Branched-chain polyamine stabilizes RNA polymerase at elevated temperatures in hyperthermophiles
Branched-chain polyamines (BCPAs) are unique polycations found in (hyper)thermophiles. Thermococcus kodakarensis grows optimally at 85 °C and produces the BCPA N4-bis(aminopropyl)spermidine by sequential addition of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine (SPD) by BCPA synthase A (BpsA). The T. kodakarensis bpsA deletion mutant (DBP1) did not grow at temperatures at or above 93 °C, and grew at 90 °C only after a long lag period following accumulation of excess cytoplasmic SPD. This suggests that BCPA plays an essential role in cell growth at higher temperatures and raises the possibility that BCPA is involved in controlling gene expression. To examine the effects of BCPA on transcription, the RNA polymerase (RNAP) core fraction was extracted from another bpsA deletion mutant, DBP4 (RNAPDBP4), which carried a His-tagged rpoL, and its enzymatic properties were compared with those of RNAP from wild-type (WT) cells (RNAPWT). LC–MS analysis revealed that nine ribosomal proteins were detected from RNAPWT but only one form RNAPDBP4. These results suggest that BCPA increases the linkage between RNAP and ribosomes to achieve efficient coupling of transcription and translation. Both RNAPs exhibited highest transcription activity in vitro at 80 °C, but the specific activity of RNAPDBP4 was lower than that of RNAPWT. Upon addition of SPD and BCPA, both increased the transcriptional activity of RNAPDBP4; however, elevation by BCPA was achieved at a tenfold lower concentration. Addition of BCPA also protected RNAPDBP4 against thermal inactivation at 90 °C. These results suggest that BCPA increases transcriptional activity in T. kodakarensis by stabilizing the RNAP complex at high temperatures.
KeywordsBranched-chain polyamine Polyamine RNA polymerase Transcription Archaea Hyperthermophile
Branched-chain polyamine synthase A
High-performance liquid chromatography
Liquid chromatography–mass spectrometry
Poly-acrylamide gel electrophoresis
Sodium dodecyl sulfate
TATA-box binding protein
Transcription factor B
This study was mainly supported by a grant from the Japan Society for the Promotion of Science (JSPS) KAKENHI (18K19191). Bioinformatic analysis was supported by a Grant for Individual Special Research, provided by Kwansei-Gakuin University.
All the authors contributed to study design. YY, MH, RH, MF, and SF performed the experiments. YY, FW, HA, and SF wrote the manuscript. All authors reviewed and approved the final draft.
This study was supported by a Grant from the Japan Society for the Promotion of Science (JSPS) KAKENHI (18K19191).
Compliance with ethical standards
Conflict of interest
The authors declare no conflicts of interest.
- Fujiwara S, Aki R, Yoshida M, Higashibata H, Imanaka T, Fukuda W (2008) Expression profiles and physiological roles of two types of molecular chaperonins from the hyperthermophilic archaeon Thermococcus kodakarensis. Appl Environ Microbiol 74:7306–7312. https://doi.org/10.1128/AEM.01245-08 CrossRefGoogle Scholar
- Fukuda W, Yamori Y, Hamakawa M, Hidese R, Kanesaki Y, Kainuma (Okamoto) A, Kato S, Fujiwara S (2019) Genes regulated by branched-chain polyamine in the hyperthermophilic archaeon Thermococcus kodakarensis. Submitted to Amino Acids Google Scholar
- Hamana K, Hamana H, Niitsu M, Samejima K, Sakane T, Yokota A (1993) Tertiary and quaternary branched polyamines distributed in thermophilic Saccharococcus and Bacillus. Microbios 75:23–32Google Scholar
- Hamana K, Hamana H, Niitsu M, Samejima K, Sakane T, Yokota A (1994) Occurrence of tertiary and quaternary branched polyamines in thermophilic archaebacteria. Microbios 79:109–119Google Scholar
- Hamana K, Niitsu M, Samejima K, Itoh T (2001) Polyamines of the thermophilic eubacteria belonging to the genera Thermosipho, Thermaerobacter and Caldicellulosiruptor. Microbios 104:177–185Google Scholar
- Hamana K, Tanaka T, Hosoya R, Niitsu M, Itoh T (2003) Cellular polyamines of the acidophilic, thermophilic and thermoacidophilic archaebacteria, Acidilobus, Ferroplasma, Pyrobaculum, Pyrococcus, Staphylothermus, Thermococcus, Thermodiscus and Vulcanisaeta. J Gen Appl Microbiol 49:287–293CrossRefGoogle Scholar
- Hidese R, Nishikawa R, Gao L, Katano M, Imai T, Kato S, Kanai T, Atomi H, Imanaka T, Fujiwara S (2014) Different roles of two transcription factor B proteins in the hyperthermophilic archaeon Thermococcus kodakarensis. Extremophiles 18:573–588. https://doi.org/10.1007/s00792-014-0638-9 CrossRefGoogle Scholar
- Hidese R, Tse KM, Kimura S, Mizohata E, Fujita J, Horai Y, Umezawa N, Higuchi T, Niitsu M, Oshima T, Imanaka T, Inoue T, Fujiwara S (2017) Active site geometry of a novel aminopropyltransferase for biosynthesis of hyperthermophile-specific branched-chain polyamine. FEBS J 284:3684–3701. https://doi.org/10.1111/febs.14262 Google Scholar
- Hirata A, Kanai T, Santangelo TJ, Tajiri M, Manabe K, Reeve JN, Imanaka T, Murakami KS (2008) Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature-sensitive. Mol Microbiol 70:623–633. https://doi.org/10.1111/j.1365-2958.2008.06430.x CrossRefGoogle Scholar
- Hosoya R, Hamana K, Niitsu M, Itoh T (2004) Polyamine analysis for chemotaxonomy of thermophilic eubacteria: Polyamine distribution profiles within the orders Aquificales, Thermotogales, Thermodesulfobacteriales, Thermales, Thermoanaerobacteriales, Clostridiales and Bacillales. J Gen Appl Microbiol 50:271–287CrossRefGoogle Scholar
- Morikawa M, Izawa Y, Rashid N, Hoaki T, Imanaka T (1994) Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp. Appl Environ Microbiol 60:4559–4566Google Scholar
- Morimoto N, Fukuda W, Nakajima N, Masuda T, Terui Y, Kanai T, Oshima T, Imanaka T, Fujiwara S (2010) Dual biosynthesis pathway for longer-chain polyamines in the hyperthermophilic archaeon Thermococcus kodakarensis. J Bacteriol 192:4991–5001. https://doi.org/10.1128/JB.00279-10 CrossRefGoogle Scholar
- Muramatsu A, Shimizu Y, Yoshikawa Y, Fukuda W, Umezawa N, Horai Y, Higuchi T, Fujiwara S, Imanaka T, Yoshikawa K (2016) Naturally occurring branched-chain polyamines induce a crosslinked meshwork structure in a giant DNA. J Chem Phys 145:235103. https://doi.org/10.1063/1.4972066 CrossRefGoogle Scholar
- Nishio T, Yoshikawa Y, Fukuda W, Umezawa N, Higuchi T, Fujiwara S, Imanaka T, Yoshikawa K (2018) Branched-chain polyamine found in hyperthermophiles induces unique temperature-dependent structural changes in genome-size DNA. Chem Phys Chem 19:2299–2304. https://doi.org/10.1002/cphc.201800396 CrossRefGoogle Scholar
- Okada K, Hidese R, Fukuda W, Niitsu M, Takao K, Horai Y, Umezawa N, Higuchi T, Oshima T, Yoshikawa Y, Imanaka T, Fujiwara S (2014) Identification of a novel aminopropyltransferase involved in the synthesis of branched-chain polyamines in hyperthermophiles. J Bacteriol 196:1866–1876. https://doi.org/10.1128/JB.01515-14 CrossRefGoogle Scholar
- Oshima T, Hamasaki N, Senshu M, Kakinuma K, Kuwajima I (1987) A new naturally occurring polyamine containing a quaternary ammonium nitrogen. J Biol Chem 262:11979–11981Google Scholar
- Tabor CW, Tabor H (1976) 1,4-Diaminobutane (putrescine), spermidine, and spermine. Annu Rev Biochem 45:285–306. https://doi.org/10.1146/annurev.bi.45.070176.001441 CrossRefGoogle Scholar
- Tabor CW, Tabor H (1984) Polyamines. Annu Rev Biochem 53:749–790. https://doi.org/10.1146/annurev.bi.53.070184.003533 CrossRefGoogle Scholar