Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

Woody species do not differ in dormancy progression: Differences in time to budbreak due to forcing and cold hardiness.

Budbreak is one of the most observed and studied phenological phases in perennial plants, but predictions remain a challenge, largely due to our poor understanding of dormancy. Two dimensions of exposure to temperature are generally used to model budbreak: accumulation of time spent at low temperatures (chilling) and accumulation of heat units (forcing). These two effects have a well-established negative correlation; with more chilling, less forcing is required for budbreak. Furthermore, temperate plant species are assumed to vary in chilling requirements for dormancy completion allowing proper budbreak. Here, dormancy is investigated from the cold hardiness standpoint across many species, demonstrating that it should be accounted for to study dormancy and accurately predict budbreak. Most cold hardiness is lost prior to budbreak, but rates of cold hardiness loss (deacclimation) vary among species, leading to different times to budbreak. Within a species, deacclimation rate increases with accumulation of chill. When inherent differences between species in deacclimation rate are accounted for by normalizing rates throughout winter by the maximum rate observed, a standardized deacclimation potential is produced. Deacclimation potential is a quantitative measurement of dormancy progression based on responsiveness to forcing as chill accumulates, which increases similarly for all species, contradicting estimations of dormancy transition based on budbreak assays. This finding indicates that comparisons of physiologic and genetic control of dormancy require an understanding of cold hardiness dynamics. Thus, an updated framework for studying dormancy and its effects on spring phenology is suggested where cold hardiness in lieu of (or in addition to) budbreak is used.

Electrical-Controllable Antiferromagnet-Based Tunnel Junction.

An electrical-controllable antiferromagnet tunnel junction is a key goal in spintronics, holding immense promise for ultradense and ultrastable antiferromagnetic memory with high processing speed for modern information technology. Here, we have advanced toward this goal by achieving an electrical-controllable antiferromagnet-based tunnel junction of Pt/Co/Pt/Co/IrMn/MgO/Pt. The exchange coupling between antiferromagnetic IrMn and Co/Pt perpendicular magnetic multilayers results in the formation of an interfacial exchange bias and exchange spring in IrMn. Encoding information states "0" and "1" is realized through the exchange spring in IrMn, which can be electrically written by spin-orbit torque switching with high cyclability and electrically read by antiferromagnetic tunneling anisotropic magnetoresistance. Combining spin-orbit torque switching of both exchange spring and exchange bias, a 16 Boolean logic operation is successfully demonstrated. With both memory and logic functionalities integrated into our electrically controllable antiferromagnetic-based tunnel junction, we chart the course toward high-performance antiferromagnetic logic-in-memory.

Genome assembly of Medicago truncatula accession SA27063 provides insight into spring black stem and leaf spot disease resistance.

Medicago truncatula, model legume and alfalfa relative, has served as an essential resource for advancing our understanding of legume physiology, functional genetics, and crop improvement traits. Necrotrophic fungus, Ascochyta medicaginicola, the causal agent of spring black stem (SBS) and leaf spot is a devasting foliar disease of alfalfa affecting stand survival, yield, and forage quality. Host resistance to SBS disease is poorly understood, and control methods rely on cultural practices. Resistance has been observed in M. truncatula accession SA27063 (HM078) with two recessively inherited quantitative-trait loci (QTL), rnpm1 and rnpm2, previously reported. To shed light on host resistance, we carried out a de novo genome assembly of HM078. The genome, referred to as MtHM078 v1.0, is comprised of 23 contigs totaling 481.19 Mbp. Notably, this assembly contains a substantial amount of novel centromere-related repeat sequences due to deep long-read sequencing. Genome annotation resulted in 98.4% of BUSCO fabales proteins being complete. The assembly enabled sequence-level analysis of rnpm1 and rnpm2 for gene content, synteny, and structural variation between SBS-resistant accession SA27063 (HM078) and SBS-susceptible accession A17 (HM101). Fourteen candidate genes were identified, and some have been implicated in resistance to necrotrophic fungi. Especially interesting candidates include loss-of-function events in HM078 because they fit the inverse gene-for-gene model, where resistance is recessively inherited. In rnpm1, these include a loss-of-function in a disease resistance gene due to a premature stop codon, and a 10.85 kbp retrotransposon-like insertion disrupting a ubiquitin conjugating E2. In rnpm2, we identified a frameshift mutation causing a loss-of-function in a glycosidase, as well as a missense and frameshift mutation altering an F-box family protein. This study generated a high-quality genome of HM078 and has identified promising candidates, that once validated, could be further studied in alfalfa to enhance disease resistance.

Duration and variability of spring green-up mediate population consequences of climate change.

Single phenological measures, like the average rate of phenological advancement, may be insufficient to explain how climate change is driving trends in animal populations. Here, we develop a multifactorial concept of spring phenology-including the onset of spring, spring duration, interannual variability, and their temporal changes-as a driver for population dynamics of migratory terrestrial species in seasonal environments. Using this conceptual model, we found that effects of advancing spring phenology on animal populations may be buffered or amplified depending on the duration and interannual variability of spring green-up, and those effects are modified by evolutionary and plastic adaptations of species. Furthermore, we compared our modelling results with empirical data on normalized difference vegetation index-based spring green-up phenology and population trends of 106 European landbird finding similar associations. We conclude how phenological changes are expected to affect migratory bird populations across Europe and identify regions that are particularly prone to suffer population declines.

Phenotyping and Exploitation of Kompetitive Allele-Specific PCR Assays for Genes Underpinning Leaf Rust Resistance in New Spring Wheat Mutant Lines.

Leaf rust (Puccinia triticina Eriks) is a wheat disease causing substantial yield losses in wheat production globally. The identification of genetic resources with permanently effective resistance genes and the generation of mutant lines showing increased levels of resistance allow the efficient incorporation of these target genes into germplasm pools by marker-assisted breeding. In this study, new mutant (M3 generation) lines generated from the rust-resistant variety Kazakhstanskaya-19 were developed using gamma-induced mutagenesis through 300-, 350-, and 400-Gy doses. In field trials after leaf rust inoculation, 75 mutant lines showed adult plant resistance. These lines were evaluated for resistance at the seedling stage via microscopy in greenhouse experiments. Most of these lines (89.33%) were characterized as resistant at both developmental stages. Hyperspectral imaging analysis indicated that infected leaves of wheat genotypes showed increased relative reflectance in visible and near-infrared light compared to the non-infected genotypes, with peak means at 462 and 644 nm, and 1936 and 2392 nm, respectively. Five spectral indexes, including red edge normalized difference vegetation index (RNDVI), structure-insensitive pigment index (SIPI), ratio vegetation index (RVSI), water index (WI), and normalized difference water index (NDWI), demonstrated significant potential for determining disease severity at the seedling stage. The most significant differences in reflectance between susceptible and resistant mutant lines appeared at 694.57 and 987.51 nm. The mutant lines developed were also used for the development and validation of KASP markers for leaf rust resistance genes Lr1, Lr2a, Lr3, Lr9, Lr10, and Lr17. The mutant lines had high frequencies of "a" resistance alleles (0.88) in all six Lr genes, which were significantly associated with seedling resistance and suggest the potential of favorable haplotype introgression through functional markers. Nine mutant lines characterized by the presence of "b" alleles in Lr9 and Lr10-except for one line with allele "a" in Lr9 and three mutant lines with allele "a" in Lr10-showed the progressive development of fungal haustorial mother cells 72 h after inoculation. One line from 300-Gy-dosed mutant germplasm with "b" alleles in Lr1, Lr2a, Lr10, and Lr17 and "a" alleles in Lr3 and Lr9 was characterized as resistant based on the low number of haustorial mother cells, suggesting the contribution of the "a" alleles of Lr3 and Lr9.

Structure of the Spring Viraemia of Carp Virus Ribonucleoprotein Complex Reveals Its Assembly Mechanism and Application in Antiviral Drug Screening.

Spring viremia of carp virus (SVCV) is a highly pathogenic Vesiculovirus infecting the common carp, yet neither a vaccine nor effective therapies are available to treat spring viremia of carp (SVC). Like all negative-sense viruses, SVCV contains an RNA genome that is encapsidated by the nucleoprotein (N) in the form of a ribonucleoprotein (RNP) complex, which serves as the template for viral replication and transcription. Here, the three-dimensional (3D) structure of SVCV RNP was resolved through cryo-electron microscopy (cryo-EM) at a resolution of 3.7 Å. RNP assembly was stabilized by N and C loops; RNA was wrapped in the groove between the N and C lobes with 9 nt nucleotide per protomer. Combined with mutational analysis, our results elucidated the mechanism of RNP formation. The RNA binding groove of SVCV N was used as a target for drug virtual screening, and it was found suramin had a good antiviral effect. This study provided insights into RNP assembly, and anti-SVCV drug screening was performed on the basis of this structure, providing a theoretical basis and efficient drug screening method for the prevention and treatment of SVC. IMPORTANCE Aquaculture accounts for about 70% of global aquatic products, and viral diseases severely harm the development of aquaculture industry. Spring viremia of carp virus (SVCV) is the pathogen causing highly contagious spring viremia of carp (SVC) disease in cyprinids, especially common carp (Cyprinus carpio), yet neither a vaccine nor effective therapies are available to treat this disease. In this study, we have elucidated the mechanism of SVCV ribonucleoprotein complex (RNP) formation by resolving the 3D structure of SVCV RNP and screened antiviral drugs based on the structure. It is found that suramin could competitively bind to the RNA binding groove and has good antiviral effects both in vivo and in vitro. Our study provides a template for rational drug discovery efforts to treat and prevent SVCV infections.

Nondestructive and three-dimensional visualization by identifying elements using synchrotron radiation microscale X-ray CT reveals microbial and cavity distributions in anaerobic granular sludge.

We developed a nondestructive three-dimensional microbial visualization method utilizing synchrotron radiation X-ray microscale computed tomography to better understand the relationship between microorganisms and their surrounding habitats. The method was tested and optimized using a mixture of axenic Escherichia coli and Comamonas testosteroni. The osmium-thiocarbohydrazide-osmium method was used to stain all the microbial cells, and gold in situ hybridization was used to detect specific phylogenetic microbial groups. The stained samples were embedded in epoxy resin for microtomographic analysis. Differences in X-ray absorbances were calculated by subtracting the pre-L3-edge images from the post-L3-edge images to visualize the osmium and gold signals. Although we successfully detected cells stained with osmium, those labeled with gold were not detected, probably because of the insufficient density of gold atoms in the microbial cells. We then applied the developed technique to anaerobic granules and visualized the distribution of microbial cells and extracellular polymeric substances. Empty spaces were highlighted to determine the cavity distribution in granules. Numerous independent cavities of different sizes were identified in the granules. The developed method can be applied to various environmental samples for deeper insights into microbial life in their habitats. IMPORTANCE: Microorganisms inhabit diverse environments and often form biofilms. One factor that affects their community structure is the surrounding physical environment. The arrangement of residential space within the formed biofilm plays a crucial role in the supply and transportation of substances, as well as the discharge of metabolites. Conventional approaches, such as scanning electron microscopy and confocal laser scanning microscopy combined with fluorescence in situ hybridization, have limitations as they provide information primarily from the biofilm surface and cross-sections. In this study, we developed a method for detecting microorganisms in biofilms using synchrotron radiation X-ray microscale computer tomography. The developed method allows nondestructive three-dimensional observation of biofilms at a single-cell resolution (voxel size of approximately 200 nm), facilitating an understanding of the relationship between microorganisms and their physical habitats.

Weak evidence of provenance effects in spring phenology across Europe and North America.

Forecasting the biological impacts of climate change requires understanding how species respond to warmer temperatures through interannual flexible variation vs through adaptation to local conditions. Yet, we often lack this information entirely or find conflicting evidence across studies, which is the case for spring phenology. We synthesized common garden studies across Europe and North America that reported spring event dates for a mix of angiosperm and gymnosperm tree species in the northern hemisphere, capturing data from 384 North American and 101 European provenances (i.e. populations) with observations from 1962 to 2019, alongside autumn event data when provided. Across continents, we found no evidence of provenance effects in spring phenology, but strong clines with latitude and mean annual temperature in autumn. These effects, however, appeared to diverge by continent and species type (gymnosperm vs angiosperm), with particularly pronounced clines in North America in autumn events. Our results suggest flexible, likely plastic responses, in spring phenology with warming, and potential limits - at least in the short term - due to provenance effects for autumn phenology. They also highlight that, after over 250 yr of common garden studies on tree phenology, we still lack a holistic predictive model of clines across species and phenological events.

The potential for an increasing threat of unseasonal temperature cycles to dormant plants.

Two functional responses largely guide woody plants' survival to winter conditions: cold hardiness and dormancy. Dormancy affects budbreak timing based on chill accumulation. Effects of warming on dormancy may appear time-shifted: fall and winter warming events decrease chill accumulation, delaying budbreak observed in spring. The same warming events also affect cold hardiness dynamics, having immediate implications. As cold deacclimation rates increase with dormancy progression, the same amount of warming has greater damage risk the later it occurs in the season, depending on return of low temperatures. Should frequency of erratic weather increase with climate change, more instances of risk are expected. However, understanding how plants fare through seasons now and in future climates still requires better knowledge of winter physiology.

Temperature sum models in plant spring phenology studies: two commonly used methods have different fields of application.

In studies of plant spring phenology, temperature sum models are traditional tools. They are used to quantify plant development in terms of accumulation of temperature-dependent developmental units, such as Growing Degree Hours, GDHs. A key parameter in these models is the threshold (or base) temperature, Tthr, representing the lower thermal limit for the development to occur. The parameter can be either estimated when the model is fitted into the data or fixed a priori. Here we examine the limitations of both methods and identify fields of applications for each of them.

Temperature dependence of spring carbon uptake in northern high latitudes during the past four decades.

In the northern high latitudes, warmer spring temperatures generally lead to earlier leaf onsets, higher vegetation production, and enhanced spring carbon uptake. Yet, whether this positive linkage has diminished under climate change remains debated. Here, we used atmospheric CO2 measurements at Barrow (Alaska) during 1979-2020 to investigate the strength of temperature dependence of spring carbon uptake reflected by two indicators, spring zero-crossing date (SZC) and CO2 drawdown (SCC). We found a fall and rise in the interannual correlation of temperature with SZC and SCC (RSZC-T and RSCC-T ), showing a recent reversal of the previously reported weakening trend of RSZC-T and RSCC-T . We used a terrestrial biosphere model coupled with an atmospheric transport model to reproduce this fall and rise phenomenon and conducted factorial simulations to explore its potential causes. We found that a strong-weak-strong spatial synchrony of spring temperature anomalies per se has contributed to the fall and rise trend in RSZC-T and RSCC-T , despite an overall unbroken temperature control on net ecosystem CO2 fluxes at local scale. Our results provide an alternative explanation for the apparent drop of RSZC-T and RSCC-T during the late 1990s and 2000s, and suggest a continued positive linkage between spring carbon uptake and temperature during the past four decades. We thus caution the interpretation of apparent climate sensitivities of carbon cycle retrieved from spatially aggregated signals.

Active around the year: Butterflies and moths adapt their life cycles to a warming world.

Living in a warming world requires adaptations to altered annual temperature regimes. In Europe, spring is starting earlier, and the vegetation period is ending later in the year. These climatic changes are leading not only to shifts in distribution ranges of flora and fauna, but also to phenological shifts. Using long-term observation data of butterflies and moths collected during the past decades across northern Austria, we test for phenological shifts over time and changes in the number of generations. On average, Lepidoptera adults emerged earlier in the year and tended to extend their flight periods in autumn. Many species increased the annual number of generations. These changes were more pronounced at lower altitudes than at higher altitudes, leading to an altered phenological zonation. Our findings indicate that climate change does not only affect community composition but also the life history of insects. Increased activity and reproductive periods might alter Lepidoptera-host plant associations and food webs.

Effect of a low-mineralized thermal spring water on skin barrier mechanical properties using atomic force microscopy.

The mineral content of thermal spring water (TSW) applied to the skin surface can directly influence the skin barrier. Indeed, our previous study showed that Avène TSW (ATSW), a low mineral content thermal spring water, protects the stratum corneum from dehydration compared to a mineral-rich TSW (MR-TSW) and maintains skin surface ultrastructure. While many TSWs have been recognized to have beneficial effects on skin, little is known about their localized and specific effects on skin barrier biomechanics at the nanometric scale. The aim of this study was to compare the effects of ATSW with a reference, MR-TSW, on the biomechanical barrier properties of the skin under homeostasis conditions using atomic force microscopy (AFM). AFM was used to obtain a precise nanomechanical mapping of the skin surface after three applications of both TSW. This provides specific information on the skin topographical profile and elasticity. The topographic profile of skin samples showed a specific compaction of the skin layers after application of MR-TSW, characterized by an increase of the total number of external skin layers, compared to non-treated samples. By contrast, ATSW did not modify the skin topographic profile. High-resolution force/volume acquisitions to capture the elastic modulus showed that it was directly correlated with skin rigidity. The elastic modulus strongly and significantly increased after MR-TSW application compared to non-treated skin. By contrast, applications of ATSW did not increase elastic modulus. These data demonstrate that applications of MR-TSW significantly modified skin barrier properties by increasing skin surface layer compaction and skin rigidity. By contrast, ATSW did not modify the topographical profile of skin explants nor induce mechanical stress at the level of the stratum corneum, indicating it does not disrupt the biophysical properties linked to skin surface integrity.

Four phases of a force transient emerge from a binary mechanical system.

Accurate models of muscle contraction are important for understanding both muscle performance and the therapeutics that enhance physiological function. However, models are only accurate and meaningful if they are consistent with physical laws. A single muscle fiber contains billions of randomly fluctuating atoms that on the spatial scale of a muscle fiber generate unidirectional force and power output. This thermal system is formally constrained by the laws of thermodynamics, and a recently developed thermodynamic model of muscle force generation provides qualitative descriptions of the muscle force-velocity relationship, muscle force generation, muscle force transients, and the thermodynamic work loop of muscle with a thermodynamic (not molecular) power stroke mechanism. To demonstrate the accuracy of this model requires that its outputs be quantitatively compared with experimentally observed muscle function. Here I show that a two-state thermodynamic model accurately describes the experimentally observed four-phase force transient response to both mechanical and chemical perturbations. This is the simplest possible model of one of the most complex characteristic signatures of muscle mechanics.

Integrative Salt Selection and Formulation Optimization: Perspectives of Disproportionation and Microenvironmental pH Modulation.

We report a novel utilization of a pH modifier as a disproportionation retardant in a tablet formulation. The drug molecule of interest has significant bioavailability challenges that require solubility enhancement. In addition to limited salt/cocrystal options, disproportionation of the potential salt(s) was identified as a substantial risk. Using a combination of Raman spectroscopy with chemometrics and quantitative X-ray diffraction in specially designed stress testing, we investigated the disproportionation phenomena. The learnings and insight drawn from crystallography drove the selection of the maleate form as the target API. Inspired by the fumarate form's unique stability and solubility characteristics, we used fumaric acid as the microenvironmental pH modulator. Proof-of-concept experiments with high-risk (HCl) and moderate-risk (maleate) scenarios confirmed the synergistic advantage of fumaric acid, which interacts with the freebase released by disproportionation to form a more soluble species. The resultant hemifumarate helps maintain the solubility at an elevated level. This work demonstrates an innovative technique to mediate the solubility drop during the "parachute" phase of drug absorption using compendial excipients, and this approach can potentially serve as an effective risk-mitigating strategy for salt disproportionation.

Time to budbreak is not enough: cold hardiness evaluation is necessary in dormancy and spring phenology studies.

BACKGROUND: Dormancy of buds is an important phase in the life cycle of perennial plants growing in environments where unsuitable growth conditions occur seasonally. In regions where low temperature defines these unsuitable conditions, the attainment of cold hardiness is also required for survival. The end of the dormant period culminates in budbreak and flower emergence, or spring phenology, one of the most appreciated and studied phenological events - a time also understood to be most sensitive to low-temperature damage. Despite this, we have a limited physiological and molecular understanding of dormancy, which has negatively affected our ability to model budbreak. This is also true for cold hardiness. SCOPE: Here we highlight the importance of including cold hardiness in dormancy studies that typically only characterize time to budbreak. We show how different temperature treatments may lead to increases in cold hardiness, and by doing so also (potentially inadvertently) increase time to budbreak. CONCLUSIONS: We present a theory that describes evaluation of cold hardiness as being key to clarifying physiological changes throughout the dormant period, delineating dormancy statuses, and improving both chill and phenology models. Erroneous interpretations of budbreak datasets are possible by not phenotyping cold hardiness. Changes in cold hardiness were very probably present in previous experiments that studied dormancy, especially when those included below-freezing temperature treatments. Separating the effects between chilling accumulation and cold acclimation in future studies will be essential for increasing our understanding of dormancy and spring phenology in plants.

Floral freezing tolerance is tied to flowering time in North American woody plant species.

BACKGROUND AND AIMS: As winter and spring temperatures continue to increase, the timing of flowering and leaf out is advancing in many seasonally cold regions. This advancement could put plants that flower early in the spring at risk of decreased reproduction in years when there are late freeze events. Unfortunately, relatively little is known about floral freezing tolerance in forest communities. In this study, we examined the impact of freezing temperatures on the flowers of woody plants in a region where there is rapid winter warming in North America. METHODS: We subjected the flowers of twenty-five woody species to a hard (-5ºC) and a light freeze (0ºC). We assessed tissue damage using electrolyte leakage. In a subset of species, we also examined the impact of a hard freeze on pollen tube growth. To determine if the vulnerability of flowers to freezing damage relates to flowering time and to examine the responsiveness of flowering time to spring temperature, we recorded the date of first flower for our study species for three years. KEY RESULTS AND CONCLUSIONS: Across species, we found that floral freezing tolerance was strongly tied to flowering time with the highest freezing tolerance occurring in plants that bloomed earlier in the year. We hypothesize that these early blooming species are unlikely to be impacted by a false spring. Instead, the most vulnerable species to a false spring should be those that bloom later in the season. The flowering time in these species is also more sensitive to temperature, putting them at a great risk of experiencing a false spring. Ultimately, floral damage in one year will not have a large impact on species fitness, but if false springs become more frequent, there could be long-term impacts on reproduction of vulnerable species.

Polycladomyces zharkentensis sp. nov., a novel thermophilic cellulose- and starch-degrading member of the Bacillota from a geothermal aquifer in Kazakhstan.

A thermophilic, aerobic and heterotrophic filamentous bacterium, designated strain ZKZ2T, was isolated from a pipeline producing hydrothermal water originating from a >2.3 km deep subsurface geothermal source in Zharkent, Almaty region, Kazakhstan. The isolate was Gram-stain-positive, non-motile, heat-resistant and capable of producing a variety of extracellular hydrolases. Growth occurred at temperatures between 55 and 75 °C, with an optimum around 70 °C, and at pH values between 5.5 and 9.0, with an optimum at pH 7.0-7.5 with the formation of aerial mycelia; endospores were produced along the aerial mycelium. The isolate was able to utilize the following substrates for growth: glycerol, l-arabinose, ribose, d-xylose, d-glucose, d-fructose, d-mannose, rhamnose, d-mannitol, methyl-d-glucopyranoside, aesculin, salicin, cellobiose, maltose, melibiose, sucrose, trehalose, melezitose, raffinose, starch, turanose and 5-keto-gluconate. Furthermore, it was able to hydrolyse carboxymethylcellulose, starch, skimmed milk, Tween 60 and Tween 80. The major cellular fatty acids were iso-C15 : 0, iso-C17 : 0, iso-C16 : 0 and C16 : 0. Our 16S rRNA gene sequence analysis placed ZKZ2T within the genus Polycladomyces, family Thermoactinomycetaceae, with the highest similarity to the type species Polycladomyces abyssicola JIR-001T (99.18 % sequence identity). Our draft genome sequence analysis revealed a genome size of 3.3 Mbp with a G+C value of 52.5 mol%. The orthologous average nucleotide identity value as compared to that of its closest relative, P. abyssicola JIR-001T, was 90.23 %, with an in silico DNA-DNA hybridization value of 40.7 %, indicating that ZKZ2T represents a separate genome species. Based on the phenotypic and genome sequence differences from the other two Polycladomyces species, we propose that strain ZKZ2T represents a novel species, for which we propose the name Polycladomyces zharkentensis sp. nov. The type strain is ZKZ2T (=CECT 30708T=KCTC 43421T).

Six novel species of the genus Methanoculleus isolated from various environments in Taiwan.

Taiwan is situated in the subtropical region and its geographical location and topographical features contribute to a rich ecological diversity and scenic landscapes. We investigated the diversity of methanogens in different environments of Taiwan using a culture-dependent method. This report presents the characterization and taxonomy of six hydrogenotrophic methanogens obtained from cold seep sediments (strain FWC-SCC1T and FWC-SCC3T), marine sediments (strain CWC-02T and YWC-01T), estuarine sediments (strain Afa-1T), and a hot spring well (strain Wushi-C6T) in Taiwan. The proposed names of the six novel species are Methanoculleus frigidifontis (type strain FWC-SCC1T=BCRC AR10056T=NBRC 113993T), Methanoculleus oceani (CWC-02T=BCRC AR10055T=NBRC 113992T), Methanoculleus methanifontis (FWC-SCC3T=BCRC AR10057T=NBRC 113994T), Methanoculleus nereidis (YWC-01T=BCRC AR10060T=NBRC 114597T), Methanoculleus formosensis (Afa-1T=BCRC AR10054T=NBRC 113995T), and Methanoculleus caldifontis (Wushi-06T=BCRC AR10059T= NBRC 114596T).