Close Phylogenetic Relationship Research Articles

Cultivation of deep-sea bacteria from the Northwest Pacific Ocean and characterization of Limnobacter profundi sp. nov., a phenol-degrading bacterium

Despite previous culture-independent studies highlighting the prevalence of the order Burkholderiales in deep-sea environments, the cultivation and characterization of deep-sea Burkholderiales have been infrequent. A total of 243 deep-sea bacterial strains were isolated from various depths in the Northwest Pacific Ocean, with 33 isolates (13.6%) from a depth of 4000 m classified into Burkholderiales. Herein, we report the isolation and genome characteristics of strain SAORIC-580T, from a depth of 4000 m in the Northwest Pacific Ocean. The strain showed a close phylogenetic relationship with Limnobacter thiooxidans CS-K2T, sharing 99.9% 16S rRNA gene sequence identity. The complete whole-genome sequence of strain SAORIC-580T comprised 3.3 Mbp with a DNA G+C content of 52.5%. Comparative genomic analysis revealed average nucleotide identities between 79.4–85.7% and digital DNA-DNA hybridization values of 19.9–29.5% when compared to other Limnobacter genomes, indicating that the strain represents a novel species within the genus. Genomic analysis revealed unique adaptations to deep-sea conditions, including genes associated with phenol degradation, stress responses, cold adaptation, heavy metal resistance, signal transduction, and carbohydrate metabolism. The SAORIC-580T genome was found to be more abundant in the deep sea than at the surface in the trenches of the Northwest Pacific Ocean, suggesting adaptations to the deep-sea environment. Phenotypic characterization highlighted distinct differences from other Limnobacter species, including variations in growth conditions, enzyme activities, and phenol degradation capabilities. Chemotaxonomic markers of the strain included ubiquinone-10, major fatty acids such as C16:0, C16:1, and C18:1, and major polar lipids including phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. Based on the polyphasic taxonomic data, it is concluded that strain SAORIC-580T (= KACC 21440T = NBRC 114111T) represents a novel species, for which the name Limnobacter profundi sp. nov. is proposed.

Role of chitin synthases CHS1 and CHS2 in biosynthesis of the cyst wall of Cryptocaryon irritans

Cryptocaryon irritans, a protozoan parasite that infects marine fish, is characterized by a complex life cycle that includes a cyst-forming reproductive phase. However, the composition of the cyst wall and mechanism of its formation remain unclear. In this study, we identified chitin as a key component of the cyst wall using calcofluor white and wheat germ agglutinin, with Fourier-transform infrared spectroscopy confirming its β-form structure. Two chitin synthase genes, CHS1 and CHS2, were identified as being expressed throughout the life cycle and show close phylogenetic relationships with chitin synthase from ciliates. Incubation with specific anti-CHS1 and -CHS2 antibodies significantly reduced both the thickness and chitin content of the cyst wall, highlighting the critical role of these enzymes in chitin biosynthesis. Treatment with benzoylureas, which inhibit chitin synthesis, caused thinning of the cyst wall and downregulation of CHS gene expression, resulting in an 84 % reduction in the hatching rate after treatment with 0.01 mM CuSO4 compared with control tomonts. Western blot analysis demonstrated that recombinant CHS proteins are immunogenic, and tomonts from CHS-immunized grouper exhibited reduced size. These findings bridge a crucial knowledge gap in understanding of the C. irritans cyst wall and highlight promising targets for infection prevention and control strategies.

Canker and dieback of Alnus rubra is caused by Lonsdalea quercina.

Understanding the ecology of pathogens is important for disease management. Recently a devastating canker disease was found on red alder (Alnus rubra) planted as landscape trees. Bacteria were isolated from two groups of symptomatic trees located approximately 1 kilometer apart and one strain from each group was used to complete Koch's postulates. Results showed that these bacteria can not only cause disease on red alder but also on two other alder species. Unexpectedly, analyses of genome sequences of bacterial strains identified them as Lonsdalea quercina, a pathogenic species previously known to cause dieback of oak species, but not alder. Additionally, a core genome phylogeny clustered bacterial strains isolated from red alder within a subclade of L. quercina strains isolated from symptomatic oak trees. Consistent with the close phylogenetic relationship, there was no obvious evidence for divergence in genome composition of strains isolated from red alder and oak. Altogether, findings indicate that L. quercina is a potential threat to Alnus species.

Genomic analysis of Salmonella Heidelberg isolated from the Brazilian poultry farms.

The rapid expansion of broiler chicken production in Brazil has presented significant sanitation challenges within the poultry industry. Among these challenges, Salmonella enterica subsp. enterica serotype Heidelberg stands as a contributor to global salmonellosis outbreaks. This study analyzed 13 draft genomes of Salmonella Heidelberg isolated from the pre-slaughter broiler chickens farms in Brazil. By conducting in silico analysis of these genomes, the study investigated genome similarity based on single nucleotide polymorphisms (SNPs) and identified genes encoding resistance to antimicrobials, sanitizers, and virulence factors. Furthermore, mobile genetic elements (MGE) were identified to assess their potential role in propagating genes through horizontal gene transfer. A risk classification was also applied based on the resistomes. The genomes revealed a high prevalence of genes conferring resistance to aminoglycosides, fosfomycin, sulfonamides, tetracycline, and genes linked to quaternary ammonium resistance. The study also uncovered six Salmonella pathogenicity islands (SPI) and over 100 genes encoding virulence factors. The association of MGE with antibiotic-resistant genes sul2 and blaCMY-2 raised concerns about the potential transfer to other bacteria, posing a substantial risk for spreading resistance mechanisms according to established risk protocols. Additionally, SNP analysis indicated close phylogenetic relationships among some isolates, suggesting a common origin. This study enhances our understanding of Salmonella Heidelberg strains by identifying key risk factors for transmission and revealing the association between resistance genes and MGEs. This insight provides a foundation for developing and implementing effective control, monitoring, and treatment strategies in the poultry industry.

Phylogenomics and phylogeographic model testing using convolutional neural networks reveal a history of recent admixture in the Canarian Kleinia neriifolia.

Multiple-island endemics (MIE) are considered ideal natural subjects to study patterns of island colonization that involve recent population-level genetic processes. Kleinia neriifolia is a Canarian MIE widespread across the archipelago, which exhibits a close phylogenetic relationship with species in northwest Africa and at the other side of the Sahara Desert. Here, we used target sequencing with plastid skimming (Hyb-Seq), a dense population-level sampling of K. neriifolia, and representatives of its African-southern Arabian relatives to infer phylogenetic relationships and divergence times at the species and population levels. Using population genetic techniques and machine learning (convolutional neural networks [CNNs]), we reconstructed phylogeographic relationships and patterns of genetic admixture based on a multilocus SNP nuclear dataset. Phylogenomic analysis based on the nuclear dataset identifies the northwestern African Kleinia anteuphorbium as the sister species of K. neriifolia, with divergence starting in the early Pliocene. Divergence from its sister clade, comprising species from the Horn of Africa and southern Arabia, is dated to the arid Messinian period, lending support to the climatic vicariance origin of the Rand Flora. Phylogeographic model testing with CNNs supports an initial colonization of the central island of Tenerife followed by eastward and westward migration across the archipelago, which resulted in the observed east/west phylogeographic split. Subsequent population extinctions linked to aridification events, and recolonization from Tenerife, are proposed to explain the patterns of genetic admixture in the eastern Canary Islands. We demonstrate that CNNs based on SNPs can be used to discriminate among complex scenarios of island migration and colonization.

Endemic maintenance of human-related hepatitis E virus strains in synurbic wild boars, Barcelona Metropolitan Area, Spain

Hepatitis E virus (HEV), shared by humans, domestic animals, and wildlife, is an emerging global public health threat. Because wild boars are a major reservoir of HEV, the new zoonotic interfaces resulting from wild boar population increase and synurbization significantly contribute to increasing the risk of zoonotic transmission of HEV.This study characterizes HEV strains of synurbic wild boars and assesses their relationship with sympatric human and domestic swine HEV strains. We analyzed the faeces of 312 synurbic wild boars collected from 2016 to 2021 in the Barcelona Metropolitan Area (BMA), where there is a high density of wild boars, and found 7 HEV-positive samples among those collected between 2019 and 2020. The molecular analysis of these isolates, along with 6 additional wild boar HEV isolates from a previous study, allowed us to establish a close phylogenetic relationship between these HEV strains and human HEV isolates from sympatric blood donors and domestic pigs from Catalonia. HEV-positive wild boar samples belonged to piglet, juvenile and yearling individuals, but not adults, indicating the endemic maintenance of HEV in the wild boar population of the BMA by naïve young individuals. All wild boar HEV isolates in this study classified within HEV genotype 3.The results show, for the first time, a close molecular similarity between the HEV strains endemically maintained by the synurbic wild boars in the BMA and citizens from the same area and period. The data could also indicate that HEV infection presents a seasonal and interannual variability in wild boars of BMA. Further investigation is required to unveil the HEV transmission routes between synurbic wild boars and sympatric citizens. These findings can serve in other synurbic wildlife-human interfaces throughout the world.

Enhancing Variant Calling in Whole-Exome Sequencing Data Using Population-Matched Reference Genomes.

Whole-exome sequencing (WES) data are frequently used for cancer diagnosis and genome-wide association studies (GWAS), based on high-coverage read mapping, informative variant calling, and high-quality reference genomes. The center position of the currently used genome assembly, GRCh38, is now challenged by two newly published telomere-to-telomere (T2T) genomes, T2T-CHM13 and T2T-YAO, and it becomes urgent to have a comparative study to test population specificity using the three reference genomes based on real case WES data. Here we report our analysis along this line for 19 tumor samples collected from Chinese patients. The primary comparison of the exon regions among the three references reveals that the sequences in up to ∼ 1% target regions in T2T-YAO are widely diversified from GRCh38 and may lead to off-target in sequence capture. However, T2T-YAO still outperforms GRCh38 genomes by obtaining 7.41% more mapped reads. Due to more reliable read-mapping and closer phylogenetic relationship with the samples than GRCh38, T2T-YAO reduces half of variant calls of clinical significance which are mostly benign, while maintaining sensitivity in identifying pathogenic variants. T2T-YAO also outperforms T2T-CHM13 in reducing calls of Chinese-specific variants. Our findings highlight the critical need for employing population-specific reference genomes in genomic analysis to ensure accurate variant analysis and the significant benefits of tailoring these approaches to the unique genetic backgrounds of each ethnic group.

Molecular epidemiology of avian influenza viruses and avian coronaviruses in environmental samples from migratory bird inhabitants in Bangladesh.

Migratory birds are a natural reservoir for major respiratory viruses such as the avian influenza virus (AIV) and the avian coronavirus (AvCoV). Transmission of these viruses from migratory birds to domestic birds increases the prevalence of those diseases that cause severe economic and public health concerns in Bangladesh. The study focused on active surveillance of major respiratory viral pathogens in migratory birds, molecular identification of the viruses, and their phylogenetic origin. To conduct this study, 850 environmental samples (830 fecal samples, 10 soil samples, and 10 water samples) were collected during three consecutive winter seasons from three divisions (Dhaka, Sylhet, and Mymensingh) and pooled according to the year of collection and locations, resulting in a total of 184 tested samples. Using gene-specific primers and probes in TaqMan-and SYBR Green-based RT-qPCR assays, the samples were screened for AIV and AvCoV, respectively. Out of the 184 pooled samples, 37 were found to be positive for these respiratory pathogens. Furthermore, out of the 37 (20.11%) positive respiratory pathogens, 11.96% were AIV (n = 22) and 8.15% were AvCoV (n = 15). For the first time in Bangladesh, AIV H4N2, H4N6, and AvCoVs have been found in fecal samples from migratory birds through surveillance. Phylogenetic analyses of the HA and NA genes of AIV and the polymerase gene (Orf 1) of AvCoV revealed that these strains share a close phylogenetic relationship with the isolates from wild birds in Europe and Asia. The Bangladeshi strains with Eurasian ancestry might pose a significant threat to migratory birds flying through the Asian flyways. They might also be a potential source of virus introduction and spread to poultry raised on land. These findings emphasize the significance of ongoing AIV and AvCoV surveillance in migratory birds in Bangladesh.

Genome characterization of Shewanella algae in Hainan Province, China.

Shewanella algae is an emerging marine zoonotic pathogen. In this study, we first reported the Shewanella algae infections in patients and animals in Hainan Province, China. Currently, there is still relatively little known about the whole-genome characteristics of Shewanella algae in most tropical regions, including in southern China. Here, we sequenced the 62 Shewanella algae strains isolated from Hainan Province and combined with the whole genomes sequences of 144 Shewanella algae genomes from public databases to analyze genomic features. Phylogenetic analysis revealed that Shewanella algae is widely distributed in the marine environments of both temperate and tropical countries, exhibiting close phylogenetic relationships with genomes isolated from patients, animals, and plants. Thereby confirming that exposure to marine environments is a risk factor for Shewanella algae infections. Average nucleotide identity analysis indicated that the clonally identical genomes could be isolated from patients with different sample types at different times. Pan-genome analysis identified a total of 21,909 genes, including 1,563 core genes, 8,292 strain-specific genes, and 12,054 accessory genes. Multiple putative virulence-associated genes were identified, encompassing 14 categories and 16 subcategories, with 171 distinct virulence factors. Three different plasmid replicon types were detected in 33 genomes. Eleven classes of antibiotic resistance genes and 352 integrons were identified. Antimicrobial susceptibility testing revealed a high resistance rate to imipenem and colistin among the strains studied, with 5 strains exhibiting multidrug resistance. However, they were all sensitive to amikacin, minocycline, and tigecycline. Our findings clarify the genomic characteristics and population structure of Shewanella algae in Hainan Province. The results offer insights into the genetic basis of pathogenicity in Shewanella algae and enhance our understanding of its global phylogeography.

The genome of a giant clam zooxanthella (Cladocopium infistulum) offers few clues to adaptation as an extracellular symbiont with high thermotolerance

BackgroundCladocopium infistulum (Symbiodiniaceae) is a dinoflagellate specialized to live in symbiosis with western Pacific giant clams (Tridacnidae). Unlike coral-associated symbionts, which reside within the host cells, C. infistulum inhabits the extracellular spaces of the clam’s digestive diverticula. It is phylogenetically basal to a large species complex of stress-tolerant Cladocopium, many of which are associated with important reef-building corals in the genus Porites. This close phylogenetic relationship may explain why C. infistulum exhibits high thermotolerance relative to other tridacnid symbionts. Moreover, past analyses of microsatellite loci indicated that Cladocopium underwent whole-genome duplication prior to the adaptive radiations that led to its present diversity.ResultsA draft genome assembly of C. infistulum was produced using long- and short-read sequences to explore the genomic basis for adaptations underlying thermotolerance and extracellular symbiosis among dinoflagellates and to look for evidence of genome duplication. Comparison to three other Cladocopium genomes revealed no obvious over-representation of gene groups or families whose functions would be important for maintaining C. infistulum’s unique physiological and ecological properties. Preliminary analyses support the existence of partial or whole-genome duplication among Cladocopium, but additional high-quality genomes are required to substantiate these findings.ConclusionAlthough this investigation of Cladocopium infistulum revealed no patterns diagnostic of heat tolerance or extracellular symbiosis in terms of overrepresentation of gene functions or genes under selection, it provided a valuable genomic resource for comparative analyses. It also indicates that ecological divergence among Cladocopium species, and potentially among other dinoflagellates, is partially governed by mechanisms other than gene content. Thus, additional high-quality, multiomic data are needed to explore the molecular basis of key phenotypes among symbiotic microalgae.

A new species of Dainty Frog (Anura: Pyxicephalidae: Cacosternum) and the first endemic anuran to the Cederberg region of South Africa

Within the genus Cacosternum, the species C. capense and C. namaquense form a clade defined by their robust morphology, large size, and distinct dorsolateral glands. In this study, we describe a third member of the group endemic to the Cederberg region of South Africa. Specimens from this area have historically been assigned to C. karooicum, but here we show that this population is genetically unrelated. Using a combination of molecular, morphological, and acoustic data, we provide evidence to show that the Cederberg population of C. karooicum represents an undescribed species within the C. capense group. The new species can be diagnosed from its closest phylogenetic relatives, C. capense and C. namaquense, by the absence of a dark streak running downwards from the pupil into the iris, the presence of large rictal glands that form an uninterrupted arc with the angle of the jaw, heart-shaped palmar tubercles, and an advertisement call with a dominant frequency below 2 kHz. We further discuss their natural history, distribution, and conservation.

Complete mitochondrial genome and phylogenetic analysis of Dollfustrema vaneyi (Trematoda: Bucephalidae)

BackgroundThe Bucephalidae is a large family of digenean trematodes but most previous analyses of its phylogenetic position have relied on a single mitochondrial gene or morphological features. Mitochondrial genomes (mitogenomes) remain unavailable for the entire family. To address this, we sequenced the complete mitogenome of Dollfustrema vaneyi and analyzed the phylogenetic relationships with other trematodes.ResultsThe circular genome of Dollfustrema vaneyi spanned 14,959 bp and contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a major non-coding region. We used concatenated amino acid and nucleotide sequences of all 36 genes for phylogenetic analyses, conducted using MrBayes, IQ-TREE and PhyloBayes. We identified pronounced topological instability across different analyses. The addition of recently sequenced two mitogenomes for the Aspidogastrea subclass along with the use of a site-heterogeneous model stabilized the topology, particularly the positions of Azygiidae and Bucephalidae. The stabilized results indicated that Azygiidae was the closest lineage to Bucephalidae in the available dataset, and together, they clustered at the base of the Plagiorchiida.ConclusionsOur study provides the first comprehensive description and annotation of the mitochondrial genome for the Bucephalidae family. The results indicate a close phylogenetic relationship between Azygiidae and Bucephalidae, and reveal their basal placement within the order Plagiorchiida. Furthermore, the inclusion of Aspidogastrea mitogenomes and the site-heterogeneous model significantly improved the topological stability. These data will provide key molecular resources for future taxonomic and phylogenetic studies of the family Bucephalidae.

Chloroplast genomes of Simarouba Aubl., molecular evolution and comparative analyses within Sapindales

Simarouba, a neotropical genus in the family Simaroubaceae, currently lacks comprehensive genomic data in existing databases. This study aims to fill this gap by providing genomic resources for three Simarouba species, S. amara, S. versicolor, and S. glauca. It also aims to perform comparative molecular evolutionary analyses in relation to other species within the order Sapindales. The analysis of these three Simarouba species revealed the presence of the typical quadripartite structure expected in plastomes. However, some pseudogenization events were identified in the psbC, infA, rpl22, and ycf1 genes. In particular, the CDS of the psbC gene in S. amara was reduced from 1422 bp to 584 bp due to a premature stop codon. Nucleotide diversity data pointed to gene and intergenic regions as promising candidates for species and family discrimination within the group, specifically matK, ycf1, ndhF, rpl32, petA-psbJ, and trnS-trnG. Selection signal analyses showed strong evidence for positive selection on the rpl23 gene. Phylogenetic analyses indicated that S. versicolor and S. glauca have a closer phylogenetic relationship than S. amara. We provide chloroplast genomes of three Simaruba species and use them to elucidate plastome evolution, highlight the presence of pseudogenization, and identify potential DNA barcode regions.

Constitutive BoHAK5 expression and early robust induction of salicylic acid biosynthesis genes as candidates to explain the tolerance of broccoli to limiting potassium

Potassium (K+) is vital for optimum plant growth and crop yield, and it is an important component of fertilizers. However, our knowledge of the physiological and molecular response to limiting K+ conditions is incomplete. Despite their close phylogenetic relationship, we observed that broccoli is more tolerant to limiting K+ conditions than Arabidopsis. For this reason, we performed a detailed physiological and transcriptomic analysis to compare and contrast their response to this abiotic stress. Our results show that the K+ content of broccoli roots decreases at a slower rate than Arabidopsis. Both species exhibited characteristic responses observed in other plants, such as those related to oxidative stress, hypoxia and jasmonic and abscisic acid signaling. However, we observed notable differences in their responses, especially in the expression of BoHAK5 and the early and strong induction of isoforms of salicylic acid (SA) biosynthesis genes in broccoli, which was reflected in the increased SA accumulation in broccoli leaves. We also observed alterations in the gene expression patterns of enzymes and in the levels of intermediates involved in the biosynthesis of glucosinolates, which are important molecules contributing to the added nutritional value of broccoli. Lastly, we provide evidence for concomitant alterations in the expression patterns of genes encoding transporters of several other ions, such as Fe2+, PO43− and NO3−. Our data provide insight into the possible mechanisms of broccoli’s tolerance to limiting K+ conditions and identify specific targets for the development of crop plants with reduced fertilization requirements.

The immune function of TLR4-1 gene in Octopus sinensis revealed by RNAi and RNA-seq

Toll-like receptors (TLRs) are a class of conserved pattern recognition receptors (PRRs) that are crucial for initiating the innate immune response and aiding in the clearance of pathogenic organisms. Many studies have identified TLR4 as a distinctive member of the TLR family, capable of activating both the Myeloid differentiation factor 88-dependent signaling pathway (MyD88-dependent) and the TIR-domain-containing adaptor inducing IFN-β dependent signaling pathway (TRIF-dependent). Nevertheless, the role of TLR4 in Cephalopoda is still largely unexplored. To elucidate the immune function of the OsTLR4-1 gene in Octopus sinensis, the OsTLR4-1 gene was first validated and analyzed in this study. The cDNA comprises a 2475 bp ORF region, encoding 824 amino acids. Evolutionary tree analysis indicated a high homology and a close phylogenetic relationship between the Octopus sinensis and other mollusks. RNA interference (RNAi) experiments demonstrated that the expression level of OsTLR4-1 gene and its protein in the lymphocytes of the RNAi group treated with OsTLR4-1 dsRNA was extremely significantly lower than that of the blank control group and negative control group (P < 0.01), and the expression of downstream genes of OsTLR4-1, including ligand MyD88, IRAK4, TRAF6, MKK6, Hsp90, COX2, TRAF3, and RIP1, were significantly down-regulated compared to the blank and negative control group (P < 0.01). Additionally, OsTLR4-1 expression in lymphocytes was highly significantly up-regulated in the LPS-treated group compared to the blank control group (P < 0.01), while its expression was extremely significantly lower in the LPS-treated group after OsTLR4-1 interference than in the blank control group (P < 0.01). The expression of its downstream effector genes Big Defensin (Big-Def) and histone H2A.V (H2A.V) was highly significantly up-regulated in lymphocytes in the LPS-treated group compared to the blank control group (P < 0.01), while their expression in the LPS-treated group after OsTLR4-1 interference was extremely significantly lower than that in the blank control group (P < 0.01). Through comparative transcriptome analysis of the RNAi group and the blank control group, it was found that differentially expressed genes were enriched in the Toll-like receptor signaling pathway, PI3K-AKT signaling pathway, P53 signaling pathway, MAPK signaling pathway, and NF-κB signaling pathway. qRT-PCR results of key genes in these pathways revealed a decrease in all genes except IκB and Jun2 genes. This study enhances our understanding of the immune function of the TLR gene family in O. sinensis and provides a foundation for further research into innate immune signaling pathways in cephalopods.

The complete mitochondrial genome and phylogenetic analysis of Rattus tanezumi (Niethammer, 1975), captured from North China

Rattus tanezumi (Niethammer, 1975) is one of the commensal rodent species in South China. With the development of transportation and climate change, R. tanezumi has gradually migrated north and become the dominant rat species for the past few years. In this study, we assembled a complete mitochondrial genome of R. tanezumi, captured from North China. The mitogenome contains 16,307 nucleotide pairs, including 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes, as well as one non-coding control region. Based on whole mitogenome phylogenetical analysis showed that R. tanezumi captured from North China had a close phylogenetic relationship with that from Japan and South Korea. These findings are valuable for further studies on the evolution, genetic diversity, and taxonomy of Asian commensal rodent.

New evidence supports the prophage origin of RcGTA.

Gene transfer agents (GTAs) are phage-like entities that package and transfer random host genome fragments between prokaryotes. RcGTA, produced by Rhodobacter capsulatus, is hypothesized to originate from a prophage ancestor. Most of the evidence supporting this hypothesis came from the finding of RcGTA-like genes in phages. More than 75% of the RcGTA genes have a phage homolog. However, only a few RcGTA homologs have been identified in a (pro)phage genome, leaving the hypothesis that GTAs evolved from prophages through gene loss with only weak evidence. We herein report the discovery of an inducible prophage (vB_MseS-P1) from a Mesorhizobium sediminum strain that contains the largest number (12) of RcGTA homologs found in a phage genome to date. We also identified three putative prophages and two prophage remnants harboring 12-14 RcGTA homologs in a Methylobacterium nodulans strain. The protein remote homology detection also revealed more RcGTA homologs from other phages than we previously thought. Moreover, the head-tail gene architecture of these newly discovered prophage-related elements closely resembles that of RcGTA. Furthermore, vB_MseS-P1 virions have structural proteins similar to RcGTA particles. Close phylogenetic relationships between certain prophage genes and RcGTA-like genes in Alphaproteobacteria further support the shared ancestry between RcGTA and prophages. Our findings provide new relatively direct evidence of the origin of RcGTA from a prophage progenitor.IMPORTANCEGTAs are important genetic elements in certain groups of bacteria and contribute to the genetic diversification, evolution, and ecological adaptation of bacteria. RcGTA, a common type of GTA, is known to package and transfer random fragments of the bacterial genome to recipient cells. However, the origin of RcGTA is still elusive. It has been hypothesized that RcGTA evolved from a prophage ancestor through gene loss. However, the few RcGTA homologs identified in a (pro)phage genome leave the hypothesis lacking direct evidence. This study uncovers the presence of a large number of RcGTA homologs in an inducible prophage and several putative prophages. The similar head-tail gene architecture and structural protein compositions of these newly discovered prophage-related elements and RcGTA further demonstrate an unprecedentedly observed close evolutionary relationship between prophages and RcGTA. Together, our findings provide more direct evidence supporting the origin of RcGTA from prophage.

Sampling of Culicoides with nontraditional methods provides unusual species composition and new records for southern Spain

BackgroundCulicoides midges have been well-studied in Spain, particularly over the last 20 years, mainly because of their role as vectors of arboviral diseases that affect livestock. Most studies on Culicoides are conducted using suction light traps in farmed environments, but studies employing alternative trapping techniques or focusing on natural habitats are scarce.MethodsIn the present study, we analyze Culicoides captured in 2023 at 476 sites in western Andalusia (southern Spain) using carbon dioxide-baited Biogents (BG)-sentinel traps across different ecosystems.ResultsWe collected 3,084 Culicoides midges (3060 females and 24 males) belonging to 23 species, including the new species Culicoides grandifovea sp. nov. and the first record of Culicoides pseudolangeroni for Europe. Both species were described with morphological and molecular methods and detailed data on spatial distribution was also recorded. The new species showed close phylogenetic relations with sequences from an unidentified Culicoides from Morocco (92.6% similarity) and with Culicoides kurensis. Culicoides imicola was the most abundant species (17.4%), followed by Culicoides grandifovea sp. nov. (14.6%) and Culicoides kurensis (11.9%). Interestingly, Culicoides montanus was the only species of the obsoletus and pulicaris species complexes captured, representing the first record of this species in southern Spain. A total of 53 valid Culicoides species have been reported in the area, with 48 already reported in literature records and 5 more added in the present study. Information on the flight period for the most common Culicoides species is also provided.ConclusionsTo the best of our knowledge, our study represents the most comprehensive effort ever done on nonfarmland habitats using carbon-dioxide baited suction traps for collecting Culicoides. Our data suggests that using carbon dioxide traps offers a completely different perspective on Culicoides communities compared with routinely used light traps, including the discovery of previously unrecorded species.Graphical

In silico and experimental characterization of a new polyextremophilic subtilisin-like protease from Microbacterium metallidurans and its application as a laundry detergent additive.

Considering the current growing interest in new and improved enzymes for use in a variety of applications, the present study aimed to characterize a novel detergent-stable serine alkaline protease from the extremophilic actinobacterium Microbacterium metallidurans TL13 (MmSP) using a combined in silico and experimental approach. The MmSP showed a close phylogenetic relationship with high molecular weight S8 peptidases of Microbacterium species. Moreover, its physical and chemical parameters computed using Expasy's ProtParam tool revealed that MmSP is hydrophilic, halophilic and thermo-alkali stable. 3D structure modelling and functional prediction of TL13 serine protease resulted in the detection of five characteristic domains: [catalytic subtilase domain, fibronectin (Fn) type-III domain, peptidase inhibitor I9, protease-associated (PA) domain and bacterial Ig-like domain (group 3)], as well as the three amino acid residues [aspartate (D182), histidine (H272) and serine (S604)] in the catalytic subtilase domain. The extremophilic strain TL13 was tested for protease production using agricultural wastes/by-products as carbon substrates. Maximum enzyme activity (390U/gds) was obtained at 8th day fermentation on potato peel medium. Extracellular extract was concentrated and partially purified using ammonium sulfate precipitation methodology (1.58 folds purification fold). The optimal pH, temperature and salinity of MmSP were 9, 60°C and 1M NaCl, respectively. The MmSP protease showed broad pH stability, thermal stability, salt tolerance and detergent compatibility. In order to achieve the maximum stain removal efficacy by the TL 13 serine protease, the operation conditions were optimized using a Box-Behnken Design (BBD) with four variables, namely, time (15-75min), temperature (30-60°C), MmSP enzyme concentration (5-10U/mL) and pH (7-11). The maximum stain removal yield (95 ± 4%) obtained under the optimal enzymatic operation conditions (treatment with 7.5U/mL of MmSP during 30min at 32°C and pH9) was in good agreement with the value predicted by the regression model (98 ± %), which prove the validity of the fitted model. In conclusion, MmSP appears to be a good candidate for industrial applications, particularly in laundry detergent formulations, due to its high hydrophilicity, alkali-halo-stability, detergent compatibility and stain removal efficiency.

Molecular characterization and identification of African Swine Fever virus isolates from emerging cases of infection in Central Luzon, Philippines

A study that evaluated the molecular characteristics to confirm the identity of the African Swine Fever (ASF) pathogen from emerging cases of infection in Central Luzon, Philippines was initiated. The study involved the collection of 125 blood samples from pigs during the four-month duration of the study when clinical signs of ASF was prevalent in the four out of seven provinces of Central Luzon, Philippines. This work included the amplification of the p72 gene in DNA samples through PCR and confirmation of the identity of the ASF pathogen through DNA sequencing. The DNA sequences of the p72 gene of the ASF pathogen are made up of 412 nucleotides and used as a basis in identifying the Philippine ASF pathogens as ASF virus PH/CL 4/2022, ASF virus PH/CL 3/2022, ASF virus PH/CL 2/2022 and ASF virus PH/CL 1/2022. Philippines ASF virus isolates showed 100% sequence similarity with sequences reported from Malaysia ASF isolate MVZT218/2021, Vietnam ASF isolate D/ASFV/VN/DN-VC/2019, and India ASF isolate ASF/MZ/IND/3 based on BLASTN homology search in the NCBI database. The results of the study revealed close phylogenetic relationships among ASF isolates from Vietnam, Malaysia, India and the Philippines.