Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus.

Methanothermococcus thermolithotrophicus is the only known methanogen that grows on sulfate as its sole sulfur source, uniquely uniting methanogenesis and sulfate reduction. Here we use physiological, biochemical and structural analyses to provide a snapshot of the complete sulfate reduction pathway of this methanogenic archaeon. We find that later steps in this pathway are catalysed by atypical enzymes. PAPS (3'-phosphoadenosine 5'-phosphosulfate) released by APS kinase is converted into sulfite and 3'-phosphoadenosine 5'-phosphate (PAP) by a PAPS reductase that is similar to the APS reductases of dissimilatory sulfate reduction. A non-canonical PAP phosphatase then hydrolyses PAP. Finally, the F420-dependent sulfite reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that the sulfate reduction pathway is present in several methanogens, the sulfate assimilation pathway in M. thermolithotrophicus is distinct. We propose that this pathway was 'mix-and-matched' through the acquisition of assimilatory and dissimilatory enzymes from other microorganisms and then repurposed to fill a unique metabolic role.

Comparative Transcriptomics Sheds Light on Remodeling of Gene Expression during Diazotrophy in the Thermophilic Methanogen Methanothermococcus thermolithotrophicus.

Some marine thermophilic methanogens are able to perform energy-consuming nitrogen fixation despite deriving only little energy from hydrogenotrophic methanogenesis. We studied this process in Methanothermococcus thermolithotrophicus DSM 2095, a methanogenic archaeon of the order Methanococcales that contributes to the nitrogen pool in some marine environments. We successfully grew this archaeon under diazotrophic conditions in both batch and fermenter cultures, reaching the highest cell density reported so far. Diazotrophic growth depended strictly on molybdenum and, in contrast to other diazotrophs, was not inhibited by tungstate or vanadium. This suggests an elaborate control of metal uptake and a specific metal recognition system for the insertion into the nitrogenase cofactor. Differential transcriptomics of M. thermolithotrophicus grown under diazotrophic conditions with ammonium-fed cultures as controls revealed upregulation of the nitrogenase machinery, including chaperones, regulators, and molybdate importers, as well as simultaneous upregulation of an ammonium transporter and a putative pathway for nitrate and nitrite utilization. The organism thus employs multiple synergistic strategies for uptake of nitrogen nutrients during the early exponential growth phase without altering transcription levels for genes involved in methanogenesis. As a counterpart, genes coding for transcription and translation processes were downregulated, highlighting the maintenance of an intricate metabolic balance to deal with energy constraints and nutrient limitations imposed by diazotrophy. This switch in the metabolic balance included unexpected processes, such as upregulation of the CRISPR-Cas system, probably caused by drastic changes in transcription levels of putative mobile and virus-like elements. IMPORTANCE The thermophilic anaerobic archaeon M. thermolithotrophicus is a particularly suitable model organism to study the coupling of methanogenesis to diazotrophy. Likewise, its capability of simultaneously reducing N2 and CO2 into NH3 and CH4 with H2 makes it a viable target for biofuel production. We optimized M. thermolithotrophicus cultivation, resulting in considerably higher cell yields and enabling the successful establishment of N2-fixing bioreactors. Improved understanding of the N2 fixation process would provide novel insights into metabolic adaptations that allow this energy-limited extremophile to thrive under diazotrophy, for instance, by investigating its physiology and uncharacterized nitrogenase. We demonstrated that diazotrophic growth of M. thermolithotrophicus is exclusively dependent on molybdenum, and complementary transcriptomics corroborated the expression of the molybdenum nitrogenase system. Further analyses of differentially expressed genes during diazotrophy across three cultivation time points revealed insights into the response to nitrogen limitation and the coordination of core metabolic processes.

Biological methane production under putative Enceladus-like conditions.

The detection of silica-rich dust particles, as an indication for ongoing hydrothermal activity, and the presence of water and organic molecules in the plume of Enceladus, have made Saturn's icy moon a hot spot in the search for potential extraterrestrial life. Methanogenic archaea are among the organisms that could potentially thrive under the predicted conditions on Enceladus, considering that both molecular hydrogen (H2) and methane (CH4) have been detected in the plume. Here we show that a methanogenic archaeon, Methanothermococcus okinawensis, can produce CH4 under physicochemical conditions extrapolated for Enceladus. Up to 72% carbon dioxide to CH4 conversion is reached at 50 bar in the presence of potential inhibitors. Furthermore, kinetic and thermodynamic computations of low-temperature serpentinization indicate that there may be sufficient H2 gas production to serve as a substrate for CH4 production on Enceladus. We conclude that some of the CH4 detected in the plume of Enceladus might, in principle, be produced by methanogens.

Profiling of Indigenous Microbial Community Dynamics and Metabolic Activity During Enrichment in Molasses-Supplemented Crude Oil-Brine Mixtures for Improved Understanding of Microbial Enhanced Oil Recovery.

Anaerobic incubations using crude oil and brine from a North Sea reservoir were conducted to gain increased understanding of indigenous microbial community development, metabolite production, and the effects on the oil-brine system after addition of a complex carbon source, molasses, with or without nitrate to boost microbial growth. Growth of the indigenous microbes was stimulated by addition of molasses. Pyrosequencing showed that specifically Anaerobaculum, Petrotoga, and Methanothermococcus were enriched. Addition of nitrate favored the growth of Petrotoga over Anaerobaculum. The microbial growth caused changes in the crude oil-brine system: formation of oil emulsions, and reduction of interfacial tension (IFT). Reduction in IFT was associated with microbes being present at the oil-brine interphase. These findings suggest that stimulation of indigenous microbial growth by addition of molasses has potential as microbial enhanced oil recovery (MEOR) strategy in North Sea oil reservoirs.

Linking energy production and protein synthesis in hydrogenotrophic methanogens.

Hydrogenotrophic methanogens possessing the hydrogen-dependent dehydrogenase Hmd also encode paralogs of this protein whose function is poorly understood. Here we present biochemical evidence that the two inactive Hmd paralogs of Methanocaldococcus jannaschii, HmdII and HmdIII, form binary and ternary complexes with several components of the protein translation apparatus. HmdII and HmdIII, but not the active dehydrogenase Hmd, bind with micromolar binding affinities to a number of tRNAs and form ternary complexes with tRNA(Pro) and prolyl-tRNA synthetase (ProRS). Fluorescence spectroscopy experiments also suggest that binding of HmdII and ProRS involves distinct binding determinants on the tRNA. These biochemical data suggest the possibility of a regulatory link between energy production and protein translation pathways that may allow a rapid cellular response to altered environmental conditions.

Growth of methanogens under defined hydrogen conditions.

Hydrogen (H(2)) is a primary electron donor for methanogenesis and its availability can have profound effects on gene expression and the physiology of energy conservation. The rigorous evaluation of the effects of hydrogen conditions requires the comparison of cultures that are grown under hydrogen limitation and hydrogen excess. The growth of methanogens under defined hydrogen conditions is complicated by the dynamics of hydrogen dissolution and its utilization by the cells. In batch culture, gassing and agitation conditions must be carefully calibrated, and even then variations in growth rate and cell density are hard to avoid. Using chemostats, continuous cultures can be achieved whose nutritional states are known, while growth rate and cell density are invariant. Cultures whose growth is limited by hydrogen can be compared to cultures whose growth is limited by some other nutrient and are therefore under hydrogen excess.

The crystal structure of protein MJ1225 from Methanocaldococcus jannaschii shows strong conservation of key structural features seen in the eukaryal gamma-AMPK.

In mammals, 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (alpha) and two regulatory subunits (beta and gamma). The gamma-subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. We describe herein the crystal structure of protein MJ1225 from Methanocaldococcus jannaschii complexed to AMP, ADP, and ATP. Our data provide evidence of a strong conservation of the key functional features seen in the gamma-subunit of the eukaryotic AMPK, and more importantly, it reveals a novel AMP binding site, herein denoted as site E, which had not been previously described in cystathionine beta-synthase domains so far. Site E is located in a small cavity existing between the alpha-helices structurally equivalent to those disrupting the internal symmetry of each Bateman domain in gamma-AMPKs and shows striking similarities with a symmetry-related crevice of the mammalian enzyme that hosts the pathological mutation N488I.

Hybrid Ptr2-like activators of archaeal transcription.

Methanocaldococcus jannaschii Ptr2, a member of the Lrp/AsnC family of bacterial DNA-binding proteins, is an activator of its eukaryal-type core transcription apparatus. In Lrp-family proteins, an N-terminal helix-turn-helix DNA-binding and dimerizing domain is joined to a C-terminal effector and multimerizing domain. A cysteine-scanning surface mutagenesis shows that the C-terminal domain of Ptr2 is responsible for transcriptional activation; two types of DNA binding-positive but activation-defective mutants are found: those unable to recruit the TBP and TFB initiation factors to the promoter, and those failing at a post-recruitment step. Transcriptional activation through the C-terminal Ptr2 effector domain is exploited in a screen of other Lrp effector domains for activation capability by constructing hybrid proteins with the N-terminal DNA-binding domain of Ptr2. Two hybrid proteins are effective activators: Ptr-H10, fusing the effector domain of Pyrococcus furiosus LrpA, and Ptr-H16, fusing the P. furiosus ORF1231 effector domain. Both new activators exhibit distinguishing characteristics: unlike octameric Ptr2, Ptr-H10 is a dimer; unlike Ptr2, the octameric Ptr-H16 poorly recruits TBP to the promoter, but more effectively co-recruits TFB with TBP. In contrast, the effector domain of Ptr1, the M. jannaschii Ptr2 paralogue, yields only very weak activation.

Concerted bifunctionality of the dCTP deaminase-dUTPase from Methanocaldococcus jannaschii: a structural and pre-steady state kinetic analysis.

Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue alpha,beta-imido-dUTP and E145Q in complex with diphosphate. Both mutant enzymes were defect in the deaminase reaction and had reduced dUTPase activity. In the structure of E145Q in complex with diphosphate, the diphosphate occupied the same position as the beta- and gamma-phosphoryls of the nucleotide analogue in the E145A complex. The C-terminal region that is unresolved in the apo-form of the enzyme was ordered in both complexes and closed over the active site by interacting with the phosphate backbone of the nucleotide or with the diphosphate. A magnesium ion was readily observed to complex with all three phosphoryls in the nucleotide complex or with the diphosphate. A water molecule that is likely to be involved in the nucleotidyl diphosphorylase reaction was observed in the E145A:alpha,beta-imido-dUTP complex and positioned similarly as in the monofunctional trimeric dUTPase. A comparison of the active sites of the bifunctional enzyme and the monofunctional family members, dCTP deaminase and dUTPase, suggests similar reaction mechanisms. The similar side chain conformations in the deaminase site between the nucleotide and diphosphate complexes indicated a concerted re-arrangement, or induced fit, of the whole active site promoted by enzyme and nucleotide phosphoryl interactions. A pre-steady state kinetic analysis of the bifunctional reaction and the dUTPase half-reaction supported a conformational change upon substrate binding in both reactions and a concerted catalytic step for the bifunctional reaction.

An iron(II) dependent formamide hydrolase catalyzes the second step in the archaeal biosynthetic pathway to riboflavin and 7,8-didemethyl-8-hydroxy-5-deazariboflavin.

The early steps in the biosynthesis of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) and riboflavin in the archaea differ from the established eukaryotic and bacterial pathways. The archaeal pathway has been proposed to begin with an archaeal-specific GTP cyclohydrolase III that hydrolyzes the imidazole ring of GTP but does not remove the resulting formyl group from the formamide [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084 ]. This enzyme is different than the bacterial GTP cyclohydrolase II which catalyzes both reactions. Here we describe the identification and characterization of the formamide hydrolase that catalyzes the second step in the archaeal Fo and riboflavin biosynthetic pathway. The Methanocaldococcus jannaschii MJ0116 gene was cloned and heterologously expressed, and the resulting enzyme was shown to catalyze the formation of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (APy) and formate from 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-monophosphate (FAPy). The MJ0116-derived protein has been named ArfB to indicate that it catalyzes the second step in archaeal riboflavin and Fo biosynthesis. ArfB was found to require ferrous iron for activity although metal analysis by ICP indicated the presence of zinc as well as iron in the purified protein. The identification of this enzyme confirms the involvement of GTP cyclohydrolase III (ArfA) in archaeal riboflavin and Fo biosynthesis.

Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography.

We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-L-tyrosine. The 1.7 A crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-L-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-L-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Angstroms resolutions by SAD phasing at CuK alpha and CrK alpha wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.

Small molecule inhibition of a Group II chaperonin: pinpointing a loop region within the equatorial domain as necessary for protein refolding.

The functionality of regions within the equatorial domain of Group II chaperonins is poorly understood. Previously we showed that a 70 amino acid sequence within this domain on the single-subunit recombinant thermosome from Methanocaldococcus jannaschii (rTHS) contains residues directly responsible for refolding protein substrates [L.M. Bergeron, C. Lee, D.S. Clark, Identification of a critical chaperoning region on an archaeal recombinant thermosome, Biochem. Biophys. Res. Commun. 369 (2008) 707-711]. In the present study, 6-aminopenicillanic acid (6-APA) was found to bind to rTHS and inhibit it from refolding proteins. Fluorescence anisotropy was used to measure a 6-APA/rTHS dissociation constant of 17.1 microM and verify that the binding site is within the first 70 amino-terminal rTHS residues. Docking simulations point to a specific loop region at residues 53-57 on rTHS as the most likely binding region. This loop region is located within the oligomeric association sites of the wild-type thermosome. These results implicate a specific equatorial region of Group II chaperonins in the refolding of proteins, and suggest its importance in conformational changes that accompany chaperone function.

Engineering of functional replication protein a homologs based on insights into the evolution of oligonucleotide/oligosaccharide-binding folds.

The bacterial single-stranded DNA-binding protein (SSB) and the archaeal/eukaryotic functional homolog, replication protein A (RPA), are essential for most aspects of DNA metabolism. Structural analyses of the architecture of SSB and RPA suggest that they are composed of different combinations of a module called the oligonucleotide/oligosaccharide-binding (OB) fold. Members of the domains Bacteria and Eukarya, in general, contain one type of SSB or RPA. In contrast, organisms in the archaeal domain have different RPAs made up of different organizations of OB folds. Interestingly, the euryarchaeon Methanosarcina acetivorans harbors multiple functional RPAs named MacRPA1 (for M. acetivorans RPA 1), MacRPA2, and MacRPA3. Comparison of MacRPA1 with related proteins in the publicly available databases suggested that intramolecular homologous recombination might play an important role in generating some of the diversity of OB folds in archaeal cells. On the basis of this information, from a four-OB-fold-containing RPA, we engineered chimeric modules to create three-OB-fold-containing RPAs to mimic a novel form of RPA found in Methanococcoides burtonii and Methanosaeta thermophila. We further created two RPAs that mimicked the RPAs in Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus through fusions of modules from MacRPA1 and M. thermautotrophicus RPA. Functional studies of these engineered proteins suggested that fusion and shuffling of OB folds can lead to well-folded polypeptides with most of the known properties of SSB and RPAs. On the basis of these results, different models that attempt to explain how intramolecular and intermolecular homologous recombination can generate novel forms of SSB or RPAs are proposed.

Aminoacylation of tRNA with phosphoserine for synthesis of cysteinyl-tRNA(Cys).

Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNA(Cys) by an indirect pathway, whereby O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA(Cys) with phosphoserine (Sep), and Sep-tRNA-Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNA(Cys) tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1alpha or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNA(Cys).

Promiscuous anaerobes: new and unconventional metabolism in methanogenic archaea.

The development of an oxygenated atmosphere on earth resulted in the polarization of life into two major groups, those that could live in the presence of oxygen and those that could not-the aerobes and the anaerobes. The evolution of aerobes from the earliest anaerobic prokaryotes resulted in a variety of metabolic adaptations. Many of these adaptations center on the need to sustain oxygen-sensitive reactions and cofactors to function in the new oxygen-containing atmosphere. Still other metabolic pathways that were not sensitive to oxygen also diverged. This is likely due to the physical separation of the organisms, based on their ability to live in the presence of oxygen, which allowed for the independent evolution of the pathways. Through the study of metabolic pathways in anaerobes and comparison to the more established pathways from aerobes, insight into metabolic evolution can be gained. This, in turn, can allow for extra- polation to those metabolic pathways occurring in the Last Universal Common Ancestor (LUCA). Some of the unique and uncanonical metabolic pathways that have been identified in the archaea with emphasis on the biochemistry of an obligate anaerobic methanogen, Methanocaldococcus jannaschii are reviewed.

Crystal structure and function of 5-formaminoimidazole-4-carboxamide ribonucleotide synthetase from Methanocaldococcus jannaschii.

Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.

Methane production by methanogens following an aerobic washing procedure: simplifying methods for manipulation.

The recent discovery of methane in the martian atmosphere is arguably one of the most important discoveries in the field of astrobiology. One possible source of this methane could be a microorganism analogous to those on Earth in the domain Archaea known as methanogens. Methanogens are described as obligately anaerobic, and methods developed to work with methanogens typically include anaerobic media and buffers, gassing manifolds, and possibly anaerobic chambers. To determine if the time, effort, and supplies required to maintain anaerobic conditions are necessary to maintain viability, we compared anaerobically washed cells with cells that were washed in the presence of atmospheric oxygen. Anaerobic tubes were opened, and cultures were poured into plastic centrifuge tubes, centrifuged, and suspended in fresh buffer, all in the presence of atmospheric oxygen. Washed cells from both aerobic and anaerobic procedures were inoculated into methanogenic growth media under anaerobic conditions and incubated at temperatures conducive to growth for each methanogenic strain tested. Methane production was measured at time intervals using a gas chromatograph. In three strains, significant differences were not seen between aerobically and anaerobically washed cells. In one strain, there was significantly less methane production observed following aerobic washing at some time points; however, substantial methane production occurred following both procedures. Thus, it appears that aerobic manipulations for relatively short periods of time with at least a few species of methanogens may not lead to loss of viability. With the discovery of methane in the martian atmosphere, it is likely that there will be an increase in astrobiology-related methanogen research. The research reported here should simplify the methodology.

Methanocaldococcus jannaschii uses a modified mevalonate pathway for biosynthesis of isopentenyl diphosphate.

Archaea have been shown to produce isoprenoids from mevalonate; however, genome analysis has failed to identify several genes in the mevalonate pathway on the basis of sequence similarity. A predicted archaeal kinase, coded for by the MJ0044 gene, was associated with other mevalonate pathway genes in the archaea and was predicted to be the "missing" phosphomevalonate kinase. The MJ0044-derived protein was tested for phosphomevalonate kinase activity and was found not to catalyze this reaction. The MJ0044 gene product was found to phosphorylate isopentenyl phosphate, generating isopentenyl diphosphate. Unlike other known kinases associated with isoprene biosynthesis, Methanocaldococcus jannaschii isopentenyl phosphate kinase is predicted to be a member of the aspartokinase superfamily.

Analysis of the differences in metabolic network expansion between prokaryotes and eukaryotes.

Recent evidence points to the existence of scale-free properties in many biological networks. By topological analysis, several models including preferential attachment and hierarchical modules have been proposed to explain how these networks are organized. On the other hand, analyses using dynamics have suggested that gene expression and metabolic networks have been organized with the scale-free property by the other models such as "rich-travel-more" and "log-normal dynamics." Because most of these approaches are based on comparative genomics of extant species, and did not consider evolutionary events such as horizontal gene transfer, gene loss and gene gain, we have analyzed transition of metabolic networks from the vertical point of view of evolution. First, to identify metabolic networks of common ancestors, we applied a parsimony algorithm for the enzymatic reaction set. Then by comparing the estimated metabolic networks among common ancestors, we investigated the transition of metabolic networks along the evolutionary process. As a result, we estimated enzymatic reaction contents of 227 common ancestors from 228 extant species, and found that links of several specific metabolites have frequently changed during the course of evolution.

Cysteinyl-tRNA(Cys) formation in Methanocaldococcus jannaschii: the mechanism is still unknown.

Most organisms form Cys-tRNA(Cys), an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. Söll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNA(Cys) formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the K(m) for cysteine and a ninefold reduction in the k(cat) for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNA(Cys) synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNA(Cys) formation in M. jannaschii still remains to be discovered.