF. Streptomyces griseus contains gcvP and the gcvT-gcvH operon, which encode three intrinsic components of the GCV system. We identified the transcriptional start sites of gcvTH and gcvP and found putative glycine riboswitches in their 5' untranslated regions (5' UTRs). The ratios of the transcripts of the gcvT and gcvP coding sequences (CDSs) to those of the respective 5' UTRs were significantly higher in the presence of glycine in the wild-type strain. However, the levels of gcvT and gcvP CDS transcripts were not increased by glycine in the respective 5' UTR deletion mutants. A reporter gene assay showed that a transcriptional terminator exists in the 5' UTR of gcvTH. Furthermore, by an in-line probing assay, we confirmed that glycine bound directly to the putative riboswitch RNAs. These results indicate that the S. griseus glycine riboswitches enhance transcriptional read-through to the downstream CDSs, like known glycine riboswitches in other bacteria. We examined the growth of three mutants in which either or both of the gcvTH and gcvP 5' UTRs were deleted. Like the wild-type strain, all mutants grew vigorously in a medium containing 0.9% glucose as a carbon source. However, the mutants showed severely restricted growth in a medium containing 0.9% glucose and 1% glycine, while the wild-type strain grew normally. This indicates that glycine has a growth-inhibitory effect and that the GCV system plays a critical role in glycine detoxification in S. griseus.
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Takeaki
T
Tezuka
Yasuo
Y
Ohnishi
D020121:5' Untranslated Regions; D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005998:Glycine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009876:Operon; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D058928:Riboswitch; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014166:Transferases / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation
D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D012329:RNA, Bacterial; D058928:Riboswitch; C064525:glycine cleavage system; D000594:Amino Acid Oxidoreductases; D014166:Transferases; D005998:Glycine