Kinase, putative Endopeptidase, and Other Proteins of Small size (KEOPS) is a multisubunit protein complex conserved in eukaryotes and archaea. It is composed of Pcc1, Kae1, Bud32, Cgi121, and Gon7 in eukaryotes and is primarily involved in N 6 -threonylcarbamoyl adenosine (t 6 A) modification of transfer RNAs (tRNAs). Recently, it was reported that KEOPS participates in homologous recombination (HR) repair in yeast. To characterize the KEOPS in archaea (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus. We show that aKEOPS also possesses five subunits, Pcc1, Kae1, Bud32, Cgi121, and Pcc1-like (or Gon7-like), just like eukaryotic KEOPS. Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1-Pcc1-Pcc1-Kae1), suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1-like, can be deleted in the wild type and in a t 6 A modification complementary strain named TsaKI, implying that the aKEOPS complex is essential for an additional cellular process in this archaeon. Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI. These results suggest that aKEOPS plays an essential role independent of the cellular t 6 A modification level. In addition, archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3ʹ CCA tail binding module. Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7. The study also reveals a possible link between the function in t 6 A modification and the additional function, presumably HR.

Plant natural products (PNPs) have shown various pharmaceutical activities, possessing great potential in global markets. Microbial cell factories (MCFs) provide an economical and sustainable alternative for the synthesis of valuable PNPs compared with traditional approaches. However, the heterologous synthetic pathways always lack native regulatory systems, bringing extra burden to PNPs production. To overcome the challenges, biosensors have been exploited and engineered as powerful tools for establishing artificial regulatory networks to control enzyme expression in response to environments. Here, we reviewed the recent progress involved in the application of biosensors that are responsive to PNPs and their precursors. Specifically, the key roles these biosensors played in PNP synthesis pathways, including isoprenoids, flavonoids, stilbenoids and alkaloids, were discussed in detail.

Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that Δ SisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in Δ SisarnA . The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro . Δ SisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro . These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo . Interestingly, Δ SisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR.

Double-stranded DNA break (DSB) repair is a fundamental process for all cellular life. Recently, KEOPS, a multiple-subunit protein complex that is conserved in eukaryotes and archaea and primarily involved in N6- threonylcarbamoyl adenosine (t6A) modification of tRNAs is reported to participate in homologous recombination in yeast. To functionally characterize archaeal KEOPS (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus. We show that aKEOPS possesses five subunits, Pcc1, Pcc1-like (or Gon7-like), Kae1, Bud32 and Cgi121, just as eukaryotic KEOPS. Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1-Pcc1-Pcc1-Kae1), suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1-like, can be deleted in the wild type and in a t6A modification complementary strain constructed, indicating that aKEOPS complex is essential in multiple cellular processes in this archaeon. Moreover, knock-down of the subunit genes leads to increase or decrease in the sensitivity of the cells to hydroxyurea and ultraviolet radiation both of which are DNA damage agents. These results indicated that aKEOPS plays an important role in DNA repair. In vitro, archaeal Cgi121 possesses dsDNA-binding activity which relies on its tRNA 3' CCA tail binding module. Our study indicates that DNA repair is an original intrinsic function of the evolutionarily conserved complex and reveals a possible link between two functions of the complex, t6A modification and DSB repair.

Single-stranded DNA binding proteins (SSBs) have been regarded as indispensable factors in all three domains of life since they play vital roles in DNA replication. Herein, we report that genes coding for the canonical SSB (SisSSB) and the non-canonical SSB (SisDBP) in the hyperthermophilic archaeon Saccharolobus islandicus REY15A can both be deleted. The growth is not affected, and the cell cycle progression and genome stability of the deletion strains is not impaired, suggesting that SisSSB and SisDBP are not essential for cell viability. Interestingly, at a lower temperature (55°C), the protein level of SisSSB increases ~1.8 fold in the wild type and the growth of ΔSisssb and ΔSisssbΔSisdbp is retarded. SisSSB exhibits melting activity on dsRNA and DNA/RNA hybrid in vitro and unwinding RNA hairpin in Escherichia coli. Furthermore, the core SisSSB domain is able to complement the absence of the cold shock proteins CspABGE in E. coli, suggesting that SisSSB functions as RNA chaperon. We show that a two-fold overexpression of SisSSB is beneficial to the cell growth at lower temperature, but it has detrimental effect on the cell growth and cell cycle progression at normal growth temperature, which differs from bacterial Csp proteins. Importantly, these in vitro and in vivo activities are conserved in SSB subtype SSB-1 in Crenarchaeota species that lack bacterial Csp homologs. Overall, we have clarified the function of the archaeal canonical SSB which does not function as a DNA processing factor, but plays a role in processes requiring dsRNA or DNA/RNA hybrid unwinding.