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Background Viruses are central to microbial community structure in all environments. The ability to generate large metagenomic assemblies of mixed microbial and viral sequences provides the opportunity to tease apart complex microbiome dynamics, but these analyses are currently limited by the tools available for analyses of viral genomes and assessing their metabolic impacts on microbiomes. Design Here we present VIBRANT, the first method to utilize a hybrid machine learning and protein similarity approach that is not reliant on sequence features for automated recovery and annotation of viruses, determination of genome quality and completeness, and characterization of viral community function from metagenomic assemblies. VIBRANT uses neural networks of protein signatures and a newly developed v-score metric that circumvents traditional boundaries to maximize identification of lytic viral genomes and integrated proviruses, including highly diverse viruses. VIBRANT highlights viral auxiliary metabolic genes and metabolic pathways, thereby serving as a user-friendly platform for evaluating viral community function. VIBRANT was trained and validated on reference virus datasets as well as microbiome and virome data. Results VIBRANT showed superior performance in recovering higher quality viruses and concurrently reduced the false identification of non-viral genome fragments in comparison to other virus identification programs, specifically VirSorter, VirFinder, and MARVEL. When applied to 120,834 metagenome-derived viral sequences representing several human and natural environments, VIBRANT recovered an average of 94% of the viruses, whereas VirFinder, VirSorter, and MARVEL achieved less powerful performance, averaging 48%, 87%, and 71%, respectively. Similarly, VIBRANT identified more total viral sequence and proteins when applied to real metagenomes. When compared to PHASTER, Prophage Hunter, and VirSorter for the ability to extract integrated provirus regions from host scaffolds, VIBRANT performed comparably and even identified proviruses that the other programs did not. To demonstrate applications of VIBRANT, we studied viromes associated with Crohn’s disease to show that specific viral groups, namely Enterobacteriales-like viruses, as well as putative dysbiosis associated viral proteins are more abundant compared to healthy individuals, providing a possible viral link to maintenance of diseased states. Conclusions The ability to accurately recover viruses and explore viral impacts on microbial community metabolism will greatly advance our understanding of microbiomes, host-microbe interactions, and ecosystem dynamics. 7uVs82Bc4tfSLymdHQuU_3 Video Abstract

Background Roseobacteraceae , often referred to as the marine roseobacter clade (MRC), are pivotal constituents of bacterial communities in coastal and pelagic marine environments. During the past two decades, 75 roseophages that infect various Roseobacteraceae lineages have been isolated. The N4-like roseophage clade, which encompasses 15 members, represents the largest clade among these roseophages. N4-like phages form a monophyletic group, classified as family Schitoviridae . And all N4-like roseophages form a unique clade within Schitoviridae and has been classified as the Rhodovirinae subfamily. Results In this study, we isolated a novel roseophage, vB_DshP-R7L, that infects Dinoroseobacter shibae DFL12 from Xiamen Bay in the East China Sea. Conserved genes of Schitoviridae have been identified in the genome of vB_DshP-R7L, and following phylogenetic analysis suggests that the newly isolated phage is a member of the Rhodovirinae subfamily and represents the sole member of a novel genus, Gonggongvirus . The genome of vB_DshP-R7L harbors six auxiliary metabolic genes (AMGs), most of which potentially enhance DNA de novo synthesis. Additionally, a gene encoding ribosomal protein was identified. Comparative genomic analysis of AMG content among Rhodovirinae indicates a distinct evolutionary history characterized by independent ancient horizontal gene transfer events. Read-mapping analysis reveals the prevalence of vB_DshP-R7L and other Rhodovirinae roseophages in estuarine waters. Conclusions Our work illustrates the genomic features of a novel roseophage clade among the subfamily Rhodovirinae. The AMG content of vB_DshP-R7L is under severe purification selection, which reveals their possible ecological importance. We also demonstrated that vB_DshP-R7L and other Rhodovirinae roseophages are only detected in estuaries. Our isolation and characterization of this novel phage expands the understanding of the phylogeny, gene transfer history, and biogeography of Rhodovirinae infecting marine Roseobacteraceae.

Aeromonas species are common pathogens of fish and some of them can opportunistically cause infectious diseases in humans. The overuse of antibiotics has led to the emergence of bacterial drug-resistance. To date, only 51 complete genome sequences of Aeromonas phages are available in GenBank. Here, we report the isolation of nine Aeromonas phages from a plateau lake in China. The protein cluster, dot plot and ANI analyses were performed on all 60 currently sequenced Aeromonas phage genomes and classified into nine clusters and thirteen singletons. Among the nine isolated phages, the DNA-packaging strategy of cluster 2L372D (including 2L372D, 2L372X, 4L372D, 4L372XY) is unknown, while the other five phages use the headful (P22/Sf6) DNA-packaging strategy. Notably, the isolated phages with larger genomes conservatively encode auxiliary metabolism genes, DNA replication and metabolism genes, while in smaller phage genomes, recombination-related genes were conserved. Finally, we propose a new classification scheme for Aeromonas phages.

Neuronal N-type (Ca V 2.2) voltage-gated calcium channels are important at the first synapse in the pain pathway. In this study, we have characterized a knockin mouse containing Ca V 2.2 with an extracellular HA tag to determine the localization of Ca V 2.2 in primary afferent pain pathways. These endogenous channels have been visualized at the plasma membrane and rigorously quantified in vivo. We examined the effect of ablation of the calcium channel auxiliary subunit α 2 δ-1 (the target of gabapentinoids) on Ca V 2.2 distribution. We found preferential cell-surface localization of Ca V 2.2 in DRG nociceptor neuron cell bodies was lost, accompanied by a dramatic reduction at dorsal horn terminals, but no effect on distribution of other spinal cord synaptic markers. The auxiliary α 2 δ calcium channel subunits play key roles in voltage-gated calcium channel function. Independent of this, α 2 δ-1 has also been suggested to be important for synaptogenesis. Using an epitope-tagged knockin mouse strategy, we examined the effect of α 2 δ-1 on Ca V 2.2 localization in the pain pathway in vivo, where Ca V 2.2 is important for nociceptive transmission and α 2 δ-1 plays a critical role in neuropathic pain. We find Ca V 2.2 is preferentially expressed on the plasma membrane of calcitonin gene-related peptide-positive small nociceptors. This is paralleled by strong presynaptic expression of Ca V 2.2 in the superficial spinal cord dorsal horn. EM-immunogold localization shows Ca V 2.2 predominantly in active zones of glomerular primary afferent terminals. Genetic ablation of α 2 δ-1 abolishes Ca V 2.2 cell-surface expression in dorsal root ganglion neurons and dramatically reduces dorsal horn expression. There was no effect of α 2 δ-1 knockout on other dorsal horn pre- and postsynaptic markers, indicating the primary afferent pathways are not otherwise affected by α 2 δ-1 ablation.

Reducing the enzyme loadings for enzymatic saccharification of lignocellulose is required for economically feasible production of biofuels and biochemicals. One strategy is addition of small amounts of synergistic proteins to cellulase mixtures. Synergistic proteins increase the activity of cellulase without causing significant hydrolysis of cellulose. Synergistic proteins exert their activity by inducing structural modifications in cellulose. Recently, synergistic proteins from various biological sources, including bacteria, fungi, and plants, were identified based on genomic data, and their synergistic activities were investigated. Currently, an up-to-date overview of several aspects of synergistic proteins, such as their functions, action mechanisms and synergistic activity, are important for future industrial application. In this review, we summarize the current state of research on four synergistic proteins: carbohydrate-binding modules, plant expansins, expansin-like proteins, and Auxiliary Activity family 9 (formerly GH61) proteins. This review provides critical information to aid in promoting research on the development of efficient and industrially feasible synergistic proteins.

Background Viral-encoded auxiliary metabolic genes (AMGs) are important toolkits for modulating their hosts’ metabolisms and the microbial-driven biogeochemical cycles. Although the functions of AMGs have been extensively reported in numerous environments, we still know little about the drivers that shape the viral community-wide AMG compositions in natural ecosystems. Exploring the drivers of viral community-wide AMG compositions is critical for a deeper understanding of the complex interplays among viruses, hosts, and the environments. Results Here, we investigated the impact of viral lifestyles (i.e., lytic and lysogenic), habitats (i.e., water, particle, and sediment), and prokaryotic hosts on viral AMG profiles by utilizing metagenomic and metatranscriptomic techniques. We found that viral lifestyles were the most important drivers, followed by habitats and host identities. Specifically, irrespective of what habitats viruses came from, lytic viruses exhibited greater AMG diversity and tended to encode AMGs for chaperone biosynthesis, signaling proteins, and lipid metabolism, which could boost progeny reproduction, whereas temperate viruses were apt to encode AMGs for host survivability. Moreover, the lytic and temperate viral communities tended to mediate the microbial-driven biogeochemical cycles, especially nitrogen metabolism, in different manners via AMGs. When focusing on each lifestyle, we further found clear dissimilarity in AMG compositions between water and sediment, as well the divergent AMGs encoded by viruses infecting different host orders. Conclusions Overall, our study provides a first systematic characterization of the drivers of viral community-wide AMG compositions and further expands our knowledge of the distinct interactions of lytic and temperate viruses with their prokaryotic hosts from an AMG perspective, which is critical for understanding virus-host-environment interactions in natural conditions. -i-eSMHy56EjkBsNfneT5s Video Abstract

Soil viruses are abundant, but the influence of the environment and climate on soil viruses remains poorly understood. Here, we addressed this gap by comparing the diversity, abundance, lifestyle, and metabolic potential of DNA viruses in three grassland soils with historical differences in average annual precipitation, low in eastern Washington (WA), high in Iowa (IA), and intermediate in Kansas (KS). Soil viruses are abundant, but the influence of the environment and climate on soil viruses remains poorly understood. Here, we addressed this gap by comparing the diversity, abundance, lifestyle, and metabolic potential of DNA viruses in three grassland soils with historical differences in average annual precipitation, low in eastern Washington (WA), high in Iowa (IA), and intermediate in Kansas (KS). Bioinformatics analyses were applied to identify a total of 2,631 viral contigs, including 14 complete viral genomes from three deep metagenomes (1 terabase [Tb] each) that were sequenced from bulk soil DNA. An additional three replicate metagenomes (∼0.5 Tb each) were obtained from each location for statistical comparisons. Identified viruses were primarily bacteriophages targeting dominant bacterial taxa. Both viral and host diversity were higher in soil with lower precipitation. Viral abundance was also significantly higher in the arid WA location than in IA and KS. More lysogenic markers and fewer clustered regularly interspaced short palindromic repeats (CRISPR) spacer hits were found in WA, reflecting more lysogeny in historically drier soil. More putative auxiliary metabolic genes (AMGs) were also detected in WA than in the historically wetter locations. The AMGs occurring in 18 pathways could potentially contribute to carbon metabolism and energy acquisition in their hosts. Structural equation modeling (SEM) suggested that historical precipitation influenced viral life cycle and selection of AMGs. The observed and predicted relationships between soil viruses and various biotic and abiotic variables have value for predicting viral responses to environmental change. IMPORTANCE Soil viruses are abundant but poorly understood. Because soil viruses regulate the dynamics of their hosts and potentially key processes in soil ecology, it is important to understand them better. Here, we leveraged massive DNA sequencing to unearth previously unknown soil viruses. We found that soil viruses differed across a historical gradient of precipitation. We compared soil viruses from Iowa, which is traditionally wetter, to those from Washington, which is traditionally drier, and from Kansas, which is intermediate. This study provides strong evidence that changes in historical precipitation impact not only the types of soil viruses but also their functional potential.

Presynaptic glutamate receptors (GluRs) modulate neurotransmitter release and are physiological targets for regulation during various forms of plasticity. Although much is known about the auxiliary subunits associated with postsynaptic GluRs, far less is understood about presynaptic auxiliary GluR subunits and their functions. At the Drosophila neuromuscular junction, a presynaptic GluR, DKaiR1D, localizes near active zones and operates as an autoreceptor to tune baseline transmission and enhance presynaptic neurotransmitter release in response to diminished postsynaptic GluR functionality, a process referred to as presynaptic homeostatic potentiation (PHP). Here, we identify an auxiliary subunit that collaborates with DKaiR1D to promote these synaptic functions. This subunit, dSol-1, is the homolog of the Caenorhabditis elegans CUB (Complement C1r/C1s, Uegf, Bmp1) domain protein Sol-1. We find that dSol-1 functions in neurons to facilitate baseline neurotransmission and to enable PHP expression, properties shared with DKaiR1D. Intriguingly, presynaptic overexpression of dSol-1 is sufficient to enhance neurotransmitter release through a DKaiR1D-dependent mechanism. Furthermore, dSol-1 is necessary to rapidly increase the abundance of DKaiR1D receptors near active zones during homeostatic signaling. Together with recent work showing the CUB domain protein Neto2 is necessary for the homeostatic modulation of postsynaptic GluRs in mammals, our data demonstrate that dSol-1 is required for the homeostatic regulation of presynaptic GluRs. Thus, we propose that CUB domain proteins are fundamental homeostatic modulators of GluRs on both sides of the synapse.

There is significant contemporary interest in the application of enzymes to replace or augment chemical reagents toward the development of more environmentally sound and sustainable processes. In particular, copper radical oxidases (CRO) from Auxiliary Activity Family 5 Subfamily 2 (AA5_2) are attractive, organic cofactor-free catalysts for the chemoselective oxidation of alcohols to the corresponding aldehydes. These enzymes were first defined by the archetypal galactose-6-oxidase (GalOx, EC 1.1.3.13) from the fungus Fusarium graminearum. The recent discovery of specific alcohol oxidases (EC 1.1.3.7) and aryl alcohol oxidases (EC 1.1.3.47) within AA5_2 has indicated a potentially broad substrate scope among fungal CROs. However, only relatively few AA5_2 members have been characterized to date. Guided by sequence similarity network and phylogenetic analysis, twelve AA5_2 homologs have been recombinantly produced and biochemically characterized in the present study. As defined by their predominant activities, these comprise four galactose 6-oxidases, two raffinose oxidases, four broad-specificity primary alcohol oxidases, and two non-carbohydrate alcohol oxidases. Of particular relevance to applications in biomass valorization, detailed product analysis revealed that two CROs produce the bioplastics monomer furan-2,5-dicarboxylic acid (FDCA) directly from 5-hydroxymethylfurfural (HMF). Furthermore, several CROs could desymmetrize glycerol (a by-product of the biodiesel industry) to d- or l-glyceraldehyde. This study furthers our understanding of CROs by doubling the number of characterized AA5_2 members, which may find future applications as biocatalysts in diverse processes.

Cellulose-degrading auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are known to be widely distributed among filamentous fungi and participate in the degradation of lignocellulose via the oxidative cleavage of celluloses, cello-oligosaccharides, or hemicelluloses. AA9 LPMOs have been reported to have extensive interactions with not only cellulases but also oxidases. The addition of AA9 LPMOs can greatly reduce the amount of cellulase needed for saccharification and increase the yield of glucose. The discovery of AA9 LPMOs has greatly changed our understanding of how fungi degrade cellulose. In this review, apart from summarizing the recent discoveries related to their catalytic reaction, functional diversity, and practical applications, the stability, expression system, and protein engineering of AA9 LPMOs are reviewed for the first time. This review may provide a reference value to further broaden the substrate range of AA9 LPMOs, expand the scope of their practical applications, and realize their customization for industrial utilization.Key Points• The stability and expression system of AA9 LPMOs are reviewed for the first time.• The protein engineering of AA9 LPMOs is systematically summarized for the first time.• The latest research results on the catalytic mechanism of AA9 LPMOs are summarized.• The application of AA9 LPMOs and their relationship with other enzymes are reviewed.