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Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.

Thermophilic microbial communities growing in low-oxygen environments often contain early-evolved archaea and bacteria, which hold clues regarding mechanisms of cellular respiration relevant to early life. Here, we conducted replicate metagenomic, metatranscriptomic, microscopic, and geochemical analyses on two hyperthermophilic (82–84 °C) filamentous microbial communities ( Conch and Octopus Springs , Yellowstone National Park, WY) to understand the role of oxygen, sulfur, and arsenic in energy conservation and community composition. We report that hyperthermophiles within the Aquificota ( Thermocrinis ), Pyropristinus ( Caldipriscus ), and Thermoproteota ( Pyrobaculum ) are abundant in both communities; however, higher oxygen results in a greater diversity of aerobic heterotrophs. Metatranscriptomics revealed major shifts in respiratory pathways of keystone chemolithotrophs due to differences in oxygen versus sulfide. Specifically, early-evolved hyperthermophiles express high levels of high-affinity cytochrome bd and CydAA’ oxidases in suboxic sulfidic environments and low-affinity heme Cu oxidases under microaerobic conditions. These energy-conservation mechanisms using cytochrome oxidases in high-temperature, low-oxygen habitats likely played a crucial role in the early evolution of microbial life. The early evolution of microbial respiration under low-oxygen conditions is still incompletely understood. Here, the authors analyzed the genomics of filamentous geothermal communities and showed that they harness energy from reduced forms of sulfur and arsenic and use low oxygen levels for respiration.

Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea ( Aeropyrum pernix and Pyrobaculum calidifontis ) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens , and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been ‘domesticated’, that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA. Bacteria can exchange DNA through extracellular appendages (‘mating pili’) in a process known as conjugation. Here, Beltran et al. determine atomic structures by cryo-electron microscopy of a bacterial conjugative pilus and two archaeal pili, showing that the archaeal pili are homologous to bacterial mating pili.

Hyperthermophilic (‘superheat-loving’) archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency ( k cat /K m ) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications. Graphical Abstract

This study aimed to address the stability limitations of third-generation biosensors using enzymes from mesophilic organisms, by engineering a stable direct electron transfer (DET)-type dehydrogenase capable of transferring electrons extracted from the substrate to the electrode. A fusion protein combining the mediated electron transfer (MET)-type aldose sugar dehydrogenase from the hyperthermophile Pyrobaculum aerophilum (PaeASD), which cannot transfer electrons generated by enzymatic reactions to the electrode without a mediator, and the natural electron transfer protein cytochrome b 562 (cyt b 562 ) was developed to investigate its potential for the DET reaction. A recombinant protein expression system was established in Escherichia coli to produce the PaeASD-cyt b 562 fusion protein, which was purified from the soluble fraction of the host cells. Intramolecular electron transfer from pyrroloquinoline quinone (PQQ) to the heme group within the PaeASD-cyt b 562 fusion protein was investigated using UV–Vis absorption spectroscopy. Upon the addition of glucose, an increase in absorption corresponding to reduced heme molecules was observed, indicating electron transfer from glucose to the heme group in the cyt b 562 component via PQQ in the PaeASD component. The DET capability of the fusion protein was evaluated using cyclic voltammetry with screen-printed carbon electrodes. A glucose concentration-dependent increase in current response confirmed DET activity. Notably, the fusion protein retained over 80% of its initial current response even after 2 months of storage at 4 °C. The novel robust PaeASD-cyt b 562 fusion protein demonstrated efficient DET capability, highlighting its high potential for application in the development of third-generation biosensors.

Ribosomes translate mRNA into protein. Despite divergence in ribosome structure over the course of evolution, the catalytic site, known as the peptidyl transferase centre (PTC), is thought to be nearly universally conserved. Here we identify clades of archaea that have highly divergent ribosomal RNA sequences in the PTC. To understand how these PTC sequences fold, we determined cryo-EM structures of the 70S and 50S ribosomes to 2.4 Å and 2 Å, respectively, from the hyperthermophilic archaeon Pyrobaculum calidifontis . PTC sequence variation leads to the rearrangement of key base triples, and differences between archaeal and bacterial ribosomal proteins enable sequence variation in archaeal PTCs. Finally, we identify an archaeal ribosome hibernation factor, Dri, that differs from known bacterial and eukaryotic hibernation factors and is found in multiple archaeal phyla. Overall, this work identifies factors that regulate ribosome function in archaea and reveals a larger diversity of the most ancient sequences in the ribosome. Sequence and structural analyses reveal a divergent peptidyl transferase centre and a hibernation factor in archaea.

RNase P RNA is an ancient, nearly universal feature of life. As part of the ribonucleoprotein RNase P complex, the RNA component catalyzes essential removal of 5' leaders in pre-tRNAs. In 2004, Li and Altman computationally identified the RNase P RNA gene in all but three sequenced microbes: Nanoarchaeum equitans, Pyrobaculum aerophilum, and Aquifex aeolicus (all hyperthermophiles) [Li Y, Altman S (2004) RNA 10:1533—1540]. A recent study concluded that N. equitans does not have or require RNase P activity because it lacks 5' tRNA leaders. The \"missing\" RNase P RNAs in the other two species is perplexing given evidence or predictions that tRNAs are trimmed in both, prompting speculation that they may have developed novel alternatives to 5' pre-tRNA processing. Using comparative genomics and improved computational methods, we have now identified a radically minimized form of the RNase P RNA in five Pyrobaculum species and the related crenarchaea Caldivirga maquilingensis and Vulcanisaeta distributa, all retaining a conventional catalytic domain, but lacking a recognizable specificity domain. We confirmed 5' tRNA processing activity by high-through-put RNA sequencing and in vitro biochemical assays. The Pyrobaculum and Caldivirga RNase P RNAs are the smallest naturally occurring form yet discovered to function as trans-acting precursor tRNA-processing ribozymes. Loss of the specificity domain in these RNAs suggests altered substrate specificity and could be a useful model for finding other potential roles of RNase P. This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene.

Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.

Actin is one of the most abundant and highly conserved eukaryotic proteins, but the basis for the exquisite sequence conservation in actin is not known. In contrast, bacterial actin-like proteins display almost no sequence conservation and form very different filaments. We have examined the filaments formed by an actin-like protein in the third kingdom of life, Archaea, and although they only have a single strand, the strand is very similar to each of the two strands in actin. This gives previously unidentified insights into the divergence of archaea and eukaryotes. The prokaryotic origins of the actin cytoskeleton have been firmly established, but it has become clear that the bacterial actins form a wide variety of different filaments, different both from each other and from eukaryotic F-actin. We have used electron cryomicroscopy (cryo-EM) to examine the filaments formed by the protein crenactin (a crenarchaeal actin) from Pyrobaculum calidifontis , an organism that grows optimally at 90 °C. Although this protein only has ∼20% sequence identity with eukaryotic actin, phylogenetic analyses have placed it much closer to eukaryotic actin than any of the bacterial homologs. It has been assumed that the crenactin filament is double-stranded, like F-actin, in part because it would be hard to imagine how a single-stranded filament would be stable at such high temperatures. We show that not only is the crenactin filament single-stranded, but that it is remarkably similar to each of the two strands in F-actin. A large insertion in the crenactin sequence would prevent the formation of an F-actin-like double-stranded filament. Further, analysis of two existing crystal structures reveals six different subunit–subunit interfaces that are filament-like, but each is different from the others in terms of significant rotations. This variability in the subunit–subunit interface, seen at atomic resolution in crystals, can explain the large variability in the crenactin filaments observed by cryo-EM and helps to explain the variability in twist that has been observed for eukaryotic actin filaments.

Genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis revealed the presence of an open reading frame, Pcal_0768, corresponding to a putative 4-α-glucanotranferase belonging to glycoside hydrolases (GH) family 77. We have produced, in Escherichia coli , and purified recombinant Pcal_0768 which exhibited high disproportionation (690 U mg −1 ) activity. To the best of our knowledge, this is the highest ever reported activity for any member of family GH77. Maltooligosaccharides, when used as sole substrates, were disproportionated into linear maltooligohomologues. The analysis of the reaction end products revealed no evidence for the production of cycloamyloses. Catalytic activity of the enzyme remained unchanged in the presence or the absence of ionic and nonionic detergents. γ-cyclodextrin, an inhibitor of 4-α-glucanotransferases, did not show any inhibitory effect on Pcal_0768 activity. These properties make Pcal_0768 a potential candidate for starch processing industry.