Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria.

Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular repABC-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, Dinoroseobacter shibae DFL 12 and Rhodobacter capsulatus B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; Ensifer meliloti 2011 and "Agrobacterium fabrum" C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (Rhodospirillum rubrum S1) as well as in one member of Caulobacterales (Caulobacter vibrioides CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in R. capsulatus.

Insights into the novel Enterococcus faecalis phage: A comprehensive genome analysis.

Enterococcus faecalis, a Gram-positive bacterium, poses a significant clinical challenge owing to its intrinsic resistance to a broad spectrum of antibiotics, warranting urgent exploration of innovative therapeutic strategies. This study investigated the viability of phage therapy as an alternative intervention for antibiotic-resistant E. faecalis, with a specific emphasis on the comprehensive genomic analysis of bacteriophage SAM-E.f 12. The investigation involved whole-genome sequencing of SAM-E.f 12 using Illumina technology, resulting in a robust dataset for detailed genomic characterization. Bioinformatics analyses were employed to predict genes and assign functional annotations. The bacteriophage SAM-E.f 12, which belongs to the Siphoviridae family, exhibited substantial potential, with a burst size of 5.7 PFU/infected cells and a latent period of 20 min. Host range determination experiments demonstrated its effectiveness against clinical E. faecalis strains, positioning SAM-E.f 12 as a precise therapeutic agent. Stability assays underscore resilience across diverse environmental conditions. This study provides a comprehensive understanding of SAM-E.f 12 genomic composition, lytic lifecycle parameters, and practical applications, particularly its efficacy in murine wound models. These results emphasize the promising role of phage therapy, specifically its targeted approach against antibiotic-resistant E. faecalis strains. The nuanced insights derived from this research will contribute to the ongoing pursuit of efficacious phage therapies and offer valuable implications for addressing the clinical challenges associated with E. faecalis infections.

UDP-glycosyltransferases act as key determinants of host plant range in generalist and specialist Spodoptera species.

Phytophagous insects have evolved sophisticated detoxification systems to overcome the antiherbivore chemical defenses produced by many plants. However, how these biotransformation systems differ in generalist and specialist insect species and their role in determining insect host plant range remains an open question. Here, we show that UDP-glucosyltransferases (UGTs) play a key role in determining the host range of insect species within the Spodoptera genus. Comparative genomic analyses of Spodoptera species that differ in host plant breadth identified a relatively conserved number of UGT genes in generalist species but high levels of UGT gene pseudogenization in the specialist Spodoptera picta. CRISPR-Cas9 knockouts of the three main UGT gene clusters of Spodoptera frugiperda revealed that UGT33 genes play an important role in allowing this species to utilize the poaceous plants maize, wheat, and rice, while UGT40 genes facilitate utilization of cotton. Further functional analyses in vivo and in vitro identified the UGT SfUGT33F32 as the key mechanism that allows generalist S. frugiperda to detoxify the benzoxazinoid DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), a potent insecticidal phytotoxin produced by poaceous plants. However, while this detoxification capacity is conserved in several generalist Spodoptera species, Spodoptera picta, which specializes on Crinum plants, is unable to detoxify DIMBOA due to a nonfunctionalizing mutation in SpUGT33F34. Collectively, these findings provide insight into the role of insect UGTs in host plant adaptation, the mechanistic basis of evolutionary transitions between generalism and specialism and offer molecular targets for controlling a group of notorious insect pests.

Large-scale genomic survey with deep learning-based method reveals strain-level phage specificity determinants.

BACKGROUND: Phage therapy, reemerging as a promising approach to counter antimicrobial-resistant infections, relies on a comprehensive understanding of the specificity of individual phages. Yet the significant diversity within phage populations presents a considerable challenge. Currently, there is a notable lack of tools designed for large-scale characterization of phage receptor-binding proteins, which are crucial in determining the phage host range. RESULTS: In this study, we present SpikeHunter, a deep learning method based on the ESM-2 protein language model. With SpikeHunter, we identified 231,965 diverse phage-encoded tailspike proteins, a crucial determinant of phage specificity that targets bacterial polysaccharide receptors, across 787,566 bacterial genomes from 5 virulent, antibiotic-resistant pathogens. Notably, 86.60% (143,200) of these proteins exhibited strong associations with specific bacterial polysaccharides. We discovered that phages with identical tailspike proteins can infect different bacterial species with similar polysaccharide receptors, underscoring the pivotal role of tailspike proteins in determining host range. The specificity is mainly attributed to the protein's C-terminal domain, which strictly correlates with host specificity during domain swapping in tailspike proteins. Importantly, our dataset-driven predictions of phage-host specificity closely match the phage-host pairs observed in real-world phage therapy cases we studied. CONCLUSIONS: Our research provides a rich resource, including both the method and a database derived from a large-scale genomics survey. This substantially enhances understanding of phage specificity determinants at the strain level and offers a valuable framework for guiding phage selection in therapeutic applications.

Highly Resolved Genomes of Two Closely Related Lineages of the Rodent Louse Polyplax serrata with Different Host Specificities.

Sucking lice of the parvorder Anoplura are permanent ectoparasites with specific lifestyle and highly derived features. Currently, genomic data are only available for a single species, the human louse Pediculus humanus. Here, we present genomes of two distinct lineages, with different host spectra, of a rodent louse Polyplax serrata. Genomes of these ecologically different lineages are closely similar in gene content and display a conserved order of genes, with the exception of a single translocation. Compared with P. humanus, the P. serrata genomes are noticeably larger (139 vs. 111 Mbp) and encode a higher number of genes. Similar to P. humanus, they are reduced in sensory-related categories such as vision and olfaction. Utilizing genome-wide data, we perform phylogenetic reconstruction and evolutionary dating of the P. serrata lineages. Obtained estimates reveal their relatively deep divergence (∼6.5 Mya), comparable with the split between the human and chimpanzee lice P. humanus and Pediculus schaeffi. This supports the view that the P. serrata lineages are likely to represent two cryptic species with different host spectra. Historical demographies show glaciation-related population size (Ne) reduction, but recent restoration of Ne was seen only in the less host-specific lineage. Together with the louse genomes, we analyze genomes of their bacterial symbiont Legionella polyplacis and evaluate their potential complementarity in synthesis of amino acids and B vitamins. We show that both systems, Polyplax/Legionella and Pediculus/Riesia, display almost identical patterns, with symbionts involved in synthesis of B vitamins but not amino acids.

A Novel Rhizobium sp. Chiba-1 Strain Exhibits a Host Range for Nodule Symbiosis in Lotus Species.

Rhizobia are soil bacteria that induce the formation of nodules in the roots of leguminous plants for mutualistic establishment. Although the symbiotic mechanism between Lotus japonicus and its major symbiotic rhizobia, Mesorhizobium loti, has been extensively characterized, our understanding of symbiotic mechanisms, such as host specificity and host ranges, remains limited. In the present study, we isolated a novel Rhizobium strain capable of forming nodules on L. burttii from agricultural soil at Iwate prefecture in Japan. We conducted genomic and host range ana-lyses of various Lotus species. The results obtained revealed that the novel isolated Rhizobium sp. Chiba-1 was closely related to R. leguminosarum and had a wide host range that induced nodule development, including L. burttii and several L. japonicus wild-type accessions. However, L. japonicus Gifu exhibited an incompatible nodule phenotype. We also identified the formation of an epidermal infection threads that was dependent on the Lotus species and independent of nodule organ development. In conclusion, this newly isolated Rhizobium strain displays a distinct nodulation phenotype from Lotus species, and the results obtained herein provide novel insights into the functional mechanisms underlying host specificity and host ranges.

First Insights into Population Structure and Genetic Diversity Versus Host Specificity in Trypanorhynch Tapeworms Using Multiplexed Shotgun Genotyping.

Theory predicts relaxed host specificity and high host vagility should contribute to reduced genetic structure in parasites while strict host specificity and low host vagility should increase genetic structure. Though these predictions are intuitive, they have never been explicitly tested in a population genomic framework. Trypanorhynch tapeworms, which parasitize sharks and rays (elasmobranchs) as definitive hosts, are the only order of elasmobranch tapeworms that exhibit considerable variability in their definitive host specificity. This allows for unique combinations of host use and geographic range, making trypanorhynchs ideal candidates for studying how these traits influence population-level structure and genetic diversity. Multiplexed shotgun genotyping (MSG) data sets were generated to characterize component population structure and infrapopulation diversity for a representative of each trypanorhynch suborder: the ray-hosted Rhinoptericola megacantha (Trypanobatoida) and the shark-hosted Callitetrarhynchus gracilis (Trypanoselachoida). Adults of R. megacantha are more host-specific and less broadly distributed than adults of C. gracilis, allowing correlation between these factors and genetic structure. Replicate tapeworm specimens were sequenced from the same host individual, from multiple conspecific hosts within and across geographic regions, and from multiple definitive host species. For R. megacantha, population structure coincided with geography rather than host species. For C. gracilis, limited population structure was found, suggesting a potential link between degree of host specificity and structure. Conspecific trypanorhynchs from the same host individual were found to be as, or more, genetically divergent from one another as from conspecifics from different host individuals. For both species, high levels of homozygosity and positive FIS values were documented.

A Zinc Finger Motif in the P1 N Terminus, Highly Conserved in a Subset of Potyviruses, Is Associated with the Host Range and Fitness of Telosma Mosaic Virus.

P1 is the first protein translated from the genomes of most viruses in the family Potyviridae, and it contains a C-terminal serine-protease domain that cis-cleaves the junction between P1 and HCPro in most cases. Intriguingly, P1 is the most divergent among all mature viral factors, and its roles during viral infection are still far from understood. In this study, we found that telosma mosaic virus (TelMV, genus Potyvirus) in passion fruit, unlike TelMV isolates present in other hosts, has two stretches at the P1 N terminus, named N1 and N2, with N1 harboring a Zn finger motif. Further analysis revealed that at least 14 different potyviruses, mostly belonging to the bean common mosaic virus subgroup, encode a domain equivalent to N1. Using the newly developed TelMV infectious cDNA clones from passion fruit, we demonstrated that N1, but not N2, is crucial for viral infection in both Nicotiana benthamiana and passion fruit. The regulatory effects of N1 domain on P1 cis cleavage, as well as the accumulation and RNA silencing suppression (RSS) activity of its cognate HCPro, were comprehensively investigated. We found that N1 deletion decreases HCPro abundance at the posttranslational level, likely by impairing P1 cis cleavage, thus reducing HCPro-mediated RSS activity. Remarkably, disruption of the Zn finger motif in N1 did not impair P1 cis cleavage and HCPro accumulation but severely debilitated TelMV fitness. Therefore, our results suggest that the Zn finger motif in P1s plays a critical role in viral infection that is independent of P1 protease activity and self-release, as well as HCPro accumulation and silencing suppression. IMPORTANCE Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy. P1, the first protein translated from most potyvirid genomes, is the most variable viral factor and has attracted great scientific interest. Here, we defined a Zn finger motif-encompassing domain (N1) at the N terminus of P1 among diverse potyviruses phylogenetically related to bean common mosaic virus. Using TelMV as a model virus, we demonstrated that the N1 domain is key for viral infection, as it is involved both in regulating the abundance of its cognate HCPro and in an as-yet-undefined key function unrelated to protease processing and RNA silencing suppression. These results advance our knowledge of the hypervariable potyvirid P1s and highlight the importance for infection of a previously unstudied Zn finger domain at the P1 N terminus.

Natural variation in Avr3D1 from Zymoseptoria sp. contributes to quantitative gene-for-gene resistance and to host specificity.

Successful host colonization by plant pathogens requires the circumvention of host defense responses, frequently through sequence modifications in secreted pathogen proteins known as avirulence factors (Avrs). Although Avr sequences are often polymorphic, the contribution of these polymorphisms to virulence diversity in natural pathogen populations remains largely unexplored. We used molecular genetic tools to determine how natural sequence polymorphisms of the avirulence factor Avr3D1 in the wheat pathogen Zymoseptoria tritici contributed to adaptive changes in virulence. We showed that there is a continuous distribution in the magnitude of resistance triggered by different Avr3D1 isoforms and demonstrated that natural variation in an Avr gene can lead to a quantitative resistance phenotype. We further showed that homologues of Avr3D1 in two nonpathogenic sister species of Z. tritici are recognized by some wheat cultivars, suggesting that Avr-R gene-for-gene interactions can contribute to nonhost resistance. We suggest that the mechanisms underlying host range, qualitative resistance, and quantitative resistance are not exclusive.

Phylogenomic analyses and host range prediction of cluster P mycobacteriophages.

Bacteriophages, infecting bacterial hosts in every environment on our planet, are a driver of adaptive evolution in bacterial communities. At the same time, the host range of many bacteriophages-and thus one of the selective pressures acting on complex microbial systems in nature-remains poorly characterized. Here, we computationally inferred the putative host ranges of 40 cluster P mycobacteriophages, including members from 6 subclusters (P1-P6). A series of comparative genomic analyses revealed that mycobacteriophages of subcluster P1 are restricted to the Mycobacterium genus, whereas mycobacteriophages of subclusters P2-P6 are likely also able to infect other genera, several of which are commonly associated with human disease. Further genomic analysis highlighted that the majority of cluster P mycobacteriophages harbor a conserved integration-dependent immunity system, hypothesized to be the ancestral state of a genetic switch that controls the shift between lytic and lysogenic life cycles-a temperate characteristic that impedes their usage in antibacterial applications.

Myxoma virus lacking the host range determinant M062 stimulates cGAS-dependent type 1 interferon response and unique transcriptomic changes in human monocytes/macrophages.

The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range C7L superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range C7L superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the C7L superfamily is the M062R gene. The M062R gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile α Motif Domain-containing 9 (SAMD9), as M062R-null (ΔM062R) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the ΔM062R. We found that the replication-defective ΔM062R activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA alone-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses.

Factors That Determine Microsporidia Infection and Host Specificity.

Microsporidia are a large phylum of obligate intracellular parasites that infect an extremely diverse range of animals and protists. In this chapter, we review what is currently known about microsporidia host specificity and what factors influence microsporidia infection. Extensive sampling in nature from related hosts has provided insight into the host range of many microsporidia species. These field studies have been supported by experiments conducted in controlled laboratory environments which have helped to demonstrate host specificity. Together, these approaches have revealed that, while examples of generalist species exist, microsporidia specificity is often narrow, and species typically infect one or several closely related hosts. For microsporidia to successfully infect and complete their life cycle within a compatible host, several steps must occur, including spore germination, host cell invasion, and proliferation of the parasite within the host tissue. Many factors influence infection, including temperature, seasonality, nutrient availability, and the presence or absence of microbes, as well as the developmental stage, sex, and genetics of the host. Several studies have identified host genomic regions that influence resistance to microsporidia, and future work is likely to uncover molecular mechanisms of microsporidia host specificity in more detail.

A genomic can of worms for schistosome host-specificity.

Understanding the genetic underpinnings of schistosome host preferences is critical. Luo et al. recently identified genes associated with intermediate and definitive host-switching based on a new chromosome-level genome for Schistosoma japonicum, population genetic comparisons, and follow-up experiments. This represents a guide to fully map-selected schistosome genes using population genetics.

The Number and Type of Chaperone-Usher Fimbriae Reflect Phylogenetic Clade Rather than Host Range in Salmonella.

Salmonella is one of the most successful foodborne pathogens worldwide, owing in part to its ability to colonize or infect a wide range of hosts. Salmonella serovars are known to encode a variety of different fimbriae (hairlike organelles that facilitate binding to surfaces); however, the distribution, number, and sequence diversity of fimbriae encoded across different lineages of Salmonella were unknown. We queried whole-genome sequence (WGS) data for 242 Salmonella enterica subsp. enterica (subspecies enterica) isolates from the top 217 serovars associated with isolation from humans and agricultural animals; this effort identified 2,894 chaperone-usher (CU)-type fimbrial usher sequences, representing the most conserved component of CU fimbriae. On average, isolates encoded 12 different CU fimbrial ushers (6 to 18 per genome), although the distribution varied significantly (P = 1.328E-08) by phylogenetic clade, with isolates in section Typhi having significantly fewer fimbrial ushers than isolates in clade A2 (medians = 10 and 12 ushers, respectively). Characterization of fimbriae in additional non-enterica subspecies genomes suggested that 8 fimbrial ushers were classified as being unique to subspecies enterica isolates, suggesting that the majority of fimbriae were most likely acquired prior to the divergence of subspecies enterica. Characterization of mobile elements suggested that plasmids represent an important vehicle facilitating the acquisition of a wide range of fimbrial ushers, particularly for the acquisition of fimbriae from other Gram-negative genera. Overall, our results suggest that differences in the number and type of fimbriae encoded most likely reflect differences in phylogenetic clade rather than differences in host range. IMPORTANCE Fimbriae of the CU assembly pathway represent important organelles that mediate Salmonella's interactions with host tissues and abiotic surfaces. Our analyses provide a comprehensive overview of the diversity of CU fimbriae in Salmonella spp., highlighting that the majority of CU fimbriae are distributed broadly across multiple subspecies and suggesting that acquisition most likely occurred prior to the divergence of subspecies enterica. Our data also suggest that plasmids represent the primary vehicles facilitating the horizontal transfer of diverse CU fimbriae in Salmonella. Finally, the observed high sequence similarity between some ushers suggests that different names may have been assigned to closely related fimbrial ushers that likely should be represented by a single designation. This highlights the need to establish standard criteria for fimbria classification and nomenclature, which will also facilitate future studies seeking to associate virulence factors with adaptation to or differences in the likelihood of causing disease in a given host.

Prediction of Prophages and Their Host Ranges in Pathogenic and Commensal Neisseria Species.

The genus Neisseria includes two pathogenic species, N. gonorrhoeae and N. meningitidis, and numerous commensal species. Neisseria species frequently exchange DNA with one another, primarily via transformation and homologous recombination and via multiple types of mobile genetic elements (MGEs). Few Neisseria bacteriophages (phages) have been identified, and their impact on bacterial physiology is poorly understood. Furthermore, little is known about the range of species that Neisseria phages can infect. In this study, we used three virus prediction tools to scan 248 genomes of 21 different Neisseria species and identified 1,302 unique predicted prophages. Using comparative genomics, we found that many predictions are dissimilar from prophages and other MGEs previously described to infect Neisseria species. We also identified similar predicted prophages in genomes of different Neisseria species. Additionally, we examined CRISPR-Cas targeting of each Neisseria genome and predicted prophage. While CRISPR targeting of chromosomal DNA appears to be common among several Neisseria species, we found that 20% of the prophages we predicted are targeted significantly more than the rest of the bacterial genome in which they were identified (i.e., backbone). Furthermore, many predicted prophages are targeted by CRISPR spacers encoded by other species. We then used these results to infer additional host species of known Neisseria prophages and predictions that are highly targeted relative to the backbone. Together, our results suggest that we have identified novel Neisseria prophages, several of which may infect multiple Neisseria species. These findings have important implications for understanding horizontal gene transfer between members of this genus. IMPORTANCE Drug-resistant Neisseria gonorrhoeae is a major threat to human health. Commensal Neisseria species are thought to serve as reservoirs of antibiotic resistance and virulence genes for the pathogenic species N. gonorrhoeae and N. meningitidis. Therefore, it is important to understand both the diversity of mobile genetic elements (MGEs) that can mediate horizontal gene transfer within this genus and the breadth of species these MGEs can infect. In particular, few bacteriophages (phages) are known to infect Neisseria species. In this study, we identified a large number of candidate phages integrated in the genomes of commensal and pathogenic Neisseria species, many of which appear to be novel phages. Importantly, we discovered extensive interspecies targeting of predicted phages by Neisseria CRISPR-Cas systems, which may reflect their movement between different species. Uncovering the diversity and host range of phages is essential for understanding how they influence the evolution of their microbial hosts.

Strain-level characterization of broad host range mobile genetic elements transferring antibiotic resistance from the human microbiome.

Mobile genetic elements (MGEs) carrying antibiotic resistance genes (ARGs) disseminate ARGs when they mobilise into new bacterial hosts. The nature of such horizontal gene transfer (HGT) events between human gut commensals and pathogens remain poorly characterised. Here, we compare 1354 cultured commensal strains (540 species) to 45,403 pathogen strains (12 species) and find 64,188 MGE-mediated ARG transfer events between the two groups using established methods. Among the 5931 MGEs, we find 15 broad host range elements predicted to have crossed different bacterial phyla while also occurring in animal and environmental microbiomes. We experimentally demonstrate that predicted broad host range MGEs can mobilise from commensals Dorea longicatena and Hungatella hathewayi to pathogen Klebsiella oxytoca, crossing phyla simultaneously. Our work establishes the MGE-mediated ARG dissemination network between human gut commensals and pathogens and highlights broad host range MGEs as targets for future ARG dissemination management.

DNA barcoding uncovers cryptic diversity in minute herbivorous mites (Acari, Eriophyoidea).

Eriophyoid mites (Acari: Eriophyoidea) are among the smallest of terrestrial arthropods and the most species-rich group of herbivorous mites with a high host specificity. However, knowledge of their species diversity has been impeded by the difficulty of their morphological differentiation. This study assembles a DNA barcode reference library that includes 1850 mitochondrial COI sequences which provides coverage for 45% of the 930 species of eriophyoid mites known from China, and for 37 North American species. Sequence analysis showed a clear barcode gap in nearly all species, reflecting the fact that intraspecific divergences averaged 0.97% versus a mean of 18.51% for interspecific divergences (minimum nearest-neighbour distances) in taxa belonging to three families. Based on these results, we used DNA barcoding to explore the species diversity of eriophyoid mites as well as their host interactions. The 1850 sequences were assigned to 531 barcode index numbers (BINs). Analyses examining the correspondence between these BINs and species identifications based on morphology revealed that members of 45 species were assigned to two or more BINs, resulting in 1.16 times more BINs than morphospecies. Richness projections suggest that over 2345 BINs occurred at the sampled locations. Host plant analysis showed that 89% of these mites (BINs) attack only one or two congeneric host species, but the others have several hosts. Furthermore, host-mite network analyses demonstrate that eriophyoid mites are high host-specific, and modularity is high in plant-mite networks. By creating a highly effective identification system for eriophyoid mites in the Barcode of Life Data Systems database (BOLD), DNA barcoding will advance our understanding of the diversity of eriophyoid mites and their host interactions.

Directed Evolution of Replication-Competent Double-Stranded DNA Bacteriophage toward New Host Specificity.

In the fight against antimicrobial resistance, bacteriophages are a promising alternative to antibiotics. However, due to their narrow spectra, phage therapy requires the careful matching between the host and bacteriophage to be effective. Despite our best efforts, nature remains as the only source of novel phage specificity. Directed evolution can potentially open an avenue for engineering phage specificity and improving qualities of phages that are not strongly selected for in their natural environments but are important for therapeutic applications. In this work, we present a strategy that generates large libraries of replication-competent phage variants directly from synthetic DNA fragments, with no restriction on their host specificity. Using the T7 bacteriophage as a proof-of-concept, we created a large library of tail fiber mutants with at least 107 unique variants. From this library, we identified mutants that have broadened specificity as evidenced by their novel lytic activity against Yersinia enterocolitica, a strain that the wild-type T7 was unable to lyse. Using the same concept, mutants with improved lytic efficiency and characteristics, such as lytic condition tolerance and resistance suppression, were also identified. However, the observed limitations in altering host specificity by tail fiber mutagenesis suggest that other bottlenecks could be of equal or even greater importance.

Diversity, host specificity and biogeography in the Cladocorynidae (Hydrozoa, Capitata), with description of a new genus.

The hydrozoan family Cladocorynidae inhabits tropical to temperate waters and comprises the two genera Pteroclava and Cladocoryne. Pteroclava lives in association with some octocorals and hydrozoans, whereas Cladocoryne is more generalist in terms of substrate choice. This work provides a thorough morpho-molecular reassessment of the Cladocorynidae by presenting the first well-supported phylogeny of the family based on the analyses of three mitochondrial and four nuclear markers. Notably, the two nominal genera were confirmed to be monophyletic and both morphological and genetic data led to the formal description of a new genus exclusively associated with octocorals, Pseudozanclea gen. nov. Maggioni & Montano. Accordingly, the diagnosis of the family was updated. The ancestral state reconstruction of selected characters revealed that the symbiosis with octocorals likely appeared in the most recent common ancestor of Pteroclava and Pseudozanclea. Additionally, the presence of euryteles aggregation in the polyp stage and the exumbrellar nematocyst pouches with euryteles represent synapomorphies of all cladocorynid taxa and probably emerged in their most recent common ancestor. The analysis of several Pteroclava krempfi colonies from Indo-Pacific and Caribbean localities associated with several host octocorals revealed a high intra-specific genetic variability. Single- and multi-locus species delimitations resulted in three to five species hypotheses, but the statistical analysis of morphometric data showed only limited distinction among the clades of P. krempfi. However, P. krempfi clades showed differences in both host specificity, mostly at the octocoral family level, and geographic distribution, with one clade found exclusively in the Caribbean Sea and the others found in the Indo-Pacific.

Molecular dynamic simulation analysis of SARS-CoV-2 spike mutations and evaluation of ACE2 from pets and wild animals for infection risk.

Coronavirus Disease 2019 (COVID-19) is an ongoing global health emergency that has caused tremendous stress and loss of life worldwide. The viral spike glycoprotein is a critical molecule mediating transmission of SARS-CoV-2 by interacting with human ACE2. However, through the course of the pandemics, there has not been a thorough analysis of the spike protein mutations, and on how these mutants influence the transmission of SARS-CoV-2. Besides, cases of SARS-CoV-2 infection among pets and wild animals have been reported, so the susceptibility of these animals requires great attention to investigate, as they may also link to the renewed question of a possible intermediate host for SARS-CoV-2 before it was transmitted to humans. With over 226,000 SARS-CoV-2 sequences obtained, we found 1573 missense mutations in the spike gene, and 226 of them were within the receptor-binding domain (RBD) region that directly interacts with human ACE2. Modeling the interactions between SARS-CoV-2 spike mutants and ACE2 molecules showed that most of the 74 missense mutations in the RBD region of the interaction interface had little impact on spike binding to ACE2, whereas several within the spike RBD increased the binding affinity toward human ACE2 thus making the virus likely more contagious. On the other hand, modeling the interactions between animal ACE2 molecules and SARS-CoV-2 spike revealed that many pets and wild animals' ACE2 had a variable binding ability. Particularly, ACE2 of bamboo rat had stronger binding to SARS-CoV-2 spike protein, whereas that of mole, vole, Mus pahari, palm civet, and pangolin had a weaker binding compared to human ACE2. Our results provide structural insights into the impact on interactions of the SARS-CoV-2 spike mutants to human ACE2, and shed light on SARS-CoV-2 transmission in pets and wild animals, and possible clues to the intermediate host(s) for SARS-CoV-2.