[P004] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P02-252] Functional analysis of a transcriptional regulator involved in arginine metabolism in the hyperthermophilic archaeon Thermococcus kodakarensis

○Naoki Ohashi¹, Yuta Michimori¹, Haruyuki Atomi^1,2 (1. Graduate School of Engineering, Kyoto University (Japan), 2. Integrated Research Center for Carbon Negative Science, Institute of Advanced Energy, Kyoto University (Japan))

Keywords:

archaea,transcription,regulation,arginine

A novel arginine catabolic pathway, designated the arginine synthetase pathway, was recently discovered in the hyperthermophilic archaeon Thermococcus kodakarensis. This pathway consists of arginine synthetase (ArcE), a novel enzyme that catalyzes the energy-conserving conversion between arginine and citrulline, ornithine transcarbamoylase (ArcB), and carbamate kinase (ArcC). The ornithine generated from the ArcB reaction is further converted to glutamate semialdehyde by ornithine ω-aminotransferase (OrnAT) and eventually to proline. T. kodakarensis is known to be an arginine auxotroph, and therefore, arginine degradation should be strictly regulated in order to avoid its depletion. In this study, we searched for a transcriptional regulator governing arginine metabolism in T. kodakarensis.
We searched for a binding site in the promoter regions of genes encoding enzymes involved in arginine metabolism. We found a common palindromic motif in the promoters of genes encoding ArcE, OrnAT, and a putative Lrp/AsnC transcriptional regulator, which we designate as ArcR. Assuming that this putative regulator is involved in the regulation of arginine metabolism, we examined the function of ArcR. Lrp/AsnC-type transcriptional regulators are commonly found in prokaryotes and are known to regulate the transcription levels of genes involved in amino acid metabolism. However, the physiological role of each regulator is often difficult to predict based on primary structure alone.
Recombinant ArcR produced in Escherichia coli was purified. Electro Mobility Shift Assays (EMSA) were performed using the purified ArcR protein and DNA fragments containing promoter regions of genes involved in arginine metabolism. ArcR displayed specific binding to promoters of genes encoding ArcE and OrnAT.
We constructed an arcR gene disruption strain (ΔarcR). The mRNA transcript levels of ΔarcR and the host strain KUR1 were compared. To examine the physiological role of ArcR in T. kodakarensis, we carried out growth experiments of the ΔarcR strain in synthetic media with varying concentrations of amino acids. Based on our results, the physiological roles of ArcR in arginine metabolism in T. kodakarensis will be discussed.