Uncharacterized Family Research Articles

COG database update 2024.

The Clusters of Orthologous Genes (COG) database, originally created in 1997, has been updated to reflect the constantly growing collection of completely sequenced prokaryotic genomes. This update increased the genome coverage from 1309 to 2296 species, including 2103 bacteria and 193 archaea, in most cases, with a single representative genome per genus. This set covers all genera of bacteria and archaea that included organisms with 'complete genomes' as per NCBI databases in November 2023. The number of COGs has been expanded from 4877 to 4981, primarily by including protein families involved in bacterial protein secretion. Accordingly, COG pathways and functional groups now include secretion systems of types II through X, as well as Flp/Tad and type IV pili. These groupings allow straightforward identification and examination of the prokaryotic lineages that encompass-or lack-a particular secretion system. Other developments include improved annotations for the rRNA and tRNA modification proteins, multi-domain signal transduction proteins, and some previously uncharacterized protein families. The new version of COGs is available at https://www.ncbi.nlm.nih.gov/research/COG, as well as on the NCBI FTP site https://ftp.ncbi.nlm.nih.gov/pub/COG/, which also provides archived data from previous COG releases.

Surface exclusion of IncC conjugative plasmids and their relatives.

The phenomenon of exclusion allows conjugative plasmids to selectively impede the entry of identical or related elements into their host cell to prevent the resulting instability. Entry exclusion blocks DNA translocation into the recipient cell, whereas surface exclusion destabilizes the mating pair. IncC conjugative plasmids largely contribute to the dissemination of antibiotic-resistance genes in Gammaproteobacteria. IncC plasmids are known to exert exclusion against their relatives, including IncC and IncA plasmids, yet the entry exclusion factor eexC alone does not account for the totality of the exclusion phenotype. In this study, a transposon-directed insertion sequencing approach identified sfx as necessary and sufficient for the remaining exclusion phenotype. Sfx is an exclusion factor unrelated to the ones described to date. A cell fractionation assay localized Sfx in the outer membrane. Reverse transcription PCR and beta-galactosidase experiments showed that sfx is expressed constitutively at a higher level than eexC. A search in Gammaproteobacteria genomes identified Sfx homologs encoded by IncC, IncA and related, untyped conjugative plasmids and an uncharacterized family of integrative and mobilizable elements that likely rely on IncC plasmids for their mobility. Mating assays demonstrated that sfx is not required in the donor for exclusion, ruling out Sfx as the exclusion target. Instead, complementation assays revealed that the putative adhesin TraN in the donor mediates the specificity of surface exclusion. Mating assays with TraN homologs from related untyped plasmids from Aeromonas spp. and Photobacterium damselae identified two surface exclusion groups, with each Sfx being specific of TraN homologs from the same group. Together, these results allow us to better understand the apparent incompatibility between IncA and IncC plasmids and to propose a mechanistic model for surface exclusion mediated by Sfx in IncC plasmids and related elements, with implications for the rampant dissemination of antibiotic resistance.

Metatranscriptomics-guided discovery and characterization of a polyphenol-metabolizing gut microbial enzyme.

Gut microbial catechol dehydroxylases are a largely uncharacterized family of metalloenzymes that potentially impact human health by metabolizing dietary polyphenols. Here, we use metatranscriptomics (MTX) to identify highly transcribed catechol-dehydroxylase-encoding genes in human gut microbiomes. We discover a prevalent, previously uncharacterized catechol dehydroxylase (Gp Hcdh) from Gordonibacter pamelaeae that dehydroxylates hydrocaffeic acid (HCA), an anti-inflammatory gut microbial metabolite derived from plant-based foods. Further analyses suggest that the activity of Gp Hcdh may reduce anti-inflammatory benefits of polyphenol-rich foods. Together, these results show the utility of combining MTX analysis and biochemical characterization for gut microbial enzyme discovery and reveal a potential link between host inflammation and a specific polyphenol-metabolizing gut microbial enzyme.

Investigation of protein family relationships with deep learning.

In this article, we propose a method for finding similarities between Pfam families based on the pre-trained neural network ProtENN2. We use the model ProtENN2 per-residue embeddings to produce new high-dimensional per-family embeddings and develop an approach for calculating inter-family similarity scores based on these embeddings, and evaluate its predictions using structure comparison. We apply our method toPfam annotation by refining clan membership for Pfam families, suggesting both new members of existing clans and potential new clans for future Pfam releases. We investigate some of the failure modes of our approach, which suggests directions for future improvements. Our method is relatively simple with few parameters and could be applied to other protein family classification models. Overall, our work suggests potential benefits of employing deep learning for improving our understanding of protein family relationships and functions of previously uncharacterized families. github.com/iponamareva/ProtCNNSim, 10.5281/zenodo.10091909.

Diversity and evolution of nitric oxide reduction in bacteria and archaea

Nitrous oxide is a potent greenhouse gas whose production is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) enzyme superfamily. We identified several previously uncharacterized HCO families, four of which (eNOR, sNOR, gNOR, and nNOR) appear to perform NO reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple NO reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from the bacterium Rhodothermus marinus and found that it performs NO reduction. These recently identified NORs exhibited broad phylogenetic and environmental distributions, greatly expanding the diversity of microbes in nature capable of NO reduction. Phylogenetic analyses further demonstrated that NORs evolved multiple times independently from oxygen reductases, supporting the view that complete denitrification evolved after aerobic respiration.

Exploring the roles of ribosomal peptides in prokaryote-phage interactions through deep learning-enabled metagenome mining

BackgroundMicrobial secondary metabolites play a crucial role in the intricate interactions within the natural environment. Among these metabolites, ribosomally synthesized and post-translationally modified peptides (RiPPs) are becoming a promising source of therapeutic agents due to their structural diversity and functional versatility. However, their biosynthetic capacity and ecological functions remain largely underexplored.ResultsHere, we aim to explore the biosynthetic profile of RiPPs and their potential roles in the interactions between microbes and viruses in the ocean, which encompasses a vast diversity of unique biomes that are rich in interactions and remains chemically underexplored. We first developed TrRiPP to identify RiPPs from ocean metagenomes, a deep learning method that detects RiPP precursors in a hallmark gene-independent manner to overcome the limitations of classic methods in processing highly fragmented metagenomic data. Applying this method to metagenomes from the global ocean microbiome, we uncover a diverse array of previously uncharacterized putative RiPP families with great novelty and diversity. Through correlation analysis based on metatranscriptomic data, we observed a high prevalence of antiphage defense-related and phage-related protein families that were co-expressed with RiPP families. Based on this putative association between RiPPs and phage infection, we constructed an Ocean Virus Database (OVD) and established a RiPP-involving host-phage interaction network through host prediction and co-expression analysis, revealing complex connectivities linking RiPP-encoding prokaryotes, RiPP families, viral protein families, and phages. These findings highlight the potential of RiPP families involved in prokaryote-phage interactions and coevolution, providing insights into their ecological functions in the ocean microbiome.ConclusionsThis study provides a systematic investigation of the biosynthetic potential of RiPPs from the ocean microbiome at a global scale, shedding light on the essential insights into the ecological functions of RiPPs in prokaryote-phage interactions through the integration of deep learning approaches, metatranscriptomic data, and host-phage connectivity. This study serves as a valuable example of exploring the ecological functions of bacterial secondary metabolites, particularly their associations with unexplored microbial interactions.CSrTVep6AzTb_yem78LPYEVideo

The Plasmodiophora brassicae Golgi-localized UPF0016 protein PbGDT1 mediates calcium but not manganese transport in yeast and Nicotiana benthamiana

Manganese and calcium homeostasis and signalling, in eukaryotic organisms, are regulated through membrane located pumps, channels and exchangers, including the Mn2+/Ca2+ uncharacterized protein family 0016 (UPF0016). Here we show that Plasmodiophora brassicae PbGDT1 is a member of the UPF0016 and an ortholog of Saccharomyces cerevisiae Gdt1p (GCR Dependent Translation Factor 1) protein involved in manganese homeostasis as well as the calcium mediated stress response in yeast. PbGDT1 complemented the ScGdt1p and ScPMR1 (Ca2+ ATPase) double null mutant under elevated calcium stress but not under elevated manganese conditions. In both yeast and Nicotiana benthamiana, PbGDT1 localizes to the Golgi apparatus, with additional ER association in N. benthamiana. Expression of PbGDT1 in N. benthamiana, suppresses BAX-triggered cell death, further highlighting the importance of calcium homeostasis in maintaining cell physiology and integrity in a stress environment.

The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types

Mosquitoes are prolific vectors of human pathogens, therefore a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.

Identification of a novel Golgi-localized putative glycosyltransferase protein in Arabidopsis thaliana.

SNAREs play an important role in the process of membrane trafficking. In the present research, we investigated subcellular localization of an uncharacterized Arabidopsis thaliana protein reported to interact with a trans-Golgi network-localized Qa-SNARE, SYNTAXIN OF PLANTS 43. Based on the similarity of its amino acid sequence to metazoan fucosyltransferases, we have named this novel protein AtGTLP (Arabidopsis thaliana GlycosylTransferase-Like Protein) and predicted that it should be a member of yet uncharacterized family of Arabidopsis fucosyltransferases, as it shows no significant sequence similarity to fucosyltransferases previously identified in Arabidopsis. AtGTLP is a membrane-anchored protein, which exhibits a type II-like topology, with a single transmembrane helix and a globular domain in the C-terminal part of its amino acid sequence. Colocalization data we collected suggest that AtGTLP should localize mainly to Golgi apparatus, especially to certain zones of trans-Golgi. As single atgtlp-/- mutants showed no obvious difference in phenotype (primary root length and fresh mass), AtGTLP and proteins related to AtGTLP with high similarity in amino acid sequences may have redundant functions.

Dual-wield NTPases: A novel protein family mined from AlphaFold DB.

AlphaFold protein structure database (AlphaFold DB) archives a vast number of predicted models. We conducted systematic data mining against AlphaFold DB and discovered an uncharacterized P-loop NTPase family. The structure of the protein family was surprisingly novel, showing an atypical topology for P-loop NTPases, noticeable twofold symmetry, and two pairs of independent putative active sites. Our findings show that structural data mining is a powerful approach to identifying undiscovered protein families.

A first complete catalog of highly expressed genes in eight chicken tissues reveals uncharacterized gene families specific for the chicken testis.

Based on next-generation sequencing, we established a repertoire of differentially overexpressed genes (DoEGs) in eight adult chicken tissues: the testis, brain, lung, liver, kidney, muscle, heart, and intestine. With 4,499 DoEGs, the testis had the highest number and proportion of DoEGs compared with the seven somatic tissues. The testis DoEG set included the highest proportion of long noncoding RNAs (lncRNAs; 1,851, representing 32% of the lncRNA genes in the whole genome) and the highest proportion of protein-coding genes (2,648, representing 14.7% of the protein-coding genes in the whole genome). The main significantly enriched Gene Ontology terms related to the protein-coding genes were "reproductive process," "tubulin binding," and "microtubule cytoskeleton." Using real-time quantitative reverse transcription-polymerase chain reaction, we confirmed the overexpression of genes that encode proteins already described in chicken sperm [such as calcium binding tyrosine phosphorylation regulated (CABYR), spermatogenesis associated 18 (SPATA18), and CDK5 regulatory subunit associated protein (CDK5RAP2)] but whose testis origin had not been previously confirmed. Moreover, we demonstrated the overexpression of vertebrate orthologs of testis genes not yet described in the adult chicken testis [such as NIMA related kinase 2 (NEK2), adenylate kinase 7 (AK7), and CCNE2]. Using clustering according to primary sequence homology, we found that 1,737 of the 2,648 (67%) testis protein-coding genes were unique genes. This proportion was significantly higher than the somatic tissues except muscle. We clustered the other 911 testis protein-coding genes into 495 families, from which 47 had all paralogs overexpressed in the testis. Among these 47 testis-specific families, eight contained uncharacterized duplicated paralogs without orthologs in other metazoans except birds: these families are thus specific for chickens/birds.NEW & NOTEWORTHY Comparative next-generation sequencing analysis of eight chicken tissues showed that the testis has highest proportion of long noncoding RNA and protein-coding genes of the whole genome. We identified new genes in the chicken testis, including orthologs of known mammalian testicular genes. We also identified 47 gene families in which all the members were overexpressed, if not exclusive, in the testis. Eight families, organized in duplication clusters, were unknown, without orthologs in metazoans except birds, and are thus specific for chickens/birds.

A loss of function mutation in CLDN25 causing Pelizaeus-Merzbacher-like leukodystrophy.

Claudin-25 (CLDN-25), also known as Claudin containing domain 1, is an uncharacterized claudin family member. It has less conserved amino acid sequences when compared to other claudins. It also has a very broad tissue expression profile and there is currently a lack of functional information from murine knockout models. Here, we report a de novo missense heterozygous variant in CLDN25 (c. 745G>C, p. A249P) found in a patient diagnosed with Pelizaeus-Merzbacher-like leukodystrophy and presenting with symptoms such as delayed motor development, several episodes of tonic absent seizures and generalized dystonia. The variant protein does not localize to the cell-cell borders where it would normally be expected to be expressed. Amino acid position 249 is located 4 amino acids from the C-terminal end of the protein where most claudin family members have a conserved binding motif for the key scaffolding protein ZO-1. However, CLDN-25 does not contain this motif. Here, we show that the C-terminal end of CLDN-25 is required for its junctional localization in a ZO-1 independent manner. The A249P mutant protein as well as a deletion mutant lacking its last 5 C-terminal amino acids also failed to localize to the cell-cell border in vitro. Intriguingly, cellular knockout of CLDN25, in vitro, appeared to increase the integrity of the tight junction between 2 contacting cells, while driving highly unusual increased movement of solutes between cells. We propose that the barrier function of CLDN-25 is akin to a decoy claudin, whereby decreasing its expression in "leaky" epithelial cells and endothelial cells will drive dynamic changes in the adhesion and interaction capacity of cell-cell contact points. While it remains unclear how this de novo CLDN-25 mutant induces leukodystrophy, our findings strongly suggest that this mutation induces haploinsufficiency of CLDN-25. Elucidating the function of this uncharacterized claudin protein will lead to a better understanding of the role of claudin proteins in health and disease.

Characterization of the DUF868 gene family in Nicotiana and functional analysis of NtDUF868-E5 involved in pigment metabolism

Domains of unknown function (DUF) proteins represent a large group of uncharacterized protein families. The DUF868 gene family in Nicotiana has not yet been described. In the present study, we identified 12, 11, and 25 DUF868 family members in the genome of Nicotiana sylvestris, N. tomentosiformis, and N. tabacum, respectively. Based on phylogenetic analysis, these were categorized into five groups (A-E). Within each group, the gene structures, motifs, and tertiary structures showed high similarity. NtDUF868 family expansion during evolution was mainly driven by segmental duplication events. MicroRNA (miRNA) target site prediction identified 12 miRNA members that target 16 NtDUF868 family genes. The promoters of these genes contain cis-regulatory elements responsive to light, phytohormones, and abiotic stresses. Expression profiling revealed their tissue- and stage-specific expression patterns. RNA-sequencing and quantitative reverse transcription PCR revealed that the NtDUF868 family genes are potentially involved in the response to abiotic and biotic stresses, particularly drought and hormone stresses, and in the resistance to black shank and bacterial wilt. We generated transformed plants using NtDUF868-E5 overexpression and gene-editing vectors. NtDUF868-E5 overexpression resulted in enhanced tobacco plant growth and development, leading to increased leaf photosynthetic capacity and higher chlorophyll and carotenoid contents. This study provided a comprehensive genome-wide analysis of the DUF868 gene family, shedding light on their potential roles in plant growth and stress responses.

Cryo-EM analysis of S. aureus TarL, a polymerase in wall teichoic acid biogenesis central to virulence and antibiotic resistance.

Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of the Staphylococcus aureus WTA ribitol-phosphate chain is catalyzed by TarL, a member of the largely uncharacterized TagF-like family of membrane-associated enzymes. We report the cryo-electron microscopy structure of TarL, showing a tetramer that forms an extensive membrane-binding platform of monotopic helices. TarL is composed of an amino-terminal immunoglobulin-like domain and a carboxyl-terminal glycosyltransferase-B domain for ribitol-phosphate polymerization. The active site of the latter is complexed to donor substrate cytidine diphosphate-ribitol, providing mechanistic insights into the catalyzed phosphotransfer reaction. Furthermore, the active site is surrounded by electropositive residues that serve to retain the lipid-linked acceptor for polymerization. Our data advance general insight into the architecture and membrane association of the still poorly characterized monotopic membrane protein class and present molecular details of ribitol-phosphate polymerization that may aid in the design of new antimicrobials.

GeNLP: a web tool for NLP-based exploration and prediction of microbial gene function.

GeNLP is a web application that enables exploring microbial gene "semantics" and predictions of uncharacterized gene families based on their genomic context. It utilizes a pre-trained language model to uncover gene relationships and allows users to access and utilize the data as well as make their own predictions through an interactive interface. The web application is accessible from all browsers at: http://gnlp.bursteinlab.org/. All source codes are freely available from GitHub under the MIT license here: https://github.com/burstein-lab/genomic-nlp-server.

A Broad-Spectrum α-Glucosidase of Glycoside Hydrolase Family 13 from Marinovum sp., a Member of the Roseobacter Clade.

Glycoside hydrolases (GHs) are a diverse group of enzymes that catalyze the hydrolysis of glycosidic bonds. The Carbohydrate-Active enZymes (CAZy) classification organizes GHs into families based on sequence data and function, with fewer than 1% of the predicted proteins characterized biochemically. Consideration of genomic context can provide clues to infer possible enzyme activities for proteins of unknown function. We used the MultiGeneBLAST tool to discover a gene cluster in Marinovum sp., a member of the marine Roseobacter clade, that encodes homologues of enzymes belonging to the sulfoquinovose monooxygenase pathway for sulfosugar catabolism. This cluster lacks a gene encoding a classical family GH31 sulfoquinovosidase candidate, but which instead includes an uncharacterized family GH13 protein (MsGH13) that we hypothesized could be a non-classical sulfoquinovosidase. Surprisingly, recombinant MsGH13 lacks sulfoquinovosidase activity and is a broad-spectrum α-glucosidase that is active on a diverse array of α-linked disaccharides, including maltose, sucrose, nigerose, trehalose, isomaltose, and kojibiose. Using AlphaFold, a 3D model for the MsGH13 enzyme was constructed that predicted its active site shared close similarity with an α-glucosidase from Halomonas sp. H11 of the same GH13 subfamily that shows narrower substrate specificity.

The Golgi-localized transporter OsPML4 contributes to manganese homeostasis in rice

Manganese (Mn), an indispensable plant micronutrient, functions as a vital enzyme co-factor in numerous biochemical reactions. In rice, the Golgi-localized PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 3 (OsPML3), a member of the UNCHARACTERIZED PROTEIN FAMILY (UPF0016), plays a pivotal role in Mn homeostasis, particularly in rapidly developing tissues. This study focused on the functional characterization of another UPF0016 family member in rice, OsPML4, to elucidate its involvement in Mn homeostasis. OsPML4 had a 73% sequence identity with OsPML3 and exhibited expression in both shoots and roots, albeit at a lower transcriptional level than OsPML3. Furthermore, subcellular localization studies confirmed that OsPML4 localizes in the Golgi apparatus. Notably, heterologous expression of OsPML4 restored growth in the Mn uptake-deficient yeast strain Δsmf1 under Mn-limited conditions. Under Mn-deficient conditions, OsPML4 knockout exacerbated the decline in shoot dry weight and intensified necrosis in young leaves of OsPML3 knockout lines, which displayed stunted growth. The Mn concentration in OsPML3PML4 double knockout lines was lower than in wild-type (WT) and OsPML3 knockout lines. At the reproductive phase, OsPML3PML4 double knockout lines exhibited reduced fertility and grain yield compared to WT and OsPML3 knockout lines. Notably, reductions were observed in the deposition of cell wall polysaccharides and the content of Lea (Lewis A structure)-containing N-glycans in the young leaves of OsPML3PML4 double knockout lines, surpassing the reductions in WT and OsPML3 knockout lines. These findings underscore the significance of OsPML4 in Mn homeostasis in the Golgi apparatus, where it co-functions with OsPML3 to regulate cell wall polysaccharide deposition and late-stage Golgi N-glycosylation.

OsPML2, a chloroplast envelope localized transporter is involved in manganese homeostasis in rice

Manganese (Mn), a vital element, plays crucial roles in various biochemical and physiological processes by serving as an essential cofactor for numerous enzymes and acting as a catalytically active metal within biological clusters. In this study, we investigate the role of PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 2 (OsPML2), a member of the UNCHARACTERIZED PROTEIN FAMILY 0016 (UPF0016) family, in regulating Mn homeostasis in rice. OsPML2 was highly expressed in young leaves, ovaries, and stigmas. Cross sections from young leaves revealed that OsPML2 was mainly expressed in the phloem region and mesophyll cells. Furthermore, heterologous expression of OsPML2 restored the growth of Mn uptake-defective yeast strain Δsmf1 under Mn-limited conditions. Subcellular localization analysis demonstrated that OsPML2 was specifically localized in the chloroplast envelope. Knockdown of OsPML2 resulted in reduced chloroplast Mn content, significantly affecting plant growth under Mn deficiency. Furthermore, analysis of isolated thylakoid membranes using blue native gels indicated a compromised accumulation of photosystem II (PSII) complexes in OsPML2 knockdown lines. Additionally, grain yield, grain length, and width were significantly reduced in OsPML2 knockdown plants. Collectively, our findings provide insights into the transport function of OsPML2, which facilitates Mn transport from the cytosol to chloroplast stroma and influences the accumulation of PSII complexes in rice.

Genome-Wide Identification, Characterization and Expression Analysis of the TaDUF724 Gene Family in Wheat (Triticum aestivum).

Unknown functional domain (DUF) proteins constitute a large number of functionally uncharacterized protein families in eukaryotes. DUF724s play crucial roles in plants. However, the insight understanding of wheat TaDUF724s is currently lacking. To explore the possible function of TaDUF724s in wheat growth and development and stress response, the family members were systematically identified and characterized. In total, 14 TaDUF724s were detected from a wheat reference genome; they are unevenly distributed across the 11 chromosomes, and, according to chromosome location, they were named TaDUF724-1 to TaDUF724-14. Evolution analysis revealed that TaDUF724s were under negative selection, and fragment replication was the main reason for family expansion. All TaDUF724s are unstable proteins; most TaDUF724s are acidic and hydrophilic. They were predicted to be located in the nucleus and chloroplast. The promoter regions of TaDUF724s were enriched with the cis-elements functionally associated with growth and development, as well as being hormone-responsive. Expression profiling showed that TaDUF724-9 was highly expressed in seedings, roots, leaves, stems, spikes and grains, and strongly expressed throughout the whole growth period. The 12 TaDUF724 were post-transcription regulated by 12 wheat MicroRNA (miRNA) through cleavage and translation. RT-qPCR showed that six TaDUF724s were regulated by biological and abiotic stresses. Conclusively, TaDUF724s were systematically analyzed using bioinformatics methods, which laid a theoretical foundation for clarifying the function of TaDUF724s in wheat.

Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola.

The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH) proteins, play central roles in the stringent response. The pathogenic oral spirochete bacterium Treponema denticola lacks a long-RSH homologue but encodes putative small alarmone synthetase (Tde-SAS, TDE1711) and small alarmone hydrolase (Tde-SAH, TDE1690) proteins. Here, we characterize the respective in vitro and in vivo activities of Tde-SAS and Tde-SAH, which respectively belong to the previously uncharacterized RSH families DsRel and ActSpo2. The tetrameric 410-amino acid (aa) Tde-SAS protein preferentially synthesizes ppGpp over pppGpp and a third alarmone, pGpp. Unlike RelQ homologues, alarmones do not allosterically stimulate the synthetic activities of Tde-SAS. The ~180 aa C-terminal tetratricopeptide repeat (TPR) domain of Tde-SAS acts as a brake on the alarmone synthesis activities of the ~220-aa N-terminal catalytic domain. Tde-SAS also synthesizes "alarmone-like" nucleotides such as adenosine tetraphosphate (ppApp), albeit at considerably lower rates. The 210-aa Tde-SAH protein efficiently hydrolyzes all guanosine and adenosine-based alarmones in a Mn(II) ion-dependent manner. Using a growth assays with a ΔrelAΔspoT strain of Escherichia coli that is deficient in pppGpp/ppGpp synthesis, we demonstrate that Tde-SAS can synthesize alarmones in vivo to restore growth in minimal media. Taken together, our results add to our holistic understanding of alarmone metabolism across diverse bacterial species. IMPORTANCE The spirochete bacterium Treponema denticola is a common component of the oral microbiota. However, it may play important pathological roles in multispecies oral infectious diseases such as periodontitis: a severe and destructive form of gum disease, which is a major cause of tooth loss in adults. The operation of the stringent response, a highly conserved survival mechanism, is known to help many bacterial species cause persistent or virulent infections. By characterizing the biochemical functions of the proteins putatively responsible for the stringent response in T. denticola, we may gain molecular insight into how this bacterium can survive within harsh oral environments and promote infection. Our results also expand our general understanding of proteins that synthesize nucleotide-based intracellular signaling molecules in bacteria.