A conserved transcription factor controls gluconeogenesis via distinct targets in hypersaline-adapted archaea with diverse metabolic capabilities.

Timely regulation of carbon metabolic pathways is essential for cellular processes and to prevent futile cycling of intracellular metabolites. In Halobacterium salinarum, a hypersaline adapted archaeon, a sugar-sensing TrmB family protein controls gluconeogenesis and other biosynthetic pathways. Notably, Hbt. salinarum does not utilize carbohydrates for energy, uncommon among Haloarchaea. We characterized a TrmB-family transcriptional regulator in a saccharolytic generalist, Haloarcula hispanica, to investigate whether the targets and function of TrmB, or its regulon, is conserved in related species with distinct metabolic capabilities. In Har. hispanica, TrmB binds to 15 sites in the genome and induces the expression of genes primarily involved in gluconeogenesis and tryptophan biosynthesis. An important regulatory control point in Hbt. salinarum, activation of ppsA and repression of pykA, is absent in Har. hispanica. Contrary to its role in Hbt. salinarum and saccharolytic hyperthermophiles, TrmB does not act as a global regulator: it does not directly repress the expression of glycolytic enzymes, peripheral pathways such as cofactor biosynthesis, or catabolism of other carbon sources in Har. hispanica. Cumulatively, these findings suggest rewiring of the TrmB regulon alongside metabolic network evolution in Haloarchaea.

Panguiarchaeum symbiosum, a potential hyperthermophilic symbiont in the TACK superphylum.

The biology of Korarchaeia remains elusive due to the lack of genome representatives. Here, we reconstruct 10 closely related metagenome-assembled genomes from hot spring habitats and place them into a single species, proposed herein as Panguiarchaeum symbiosum. Functional investigation suggests that Panguiarchaeum symbiosum is strictly anaerobic and grows exclusively in thermal habitats by fermenting peptides coupled with sulfide and hydrogen production to dispose of electrons. Due to its inability to biosynthesize archaeal membranes, amino acids, and purines, this species likely exists in a symbiotic lifestyle similar to DPANN archaea. Population metagenomics and metatranscriptomic analyses demonstrated that genes associated with amino acid/peptide uptake and cell attachment exhibited positive selection and were highly expressed, supporting the proposed proteolytic catabolism and symbiotic lifestyle. Our study sheds light on the metabolism, evolution, and potential symbiotic lifestyle of Panguiarchaeum symbiosum, which may be a unique host-dependent archaeon within the TACK superphylum.

A Practical Guide to Small Protein Discovery and Characterization Using Mass Spectrometry.

Small proteins of up to ∼50 amino acids are an abundant class of biomolecules across all domains of life. Yet due to the challenges inherent in their size, they are often missed in genome annotations, and are difficult to identify and characterize using standard experimental approaches. Consequently, we still know few small proteins even in well-studied prokaryotic model organisms. Mass spectrometry (MS) has great potential for the discovery, validation, and functional characterization of small proteins. However, standard MS approaches are poorly suited to the identification of both known and novel small proteins due to limitations at each step of a typical proteomics workflow, i.e., sample preparation, protease digestion, liquid chromatography, MS data acquisition, and data analysis. Here, we outline the major MS-based workflows and bioinformatic pipelines used for small protein discovery and validation. Special emphasis is placed on highlighting the adjustments required to improve detection and data quality for small proteins. We discuss both the unbiased detection of small proteins and the targeted analysis of small proteins of interest. Finally, we provide guidelines to prioritize novel small proteins, and an outlook on methods with particular potential to further improve comprehensive discovery and characterization of small proteins.

PRODORIC: state-of-the-art database of prokaryotic gene regulation.

PRODORIC is worldwide one of the largest collections of prokaryotic transcription factor binding sites from multiple bacterial sources with corresponding interpretation and visualization tools. With the introduction of PRODORIC2 in 2017, the transition to a modern web interface and maintainable backend was started. With this latest PRODORIC release the database backend is now fully API-based and provides programmatical access to the complete PRODORIC data. The visualization tools Genome Browser and ProdoNet from the original PRODORIC have been reintroduced and were integrated into the PRODORIC website. Missing input and output options from the original Virtual Footprint were added again for position weight matrix pattern-based searches. The whole PRODORIC dataset was reannotated. Every transcription factor binding site was re-evaluated to increase the overall database quality. During this process, additional parameters, like bound effectors, regulation type and different types of experimental evidence have been added for every transcription factor. Additionally, 109 new transcription factors and 6 new organisms have been added. PRODORIC is publicly available at https://www.prodoric.de.

Molecular Basis for the Substrate Promiscuity of Isopentenyl Phosphate Kinase from Candidatus methanomethylophilus alvus.

Isopentenyl phosphate kinases (IPKs) catalyze the ATP-dependent phosphorylation of isopentenyl monophosphate (IP) to isopentenyl diphosphate (IPP) in the alternate mevalonate pathways of the archaea and plant cytoplasm. In recent years, IPKs have also been employed in artificial biosynthetic pathways called "(iso) prenol pathways" that utilize promiscuous kinases to sequentially phosphorylate (iso) prenol and generate the isoprenoid precursors IPP and dimethylallyl diphosphate (DMAPP). Furthermore, IPKs have garnered attention for their impressive substrate promiscuity toward non-natural alkyl-monophosphates (alkyl-Ps), which has prompted their utilization as biocatalysts for the generation of novel isoprenoids. However, none of the IPK crystal structures currently available contain non-natural substrates, leaving the roles of active-site residues in substrate promiscuity ambiguous. To address this, we present herein the high-resolution crystal structures of an IPK from Candidatus methanomethylophilus alvus (CMA) in the apo form and bound to natural and non-natural substrates. Additionally, we describe active-site engineering studies leading to enzyme variants with broadened substrate scope, as well as structure determination of two such variants (Ile74Ala and Ile146Ala) bound to non-natural alkyl-Ps. Collectively, our crystallographic studies compare six structures of CMA variants in different ligand-bound forms and highlight contrasting structural dynamics of the two substrate-binding sites. Furthermore, the structural and mutational studies confirm a novel role of the highly conserved DVTGG motif in catalysis, both in CMA and in IPKs at large. As such, the current study provides a molecular basis for the substrate-binding modes and catalytic performance of CMA toward the goal of developing IPKs into useful biocatalysts.

Small Proteins in Archaea, a Mainly Unexplored World.

In recent years, increasing numbers of small proteins have moved into the focus of science. Small proteins have been identified and characterized in all three domains of life, but the majority remains functionally uncharacterized, lack secondary structure, and exhibit limited evolutionary conservation. While quite a few have already been described for bacteria and eukaryotic organisms, the amount of known and functionally analyzed archaeal small proteins is still very limited. In this review, we compile the current state of research, show strategies for systematic approaches for global identification of small archaeal proteins, and address selected functionally characterized examples. Besides, we document exemplarily for one archaeon the tool development and optimization to identify small proteins using genome-wide approaches.

An archaeal histone-like protein regulates gene expression in response to salt stress.

Histones, ubiquitous in eukaryotes as DNA-packing proteins, find their evolutionary origins in archaea. Unlike the characterized histone proteins of a number of methanogenic and themophilic archaea, previous research indicated that HpyA, the sole histone encoded in the model halophile Halobacterium salinarum, is not involved in DNA packaging. Instead, it was found to have widespread but subtle effects on gene expression and to maintain wild type cell morphology. However, the precise function of halophilic histone-like proteins remain unclear. Here we use quantitative phenotyping, genetics, and functional genomics to investigate HpyA function. These experiments revealed that HpyA is important for growth and rod-shaped morphology in reduced salinity. HpyA preferentially binds DNA at discrete genomic sites under low salt to regulate expression of ion uptake, particularly iron. HpyA also globally but indirectly activates other ion uptake and nucleotide biosynthesis pathways in a salt-dependent manner. Taken together, these results demonstrate an alternative function for an archaeal histone-like protein as a transcriptional regulator, with its function tuned to the physiological stressors of the hypersaline environment.

The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase.

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively supercoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes.

Novel Glutamate-Putrescine Ligase Activity in Haloferax mediterranei: A New Function for glnA-2 Gene.

The genome of the halophilic archaea Haloferax mediterranei contains three ORFs that show homology with glutamine synthetase (GS) (glnA-1, glnA-2, and glnA-3). Previous studies have focused on the role of GlnA-1, suggesting that proteins GlnA-2 and GlnA-3 could play a different role to that of GS. Glutamine synthetase (EC 6.3.1.2) belongs to the class of ligases, including 20 subclasses of other different enzymes, such as aspartate-ammonia ligase (EC 6.3.1.1), glutamate-ethylamine ligase (EC 6.3.1.6), and glutamate-putrescine ligase (EC 6.3.1.11). The reaction catalyzed by glutamate-putrescine ligase is comparable to the reaction catalyzed by glutamine synthetase (GS). Both enzymes can bind a glutamate molecule to an amino group: ammonium (GS) or putrescine (glutamate-putrescine ligase). In addition, they present the characteristic catalytic domain of GS, showing significant similarities in their structure. Although these proteins are annotated as GS, the bioinformatics and experimental results obtained in this work indicate that the GlnA-2 protein (HFX_1688) is a glutamate-putrescine ligase, involved in polyamine catabolism. The most significant results are those related to glutamate-putrescine ligase's activity and the analysis of the transcriptional and translational expression of the glnA-2 gene in the presence of different nitrogen sources. This work confirms a new metabolic pathway in the Archaea domain which extends the knowledge regarding the utilization of alternative nitrogen sources in this domain.

Halobacterium salinarum and Haloferax volcanii Comparative Transcriptomics Reveals Conserved Transcriptional Processing Sites.

Post-transcriptional processing of messenger RNA is an important regulatory strategy that allows relatively fast responses to changes in environmental conditions. In halophile systems biology, the protein perspective of this problem (i.e., ribonucleases which implement the cleavages) is generally more studied than the RNA perspective (i.e., processing sites). In the present in silico work, we mapped genome-wide transcriptional processing sites (TPS) in two halophilic model organisms, Halobacterium salinarum NRC-1 and Haloferax volcanii DS2. TPS were established by reanalysis of publicly available differential RNA-seq (dRNA-seq) data, searching for non-primary (monophosphorylated RNAs) enrichment. We found 2093 TPS in 43% of H. salinarum genes and 3515 TPS in 49% of H. volcanii chromosomal genes. Of the 244 conserved TPS sites found, the majority were located around start and stop codons of orthologous genes. Specific genes are highlighted when discussing antisense, ribosome and insertion sequence associated TPS. Examples include the cell division gene ftsZ2, whose differential processing signal along growth was detected and correlated with post-transcriptional regulation, and biogenesis of sense overlapping transcripts associated with IS200/IS605. We hereby present the comparative, transcriptomics-based processing site maps with a companion browsing interface.

Early response of methanogenic archaea to H2 as evaluated by metagenomics and metatranscriptomics.

BACKGROUND: The molecular machinery of the complex microbiological cell factory of biomethane production is not fully understood. One of the process control elements is the regulatory role of hydrogen (H2). Reduction of carbon dioxide (CO2) by H2 is rate limiting factor in methanogenesis, but the community intends to keep H2 concentration low in order to maintain the redox balance of the overall system. H2 metabolism in methanogens becomes increasingly important in the Power-to-Gas renewable energy conversion and storage technologies. RESULTS: The early response of the mixed mesophilic microbial community to H2 gas injection was investigated with the goal of uncovering the first responses of the microbial community in the CH4 formation and CO2 mitigation Power-to-Gas process. The overall microbial composition changes, following a 10 min excessive bubbling of H2 through the reactor, was investigated via metagenome and metatranscriptome sequencing. The overall composition and taxonomic abundance of the biogas producing anaerobic community did not change appreciably 2 hours after the H2 treatment, indicating that this time period was too short to display differences in the proliferation of the members of the microbial community. There was, however, a substantial increase in the expression of genes related to hydrogenotrophic methanogenesis of certain groups of Archaea. As an early response to H2 exposure the activity of the hydrogenotrophic methanogenesis in the genus Methanoculleus was upregulated but the hydrogenotrophic pathway in genus Methanosarcina was downregulated. The RT-qPCR data corroborated the metatranscriptomic RESULTS: H2 injection also altered the metabolism of a number of microbes belonging in the kingdom Bacteria. Many Bacteria possess the enzyme sets for the Wood-Ljungdahl pathway. These and the homoacetogens are partners for syntrophic community interactions between the distinct kingdoms of Archaea and Bacteria. CONCLUSIONS: External H2 regulates the functional activity of certain Bacteria and Archaea. The syntrophic cross-kingdom interactions in H2 metabolism are important for the efficient operation of the Power-to-Gas process. Therefore, mixed communities are recommended for the large scale Power-to-Gas process rather than single hydrogenotrophic methanogen strains. Fast and reproducible response from the microbial community can be exploited in turn-off and turn-on of the Power-to-Gas microbial cell factories.

CdrS Is a Global Transcriptional Regulator Influencing Cell Division in Haloferax volcanii.

Transcriptional regulators that integrate cellular and environmental signals to control cell division are well known in bacteria and eukaryotes, but their existence is poorly understood in archaea. We identified a conserved gene (cdrS) that encodes a small protein and is highly transcribed in the model archaeon Haloferax volcanii. The cdrS gene could not be deleted, but CRISPR interference (CRISPRi)-mediated repression of the cdrS gene caused slow growth and cell division defects and changed the expression of multiple genes and their products associated with cell division, protein degradation, and metabolism. Consistent with this complex regulatory network, overexpression of cdrS inhibited cell division, whereas overexpression of the operon encoding both CdrS and a tubulin-like cell division protein (FtsZ2) stimulated division. Chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) identified 18 DNA-binding sites of the CdrS protein, including one upstream of the promoter for a cell division gene, ftsZ1, and another upstream of the essential gene dacZ, encoding diadenylate cyclase involved in c-di-AMP signaling, which is implicated in the regulation of cell division. These findings suggest that CdrS is a transcription factor that plays a central role in a regulatory network coordinating metabolism and cell division. IMPORTANCE Cell division is a central mechanism of life and is essential for growth and development. Members of the Bacteria and Eukarya have different mechanisms for cell division, which have been studied in detail. In contrast, cell division in members of the Archaea is still understudied, and its regulation is poorly understood. Interestingly, different cell division machineries appear in members of the Archaea, with the Euryarchaeota using a cell division apparatus based on the tubulin-like cytoskeletal protein FtsZ, as in bacteria. Here, we identify the small protein CdrS as essential for survival and a central regulator of cell division in the euryarchaeon Haloferax volcanii. CdrS also appears to coordinate other cellular pathways, including synthesis of signaling molecules and protein degradation. Our results show that CdrS plays a sophisticated role in cell division, including regulation of numerous associated genes. These findings are expected to initiate investigations into conditional regulation of division in archaea.

Analysis of Haloferax mediterranei Lrp Transcriptional Regulator.

Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.

Functional analysis of Lsm protein under multiple stress conditions in the extreme haloarchaeon Haloferax mediterranei.

The Sm, like-Sm, and Hfq proteins belonging to the Sm superfamily of proteins are represented in all domains of life. These proteins are involved in several RNA metabolism pathways. The functions of bacterial Hfq and eukaryotic Sm proteins have been described, but knowledge about the in vivo functions of archaeal Sm proteins remains limited. This study aims to improve the understanding of Lsm proteins and their role using the haloarchaeon Haloferax mediterranei as a model microorganism. The Haloferax mediterranei genome contains one lsm gene that overlaps with the rpl37e gene. To determine the expression of lsm and rpl37e genes and the co-transcription of both, reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed under different standard and stress conditions. The results suggest that the expression of lsm and rpl37e is constitutive. Co-transcription occurs at sub-optimal salt concentrations and temperatures, depending on the growth phase. The halophilic Lsm protein contains two Sm motifs, Sm1 and Sm2, and the sequence encoding the Sm2 motif also constitutes the promoter of the rpl37e gene. To investigate their biological functions, the lsm deletion mutant and the Sm1 motif deletion mutant, where the Sm2 motif remained intact, were generated and characterised. Comparison of the lsm deletion mutant, Sm1 deletion mutant, and the parental strain HM26 under standard and stress growth conditions revealed growth differences. Finally, swarming assays in complex and defined media showed greater swarming capacity in the deletion mutants.

Characterization of Regulatory Elements of L11 and L1 Operons in Thermophilic Bacteria and Archaea.

Ribosomal protein L1 is a conserved two-domain protein that is involved in formation of the L1 stalk of the large ribosomal subunit. When there are no free binding sites available on the ribosomal 23S RNA, the protein binds to the specific site on the mRNA of its own operon (L11 operon in bacteria and L1 operon in archaea) preventing translation. Here we show that the regulatory properties of the r-protein L1 and its domain I are conserved in the thermophilic bacteria Thermus and Thermotoga and in the halophilic archaeon Haloarcula marismortui. At the same time the revealed features of the operon regulation in thermophilic bacteria suggest presence of two regulatory regions.

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo.

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed "recoding". In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for -1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed -1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed.

Towards the Elucidation of Assimilative nasABC Operon Transcriptional Regulation in Haloferax mediterranei.

The assimilatory pathway of the nitrogen cycle in the haloarchaeon Haloferax mediterranei has been well described and characterized in previous studies. However, the regulatory mechanisms involved in the gene expression of this pathway remain unknown in haloarchaea. This work focuses on elucidating the regulation at the transcriptional level of the assimilative nasABC operon (HFX_2002 to HFX_2004) through different approaches. Characterization of its promoter region using ß-galactosidase as a reporter gene and site-directed mutagenesis has allowed us to identify possible candidate binding regions for a transcriptional factor. The identification of a potential transcriptional regulator related to nitrogen metabolism has become a real challenge due to the lack of information on haloarchaea. The investigation of protein-DNA binding by streptavidin bead pull-down analysis combined with mass spectrometry resulted in the in vitro identification of a transcriptional regulator belonging to the Lrp/AsnC family, which binds to the nasABC operon promoter (p.nasABC). To our knowledge, this study is the first report to suggest the AsnC transcriptional regulator as a powerful candidate to play a regulatory role in nasABC gene expression in Hfx. mediterranei and, in general, in the assimilatory nitrogen pathway.

Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems.

CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.

Nitrate-Responsive Suppression of Dimethyl Sulfoxide Respiration in a Facultative Anaerobic Haloarchaeon, Haloferax volcanii.

Haloferax volcanii is a facultative anaerobic haloarchaeon that can grow using nitrate or dimethyl sulfoxide (DMSO) as a respiratory substrate under anaerobic conditions. Comparative transcriptome analysis of denitrifying and aerobic cells of H. volcanii indicated extensive changes in gene expression involving the activation of denitrification, suppression of DMSO respiration, and conversion of the heme biosynthetic pathway under denitrifying conditions. The anaerobic growth of H. volcanii by DMSO respiration was inhibited at nitrate concentrations of <1 mM, whereas nitrate-responsive growth inhibition was not observed in the ΔnarO mutant. A reporter assay demonstrated that the transcription of the dms operon was suppressed by nitrate. In contrast, the anaerobic growth of the ΔdmsR mutant by denitrification was little affected by the addition of DMSO. NarO has been identified as an activator of denitrification-related genes in response to anaerobic conditions, and here, we found that NarO is also involved in nitrate-responsive suppression of the dms operon. Nitrate-responsive suppression of DMSO respiration is known in several bacteria such as Escherichia coli and photosynthetic Rhodobacter species. This is the first report to show that a regulatory mechanism that suppresses DMSO respiration in response to nitrate exists not only in bacteria but also in haloarchaea. IMPORTANCE Haloferax volcanii can grow anaerobically by denitrification (nitrate respiration) or DMSO respiration. In facultative anaerobic bacteria that can grow by both nitrate respiration and DMSO respiration, nitrate respiration is preferentially induced when both nitrate and DMSO are available as the respiratory substrates. The results of transcriptome analysis, growth phenotyping, and reporter assays indicated that DMSO respiration is suppressed in response to nitrate in H. volcanii. The haloarchaeon-specific regulator NarO, which activates denitrification under anaerobic conditions, is suggested to be involved in the nitrate-responsive suppression of DMSO respiration.

An alternative resource allocation strategy in the chemolithoautotrophic archaeon Methanococcus maripaludis.

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h-1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h-1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.