MiST 4.0: a new release of the microbial signal transduction database, now with a metagenomic component.

Signal transduction systems in bacteria and archaea link environmental stimuli to specific adaptive cellular responses. They control gene expression, motility, biofilm formation, development and other processes that are vital to survival. The microbial signal transduction (MiST) database is an online resource that stores tens of thousands of genomes and allows users to explore their signal transduction profiles, analyze genomes in bulk using the database application programming interface (API) and make testable hypotheses about the functions of newly identified signaling systems. However, signal transduction in metagenomes remained completely unexplored. To lay the foundation for research in metagenomic signal transduction, we have prepared a new release of the MiST database, MiST 4.0, which features over 10 000 metagenome-assembled genomes (MAGs), a scaled representation of proteins and detailed BioSample information. In addition, several thousands of new genomes have been processed and stored in the database. A new interface has been developed that allows users to seamlessly switch between genomes and MAGs. MiST 4.0 is freely available at https://mistdb.com; metagenomes and MAGs can also be explored using the API available on the same page.

Amine-recognizing domain in diverse receptors from bacteria and archaea evolved from the universal amino acid sensor.

Bacteria possess various receptors that sense different signals and transmit information to enable an optimal adaptation to the environment. A major limitation in microbiology is the lack of information on the signal molecules that activate receptors. Signals recognized by sensor domains are poorly reflected in overall sequence identity, and therefore, the identification of signals from the amino acid sequence of the sensor alone presents a challenge. Biogenic amines are of great physiological importance for microorganisms and humans. They serve as substrates for aerobic and anaerobic growth and play a role of neurotransmitters and osmoprotectants. Here, we report the identification of a sequence motif that is specific for amine-sensing sensor domains that belong to the Cache superfamily of the most abundant extracellular sensors in prokaryotes. We identified approximately 13,000 sensor histidine kinases, chemoreceptors, receptors involved in second messenger homeostasis and Ser/Thr phosphatases from 8,000 bacterial and archaeal species that contain the amine-recognizing motif. The screening of compound libraries and microcalorimetric titrations of selected sensor domains confirmed their ability to specifically bind biogenic amines. Mutants in the amine-binding motif or domains that contain a single mismatch in the binding motif had either no or a largely reduced affinity for amines. We demonstrate that the amine-recognizing domain originated from the universal amino acid-sensing Cache domain, thus providing insight into receptor evolution. Our approach enables precise "wet"-lab experiments to define the function of regulatory systems and therefore holds a strong promise to enable the identification of signals stimulating numerous receptors.

Amino acid sensor conserved from bacteria to humans.

SignificanceAmino acids are the building blocks of life and important signaling molecules. Despite their common structure, no universal mechanism for amino acid recognition by cellular receptors is currently known. We discovered a simple motif, which binds amino acids in various receptor proteins from all major life-forms. In humans, this motif is found in subunits of calcium channels that are implicated in pain and neurodevelopmental disorders. Our findings suggest that γ-aminobutyric acid-derived drugs bind to the same motif in human proteins that binds natural ligands in bacterial receptors, thus enabling future improvement of important drugs.

Photoactive Yellow Protein Represents a Distinct, Evolutionarily Novel Family of PAS Domains.

Photoactive yellow protein (PYP) is a model photoreceptor. It binds a p-coumaric acid as a chromophore, thus enabling blue light sensing. The first discovered single-domain PYP from Halorhodospira halophila has been studied thoroughly in terms of its structural dynamics and photochemical properties. However, the evolutionary origins and biological role of PYP homologs are not well understood. Here, we show that PYP is an evolutionarily novel domain family of the ubiquitous PAS (Per-Arnt-Sim) superfamily. It likely originated from the phylum Myxococcota and was then horizontally transferred to representatives of a few other bacterial phyla. We show that PYP is associated with signal transduction either by domain fusion or by genome context. Key cellular functions modulated by PYP-initiated signal transduction pathways likely involve gene expression, motility, and biofilm formation. We identified three clades of the PYP family, one of which is poorly understood and potentially has novel functional properties. The Tyr42, Glu46, and Cys69 residues that are involved in p-coumaric acid binding in the model PYP from H. halophila are well conserved in the PYP family. However, we also identified cases where substitutions in these residues might have led to neofunctionalization, such as the proposed transition from light to redox sensing. Overall, this study provides definition, a newly built hidden Markov model, and the current genomic landscape of the PYP family and sets the stage for the future exploration of its signaling mechanisms and functional diversity. IMPORTANCE Photoactive yellow protein is a model bacterial photoreceptor. For many years, it was considered a prototypical model of the ubiquitous PAS domain superfamily. Here, we show that, in fact, the PYP family is evolutionarily novel, restricted to a few bacterial phyla and distinct from other PAS domains. We also reveal the diversity of PYP-containing signal transduction proteins and their potential mechanisms.

TREND: a platform for exploring protein function in prokaryotes based on phylogenetic, domain architecture and gene neighborhood analyses.

Key steps in a computational study of protein function involve analysis of (i) relationships between homologous proteins, (ii) protein domain architecture and (iii) gene neighborhoods the corresponding proteins are encoded in. Each of these steps requires a separate computational task and sets of tools. Currently in order to relate protein features and gene neighborhoods information to phylogeny, researchers need to prepare all the necessary data and combine them by hand, which is time-consuming and error-prone. Here, we present a new platform, TREND (tree-based exploration of neighborhoods and domains), which can perform all the necessary steps in automated fashion and put the derived information into phylogenomic context, thus making evolutionary based protein function analysis more efficient. A rich set of adjustable components allows a user to run the computational steps specific to his task. TREND is freely available at http://trend.zhulinlab.org.

MiST 3.0: an updated microbial signal transduction database with an emphasis on chemosensory systems.

Bacteria and archaea employ dedicated signal transduction systems that modulate gene expression, second-messenger turnover, quorum sensing, biofilm formation, motility, host-pathogen and beneficial interactions. The updated MiST database provides a comprehensive classification of microbial signal transduction systems. This update is a result of a substantial scaling to accommodate constantly growing microbial genomic data. More than 125 000 genomes, 516 million genes and almost 100 million unique protein sequences are currently stored in the database. For each bacterial and archaeal genome, MiST 3.0 provides a complete signal transduction profile, thus facilitating theoretical and experimental studies on signal transduction and gene regulation. New software infrastructure and distributed pipeline implemented in MiST 3.0 enable regular genome updates based on the NCBI RefSeq database. A novel MiST feature is the integration of unique profile HMMs to link complex chemosensory systems with corresponding chemoreceptors in bacterial and archaeal genomes. The data can be explored online or via RESTful API (freely available at https://mistdb.com).

A di-iron protein recruited as an Fe[II] and oxygen sensor for bacterial chemotaxis functions by stabilizing an iron-peroxy species.

Many bacteria contain cytoplasmic chemoreceptors that lack sensor domains. Here, we demonstrate that such cytoplasmic receptors found in 8 different bacterial and archaeal phyla genetically couple to metalloproteins related to ß-lactamases and nitric oxide reductases. We show that this oxygen-binding di-iron protein (ODP) acts as a sensor for chemotactic responses to both iron and oxygen in the human pathogen Treponema denticola (Td). The ODP di-iron site binds oxygen at high affinity to reversibly form an unusually stable µ-peroxo adduct. Crystal structures of ODP from Td and the thermophile Thermotoga maritima (Tm) in the Fe[III]2-O22-, Zn[II], and apo states display differences in subunit association, conformation, and metal coordination that indicate potential mechanisms for sensing. In reconstituted systems, iron-peroxo ODP destabilizes the phosphorylated form of the receptor-coupled histidine kinase CheA, thereby providing a biochemical link between oxygen sensing and chemotaxis in diverse prokaryotes, including anaerobes of ancient origin.

Microbial diversity in acidic thermal pools in the Uzon Caldera, Kamchatka.

Microbial communities of four acidic thermal pools in the Uzon Caldera, Kamchatka, Russia, were studied using amplification and pyrosequencing of 16S rRNA gene fragments. The sites differed in temperature and pH: 1805 (60 °C, pH 3.7), 1810 (90 °C, pH 4.1), 1818 (80 °C, pH 3.5), and 1807 (86 °C, pH 5.6). Archaea of the order Sulfolobales were present among the dominant groups in all four pools. Acidilobales dominated in pool 1818 but were a minor fraction at the higher temperature in pool 1810. Uncultivated Archaea of the Hot Thaumarchaeota-related clade were present in significant quantities in pools 1805 and 1807, but they were not abundant in pools 1810 and 1818, where high temperatures were combined with low pH. Nanoarchaeota were present in all pools, but were more abundant in pools 1810 and 1818. A similar abundance pattern was observed for Halobacteriales. Thermophilic Bacteria were less diverse and were mostly represented by aerobic hydrogen- and sulfur-oxidizers of the phylum Aquificae and sulfur-oxidising Proteobacteria of the genus Acidithiobacillus. Thus we showed that extremely acidic hot pools contain diverse microbial communities comprising different metabolic groups of prokaryotes, including putative lithoautotrophs using energy sources of volcanic origin, and various facultative and obligate heterotrophs.

A Novel Highly Thermostable Multifunctional Beta-Glycosidase from Crenarchaeon Acidilobus saccharovorans.

We expressed a putative ß-galactosidase Asac_1390 from hyperthermophilic crenarchaeon Acidilobus saccharovorans in Escherichia coli and purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity with p-nitrophenyl (pNP) substrates followed the order pNP-ß-D-galactopyranoside (328 U mg(-1)), pNP-ß-D-glucopyranoside (246 U mg(-1)), pNP-ß-D-xylopyranoside (72 U mg(-1)), and pNP-ß-D-mannopyranoside (28 U mg(-1)). Thus the enzyme was actually a multifunctional ß-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.

Microbial life in Bourlyashchy, the hottest thermal pool of Uzon Caldera, Kamchatka.

Bourlyashchy is the largest and hottest pool in the Uzon Caldera, located in the territory of Kronotsky Nature Reserve, Kamchatka, Russia, with sediment surface temperatures at the margins ranging from 86 to 97 °C, and pH from 6.0 to 7.0. The microbial communities of the pool water and sediments were studied comprehensively from 2005 to 2014. Radioisotopic tracer studies revealed the processes of inorganic carbon assimilation, sulfate reduction, lithotrophic methanogenesis and potentially very active process of acetate oxidation to CO2. The total number of microbial cells in water was different in different years ranging from 5.2 to 7.0 × 10(6); in sediments, it changed from year to year between 6.3 × 10(6) and 1.75 × 10(8), increasing with a decrease in temperature. FISH with Archaea- and Bacteria-specific probes showed that the share of Bacteria differed with year, changing from 34 to 71%. According to 16S rRNA gene pyrosequencing data, lithoautotrophs (Aquificales and Thermoproteales) predominated in water samples, while in sediments they shared the niche with organotrophic Crenarchaeota, Korarchaeota, and bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). The majority of organisms in water belonged to cultivated orders of prokaryotes; the only large uncultured group was that representing a novel order in class Thermoprotei. In sediments, unclassified Aquificeae comprised a significant part of the bacterial population. Thus, we showed that the hottest of the terrestrial hot pools studied contains numerous and active microbial populations where Bacteria represent a significant part of the microbial community, and planktonic and sediment populations differ in both composition and function.

Genome analysis of Chitinivibrio alkaliphilus gen. nov., sp. nov., a novel extremely haloalkaliphilic anaerobic chitinolytic bacterium from the candidate phylum Termite Group 3.

Anaerobic enrichments from hypersaline soda lakes with chitin as substrate yielded five closely related anaerobic haloalkaliphilic isolates growing on insoluble chitin by fermentation at pH 10 and salinities up to 3.5 M. The chitinolytic activity was exclusively cell associated. To better understand the biology and evolutionary history of this novel bacterial lineage, the genome of the type strain ACht1 was sequenced. Analysis of the 2.6 Mb draft genome revealed enzymes of chitin-degradation pathways, including secreted cell-bound chitinases. The reconstructed central metabolism revealed pathways enabling the fermentation of polysaccharides, while it lacks the genes needed for aerobic or anaerobic respiration. The Rnf-type complex, oxaloacetate decarboxylase and sodium-transporting V-type adenosine triphosphatase were identified among putative membrane-bound ion pumps. According to 16S ribosomal RNA analysis, the isolates belong to the candidate phylum Termite Group 3, representing its first culturable members. Phylogenetic analysis using ribosomal proteins and taxonomic distribution analysis of the whole proteome supported a class-level classification of ACht1 most probably affiliated to the phylum Fibribacteres. Based on phylogenetic, phenotypic and genomic analyses, the novel bacteria are proposed to be classified as Chitinivibrio alkaliphilus gen. nov., sp. nov., within a novel class Chitinivibrione.

Analysis of the complete genome of Fervidococcus fontis confirms the distinct phylogenetic position of the order Fervidicoccales and suggests its environmental function.

The complete genome of the obligately anaerobic crenarchaeote Fervidicoccus fontis Kam940(T), a terrestrial hot spring inhabitant with a growth optimum of 65-70 °C, has been sequenced and analyzed. The small 1.3-Mb genome encodes several extracellular proteases and no other extracellular hydrolases. No complete pathways of carbohydrate catabolism were found. Genes coding for enzymes necessary for amino acid transamination and further oxidative decarboxylation are present. The genome encodes no mechanisms of acyl-CoA and acetyl-CoA oxidation. Two [NiFe]-hydrogenases are encoded: a membrane-bound energy-converting hydrogenase and a cytoplasmic one. The ATP-synthase is H(+)-dependent as inferred from the amino acid sequence of the membrane rotor subunit. On the whole, genome analysis shows F. fontis to be a peptidolytic heterotroph with a restricted biosynthetic potential, which is in accordance with its phenotypic properties. The analysis of phylogenetic markers and of the distribution of best blastp hits of F. fontis proteins in the available genomes of Crenarchaeota supports distinct phylogenetic position of the order Fervidicoccales as a separate lineage adjoining the heterogeneous order Desulfurococcales. In addition, certain F. fontis genomic features correlate with its adaptation to temperatures of 60-80 °C, which are lower than temperatures preferred by Desulfurococcales.

Framework for exploring the sensory repertoire of the human gut microbiota.

Bacteria sense changes in their environment and transduce signals to adjust their cellular functions accordingly. For this purpose, bacteria employ various sensors feeding into multiple signal transduction pathways. Signal recognition by bacterial sensors is studied mainly in a few model organisms, but advances in genome sequencing and analysis offer new ways of exploring the sensory repertoire of many understudied organisms. The human gut is a natural target of this line of study: it is a nutrient-rich and dynamic environment and is home to thousands of bacterial species whose activities impact human health. Many gut commensals are also poorly studied compared to model organisms and are mainly known through their genome sequences. To begin exploring the signals human gut commensals sense and respond to, we have designed a framework that enables the identification of sensory domains, prediction of signals that they recognize, and experimental verification of these predictions. We validate this framework's functionality by systematically identifying amino acid sensors in selected bacterial genomes and metagenomes, characterizing their amino acid binding properties, and demonstrating their signal transduction potential.IMPORTANCESignal transduction is a central process governing how bacteria sense and respond to their environment. The human gut is a complex environment with many living organisms and fluctuating streams of nutrients. One gut inhabitant, Escherichia coli, is a model organism for studying signal transduction. However, E. coli is not representative of most gut microbes, and signaling pathways in the thousands of other organisms comprising the human gut microbiota remain poorly understood. This work provides a foundation for how to explore signals recognized by these organisms.

Ubiquitous purine sensor modulates diverse signal transduction pathways in bacteria.

Purines and their derivatives control intracellular energy homeostasis and nucleotide synthesis, and act as signaling molecules. Here, we combine structural and sequence information to define a purine-binding motif that is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism, and second-messenger turnover. Microcalorimetric titrations of selected sensor domains validate their ability to specifically bind purine derivatives, and evolutionary analyses indicate that purine sensors share a common ancestor with amino-acid receptors. Furthermore, we provide experimental evidence of physiological relevance of purine sensing in a second-messenger signaling system that modulates c-di-GMP levels.

Origin and functional diversification of PAS domain, a ubiquitous intracellular sensor.

Signal perception is a key function in regulating biological activities and adapting to changing environments. Per-Arnt-Sim (PAS) domains are ubiquitous sensors found in diverse receptors in bacteria, archaea, and eukaryotes, but their origins, distribution across the tree of life, and extent of their functional diversity are not fully characterized. Here, we show that using sequence conservation and structural information, it is possible to propose specific and potential functions for a large portion of nearly 3 million PAS domains. Our analysis suggests that PAS domains originated in bacteria and were horizontally transferred to archaea and eukaryotes. We reveal that gas sensing via a heme cofactor evolved independently in several lineages, whereas redox and light sensing via flavin adenine dinucleotide and flavin mononucleotide cofactors have the same origin. The close relatedness of human PAS domains to those in bacteria provides an opportunity for drug design by exploring potential natural ligands and cofactors for bacterial homologs.

Amine recognizing domain in diverse receptors from bacteria and archaea evolved from the universal amino acid sensor.

Bacteria contain many different receptor families that sense different signals permitting an optimal adaptation to the environment. A major limitation in microbiology is the lack of information on the signal molecules that activate receptors. Due to a significant sequence divergence, the signal recognized by sensor domains is only poorly reflected in overall sequence identity. Biogenic amines are of central physiological relevance for microorganisms and serve for example as substrates for aerobic and anaerobic growth, neurotransmitters or osmoprotectants. Based on protein structural information and sequence analysis, we report here the identification of a sequence motif that is specific for amine-sensing dCache sensor domains (dCache_1AM). These domains were identified in more than 13,000 proteins from 8,000 bacterial and archaeal species. dCache_1AM containing receptors were identified in all major receptor families including sensor kinases, chemoreceptors, receptors involved in second messenger homeostasis and Ser/Thr phosphatases. The screening of compound libraries and microcalorimetric titrations of selected dCache_1AM domains confirmed their capacity to specifically bind amines. Mutants in the amine binding motif or domains that contain a single mismatch in the binding motif, had either no or a largely reduced affinity for amines, illustrating the specificity of this motif. We demonstrate that the dCache_1AM domain has evolved from the universal amino acid sensing domain, providing novel insight into receptor evolution. Our approach enables precise "wet"-lab experiments to define the function of regulatory systems and thus holds a strong promise to address an important bottleneck in microbiology: the identification of signals that stimulate numerous receptors.

The importance of cache domains in α2δ proteins and the basis for their gabapentinoid selectivity.

In this hybrid review, we have first collected and reviewed available information on the structure and function of the enigmatic cache domains in α2δ proteins. These are organized into two double cache (dCache_1) domains, and they are present in all α2δ proteins. We have also included new data on the key function of these domains with respect to amino acid and gabapentinoid binding to the universal amino acid-binding pocket, which is present in α2δ-1 and α2δ-2. We have now identified the reason why α2δ-3 and α2δ-4 do not bind gabapentinoid drugs or amino acids with bulky side chains. In relation to this, we have determined that the bulky amino acids Tryptophan and Phenylalanine prevent gabapentin from inhibiting cell surface trafficking of α2δ-1. Together, these novel data shed further light on the importance of the cache domains in α2δ proteins.

Transmembrane Transcription Regulators Are Widespread in Bacteria and Archaea.

To adapt and proliferate, bacteria must sense and respond to the ever-changing environment. Transmembrane transcription regulators (TTRs) are a family of one-component transcription regulators that respond to extracellular information and influence gene expression from the cytoplasmic membrane. How TTRs function to modulate expression of their target genes while localized to the cytoplasmic membrane remains poorly understood. In part, this is due to a lack of knowledge regarding the prevalence of TTRs among prokaryotes. Here, we show that TTRs are highly diverse and prevalent throughout bacteria and archaea. Our work demonstrates that TTRs are more common than previously appreciated and are enriched within specific bacterial and archaeal phyla and that many TTRs have unique transmembrane region properties that can facilitate association with detergent-resistant membranes. IMPORTANCE One-component signal transduction systems are the major class of signal transduction systems among bacteria and are commonly cytoplasmic. TTRs are a group of unique one-component signal transduction systems that influence transcription from the cytoplasmic membrane. TTRs have been implicated in a wide array of biological pathways critical for both pathogens and human commensal organisms but were considered to be rare. Here, we demonstrate that TTRs are in fact highly diverse and broadly distributed in bacteria and archaea. Our findings suggest that transcription factors can access the chromosome and influence transcription from the membrane in both archaea and bacteria. This study challenges thus the commonly held notion that signal transduction systems require a cytoplasmic transcription factor and highlights the importance of the cytoplasmic membrane in directly influencing signal transduction.