Large-scale array for radio astronomy on the farside (LARAF).

At the Royal Society meeting in 2023, we have mainly presented our lunar orbit array concept called DSL, and also briefly introduced a concept of a lunar surface array, LARAF. As the DSL concept had been presented before, in this article, we introduce the LARAF. We propose to build an array in the far side of the Moon, with a master station which handles the data collection and processing, and 20 stations with maximum baseline of 10 km. Each station consists of 12 membrane antenna units, and the stations are connected to the master station by power line and optical fibre. The array will make interferometric observation in the 0.1-50 MHz band during the lunar night, powered by regenerated fuel cells. The whole array can be carried to the lunar surface with a heavy rocket mission, and deployed with a rover in eight months. Such an array would be an important step in the long-term development of lunar-based ultralong wavelength radio astronomy. It has a sufficiently high sensitivity to observe many radio sources in the sky, though still short of the dark age fluctuations. We discuss the possible options in the power supply, data communication, deployment etc. This article is part of a discussion meeting issue 'Astronomy from the Moon: the next decades (part 2)'.

The FHA domain protein ArnA functions as a global DNA damage response repressor in the hyperthermophilic archaeon Saccharolobus islandicus.

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 ΔSisarnA 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 ΔSisarnA. The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro. ΔSisarnB 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, ΔSisarnE 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.

The archaeal KEOPS complex possesses a functional Gon7 homolog and has an essential function independent of the cellular t6A modification level.

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 N6-threonylcarbamoyl adenosine (t6A) 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 t6A 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 t6A modification level. In addition, archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3' 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 t6A modification and the additional function, presumably HR.

The canonical single-stranded DNA-binding protein is not an essential replication factor but an RNA chaperon in Saccharolobus islandicus.

Single-stranded DNA-binding proteins (SSBs) have been regarded as indispensable replication factors. Herein, we report that the genes encoding the canonical SSB (SisSSB) and the non-canonical SSB (SisDBP) in Saccharolobus islandicus REY15A are not essential for cell viability. Interestingly, at a lower temperature (55°C), the protein level of SisSSB increases and the growth of ΔSisssb and ΔSisssbΔSisdbp is retarded. SisSSB exhibits melting activity on dsRNA and DNA/RNA hybrid in vitro and is able to melt RNA hairpin in Escherichia coli. Furthermore, the core SisSSB domain is able to complement the absence of cold-shock proteins in E. coli. Importantly, these activities are conserved in the canonical SSBs from Crenarchaeota species that lack bacterial Csp homologs. Overall, our study has clarified the function of the archaeal canonical SSBs which do not function as a DNA-processing factor, but play a role in the processes requiring melting of dsRNA or DNA/RNA hybrid.

An alkaline thermostable laccase from termite gut associated strain of Bacillus stratosphericus.

Laccase, an important oxidoreductase, is widely distributed in various organisms. Termites are known to decompose lignocellulose efficiently with the aid of gut microorganisms. However, few laccases have been characterized from termite or its gut microbes. We aimed to screen the strain capable of degrading lignocellulose from fungus-growing termites. In this study, Bacillus stratosphericus BCMC2 with lignocellulolytic activity was firstly isolated from the hindgut of fungus-growing termite Macrotermes barneyi. The laccase gene (BaCotA) was cloned both from the BCMC2 strain and termite intestinal metagenomic DNA. BaCotA was overexpressed in E. coli, and the recombinant BaCotA showed high specific activity (554.1 U/mg). BaCotA was thermostable with an optimum temperature of 70 °C, pH 5.0. Furthermore, BaCotA was resistant to alkali and organic solvents. The enzyme remained more than 70% residual activity at pH 8.0 for 120 min; and the organic solvents such as methanol, ethanol and acetone (10%) had no inhibitory effect on laccase activity. Additionally, BaCotA exhibited efficient decolorization ability towards indigo and crystal violet. The multiple enzymatic properties suggested the presented laccase as a potential candidate for industrial applications. Moreover, this study highlighted that termite intestine is a good resource for either new strains or enzymes.

Wolfberry genomes and the evolution of Lycium (Solanaceae).

Wolfberry Lycium, an economically important genus of the Solanaceae family, contains approximately 80 species and shows a fragmented distribution pattern among the Northern and Southern Hemispheres. Although several herbaceous species of Solanaceae have been subjected to genome sequencing, thus far, no genome sequences of woody representatives have been available. Here, we sequenced the genomes of 13 perennial woody species of Lycium, with a focus on Lycium barbarum. Integration with other genomes provides clear evidence supporting a whole-genome triplication (WGT) event shared by all hitherto sequenced solanaceous plants, which occurred shortly after the divergence of Solanaceae and Convolvulaceae. We identified new gene families and gene family expansions and contractions that first appeared in Solanaceae. Based on the identification of self-incompatibility related-gene families, we inferred that hybridization hotspots are enriched for genes that might be functioning in gametophytic self-incompatibility pathways in wolfberry. Extremely low expression of LOCULE NUBER (LC) and COLORLESS NON-RIPENING (CNR) orthologous genes during Lycium fruit development and ripening processes suggests functional diversification of these two genes between Lycium and tomato. The existence of additional flowering locus C-like MADS-box genes might correlate with the perennial flowering cycle of Lycium. Differential gene expression involved in the lignin biosynthetic pathway between Lycium and tomato likely illustrates woody and herbaceous differentiation. We also provide evidence that Lycium migrated from Africa into Asia, and subsequently from Asia into North America. Our results provide functional insights into Solanaceae origins, evolution and diversification.

Phosphoproteomic Analysis Reveals Rio1-Related Protein Phosphorylation Changes in Response to UV Irradiation in Sulfolobus islandicus REY15A.

DNA damage response (DDR) in eukaryotes is largely regulated by protein phosphorylation. In archaea, many proteins are phosphorylated, however, it is unclear how the cells respond to DNA damage through global protein phosphorylation. We previously found that Δrio1, a Rio1 kinase homolog deletion strain of Sulfolobus islandicus REY15A, was sensitive to UV irradiation. In this study, we showed that Δrio1 grew faster than the wild type. Quantitative phosphoproteomic analysis of the wild type and Δrio1, untreated and irradiated with UV irradiation, revealed 562 phosphorylated sites (with a Ser/Thr/Tyr ratio of 65.3%/23.8%/10.9%) of 333 proteins in total. The phosphorylation levels of 35 sites of 30 proteins changed with >1.3-fold in the wild type strain upon UV irradiation. Interestingly, more than half of the UV-induced changes in the wild type did not occur in the Δrio1 strain, which were mainly associated with proteins synthesis and turnover. In addition, a protein kinase and several transcriptional regulators were differentially phosphorylated after UV treatment, and some of the changes were dependent on Rio1. Finally, many proteins involved in various cellular metabolisms exhibited Riol-related and UV-independent phosphorylation changes. Our results suggest that Rio1 is involved in the regulation of protein recycling and signal transduction in response to UV irradiation, and plays regulatory roles in multiple cellular processes in S. islandicus.