An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon Methanococcus maripaludis.

BACKGROUND: The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory. RESULTS: This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes. CONCLUSIONS: Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

A 3'UTR-derived small RNA represses pneumolysin synthesis and facilitates pneumococcal brain invasion.

Pneumolysin (Ply) of Streptococcus pneumoniae (pneumococcus) at relatively high and low levels facilitates pneumococcal invasion into the lung and brain, respectively; however, the regulatory mechanisms of Ply expression are poorly understood. Here, we find that a small RNA plyT, processed from the 3'UTR of the ply operon, is expressed higher in anaerobically- than in statically-cultured pneumococcus D39. Using bioinformatic, biochemical and genetic approaches, we reveal that PlyT inhibits Ply synthesis and hemolytic activities by pairing with an RBS-embedded intergenic region of the ply operon. The RNA-binding protein SPD_1558 facilitates the pairing. Importantly, PlyT inhibition of Ply synthesis is stronger in anaerobic culture and leads to lower Ply abundance. Deletion of plyT decreases the number of pneumococci in the infected mouse brain and reduces the virulence, demonstrating that PlyT-regulated lower Ply in oxygen-void microenvironments, such as the blood, is important for pneumococcus to cross the blood-brain barrier and invade the brain. PlyT-mediated repression of Ply synthesis at anoxic niches is also verified in pneumococcal serotype 4 and 14 strains; moreover, the ply operon with a 3'UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus.

Advancements in prokaryotic systematics and the role of Bergey's International Society for Microbial Systematicsin addressing challenges in the meta-data era.

Prokaryotes are ubiquitous in the biosphere, important for human health and drive diverse biological and environmental processes. Systematics of prokaryotes, whose origins can be traced to the discovery of microorganisms in the 17th century, has transitioned from a phenotype-based classification to a more comprehensive polyphasic taxonomy and eventually to the current genome-based taxonomic approach. This transition aligns with a foundational shift from studies focused on phenotypic traits that have limited comparative value to those using genome sequences. In this context, Bergey's Manual of Systematics of Archaea and Bacteria (BMSAB) and Bergey's International Society for Microbial Systematics (BISMiS) play a pivotal role in guiding prokaryotic systematics. This review focuses on the historical development of prokaryotic systematics with a focus on the roles of BMSAB and BISMiS. We also explore significant contributions and achievements by microbiologists, highlight the latest progress in the field and anticipate challenges and opportunities within prokaryotic systematics. Additionally, we outline five focal points of BISMiS that are aimed at addressing these challenges. In conclusion, our collaborative effort seeks to enhance ongoing advancements in prokaryotic systematics, ensuring its continued relevance and innovative characters in the contemporary landscape of genomics and bioinformatics.

Isolation of a methyl-reducing methanogen outside the Euryarchaeota.

Methanogenic archaea are main contributors to methane emissions, and have a crucial role in carbon cycling and global warming. Until recently, methanogens were confined to Euryarchaeota, but metagenomic studies revealed the presence of genes encoding the methyl coenzyme M reductase complex in other archaeal clades1-4, thereby opening up the premise that methanogenesis is taxonomically more widespread. Nevertheless, laboratory cultivation of these non-euryarchaeal methanogens was lacking to corroborate their potential methanogenic ability and physiology. Here we report the isolation of a thermophilic archaeon LWZ-6 from an oil field. This archaeon belongs to the class Methanosuratincolia (originally affiliated with 'Candidatus Verstraetearchaeota') in the phylum Thermoproteota. Methanosuratincola petrocarbonis LWZ-6 is a strict hydrogen-dependent methylotrophic methanogen. Although previous metagenomic studies speculated on the fermentative potential of Methanosuratincolia members, strain LWZ-6 does not ferment sugars, peptides or amino acids. Its energy metabolism is linked only to methanogenesis, with methanol and monomethylamine as electron acceptors and hydrogen as an electron donor. Comparative (meta)genome analysis confirmed that hydrogen-dependent methylotrophic methanogenesis is a widespread trait among Methanosuratincolia. Our findings confirm that the diversity of methanogens expands beyond the classical Euryarchaeota and imply the importance of hydrogen-dependent methylotrophic methanogenesis in global methane emissions and carbon cycle.

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.

The varied roles of pilA-N, omcE, omcS, omcT, and omcZ in extracellular electron transfer by Geobacter sulfurreducens.

Geobacter sulfurreducens mediates extracellular electron transfer (EET) reactions with different substrates, such as solid-phase Fe(III)-containing minerals, anodes and the cells of Geobacter metallireducens. To compare their roles in EET, the pilA-N, omcE, omcS, omcT and omcZ genes of G. sulfurreducens were systematically deleted. All mutants showed impaired and varied ability to form biofilms on nonconductive surface. Deletion of omcE also impaired bacterial ability to reduce ferrihydrite, but its impacts on the ability for anode reduction and the co-culture of G. metallireducens-G. sulfurreducens were minimal. The mutant without omcS showed diminished ability to reduce ferrihydrite and to form the co-culture, but was able to regain its ability to reduce anodes. Deletion of omcT, omcZ or pilA-N alone impaired bacterial ability to reduce ferrihydrite and anodes and to form the co-culture. Deletion of all tested genes abolished bacterial ability to reduce ferrihydrite and anodes. Triple-deletion of all omcS, omcT and omcZ abolished the ability of G. sulfurreducens to co-culture with G. metallireducens. However, deletion of only omcZ or pilA-N or both omcS and omcT abolished the ability of G. sulfurreducens without hydrogenase gene hybL to co-culture with G. metallireducens, which show their indispensable roles in direct electron transfer from G. metallireducens to G. sulfurreducens. Thus, the roles of pilA-N, omcE, omcS, omcT and omcZ for G. sulfurreducens in EET vary substantially, which also suggest that possession of PilA-N and multiple cytochromes of different structures enables G. sulfurreducens to mediate EET reactions efficiently with substrates of different properties.

Endonucleolytic processing plays a critical role in the maturation of ribosomal RNA in Methanococcus maripaludis.

Ribosomal RNA (rRNA) processing and maturation are fundamentally important for ribosome biogenesis, but the mechanisms in archaea, the third form of life, remains largely elusive. This study aimed to investigate the rRNA maturation process in Methanococcus maripaludis, a representative archaeon lacking known 3'-5' exonucleases. Through cleavage site identification and enzymatic assays, the splicing endonuclease EndA was determined to process the bulge-helix-bulge (BHB) motifs in 16S and 23S rRNA precursors. After splicing, the circular processing intermediates were formed and this was confirmed by quantitative RT-PCR and Northern blot. Ribonuclease assay revealed a specific cleavage at a 10-nt A/U-rich motif at the mature 5' end of pre-16S rRNA, which linearized circular pre-16S rRNA intermediate. Further 3'-RACE and ribonuclease assays determined that the endonuclease Nob1 cleaved the 3' extension of pre-16S rRNA, and so generated the mature 3' end. Circularized RT-PCR (cRT-PCR) and 5'-RACE identified two cleavage sites near helix 1 at the 5' end of 23S rRNA, indicating that an RNA structure-based endonucleolytic processing linearized the circular pre-23S rRNA intermediate. In the maturation of pre-5S rRNA, multiple endonucleolytic processing sites were determined at the 10-nt A/U-rich motif in the leader and trailer sequence. This study demonstrates that endonucleolytic processing, particularly at the 10-nt A/U-rich motifs play an essential role in the pre-rRNA maturation of M. maripaludis, indicating diverse pathways of rRNA maturation in archaeal species.

Methylotrophic methanogens and bacteria synergistically demethylate dimethylarsenate in paddy soil and alleviate rice straighthead disease.

Microorganisms play a key role in arsenic (As) biogeochemistry, transforming As species between inorganic and organic forms and different oxidation states. Microbial As methylation is enhanced in anoxic paddy soil, producing primarily dimethylarsenic (DMAs), which can cause rice straighthead disease and large yield losses. DMAs can also be demethylated in paddy soil, but the microorganisms driving this process remain unclear. In this study, we showed that the enrichment culture of methylotrophic methanogens from paddy soil demethylated pentavalent DMAs(V) efficiently. DMAs(V) was reduced to DMAs(III) before demethylation. 16S rRNA gene diversity and metagenomic analysis showed that Methanomassiliicoccus dominated in the enrichment culture, with Methanosarcina and Methanoculleus also being present. We isolated Methanomassiliicoccus luminyensis CZDD1 and Methanosarcina mazei CZ1 from the enrichment culture; the former could partially demethylate trivalent DMAs(III) but not DMAs(V) and the latter could demethylate neither. Addition of strain CZDD1 to the enrichment culture greatly accelerated DMAs(V) demethylation. Demethylation of DMAs(V) in the enrichment culture was suppressed by ampicillin, suggesting the involvement of bacteria. We isolated three anaerobic bacterial strains including Clostridium from the enrichment culture, which could produce hydrogen and reduce DMAs(V) to DMAs(III). Furthermore, augmentation of the Methanomassiliicoccus-Clostridium coculture to a paddy soil decreased DMAs accumulation by rice and alleviated straighthead disease. The results reveal a synergistic relationship whereby anaerobic bacteria reduce DMAs(V) to DMAs(III) for demethylation by Methanomassiliicoccus and also produce hydrogen to promote the growth of Methanomassiliicoccus; enhancing their populations in paddy soil can help alleviate rice straighthead disease.

A robust genetic toolbox for fine-tuning gene expression in the CO2-Fixing methanogenic archaeon Methanococcus maripaludis.

Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼104-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications.

Internal transcription termination widely regulates differential expression of operon-organized genes including ribosomal protein and RNA polymerase genes in an archaeon.

Genes organized within operons in prokaryotes benefit from coordinated expression. However, within many operons, genes are expressed at different levels, and the mechanisms for this remain obscure. By integrating PacBio-seq, dRNA-seq, Term-seq and Illumina-seq data of a representative archaeon Methanococcus maripaludis, internal transcription termination sites (ioTTSs) were identified within 38% of operons. Higher transcript and protein abundances were found for genes upstream than downstream of ioTTSs. For representative operons, these differences were confirmed by northern blotting, qRT-PCR and western blotting, demonstrating that these ioTTS terminations were functional. Of special interest, mutation of ioTTSs in ribosomal protein (RP)-RNA polymerase (RNAP) operons not only elevated expression of the downstream RNAP genes but also decreased production of the assembled RNAP complex, slowed whole cell transcription and translation, and inhibited growth. Overexpression of the RNAP subunits with a shuttle vector generated the similar physiological effects. Therefore, ioTTS termination is a general and physiologically significant regulatory mechanism of the operon gene expression. Because the RP-RNAP operons are found to be widely distributed in archaeal species, this regulatory mechanism could be commonly employed in archaea.

Proposed minimal standards for description of methanogenic archaea.

Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA-DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.

Pn-AqpC-Mediated Fermentation Pattern Coordination with the Two-Component System 07 Regulates Host N-Glycan Degradation of Streptococcus pneumoniae.

The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) is a human nasopharyngeal commensal, and host N-glycan metabolism promotes its colonization and invasion. It has been reported that glucose represses, while fetuin, a glycoconjugated model protein, induces, the genes involved in N-glycan degradation through the two-component system TCS07. However, the mechanisms of glucose repression and TCS07 induction remain unknown. Previously, we found that the pneumococcal aquaglyceroporin Pn-AqpC facilitates oxygen uptake, thereby contributing to the antioxidant potential and virulence. In this study, through Tandem Mass Tag (TMT) quantitative proteomics, we found that the deletion of Pn-aqpC caused a marked upregulation of 11 proteins involved in N-glycan degradation in glucose-grown pneumococcus R6. Both quantitative RT-PCR and GFP fluorescence reporters revealed that the upregulation of N-glycan genes was completely dependent on response regulator (RR) 07, but not on the histidine kinase HK07 of TCS07 or the phosphoryl-receiving aspartate residue of RR07 in ΔPn-aqpC, indicating that RR07 was activated in an HK07-independent manner when Pn-AqpC was absent. The deletion of Pn-aqpC also enhanced the expression of pyruvate formate lyase and increased formate production, probably due to reduced cellular oxygen content, indicating that a shunt of glucose catabolism to mixed acid fermentation occurs. Notably, formate induced the N-glycan degradation genes in glucose-grown R6, but the deletion of rr07 abolished this induction, indicating that formate activates RR07. However, the induction of N-glycan degradation proteins reduced the intraspecies competition of R6 in glucose. Therefore, although N-glycan degradation promotes pneumococcal pathogenesis, the glucose metabolites-based RR07 regulation reported here is of importance for balancing growth fitness and the pathogenicity of pneumococcus. IMPORTANCE Pneumococcus, a human opportunistic pathogen, is capable of metabolizing host complex N-glycans. N-glycan degradation promotes pneumococcus colonization in the nasopharynx as well as invasion into deeper tissues, thus significantly contributing to pathogenesis. It is known that the two-component system 07 induces the N-glycan metabolizing genes; however, how TCS07 is activated remains unknown. This study reveals that formate, the anaerobic fermentation metabolite of pneumococcus, is a novel activator of the response regulator (RR) 07. Although the high expression of N-glycan degradation genes promotes pneumococcal colonization in the nasopharynx and pathogenesis, this reduces pneumococcal growth fitness in glucose as indicated in this work. Notably, the presence of Pn-AqpC, an oxygen-transporting aquaglyceroporin, enables pneumococcus to maintain glucose homolactic acid fermentation, thus reducing formate production, maintaining RR07 inactivation, and controlling N-glycan degrading genes at a non-induced status. Thus, this study highlights a novel fermentation metabolism pattern linking TCS-regulated carbohydrate utilization strategies as a trade-off between the fitness and the pathogenicity of pneumococcus.

CRISPR-Cas9 Toolkit for Genome Editing in an Autotrophic CO2-Fixing Methanogenic Archaeon.

The CRISPR-Cas9 system is a robust genome editing tool that is widely applied in eukaryotes and bacteria. However, use of this technique has only been developed for one species of Archaea, a domain of life ranking in parallel with Eukarya and Bacteria. In this study, we applied the CRISPR-Cas9 genome editing technique to Methanococcus maripaludis, an autotrophic and hydrogenotrophic methanogenic archaeon with a remarkably polyploid genome comprising up to ~55 chromosomal copies per cell. An editing plasmid was designed that encodes small guide RNA (sgRNA), Cas9 protein and an ~1-kb repair template (donor). Highly efficient (75% to 100%) and precise genome editing was achieved following one-step transformation. Significantly, the Cas9-based system efficiently deleted one or two genes and a large DNA fragment (~9 kb) and even synchronously deleted 13 genes located at three loci in all chromosomal copies of M. maripaludis. Moreover, precise in situ genome modifications, such as gene tagging and multiple- and even single-nucleotide mutagenesis, were also introduced with high efficiency. Further, as a proof of concept, precise mutagenesis at the nucleotide level allowed the engineering of both transcriptional and translational activities. Mutations were introduced into an archaeal promoter BRE (transcription factor B [TFB] recognition element), a terminator U-tract region, and a gene coding region. Stop codon introduction into a gene through single-nucleotide substitution shut down its expression, providing an alternative strategy for gene inactivation. In conclusion, the robust CRISPR-Cas9 genetic toolkit developed in this investigation greatly facilitates the application of M. maripaludis as a model system in the study of archaeal biology and biotechnology development, particularly CO2-based biotechnologies. IMPORTANCE Archaea are prokaryotes with intriguing biological characteristics. They possess bacterial cell structures but eukaryotic homologous information processing machinery and eukaryotic featured proteins. Archaea also display excellent adaptability to extreme environments and play pivotal roles in ecological processes, thus exhibiting valuable biotechnological potential. However, the in-depth understanding and practical application of archaea are much lagging, because only a minority of pure cultures are available, and even worse, very few can be genetically manipulated. This work developed CRISPR-Cas9-based genome editing technology in Methanococcus maripaludis, a CO2-fixing methanogenic archaeon. The CRISPR-Cas9 approach developed in this study provides an elegant and efficient genome editing toolkit that can be applied in the knockout of single or multiple genes, in situ gene tagging, multiple- or single-nucleotide mutagenesis, and inactivation of gene expression by introduction of stop codons. The successful development of the CRISPR-Cas9 toolkit will facilitate the application of M. maripaludis in archaeal biology research and biotechnology development, particularly CO2-derived biotechnologies.

Anaerobic methane oxidation linked to Fe(III) reduction in a Candidatus Methanoperedens-enriched consortium from the cold Zoige wetland at Tibetan Plateau.

Anaerobic oxidation of methane (AOM) is a microbial process degrading ample methane in anoxic environments, and Ca. Methanoperedens mediated nitrate- or metal-reduction linked AOM is believed important in freshwater systems. This work, via 16S rRNA gene diversity survey and 16S rRNA quantification, found abundant Ca. Methanoperedens along with iron in the cold Zoige wetland at Tibetan Plateau. The wetland soil microcosm performed Fe(III) reduction, rather than nitrate- nor sulphate-reduction, coupled methane oxidation (3.87 µmol d-1 ) with 32.33 µmol Fe(II) accumulation per day at 18°C, but not at 30°C. A metagenome-assembled genome (MAG) recovered from the microcosm exhibits ~74% average nucleotide identity with the reported Ca. Methanoperedens spp. that perform Fe(III) reduction linked AOM, thus a novel species Ca. Methanoperedens psychrophilus was proposed. Ca. M. psychrophilus contains the whole suite of CO2 reductive methanogenic genes presumably involving in AOM via a reverse direction, and comparative genome analysis revealed its unique gene categories: the multi-heme clusters (MHCs) cytochromes, the S-layer proteins highly homologous to those recovered from lower temperature environments and type IV pili, those could confer Ca. M. psychrophilus of cold adaptability. Therefore, this work reports the first methanotroph implementing AOM in an alpine wetland.

The Archaeal Transcription Termination Factor aCPSF1 is a Robust Phylogenetic Marker for Archaeal Taxonomy.

Archaea are highly diverse and represent a primary life domain, but the majority of them remain uncultured. Currently, 16S rRNA phylogeny is widely used in archaeal taxonomy and diversity surveys. However, highly conserved sequence of 16S rRNA possibly results in generation of chimera in the amplicons and metagenome-assembled genomes (MAGs) and therefore limits its application. The newly developed phylogenomic approach has overcome these flaws, but it demands high-quality MAGs and intensive computation. In this study, we investigated the use of the archaeal transcription termination factor aCPSF1 in archaeal classification and diversity surveys. The phylogenetic analysis of 1,964 aCPSF1 orthologs retrieved from the available archaeal (meta)genomes resulted in convergent clustering patterns with those of archaeal phylogenomics and 16S rRNA phylogeny. The aCPSF1 phylogeny also displayed comparable clustering with the methanoarchaeal McrABG phylogeny and the haloarchaeal phylogenomics. Normalization of 779 aCPSF1 sequences including 261 from cultured archaeal species yielded a taxonomic ranking system with higher resolutions than that obtained with 16S rRNA for genus and species. Using the aCPSF1 taxonomy, 144 unclassified archaea in NCBI database were identified to various taxonomic ranks. Moreover, aCPSF1- and 16S rRNA-based surveys of the archaeal diversity in a sample from a South China Sea cold seep produced similar results. Our results demonstrate that aCPSF1 is an alternative archaeal phylogenetic marker, which exhibits higher resolution than 16S rRNA, and is more readily usable than phylogenomics in the taxonomic study of archaea. IMPORTANCE Archaea represent a unique type of prokaryote, which inhabit in various environments including extreme environments, and so define the boundary of biosphere, and play pivotal ecological roles, particularly in extreme environments. Since their discovery over 40 years ago, environmental archaea have been widely investigated using the 16S rRNA sequence comparison, and the recently developed phylogenomic approach because the majority of archaea are recalcitrant to laboratory cultivation. However, the highly conserved sequence of 16S rRNA and intensive bioinformatic computation of phylogenomics limit their applications in archaeal species delineation and diversity investigations. aCPSF1 is a ubiquitously distributed and vertically inherited transcription termination factor in archaea. In this study, we developed an aCPSF1-based archaeal taxonomic system which exhibits congruent phylogenic clustering patterns with archaeal phylogenomics and higher resolution than 16S rRNA in distinguishing archaea at lower taxonomic ranks. Therefore, aCPSF1 is a new phylogenetic marker in the taxonomic and diversity studies of archaea.

aCPSF1 cooperates with terminator U-tract to dictate archaeal transcription termination efficacy.

Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea; however, the working mechanism remains elusive. This work, through analyzing transcript-3'end-sequencing data of Methanococcus maripaludis, found genome-wide positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficacies (TTEs). In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding affinity, and in vivo assays demonstrated the two elements are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract cooperatively determine high TTEs. The N-terminal KH domains equip aCPSF1 with specific-binding capacity to terminator U-tract and the aCPSF1-terminator U-tract cooperation; while the nuclease activity of aCPSF1 was also required for TTEs. aCPSF1 also guarantees the terminations of transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract cooperation in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in archaea, which may be widely employed by archaeal phyla; using one trans-action factor to recognize U-rich terminator signal and cleave transcript 3'-end, the archaeal aCPSF1-dependent transcription termination may represent a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery.

RNase Z Oxidative Degradation Impedes tRNA Maturation and is Involved in Streptococcal Translation Regulation in Response to Oxidative Stress.

When encountering oxidative stress, organisms selectively upregulate antioxidant genes and simultaneously suppress the translation of most other proteins. Eukaryotes employ multiple strategies to adjust translation at both the initiation and elongation stages; however, how prokaryotes modulate translation under oxidative stress remains unclear. Here, we report that upon hydrogen peroxide (H2O2) challenge, Streptococcus oligofermentans reduced translation via RNase Z (So-RNaseZ) oxidative degradation, thus hindering tRNA maturation. S. oligofermentans encodes all CCA-less tRNAs that require So-RNaseZ for 3' end maturation. A combination of nonreducing SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC/MS-MS) assays demonstrated that H2O2 oxidation induced Cys38-Cys149 disulfide linkages in recombinant So-RNaseZ protein, and serine substitution of Cys38 or Cys149 abolished these disulfide linkages. Consistently, redox Western blotting also determined intramolecular disulfide-linked So-RNaseZ in H2O2-treated S. oligofermentans cells. The disulfide-linked So-RNaseZ and monomer were both subject to proteolysis, whereas C149S mutation alleviated oxidative degradation of So-RNaseZ, suggesting that H2O2-mediated disulfide linkages substantially contributed to So-RNaseZ degradation. Accordingly, Northern blotting determined that tRNA precursor accumulation and mature tRNA species decrease in H2O2-treated S. oligofermentans. Moreover, reduced overall protein synthesis, as indicated by puromycin incorporation, and retarded growth of S. oligofermentans occurred in an H2O2 concentration-dependent manner. Overexpression of So-RNaseZ not only elevated tRNA precursor processing and protein synthesis but also partly rescued H2O2-suppressed S. oligofermentans growth. Moreover, So-RNaseZ oxidative degradation-mediated translation repression elevated S. oligofermentans survival under high H2O2 stress. Therefore, this work found that So-RNaseZ oxidative degradation-impeded tRNA maturation contributes to streptococcal translation repression and provides the oxidative stress adaptability for S. oligofermentans. IMPORTANCE Translation regulation is a common strategy used by organisms to reduce oxidative damage. Catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports a novel translation regulation mechanism employed by Streptococcus oligofermentans in response to H2O2 challenge, in which the key tRNA endonuclease So-RNaseZ is oxidized to form Cys38-Cys149 disulfide linkages and both the disulfide-linked So-RNaseZ and monomers are subject to proteolysis; thus, tRNA maturation, protein translation, and growth are all suppressed. Notably, So-RNaseZ oxidative degradation-mediated translation repression offers oxidative adaptability to S. oligofermentans and enhances its survival against high H2O2 challenge. So-RNaseZ orthologs and H2O2-sensitive cysteines (Cys38 and Cys149) are widely distributed in Streptococcus and Lactococcus species genomes, which also encode all CCA-less tRNAs and lack catalase. Therefore, RNase Z oxidative degradation-based translation regulation could be widely employed by these lactic acid bacteria, including pathogenic streptococci, to cope with H2O2.

Bacteria and Archaea Synergistically Convert Glycine Betaine to Biogenic Methane in the Formosa Cold Seep of the South China Sea.

Cold seeps are globally widespread seafloor ecosystems that feature abundant methane production and flourishing chemotrophic benthic communities. Chemical evidence indicates that cold seep methane is largely biogenic; however, the primary methane-producing organisms and associated pathways involved in methanogenesis remain elusive. This work detected methane production when glycine betaine (GBT) or trimethylamine (TMA) was added to the sediment microcosms of the Formosa cold seep, South China Sea. The methane production was suppressed by antibiotic inhibition of bacteria, while GBT was accumulated. This suggests that the widely used osmoprotectant GBT could be converted to cold seep biogenic methane via the synergistic activity of bacteria and methanogenic archaea because archaea are not sensitive to antibiotics and no bacteria are known to produce ample methane (mM). 16S rRNA gene diversity analyses revealed that the predominant bacterial and archaeal genera in the GBT-amended methanogenic microcosms included Oceanirhabdus and Methanococcoides. Moreover, metagenomic analyses detected the presence of grdH and mtgB genes that are involved in GBT reduction and demethylation, respectively. Two novel species were obtained, including bacterium Oceanirhabdus seepicola, which reduces GBT to TMA, and a methanogenic archaeon, Methanococcoides seepicolus, which produces methane from TMA and GBT. The two strains reconstituted coculture efficiently converted GBT to methane at 18°C; however, at 4°C addition of dimethylglycine (DMG), the GBT demethylation product, was necessary. Therefore, this work demonstrated that GBT is the precursor not only of the biogenic methane but also of the cryoprotectant DMG to the microorganisms at the Formosa cold seep. IMPORTANCE Numerous cold seeps have been found in global continental margins where methane is enriched in pore waters that are forced upward from sediments. Therefore, high concerns have been focused on the methane-producing organisms and the metabolic pathways in these environments because methane is a potent greenhouse gas. In this study, GBT was identified as the main precursor for methane in the Formosa cold seep of the South China Sea. Further, synergism of bacteria and methanogenic archaea was identified in GBT conversion to methane via the GBT reduction pathway, while methanogen-mediated GBT demethylation to methane was also observed. In addition, GBT-demethylated product dimethyl glycine acted as a cryoprotectant that promoted the cold seep microorganisms at cold temperatures. GBT is an osmoprotectant that is widely used by marine organisms, and therefore, the GBT-derived methanogenic pathway reported here could be widely distributed among global cold seep environments.