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A comprehensive whole-genome analysis of gene function by transposon mutagenesis and deep sequencing methodology has been implemented successfully in a representative of the Archaea domain. Libraries of transposon mutants were generated for the hydrogenotrophic, methanogenic archaeon Methanococcus maripaludis S2 using a derivative of the Tn5 transposon. About 89,000 unique insertions were mapped to the genome, which allowed for the classification of 526 genes or about 30% of the genome as possibly essential or strongly advantageous for growth in rich medium. Many of these genes were homologous to eukaryotic genes that encode fundamental processes in replication, transcription, and translation, providing direct evidence for their importance in Archaea. Some genes classified as possibly essential were unique to the archaeal or methanococcal lineages, such as that encoding DNA polymerase PolD. In contrast, the archaeal homolog to the gene encoding DNA polymerase B was not essential for growth, a conclusion confirmed by construction of an independent deletion mutation. Thus PolD, and not PolB, likely plays a fundamental role in DNA replication in methanococci. Similarly, 121 hypothetical ORFs were classified as possibly essential and likely play fundamental roles in methanococcal information processing or metabolism that are not established outside this group of prokaryotes.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells 1 , 2 . Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg (ref. 1 ) and 303 Pg (ref. 3 ) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem 4 , 5 , 6 , 7 . Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells 7 , 8 , are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot-blot hybridization protocols support the lipid-based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.

The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage. TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis ( trp ) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis , we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli , and the protein, here designated Tar ( T hermococcales a romatic amino acid r egulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant ( K d ) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis ( tyr-phe ) and aro operons. The protein recognized a palindromic sequence (TGGACA-N 8 -TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp , tyr-phe , and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis . IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.

Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ 2603 in Pyrococcus abyssi 23S rRNA and of Ψ 55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis , we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5 , nop10 , and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ 55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ 2607 (Ψ 2603 in P . abyssi ) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P . abyssi ), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.

Methanogenic archaea survive under aerated soil conditions in paddy fields, and their community is stable under these conditions. Changes in the abundance and composition of an active community of methanogenic archaea were assessed by analyzing mcrA gene (encoding α subunit of methyl-coenzyme M reductase) and transcripts during a prolonged drained period in a paddy-upland rotational field. Paddy rice (Oryza sativa L.) was planted in the flooded field and rotated with soybean (Glycine max [L.] Merr.) under upland soil conditions. Soil samples were collected from the rotational plot in the first year, with paddy rice, and in the two successive years, with soybean, at six time points, before seeding, during cultivation, and after harvest as well as from a consecutive paddy (control) plot. By the time that soybean was grown in the second year, the methanogenic archaeal community in the rotational plot maintained high mcrA transcript levels, comparable with those of the control plot community, but the levels drastically decreased by over three orders of magnitude after 2 years of upland conversion. The composition of active methanogenic archaeal communities that survived upland conversion in the rotational plot was similar to that of the active community in the control plot. These results revealed that mcrA gene transcription of methanogenic archaeal community in the rotational field was affected by a prolonged non-flooding period, longer than 1 year, indicating that unknown mechanisms maintain the stability of methanogenic archaeal community in paddy fields last up to 1 year after the onset of drainage.

Relative RNA abundance was measured at different cell-cycle stages in synchronized cultures of the hyperthermophilic archaeon Sulfolobus acidocaldarius. Cyclic induction was observed for >160 genes, demonstrating central roles for transcriptional regulation and cell-cycle-specific gene expression in archaeal cell-cycle progression. Many replication genes were induced in a cell-cycle-specific manner, and novel replisome components are likely to be among the genes of unknown function with similar induction patterns. Candidate genes for the unknown genome segregation and cell division machineries were also identified, as well as seven transcription factors likely to be involved in cell-cycle control. Two serine-threonine protein kinases showed distinct cell-cycle-specific induction, suggesting regulation of the archaeal cell cycle also through protein modification. Two candidate recognition elements, CCR boxes, for transcription factors in control of cell-cycle regulons were identified among gene sets with similar induction kinetics. The results allow detailed characterization of the genome segregation, division, and replication processes and may, because of the extensive homologies between the archaeal and eukaryotic information machineries, also be applicable to core features of the eukaryotic cell cycle.

Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand.

We investigated expression of genes mediating elemental cycling at the microspatial scale in the ocean’s largest river plume using, to our knowledge, the first fully quantitative inventory of genes and transcripts. The bacterial and archaeal communities associated with a phytoplankton bloom in Amazon River Plume waters at the outer continental shelf in June 2010 harbored ∼1.0 × 10 ¹³ genes and 4.7 × 10 ¹¹ transcripts per liter that mapped to several thousand microbial genomes. Genomes from free-living cells were more abundant than those from particle-associated cells, and they generated more transcripts per liter for carbon fixation, heterotrophy, nitrogen and phosphorus uptake, and iron acquisition, although they had lower expression ratios (transcripts⋅gene ⁻¹) overall. Genomes from particle-associated cells contributed more transcripts for sulfur cycling, aromatic compound degradation, and the synthesis of biologically essential vitamins, with an overall twofold up-regulation of expression compared with free-living cells. Quantitatively, gene regulation differences were more important than genome abundance differences in explaining why microenvironment transcriptomes differed. Taxa contributing genomes to both free-living and particle-associated communities had up to 65% of their expressed genes regulated differently between the two, quantifying the extent of transcriptional plasticity in marine microbes in situ. In response to patchiness in carbon, nutrients, and light at the micrometer scale, Amazon Plume microbes regulated the expression of genes relevant to biogeochemical processes at the ecosystem scale.

The effects of water-saving irrigation on emissions of greenhouse gases and soil prokaryotic communities were investigated in an experimental rice field. The water layer was kept at 1–2 cm in the water-saving (WS) irrigation treatment and at 6 cm in the continuous flooding (CF) irrigation treatment. WS irrigation decreased CH₄ emissions by 78 % and increased N₂O emissions by 533 %, resulting in 78 % reduction of global warming potential compared to the CF irrigation. WS irrigation did not affect the abundance or phylogenetic distribution of bacterial/archaeal 16S rRNA genes and the abundance of bacterial/archaeal 16S rRNAs. The transcript abundance of CH₄ emission-related genes generally followed CH₄ emission patterns, but the difference in abundance between mcrA transcripts and amoA/pmoA transcripts best described the differences in CH₄ emissions between the two irrigation practices. WS irrigation increased the relative abundance of 16S rRNAs and functional gene transcripts associated with Anaeromyxobacter and Methylocystis spp., suggesting that their activities might be important in emissions of the greenhouse gases. The N₂O emission patterns were not reflected in the abundance of N₂O emission-related genes and transcripts. We showed that the alternative irrigation practice was effective for mitigating greenhouse gas emissions from rice fields and that it did not affect the overall size and structure of the soil prokaryotic community but did affect the activity of some groups.

Active expression of putative ammonia monooxygenase gene subunit A (amoA) of marine group I Crenarchaeota has been detected in the Black Sea water column. It reached its maximum, as quantified by reverse-transcription quantitative PCR, exactly at the nitrate maximum or the nitrification zone modeled in the lower oxic zone. Crenarchaeal amoA expression could explain 74.5% of the nitrite variations in the lower oxic zone. In comparison, amoA expression by γ-proteobacterial ammonia-oxidizing bacteria (AOB) showed two distinct maxima, one in the modeled nitrification zone and one in the suboxic zone. Neither the amoA expression by crenarchaea nor that by β-proteobacterial AOB was significantly elevated in this latter zone. Nitrification in the suboxic zone, most likely microaerobic in nature, was verified by ¹⁵NO[Formula: see text] and ¹⁵N¹⁵N production in ¹⁵NH[Formula: see text] incubations with no measurable oxygen. It provided a direct local source of nitrite for anammox in the suboxic zone. Both ammonia-oxidizing crenarchaea and γ-proteobacterial AOB were important nitrifiers in the Black Sea and were likely coupled to anammox in indirect and direct manners respectively. Each process supplied about half of the nitrite required by anammox, based on ¹⁵N-incubation experiments and modeled calculations. Because anammox is a major nitrogen loss in marine suboxic waters, such nitrification-anammox coupling potentially occurring also in oceanic oxygen minimum zones would act as a short circuit connecting regenerated ammonium to direct nitrogen loss, thus reducing the presumed direct contribution from deep-sea nitrate.