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.

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.

Ubiquitous purine sensor modulates diverse signal transduction pathways in bacteria.

Purines and their derivatives are key molecules for controlling intracellular energy homeostasis and nucleotide synthesis. In eukaryotes, including humans, purines also act as signaling molecules that mediate extracellular communication and control key cellular processes, such as proliferation, migration, differentiation, and apoptosis. However, the signaling role of purines in bacteria is largely unknown. Here, by combining structural and sequence information, we define a purine-binding motif, which is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism and second messenger turnover. The screening of compound libraries and microcalorimetric titrations of selected sensor domains validated their ability to specifically bind purine derivatives. The physiological relevance of purine sensing was demonstrated in a second messenger signaling system that modulates c-di-GMP levels.

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.

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.

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.

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.

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.

Phyletic Distribution and Diversification of the Phage Shock Protein Stress Response System in Bacteria and Archaea.

Maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protein protection networks that connect stress perception, transmembrane signal transduction, and mediation of cellular responses upon cell envelope stress. The phage shock protein (Psp) stress response is one such conserved protection network. Most knowledge about the Psp response derives from studies in the Gram-negative model bacterium Escherichia coli, where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of the Proteobacteria, Actinobacteria, and Firmicutes. However, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and describe that the predicted Psp systems deviate dramatically from the known prototypes. The core proteins PspA and PspC have been integrated into various (often phylum-specifically) conserved protein networks during evolution. Based on protein domain-based and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system for Psp networks in bacteria and archaea. We validate our approach by performing a comprehensive in vivo protein interaction study of Psp domains identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected protein network. Our study highlights the diversity of Psp domain organizations and potentially diverse functions across the plethora of the microbial landscape, thus laying the ground for studies beyond known Psp functions in underrepresented organisms. IMPORTANCE The PspA protein domain is found in all domains of life, highlighting its central role in Psp networks. To date, all insights into the core functions of Psp responses derive mainly from protein network blueprints representing only three bacterial phyla. Despite large overlaps in function and regulation, the evolutionary diversity of Psp networks remains largely elusive. Here, we present an unbiased protein domain- and genomic context-centered approach that describes and classifies Psp systems. Our results suggest so-far-unknown Psp-associated roles with other protein networks giving rise to new functions. We demonstrate the applicability of our approach by dissecting the Psp protein network present in Bacillus subtilis and demonstrate Psp domains working in concert with other cell envelope stress response systems. We find that the Psp-like protein universe reflects a surprising diversity within the bacterial and archaeal microbial world.

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.

Strategic traits of bacteria and archaea vary widely within substrate-use groups.

Quantitative traits such as maximum growth rate and cell radial diameter are one facet of ecological strategy variation across bacteria and archaea. Another facet is substrate-use pathways, such as iron reduction or methylotrophy. Here, we ask how these two facets intersect, using a large compilation of data for culturable species and examining seven quantitative traits (genome size, signal transduction protein count, histidine kinase count, growth temperature, temperature-adjusted maximum growth rate, cell radial diameter and 16S rRNA operon copy number). Overall, quantitative trait variation within groups of organisms possessing a particular substrate-use pathway was very broad, outweighing differences between substrate-use groups. Although some substrate-use groups had significantly different means for some quantitative traits, standard deviation of quantitative trait values within each substrate-use pathway mostly averaged between 1.6 and 1.8 times larger than standard deviation across group means. Most likely, this wide variation reflects ecological strategy: for example, fast maximum growth rate is likely to express an early successional or copiotrophic strategy, and maximum growth varies widely within most substrate-use pathways. In general, it appears that these quantitative traits express different and complementary information about ecological strategy, compared with substrate use.

Diversity of bacterial chemosensory systems.

Chemosensory system is the most complex, specialized mode of signal transduction in bacteria and archaea. It is composed of several core and auxiliary protein components that are highly organized in order to deliver a fast response to changing environmental conditions. Chemosensory pathways were studied in-depth in a handful of model organisms and experimentally characterized at least to some degree in approximately thirty other species. However, genome-wide analyses have revealed their presence in thousands of sequenced microbial genomes. Both experimental and computational studies uncovered substantial diversity in system design, functional regulation, cellular localization and phyletic distribution of chemosensory pathways. Here, we summarize advances and expose gaps in our current understanding of the diversity of chemosensory systems.

Origins and Molecular Evolution of the NusG Paralog RfaH.

The only universally conserved family of transcription factors comprises housekeeping regulators and their specialized paralogs, represented by well-studied NusG and RfaH. Despite their ubiquity, little information is available on the evolutionary origins, functions, and gene targets of the NusG family members. We built a hidden Markov model profile of RfaH and identified its homologs in sequenced genomes. While NusG is widespread among bacterial phyla and coresides with genes encoding RNA polymerase and ribosome in all except extremely reduced genomes, RfaH is mostly limited to Proteobacteria and lacks common gene neighbors. RfaH activates only a few xenogeneic operons that are otherwise silenced by NusG and Rho. Phylogenetic reconstructions reveal extensive duplications and horizontal transfer of rfaH genes, including those borne by plasmids, and the molecular evolution pathway of RfaH, from "early" exclusion of the Rho terminator and tightened RNA polymerase binding to "late" interactions with the ops DNA element and autoinhibition, which together define the RfaH regulon. Remarkably, NusG is not only ubiquitous in Bacteria but also common in plants, where it likely modulates the transcription of plastid genes.IMPORTANCE In all domains of life, NusG-like proteins make contacts similar to those of RNA polymerase and promote pause-free transcription yet may play different roles, defined by their divergent interactions with nucleic acids and accessory proteins, in the same cell. This duality is illustrated by Escherichia coli NusG and RfaH, which silence and activate xenogenes, respectively. We combined sequence analysis and recent functional and structural insights to envision the evolutionary transformation of NusG, a core regulator that we show is present in all cells using bacterial RNA polymerase, into a virulence factor, RfaH. Our results suggest a stepwise conversion of a NusG duplicate copy into a sequence-specific regulator which excludes NusG from its targets but does not compromise the regulation of housekeeping genes. We find that gene duplication and lateral transfer give rise to a surprising diversity within the only ubiquitous family of transcription factors.

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.

How Bacterial Chemoreceptors Evolve Novel Ligand Specificities.

Chemoreceptor-based signaling pathways are among the major modes of bacterial signal transduction, and Pseudomonas aeruginosa PAO1 is an important model to study their function. Of the 26 chemoreceptors of this strain, PctA has a broad ligand range and responds to most of the proteinogenic amino acids, whereas PctB and PctC have a much narrower range and show strong ligand preference for l-glutamine and γ-aminobutyrate, respectively. Using several comparative genomics approaches, we show that these receptors are paralogs: pctA gene duplication in the common ancestor of the genus Pseudomonas led to pctC, whereas pctB originated through another, independent pctA duplication in the common ancestor of P. aeruginosa Thus, the broad-range amino acid chemoreceptor was evolutionarily older, and chemoreceptors that complemented "missing" amino acid sensing abilities arose later in specific Pseudomonas lineages. Using comparative sequence analysis, newly solved crystal structures of PctA, PctB, and PctC ligand-binding domains, and their molecular dynamics simulations, we identified a conserved amino acid recognition motif and changes in the ligand-binding pocket that led to novel ligand specificities. In addition, we determined major forces driving the evolution of this group of chemoreceptors.IMPORTANCE Many bacteria possess a large number of chemoreceptors that recognize a variety of different compounds. More than 60% of the genomes analyzed in this study contain paralogous chemoreceptors, suggesting that they emerge with high frequency. We provide first insight on how paralogous receptors have evolved and show that two chemoreceptors with a narrow ligand range have evolved from an ancestral protein with a broad chemoeffector spectrum. Protein structures show that multiple changes in the ligand-binding site account for the differences in the ligand spectrum. This work lays the ground for further studies aimed at establishing whether the principles of ligand-binding evolution reported here can be generalized for a wider spectrum of sensory proteins in bacteria.

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.