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.

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.

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.

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.

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 conserved ribonuclease aCPSF1 triggers genome-wide transcription termination of Archaea via a 3'-end cleavage mode.

Transcription termination defines accurate transcript 3'-ends and ensures programmed transcriptomes, making it critical to life. However, transcription termination mechanisms remain largely unknown in Archaea. Here, we reported the physiological significance of the newly identified general transcription termination factor of Archaea, the ribonuclease aCPSF1, and elucidated its 3'-end cleavage triggered termination mechanism. The depletion of Mmp-aCPSF1 in Methanococcus maripaludis caused a genome-wide transcription termination defect and disordered transcriptome. Transcript-3'end-sequencing revealed that transcriptions primarily terminate downstream of a uridine-rich motif where Mmp-aCPSF1 performed an endoribonucleolytic cleavage, and the endoribonuclease activity was determined to be essential to the in vivo transcription termination. Co-immunoprecipitation and chromatin-immunoprecipitation detected interactions of Mmp-aCPSF1 with RNA polymerase and chromosome. Phylogenetic analysis revealed that the aCPSF1 orthologs are ubiquitously distributed among the archaeal phyla, and two aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to terminate transcription of M. maripaludis. Therefore, the aCPSF1 dependent termination mechanism could be widely employed in Archaea, including Lokiarchaeota belonging to Asgard Archaea, the postulated archaeal ancestor of Eukaryotes. Strikingly, aCPSF1-dependent archaeal transcription termination reported here exposes a similar 3'-cleavage mode as the eukaryotic RNA polymerase II termination, thus would shed lights on understanding the evolutionary linking between archaeal and eukaryotic termination machineries.

The archaeal RNA chaperone TRAM0076 shapes the transcriptome and optimizes the growth of Methanococcus maripaludis.

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5' untranslated regions (5'UTRs) of 20-60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5'UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5'UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.

Post-transcriptional regulation is involved in the cold-active methanol-based methanogenic pathway of a psychrophilic methanogen.

The methanol-derived methanogenetic pathway contributes to bulk methane production in cold regions, but the cold adaptation mechanisms are obscure. This work investigated the mechanisms using a psychrophilic methylotrophic methanogen Methanolobus psychrophilus R15. R15 possesses two mtaCB operon paralogues-encoding methanol:corrinoid methyltransferase that is key to methanol-based methanogenesis. Molecular combined methanogenic assays determined that MtaC1 is important in methanogenesis at the optimal temperature of 18°C, but MtaC2 can be a cold-adaptive paralogue by highly upregulated at 8°C. The 5'P-seq and 5'RACE all assayed that processing occurred at the 5' untranslated region (5'-UTR) of mtaC2; reporter genes detected higher protein expression, and RNA half-life experiments assayed prolonged lifespan of the processed transcript. Therefore, mtaC2 5'-UTR processing to move the bulged structure elevated both the translation efficiency and transcript stability. 5'P-seq, quantitative RT-PCR and northern blot all identified enhanced mtaC2 5'-UTR processing at 8°C, which could contribute to the upregulation of mtaC2 at cold. The R15 cell extract contains an endoribonuclease cleaving an identified 10 nt-processing motif and the native mtaC2 5'-UTR particularly folded at 8°C. Therefore, this study revealed a 5'-UTR processing mediated post-transcriptional regulation mechanism controlling the cold-adaptive methanol-supported methanogenetic pathway, which may be used by other methylotrophic methanogens.

Aurantimonas marina sp. nov., isolated from deep-sea sediment.

A novel bacterial strain, designated SW136T, was isolated from a deep-sea sediment sample collected from the South China Sea. Cells were Gram-stain-negative, aerobic, catalase-positive and oxidase-positive. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SW136T represented a novel member of the genus Aurantimonas, forming a distinct cluster with 'Aurantimonas litoralis', Aurantimonas coralicida and Aurantimonas manganoxydans (98.2, 98.1 and 97.9% sequence similarity, respectively). The predominant cellular fatty acid of strain SW136T was C18 : 1 ω7c. Strain SW136T contained ubiquinone-10 as the dominant respiratory quinone, and diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids. The genomic DNA G+C content was 64.3 mol%. The average nucleotide identity and digital DNA-DNA hybridization values of strain SW136T with A. coralicida CGMCC 1.12222T and A. manganoxydans CGMCC 1.12225T were 78.8 and 78.6 % and 21.5 and 25.5 %, respectively. On the basis of phylogenetic inference and phenotypic characteristics, we propose that strain SW136T represents a novel species of the genus Aurantimonas, with the name Aurantimonas marina sp. nov. The type strain is SW136T (=CGMCC 1.17725T=KCTC 82366T).

The Archaeal Small Heat Shock Protein Hsp17.6 Protects Proteins from Oxidative Inactivation.

Small heat shock proteins (sHsps) are widely distributed among various types of organisms and function in preventing the irreversible aggregation of thermal denaturing proteins. Here, we report that Hsp17.6 from Methanolobus psychrophilus exhibited protection of proteins from oxidation inactivation. The overexpression of Hsp17.6 in Escherichia coli markedly increased the stationary phase cell density and survivability in HClO and H2O2. Treatments with 0.2 mM HClO or 10 mM H2O2 reduced malate dehydrogenase (MDH) activity to 57% and 77%, whereas the addition of Hsp17.6 recovered the activity to 70-90% and 86-100%, respectively. A similar effect for superoxide dismutase oxidation was determined for Hsp17.6. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis assays determined that the Hsp17.6 addition decreased H2O2-caused disulfide-linking protein contents and HClO-induced degradation of MDH; meanwhile, Hsp17.6 protein appeared to be oxidized with increased molecular weights. Mass spectrometry identified oxygen atoms introduced into the larger Hsp17.6 molecules, mainly at the aspartate and methionine residues. Substitution of some aspartate residues reduced Hsp17.6 in alleviating H2O2- and HClO-caused MDH inactivation and in enhancing the E. coli survivability in H2O2 and HClO, suggesting that the archaeal Hsp17.6 oxidation protection might depend on an "oxidant sink" effect, i.e., to consume the oxidants in environments via aspartate oxidation.

A Streptococcus aquaporin acts as peroxiporin for efflux of cellular hydrogen peroxide and alleviation of oxidative stress.

Aquaporins (AQPs) are transmembrane proteins widely distributed in various organisms, and they facilitate bidirectional diffusion of water and uncharged solutes. The catalase-negative bacterium Streptococcus oligofermentans produces the highest H2O2 levels reported to date, which has to be exported to avoid oxidative stress. Here, we report that a S. oligofermentans aquaporin functions as a peroxiporin facilitating bidirectional transmembrane H2O2 transport. Knockout of this aquaporin homolog, So-AqpA, reduced H2O2 export by ∼50% and increased endogenous H2O2 retention, as indicated by the cellular H2O2 reporter HyPer. Heterologous expression of So-aqpA accelerated exogenous H2O2 influx into Saccharomyces cerevisiae and Escherichia coli cells, indicating that So-AqpA acts as an H2O2-transferring aquaporin. Alanine substitution revealed Phe-40 as a key residue for So-AqpA-mediated H2O2 transport. Northern blotting, qPCR, and luciferase reporter assays disclosed that H2O2 induces a >10-fold expression of So-aqpA Super-resolution imaging showed that H2O2 treatment increases So-AqpA protein molecules per cell by 1.6- to 3-fold. Inactivation of two redox-regulatory transcriptional repressors, PerR and MntR, reduced H2O2-induced So-aqpA expression to 1.8- and 4-fold, respectively. Electrophoretic mobility shift assays determined that MntR, but not PerR, binds to the So-aqpA promoter, indicating that MntR directly regulates H2O2-induced So-aqpA expression. Importantly, So-aqpA deletion decreased oxic growth and intraspecies competition and diminished the competitive advantages of S. oligofermentans over the caries pathogen Streptococcus mutans Of note, So-aqpA orthologs with the functionally important Phe-40 are present in all streptococci. Our work has uncovered an intrinsic, H2O2-inducible bacterial peroxiporin that has a key physiological role in H2O2 detoxification in S. oligofermentans.

The carbon catabolite repressor CcpA mediates optimal competence development in Streptococcus oligofermentans through post-transcriptional regulation.

Natural transformation increases the genetic diversity of bacteria, but is costly and must be strictly controlled. We previously found that deletion of ccpA, a key regulator of carbon catabolite repression (CCR), reduced transformation efficiency of Streptococcus oligofermentans, the current work further investigated the regulatory mechanisms of CcpA. The competence operon comCDE is subjected to basal and autoregulatory transcription. A luciferase reporter detected a transcriptional readthrough (TRT) from the upstream tRNAArg into the comCDE operon, which was induced by L -arginine. Insertion of the Escherichia coli T1T2 terminator downstream of tRNAArg abolished TRT, and reduced the basal comCDE transcription by 77% and also the transformation efficiency. Deletion of ccpA increased tRNAArg TRT and tRNAArg -comCDE polycistronic transcript by twofold. An in vitro transcription assay determined that CcpA promoted the transcription termination of tRNAArg TRT, and RNA EMSA and SPR assays detected equal binding affinity of CcpA to both the RNA and DNA of tRNAArg . These results indicate that CcpA controls the basal comCDE transcription by post-transcriptional actions. Overexpression of comDE or its phospho-mimicking mutant comDED58E reduced transformation efficiency, indicating that excessive ComE impairs competence development. CCR-regulated competence was further confirmed by higher tRNAArg TRT but lower transformation efficiency in galactose than in glucose.

Phenotypic divergence of thermotolerance: Molecular basis and cold adaptive evolution related to intrinsic DNA flexibility of glacier-inhabiting Cryobacterium strains.

The link between guanine-cytosine (GC) content and thermal adaptation is controversial. Here, we compared maximum growth temperature (TMGT ) and genomics of 78 Cryobacterium strains to avoid unreliable conclusions resulting from distantly phylogenetic groups. Phylogenomic analysis revealed this taxon had much higher diversification than we knew. Interestingly, these strains showed thermotolerance divergence with phylogenetic cohesion. A significant difference was found between TMGT ≤ 20°C strains and TMGT > 20°C strains in genomic GC content which mainly caused by variation of GC3. TMGT ≤ 20°C strains tended to use synonymous codons ended with A/U, but TMGT > 20°C strains tended to use G/C. Lower GC content at synonymous sites (≈GC3) of TMGT ≤ 20°C strains could provide lower intrinsic DNA flexibility which strongly associated with optimal molecular dynamics, and then guarantee DNA function at lower growth temperatures. This analysis of codon bias revealed close relationships for thermal adaptation, GC content at synonymous sites (≈GC3), intrinsic DNA flexibility and optimal DNA dynamics. Natural selection was main force driving this codon bias; strains with lower TMGT endured stronger natural selection. Therefore, this study provided molecular basis for bacterial adaptive evolution from moderate temperature to low temperature.

Comprehensive analysis of the pre-ribosomal RNA maturation pathway in a methanoarchaeon exposes the conserved circularization and linearization mode in archaea.

The ribosomal RNA (rRNA) genes are generally organized as an operon and cotranscribed into a polycistronic precursor; therefore, processing and maturation of pre-rRNAs are essential for ribosome biogenesis. However, rRNA maturation pathways of archaea, particularly of methanoarchaea, are scarcely known. Here, we thoroughly elucidated the maturation pathway of the rRNA operon (16S-tRNAAla-23S-tRNACys-5S) in Methanolobus psychrophilus, one representative of methanoarchaea. Enzymatic assay demonstrated that EndA, a tRNA splicing endoribonuclease, cleaved bulge-helix-bulge (BHB) motifs buried in the processing stems of pre-16S and pre-23S rRNAs. Northern blot and quantitative PCR detected splicing-coupled circularization of pre-16S and pre-23S rRNAs, which accounted for 2% and 12% of the corresponding rRNAs, respectively. Importantly, endoribonuclease Nob1 was determined to linearize circular pre-16S rRNA at the mature 3' end so to expose the anti-Shine-Dalgarno sequence, while circular pre-23S rRNA was linearized at the mature 5' end by an unknown endoribonuclease. The resultant 5' and 3' extension in linearized pre-16S and pre-23S rRNAs were finally matured through 5'-3' and 3'-5' exoribonucleolytic trimming, respectively. Additionally, a novel processing pathway of endoribonucleolysis coupled with exoribonucleolysis was identified for the pre-5S rRNA maturation in this methanogen, which could be also conserved in most methanogenic euryarchaea. Based on evaluating the phylogenetic conservation of the key elements that are involved in circularization and linearization of pre-rRNA maturation, we predict that the rRNA maturation mode revealed here could be prevalent among archaea.

A newly identified duplex RNA unwinding activity of archaeal RNase J depends on processive exoribonucleolysis coupled steric occlusion by its structural archaeal loops.

RNase J is a prokaryotic 5'-3' exo/endoribonuclease that functions in mRNA decay and rRNA maturation. Here, we report a novel duplex unwinding activity of mpy-RNase J, an archaeal RNase J from Methanolobus psychrophilus, which enables it to degrade duplex RNAs with hairpins up to 40 bp when linking a 5' single-stranded overhangs of ≥ 7 nt, corresponding to the RNA channel length. A 6-nt RNA-mpy-RNase J-S247A structure reveals the RNA-interacting residues and a steric barrier at the RNA channel entrance comprising two archaeal loops and two helices. Mutagenesis of the residues key to either exoribonucleolysis or RNA translocation diminished the duplex unwinding activity. Substitution of the residues in the steric barrier yielded stalled degradation intermediates at the duplex RNA regions. Thus, an exoribonucleolysis-driven and steric occlusion-based duplex unwinding mechanism was identified. The duplex unwinding activity confers mpy-RNase J the capability of degrading highly structured RNAs, including the bacterial REP RNA, and archaeal mRNAs, rRNAs, tRNAs, SRPs, RNase P and CD-box RNAs, providing an indicative of the potential key roles of mpy-RNase J in pleiotropic RNA metabolisms. Hydrolysis-coupled duplex unwinding activity was also detected in a bacterial RNase J, which may use a shared but slightly different unwinding mechanism from archaeal RNase Js, indicating that duplex unwinding is a common property of the prokaryotic RNase Js.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry.

Molecular Insights into Hydrogen Peroxide-sensing Mechanism of the Metalloregulator MntR in Controlling Bacterial Resistance to Oxidative Stresses.

Manganese contributes to anti-oxidative stress particularly in catalase-devoid bacteria, and DtxR family metalloregulators, through sensing cellular Mn2+ content, regulate its homeostasis. Here, we show that metalloregulator MntR (So-MntR) functions dually as Mn2+ and H2O2 sensors in mediating H2O2 resistance by an oral streptococcus. H2O2 disrupted So-MntR binding to Mn2+ transporter mntABC promoter and induced disulfide-linked dimerization of the protein. Mass spectrometry identified Cys-11/Cys-156 and Cys-11/Cys-11 disulfide-linked peptides in H2O2-treated So-MntR. Site mutagenesis of Cys-11 and Cys-156 and particularly Cys-11 abolished H2O2-induced disulfide-linked dimers and weakened H2O2 damage on So-MntR binding, indicating that H2O2 inactivates So-MntR via disulfide-linked dimerization. So-MntR C123S mutant was extremely sensitive to H2O2 oxidization in dimerization/oligomerization, probably because the mutagenesis caused a conformational change that facilitates Cys-11/Cys-156 disulfide linkage. Intermolecular Cys-11/Cys-11 disulfide was detected in C123S/C156S double mutant. Redox Western blot detected So-MntR oligomers in air-exposed cells but remarkably decreased upon H2O2 pulsing, suggesting a proteolysis of the disulfide-linked So-MntR oligomers. Remarkably, elevated C11S and C156S but much lower C123S proteins were detected in H2O2-pulsed cells, confirming Cys-11 and Cys-156 contributed to H2O2-induced oligomerization and degradation. Accordingly, in the C11S and C156S mutants, expression of mntABC and cellular Mn2+ decreased, but H2O2 susceptibility increased. In the C123S mutant, increased mntABC expression, cellular Mn2+ content, and manganese-mediated H2O2 survival were determined. Given the wide distribution of Cys-11 in streptococcal DtxR-like metalloregulators, the disclosed redox regulatory function and mechanism of So-MntR can be employed by the DtxR family proteins in bacterial resistance to oxidative stress.

New molecular insights into an archaeal RNase J reveal a conserved processive exoribonucleolysis mechanism of the RNase J family.

RNase J, a prokaryotic 5'-3' exo/endoribonuclease, contributes to mRNA decay, rRNA maturation and post-transcriptional regulation. Yet the processive-exoribonucleolysis mechanism remains obscure. Here, we solved the first RNA-free and RNA-bound structures of an archaeal RNase J, and through intensive biochemical studies provided detailed mechanistic insights into the catalysis and processivity. Distinct dimerization/tetramerization patterns were observed for archaeal and bacterial RNase Js, and unique archaeal Loops I and II were found involved in RNA interaction. A hydrogen-bond-network was identified for the first time that assists catalysis by facilitating efficient proton transfer in the catalytic center. A conserved 5'-monophosphate-binding pocket that coordinates the RNA 5'-end ensures the 5'-monophosphate preferential exoribonucleolysis. To achieve exoribonucleolytic processivity, the 5'-monophosphate-binding pocket and nucleotide +4 binding site anchor RNA within the catalytic track; the 5'-capping residue Leu37 of the sandwich pocket coupled with the 5'-monophosphate-binding pocket are dedicated to translocating and controlling the RNA orientation for each exoribonucleolytic cycle. The processive-exoribonucleolysis mechanism was verified as conserved in bacterial RNase J and also exposes striking parallels with the non-homologous eukaryotic 5'-3' exoribonuclease, Xrn1. The findings in this work shed light on not only the molecular mechanism of the RNase J family, but also the evolutionary convergence of divergent exoribonucleases.

Genomic encyclopedia of bacteria and archaea: sequencing a myriad of type strains.

Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.