Comparative Sequence Analysis Research Articles

Variability of <i>Puccinia komarovii</i> var. <i>komarovii</i> isolates from <i>Impatiens parviflora</i> DC. and its occurrence in the Drawa and Wigry National Parks in Poland

Small balsam (<i>Impatiens parviflora</i> DC.), an invasive plant native to central Asia, is currently one of the most extensive invasive plants in Central Europe, including Poland. Its expansion may affect populations of native species, such as touch-me-not balsam (Impatiens noli-tangere). It is particularly dangerous in legally protected areas, where the possibilities of eradicating it are very limited. There is some hope for stopping its expansion by using pathogens as a biological weapon. In this case, useful may be host-specific rust fungus <i>Puccinia komarovii</i> var. <i>komarovii</i>. Our three-year observations of I. parviflora infection severity by <i>P. komarovii</i> var. <i>komarovii</i> carried out in Poland in Drawa National Park (DNP) and Wigry National Park (WNP) showed the higher incidence of rust on leaves in DNP, where the median blade infestation for two years was 51% of leaf coverage. In contrast, for three years in WNP, the median blade infestation amounted to only 25%. The most severe symptoms occurred during the summer months. However the percentage of leaf coverage by pathogen exhibited fluctuations depending on the year, implying an influence of environmental conditions that stabilized the infection each season. Molecular analyses, in addition to confirming the infection of the tested plants by <i>P. komarovii</i> var.<i> komarovii</i>, enabled the characterization of its population based on comparative analysis of ITS and LSU sequences. Clear differences were found between the studied isolates and <i>P. komarovii</i> from <i>I. glanduliferae</i> and <i>I. brachycentra</i>. There was no diversity among our isolates, but they differed from the sequences of Polish isolates originating from <i>I. parviflora</i> available in GenBank, which indicates some diversity of rust in a relatively small area. It probably results from sexual reproduction and may enable rapid adaptation of the pathogen to the region or host plant, or it may indicate certain “entry points” to Poland, where the isolates arrived from their native place of occurrence.

Frankia nepalensis sp. nov., a non-infective non-nitrogen-fixing isolate from root nodules of Coriaria nepalensis Wall

Strains CN4T, CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus. The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N2-fixing vesicles and do not fix nitrogen in vitro. The peptidoglycan of strain CN4T contains meso-diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H4) and MK-9(H6) are the predominant menaquinones (>15 %), and iso-C16 : 0 and C17 : 1ω8c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4T is most closely related to Frankia saprophytica CN 3T. An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3T , Frankia asymbiotica M16386T and Frankia inefficax EuI1cT with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4T, CN6, CN7 and CNm7 had dDDH (98.6–99.6 %) and ANI values that grouped them as representing a single species. CN4T has a 10.76 Mb genome. CN4T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4T (=DSM 114740T = LMG 32595T) to a novel species, with CN4T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.

Strand’s mouse <i>Sicista strandi </i> (Rodentia, Dipodoidea) on the southwestern periphery of its range: Genetic and environmental aspects

The position of the Sicista strandi population from the southwestern periphery of its range (the mountainous Caucasus, the vicinity of the Ekiptsoko cordon, Zol’sky district of Kabardino-Balkaria, the adjacent territory with terra typica of the species) in the species structure was investigated on the basis of a comparative analysis of the nucleotide sequences of a fragment of the gene of nu-clear DNA in this population and in representatives of the species from a number of localities from the Russian Plain for the first time. It was established that according to this molecular feature, the studied Caucasian population belongs to the southern grouping of the species, covering, in addition to the Caucasus, samples from the basins of the Lower (Rostov region) and Middle Don (Lugansk region) and presumably from the Lower Volga region, and differs from samples from the northern parts of the range in the Central Chernozem Zone (Kursk and Belgorod region). In the S. strandi population from the southwestern periphery of the range, a number of environmental and biological characteristics (biotopic confinement, population composition, abundance, diurnal, seasonal activity, reproduction, and nutrition) considered in a comparative aspect in connection with the genetic differentiation of the species were studied for the first time.

Cloning, Identification, and Functional Analysis of the Foxl2 Gene in Procambarus clarkii.

Procambarus clarkii is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 (Foxl2) gene has been found to be involved in the reproductive development of many crustaceans. To understand the role of the Foxl2 gene in the gonad development of P. clarkii, we designed CDS-specific primers for the P. clarkii Foxl2 (PcFoxl2) gene and cloned its CDS sequence using RT-PCR. The nucleotide and protein sequence information was then analyzed through bioinformatics analysis. The expression and subcellular localization of PcFoxl2 in various tissues were detected using qRT-PCR and in situ hybridization. The effects of PcFoxl2 knockdown on gonad development were investigated using RNA interference. The results showed that the CDS length of the PcFoxl2 gene was 1614 bp and encoded 537 amino acids. Protein sequence comparison and phylogenetic analysis showed that PcFoxl2 was the closest relative to Crayfish. qRT-PCR analysis indicated that the expression level of PcFoxl2 in the testis was significantly higher (>40 fold) than that in the ovary (p < 0.01). The in situ hybridization results showed that PcFoxl2 was expressed in both the cytoplasm and the nucleus of egg cells, and that the expression was strongest in egg cells at the early stage of yolk synthesis, while weak in the secondary oocytes. The positive signal was strongest in the spermatocyte nucleolus, while only a trace signal was observed in the cytoplasm. After interfering with the PcFoxl2 gene using dsRNA, the expression of PcFoxl2 in the RNA interference group was significantly lower than that in the control group, and this interference effect lasted for one week. Moreover, the gonad index of the experimental group was significantly lower than that of the control group (p < 0.05) after 10 days of P. clarkii cultivation following PcFoxl2 knockdown. The expression levels of the nanos and S3a genes, which are related to gonad development, decreased significantly after PcFoxl2 gene interference. The results suggest that the Foxl2 gene is involved in the growth and development of gonads, particularly in the development of testis, and is related to the early development of oocytes. This study provides a theoretical basis for the artificial breeding of P. clarkii.

High genomic plasticity and unique features of Xanthomonas translucens pv. graminis revealed through comparative analysis of complete genome sequences

BackgroundXanthomonas translucens pv. graminis (Xtg) is a major bacterial pathogen of economically important forage grasses, causing severe yield losses. So far, genomic resources for this pathovar consisted mostly of draft genome sequences, and only one complete genome sequence was available, preventing comprehensive comparative genomic analyses. Such comparative analyses are essential in understanding the mechanisms involved in the virulence of pathogens and to identify virulence factors involved in pathogenicity.ResultsIn this study, we produced high-quality, complete genome sequences of four strains of Xtg, complementing the recently obtained complete genome sequence of the Xtg pathotype strain. These genomic resources allowed for a comprehensive comparative analysis, which revealed a high genomic plasticity with many chromosomal rearrangements, although the strains were highly related. A high number of transposases were exclusively found in Xtg and corresponded to 413 to 457 insertion/excision transposable elements per strain. These mobile genetic elements are likely to be involved in the observed genomic plasticity and may play an important role in the adaptation of Xtg. The pathovar was found to lack a type IV secretion system, and it possessed the smallest set of type III effectors in the species. However, three XopE and XopX family effectors were found, while in the other pathovars of the species two or less were present. Additional genes that were specific to the pathovar were identified, including a unique set of minor pilins of the type IV pilus, 17 TonB-dependent receptors (TBDRs), and 11 plant cell wall degradative enzymes.ConclusionThese results suggest a high adaptability of Xtg, conferred by the abundance of mobile genetic elements, which could play a crucial role in pathogen adaptation. The large amount of such elements in Xtg compared to other pathovars of the species could, at least partially, explain its high virulence and broad host range. Conserved features that were specific to Xtg were identified, and further investigation will help to determine genes that are essential to pathogenicity and host adaptation of Xtg.

Molecular characterization and in-depth genomic analysis to unravel the pathogenic features of an environmental isolate Enterobacter sp. S-33.

Enterobacter species represent widely distributed opportunistic pathogens, commonly associated with plants and humans. In the present study, we performed a detailed molecular characterization as well as genomic study of a type VI secretion system (T6SS) bacterium belonging to member of the family Enterobacteriaceae and named Enterobacter sp. S-33. The comparative sequence analysis of the 16S rRNA gene showed that the strain was closely related to other Enterobacter species. The complete genome of the strain with a genome size of 4.6 Mbp and GC-content of 55.63% was obtained through high-quality sequencing. The genomic analysis with online tools unravelled the various genes belonging to the bacterial secretion system, antibiotic resistance, virulence, efflux pumps, etc. The isolate showed the motility behavior that contributes to Enterobacter persistence in a stressed environment and further supports infections. PCR amplification and further sequencing confirmed the presence of drug-efflux genes acrA, acrB, and outer membrane genes, viz. OmpA, OmpC, and OmpF. The cell surface hydrophobicity and co-aggregation assay against different bacterial strains illustrated its putative pathogenic nature. Genome mining identified various biosynthetic gene clusters (BGCs) corresponding to non-ribosomal proteins (NRPS), siderophore, and arylpolyene production. Briefly, genome sequencing and detailed characterization of environmental Enterobacter isolate will assist in understanding the epidemiology of Enterobacter species, and the further prevention and treatment of infectious diseases caused by these broad-host range species.

WITHDRAWN: Molecular identification of myxozoans and helminths in the black goby Gobius niger L. (Gobiiformes: Gobiidae) from the Finnish Archipelago, Baltic sea

Black gobies (Gobius niger) from the Finnish Archipelago, Baltic Sea, were screened for helminth and myxozoan infections in summer 2020. Helminths were identified morphologically and/or molecularly, myxozoan DNA was only detected during molecular screening. Altogether 30 novel sequences were generated and analysed using maximum likelihood estimation. Morphological and phylogenetic analyses based on SSU rDNA (myxozoans) and mitochondrial genes (helminths) revealed the presence of 10 species: two species of Myxozoa (a putative new species of Myxobolus and Tetracapsuloides bryosalmonae), and eight helminth species belonging to the Digenea (Diplostomum mergi Lineage 3), Cestoda (Bothriocephalus scorpii), Nematoda (Contracaecum rudolphii A, Cucullanus sp. and Hysterothylacium aduncum), and Acanthocephala (a putative new species of Corynosoma, Corynosoma semerme and Neoechinorhynchus sp.). Phylogenetic and comparative sequence analyses revealed that the putative new acanthocephalan species is closely related to C. neostrumosum in the Caspian Sea. Myxobolus sp. was resolved as closely related but distinct from a species described from freshwater fish in Lake Balaton, Hungary. In addition to these two putative new species, the black goby represents a new host record for four parasite species (Tetracapsuloides bryosalmonae, Diplostomum mergi Lineage 3, Contracaecum rudolphii A and Corynosoma semerme). The Finnish Archipelago is a novel locality record for four species (Myxobolus sp., Corynosoma sp., Diplostomum mergi Lineage 3 and Bothriocephalus scorpii).

Identification of stem rot of stock (Matthiola incana) caused by Sclerotinia sclerotiorum in China

The ornamental plant stock flower (Matthiola incana (L.) R. Br.) has been widely cultivated in China. In 2021, stem rot on M. incana, which caused plant death, was first observed in Shanghai, China. Field surveys indicated a disease incidence of 15%–20%. Following pathogen isolation, identification was performed by combining morphological observation of colonies, sclerotia and apothecia, with molecular methods, including DNA sequencing and phylogenetic analysis. Morphological characteristics of the isolates, including white cottony mycelium, blackish globular to irregularly shaped sclerotia and apothecia formation, were consistent with Sclerotinia sclerotiorum. Results of sequence comparison and phylogenetic analysis based on the internal transcribed spacer (ITS) sequences supported morphological data. Pathogenicity assays were conducted successfully according to Koch's postulates. Considering the results of the morphological and molecular identification, along with the pathogenicity assessment, S. sclerotiorum was confirmed as the causal agent of stem rot of stock flower in Shanghai, China. A combination of cultural practices, rotation of resistant ornamentals, and biological and chemical treatments is recommended to control stem rot of stock.

First specific detection and validation of tomato wilt caused by Fusarium brachygibbosum using a PCR assay.

Tomato wilt is a widespread soilborne disease of tomato that has caused significant yield losses in many tomato growing regions of the world. Previously, it was reported that tomato wilt can be caused by many pathogens, such as Fusarium oxysporum, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Fusarium acuminatum, and Plectosphaerella cucumerina. In addition, we have already reported that Fusarium brachygibbosum caused symptomatic disease of tomato wilt for the first time in China. The symptoms of tomato wilt caused by these pathogens are similar, making it difficult to distinguish them in the field. However, F. brachygibbosum specific identification method has not been reported. Therefore, it is of great importance to develop a rapid and reliable diagnostic method for Fusarium brachygibbosum to establish a more effective plan to control the disease. In this study, we designed F. brachygibbosum-specific forward primers and reverse primers with a fragment size of 283bp located in the gene encoding carbamoyl phosphate synthase arginine-specific large chain by whole genome sequence comparison analysis of the genomes of eight Fusarium spp.. We then tested different dNTP, Mg2+ concentrations, and annealing temperatures to determine the optimal parameters for the PCR system. We evaluated the specificity, sensitivity and stability of the PCR system based on the optimized reaction system and conditions. The PCR system can specifically identify the target pathogens from different fungal pathogens, and the lower detection limit of the target pathogens is at concentrations of 10 pg/uL. In addition, we can accurately identify F. brachygibbosum in tomato samples using the optimized PCR method. These results prove that the PCR method developed in this study can accurately identify and diagnose F. brachygibbosum.

Phylodynamic characteristics of the LMP-1 gene of the Epstein–Barr virus isolated in the Nizhny Novgorod region

Introduction. Epstein–Barr virus (EBV) is one of the most common herpesviruses and has a pronounced genetic polymorphism. The study of the phylodynamic characteristics of the virus is an important aspect of the study of evolutionary changes in the LMP-1 gene and their consequences.&#x0D; The aim of the work was a philodynamic analysis of EBV isolates from Nizhny Novgorod region based on the C-terminal fragment of the LMP-1 gene.&#x0D; Materials and methods. The study included 158 EBV isolates obtained from blood leukocytes and saliva of children aged 1–17 years with a diagnosis of infectious mononucleosis caused by EBV (n = 68) and apparently healthy children of comparable sex and age (n = 29). LMP-1 genovariants were obtained using the Sanger sequencing method. Comparative analysis of amino acid sequences was performed using the MEGA X program. Philodynamic analysis of the obtained nucleotide sequences and isolates deposited in GenBank was carried out using the BEAST v. 1.10.4 software package. Recombination analysis was performed using the Simplot program.&#x0D; Results. 158 nucleotide sequences of the C-terminal fragment of the LMP-1 gene from Nizhny Novgorod region EBV isolates were obtained and deposited in the GenBank database. The circulation time of the nearest common ancestor for the modified B95-8 genovariants with G212S + E328Q + S366T and NC mutations with the D250N substitution has been established dating back to 1994 and 1923. The rate of evolution of these genovariants was the highest and amounted to 1.298 × 10–4 and 7.868 × 10–4 nucleotide substitutions/site/year. Recombinations were detected in the Nizhny Novgorod region sequences Med-, B95-8, China 1 with mutations G212S, G212S, E214Q, respectively.&#x0D; Conclusion. For the first time, a phylodynamic characterization of Nizhny Novgorod region isolates and LMP-1 EBV genovariants isolated in various regions of the world is given. The data obtained expand the existing understanding of the circulation of EBV LMP-1 genovariants in the territory of the European part of Russia.

Low prevalence of Babesia hongkongensis infection in community and privately-owned cats in Hong Kong

Domestic cats are susceptible to infection with at least 11 species of Babesia. In Hong Kong, where dogs are commonly infected with B. gibsoni, a single infection in a cat by a novel species, B. hongkongensis, was reported previously. The aim of this study was to investigate the frequency of Babesia spp. detection in cats in Hong Kong. Residual blood-derived DNA from healthy free-roaming community cats (n = 239), and privately-owned cats with and without anaemia undergoing diagnostic investigations (n = 125) was tested for Babesia spp. DNA using a pan-Babesia PCR targeting mitochondrial Cytochrome B, and a B. hongkongensis specific PCR targeting 18S rRNA. Positive samples were confirmed by sequencing and comparative sequence analysis against the GenBank nucleotide database. Babesia hongkongensis was detected in 4/239 (1.7 %) community cats, and 0/125 (0.0 %) privately-owned cats. Babesia gibsoni was detected in 0/239 community cats and 1/125 (0.8 %) privately-owned cats. Cats infected with B. hongkongensis were clinically healthy at the time of sampling. The B. gibsoni-infected cat was anaemic and thrombocytopenic. Cats in Hong Kong can be infected with B. hongkongensis and B. gibsoni, albeit at low frequency. The tick vector for B. hongkongensis is yet to be identified.

Phylogenetics Study to Compare Chloroplast Genomes in Four Magnoliaceae Species.

Magnoliaceae, a family of perennial woody plants, contains several endangered species whose taxonomic status remains ambiguous. The study of chloroplast genome information can help in the protection of Magnoliaceae plants and confirmation of their phylogenetic relationships. In this study, the chloroplast genomes were sequenced, assembled, and annotated in Woonyoungia septentrionalis and three Michelia species (Michelia champaca, Michelia figo, and Michelia macclurei). Comparative analyses of genomic characteristics, repetitive sequences, and sequence differences were performed among the four Magnoliaceae plants, and phylogenetic relationships were constructed with twenty different magnolia species. The length of the chloroplast genomes varied among the four studied species ranging from 159,838 bp (Woonyoungia septentrionalis) to 160,127 bp (Michelia macclurei). Four distinct hotspot regions were identified based on nucleotide polymorphism analysis. They were petA-psbJ, psbJ-psbE, ndhD-ndhE, and rps15-ycf1. These gene fragments may be developed and utilized as new molecular marker primers. By using Liriodendron tulipifera and Liriodendron chinense as outgroups reference, a phylogenetic tree of the four Magnoliaceae species and eighteen other Magnoliaceae species was constructed with the method of Shared Coding Sequences (CDS). Results showed that the endangered species, W. septentrionalis, is relatively genetically distinct from the other three species, indicating the different phylogenetic processes among Magnoliaceae plants. Therefore, further genetic information is required to determine the relationships within Magnoliaceae. Overall, complete chloroplast genome sequences for four Magnoliaceae species reported in this paper have shed more light on phylogenetic relationships within the botanical group.

Overexpression of VEGFA mediated by HIF-1 is associated with higher rate of spread through air spaces in resected lung adenocarcinomas.

Spread through air spaces (STAS), a newly identified pattern of invasion in lung adenocarcinomas (LACs), is an unfavorable prognostic factor for patients with LAC, but the molecular characteristics and mechanisms underlying STAS have not been adequately explored. In total, 650 pathologically confirmed invasive LAC patients who underwent curative resection between December 2019 and April 2020 were reviewed. Disease-free survival (DFS) and overall survival (OS) were analyzed using the log-rank test and the Cox proportional hazards model. A comparative deep sequencing analysis was conducted to explore the molecular characteristics underlying STAS. Vascular endothelial growth factor A (VEGFA) expression was evaluated by immunoblotting and immunohistochemical analysis using fresh tumor tissue and tissue microarray. STAS was more prevalent in patients with a smoking history (p < 0.001), high pathological TNM stage (p < 0.001), lymphovascular invasion (p < 0.001), visceral pleural invasion (p < 0.001) and micropapillary/solid histological subtypes (p < 0.001). STAS-negative patients had better DFS (p < 0.001) and OS (p = 0.003) compared to STAS-positive patients with invasive LACs, especially in the lymph node-negative population (p < 0.001). After RNA-sequencing analysis, hypoxia-inducible factor-1 (HIF-1) signaling was enriched and appeared to be strongly correlated with STAS, and more STAS-positive individuals were detected in the higher VEGFA-expressing group (p = 0.042). We demonstrated that STAS was an independent prognostic marker of poor clinical outcome, especially in lymph node-negative patients, and that higher VEGFA expression mediated by HIF-1 signaling was associated with an increased STAS rate.

Identification and characteristics of temperature-sensitive sites of Pseudomonas plecoglossicida based on aptamers

Pseudomonas plecoglossicida can infect Pseudosciaena crocea and cause visceral white spot disease, resulting in substantial economic losses to the P. crocea industry. Pseudomonas plecoglossicida is a temperate-sensitive bacterium and only infects P. crocea below 22 ℃. Aptamers are oligonucleotide molecules selected by the systematic evolution of ligands by exponential enrichment (SELEX) and can identify all kinds of targets. In the present study, five aptamers (M17, M19, M48, M56 and M81) for P. plecoglossicida cultured at 18 ℃ were selected by SELEX and based on comparative analysis of the high frequency sequences. The five aptamers had significantly higher affinities for the pathogenic P. plecoglossicida at 18 ℃ than for the nonpathogenic P. plecoglossicida at 28 ℃ and the other six bacteria (Escherichia coli, Edwardsiella tarda, Aeromonas hydrophila, Vibiro harveryi, Vibiro alginolyticus, Pseudomonas aeruginosa) (P < 0.01). The affinity constants (Kd) and maximum affinity (Am) of these aptamers for P. plecoglossicida were determined, with Kd values of M17 (23.35 ± 6.40), M19 (66.37 ± 14.73), M48 (105.91 ± 21.41), M56 (31.16 ± 5.03) and M81 (52.63 ± 13.82) nmol/L and with Am values of M17 (1041.26 ± 57.08), M19 (2291.76 ± 171.96), M48 (1258.51 ± 21.41), M56 (1307.70 ± 54.01) and M81 (543.27 ± 44.16) nmol/L, respectively. The secondary structures of the aptamers were analyzed, and the aptamer binding proteins of the pathogenic P. plecoglossicida were isolated by magnetic separation and purified by polyacrylamide gel electrophoresis. Mass spectrometry revealed ribosomal proteins to be the main aptamer binding proteins. The secondary and tertiary structures and subcellular localization of these aptamer binding proteins were also studied and analyzed. These findings provide a foundation for the temperature-sensitive pathogenic mechanism of P. plecoglossicida in P. crocea. This is the first report on aptamers that recognize temperature-sensitive sites in P. plecoglossicida.

Solibacillus daqui sp. nov., isolated from high-temperature Daqu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS111008T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu, and was characterized by polyphasic taxonomy. This novel isolate grew in the presence of 0-5 % (w/v) NaCl, at pH 6.0-9.0 and 25-45 °C; optimum growth was observed with 1 % (w/v) NaCl, at pH 8.0 and 30 °C. A comparative analysis of the 16S rRNA gene sequence (1461 bp) of strain ZS111008T showed highest similarity to Solibacillus silvestris DSM12223T (96.7%), followed by Solibacillus cecembensis PN5T (96.6%) and Solibacillus isronensis AMCK01000046 (96.5%). The DNA G+C content of strain ZS111008T was 37.21 mol%. The respiratory quinone was identified as menaquinone-7 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and one unknown phospholipid. Lys was detected as the diagnostic diamino acid in the cell wall. Based on morphological characteristics, chemotaxonomic characteristics and physiological properties, strain ZS111008T represents a novel species of the genus Solibacillus, for which the name Solibacillus daqui sp. nov. is proposed. The type strain for this proposed species is ZS111008T (=CGMCC 1.19455T=JCM 35214T).

Aerococcus agrisoli sp. nov., isolated from paddy soil.

A non-spore-forming, Gram-stain-positive, short rod-shaped strain, designated SJQ22T, was isolated from a paddy soil sample collected in Shanghai, PR China. A comparative analysis of 16S rRNA gene sequences showed that strain SJQ22T fell within the genus Aerococcus, forming a clear cluster with the type strains of Aerococcus viridans (98.6 % sequence similarity) and Aerococcus urinaeequi (98.5 % sequence similarity). Strain SJQ22T grew at 30-45 °C (optimum, 30 °C), pH 6.0-8.0 (optimum, pH 7.0) and with a NaCl concentration of 0-4 % (optimum, 1 %). Cells were negative for oxidase and catalase activity. Chemotaxonomic analysis showed that strain SJQ22T possessed C16:0 and C18:1 ω9c as the predominant fatty acids. The DNA G + C content was 39.0 mol%. Strain SJQ22T exhibited DNA-DNA relatedness levels of 13±2 % with A. viridans ATCC 11563T and 9±2 % with A. urinaeequi IFO 12173T. Based on the data obtained, strain SJQ22T represents a novel species of the genus Aerococcus, for which the name Aerococcus agrisoli sp. nov. is proposed. The type strain is SJQ22T (=JCM 33111T=CCTCC AB 2018283T).

Phycicoccus sonneraticus sp. nov., a Novel Endophytic Actinobacterium Isolated from the Bark of Sonneratia apetala.

A novel endophytic actinobacterial strain, designated MQZ13P-5T, was isolated from a piece of bark of Sonneratia apetala, collected from Guangxi Zhuang Autonomous Region, China. This strain was Gram-stain positive, aerobic, non-spore-forming, non-motile and rod-shaped. Comparative 16S rRNA gene sequence analysis showed that strain MQZ13P-5T was related to the genus Phycicoccus with exhibiting the highest similarity (98.0%) to Phycicoccus endophyticus IP6SC6T. The phylogenetic trees based on 16S rRNA gene sequences and core genes indicated that strain MQZ13P-5T belonged to the genus Phycicoccus and could not be assigned to any described species. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain MQZ13P-5T and type strains of Phycicoccus species were less than 84% and 27%, respectively, below the thresholds for species delineation. This strain showed chemotaxonomic and phenotypic properties consistent with its classification in the genus Phycicoccus. Based on the taxonomic data, strain MQZ13P-5T should represent a novel species of the genus Phycicoccus, for which the name Phycicoccus sonneraticus sp. nov. is proposed, with the type strain MQZ13P-5T (= CGMCC 1.18744T = JCM 34337T).

The Arabidopsis SWEET1 and SWEET2 uniporters recognize similar substrates while differing in subcellular localization

Sugars Will Eventually be Exported Transporters (SWEETs) are central for sugar allocation in plants. The SWEET family has approximately 20 homologs in most plant genomes, and despite extensive research on their structures and molecular functions, it is still unclear how diverse SWEETs recognize different substrates. Previous work using SweetTrac1, a biosensor constructed by the intramolecular fusion of a conformation-sensitive fluorescent protein in the plasma membrane transporter SWEET1 from Arabidopsis thaliana, identified common features in the transporter's substrates. Here, we report SweetTrac2, a new biosensor based on the Arabidopsis vacuole membrane transporter SWEET2, and use it to explore the substrate specificity of this second protein. Our results show that SWEET1 and SWEET2 recognize similar substrates but some with different affinities. Sequence comparison and mutagenesis analysis support the conclusion that the differences in affinity depend on nonspecific interactions involving previously uncharacterized residues in the substrate-binding pocket. Furthermore, SweetTrac2 can be an effective tool for monitoring sugar transport at vacuolar membranes that would be otherwise challenging to study.

Prevalence and Molecular Characteristics of FAdV-4 from Indigenous Chicken Breeds in Yunnan Province, Southwestern China.

Fowl adenovirus-induced hepatitis-pericardial effusion syndrome outbreaks have been increasingly reported in China since 2015, resulting in substantial economic losses to the poultry industry. The genetic diversity of indigenous chicken results in different immune traits, affecting the evolution of these viruses. Although the molecular epidemiology of fowl adenovirus serotype 4 (FAdV-4) has been well studied in commercial broiler and layer chickens, the prevalence and genetic characteristics of FAdV-4 in indigenous chickens remain largely unknown. In this study, samples were collected from six indigenous chicken breeds in Yunnan province, China. FAdV-positive samples were identified in five of the six indigenous chicken populations via PCR and 10 isolates were obtained. All FAdVs belonged to serotype FAdV-4 and species FAdV-C. The hexon, fiber, and penton gene sequence comparison analysis demonstrated that the prevalence of FAdV-4 isolates in these chickens might have originated from other provinces that exported chicks and poultry products to Yunnan province. Moreover, several distinct amino acid mutations were firstly identified in the major structural proteins. Our findings highlighted the need to decrease inter-regional movements of live poultry to protect indigenous chicken genetic resources and that the immune traits of these indigenous chickens might result in new mutations of FAdV-4 strains.

Genetic and phenotypic characterizations of IncX3 plasmids harboring bla NDM-5 and bla NDM-16b in Japan.

The spread of the gene encoding the NDM-type carbapenemase, bla NDM, poses a serious threat to clinical practice and public health. This gene is usually carried by transferable plasmids, which facilitates its rapid spread among various species of Enterobacterales. Plasmids with the IncX3 replicon play a major role in the spread of bla NDM in Asian countries. In this study, we conducted genomic sequencing and a comparative analysis of the IncX3-type plasmids harboring bla NDM-5 and bla NDM-16b, a single-nucleotide variant of bla NDM-5, isolated in a national antimicrobial-resistant bacterial genomic surveillance in Japan (Japan Antimicrobial Resistant Bacterial Surveillance), which was carried out during 2019-2020. We compared five plasmids harboring bla NDM-5 and one plasmid harboring bla NDM-16b. They were highly identical and differed in several nucleotide substitutions or variations in insertion sequences, although they were carried by different species or genotypes. Notably, bla NDM-16b-carrying pJBBDACG-19-0070 was identical to a plasmid carrying bla NDM-5 except for one nucleotide substitution in the bla NDM gene throughout the 46 kb sequences. Transformants carrying these two plasmids showed comparative resistance to carbapenems, indicating that a substitution on the NDM enzyme (Ala233 to Val) did not affect carbapenem resistance, at least under normal experimental conditions. A database search revealed that several identical plasmids carrying bla NDM-5 were reported from neighboring countries, mainly China, before our surveillance period. Our analysis suggested that the IncX3 plasmid harboring bla NDM-5 is spreading in Japan, and bla NDM-16b emerged from a single nucleotide substitution on the plasmid, with widespread dissemination in Asian countries. IMPORTANCE IncX3 plasmids harboring bla NDM-5 play a major role in the spread of carbapenem resistance in Asia, particularly in China, in clinical and environmental settings. In this study, we present that Enterobacterales isolates carrying IncX3 plasmids harboring bla NDM-5 have been disseminated in Japan, where their identification was previously rare. In addition, bla NDM-16b, a single-nucleotide variant of bla NDM-5, was found to be carried by an identical IncX3 plasmid. A comparative sequence analysis revealed that the bla NDM-16b gene emerged from a single nucleotide substitution on an IncX3 plasmid harboring bla NDM-5. The bla NDM-16b gene did not confer elevated carbapenem resistance compared to bla NDM-5 in our assay using transformants carrying the plasmid harboring either of these genes, although the A233V substitution was reported to confer stability to the enzyme in ion-depleted conditions. Nevertheless, vigilance regarding the emergence of novel variants is required.