Low Sequence Research Articles

Re-engineering of a carotenoid-binding protein based on NMR structure.

Recently, a number of message passing neural network (MPNN)-based methods have been introduced that, based on backbone atom coordinates, efficiently recover native amino acid sequences of proteins and predict modifications that result in better expressing, more soluble, and stable variants. However, usually, X-ray structures, or artificial structures generated by algorithms trained on X-ray structures, were employed to define target backbone conformations. Here, we show that commonly used algorithms ProteinMPNN and SolubleMPNN display low sequence recovery on structures determined using NMR. We subsequently propose a computational approach that we successfully apply to re-engineer AstaP, a protein that natively binds a large hydrophobic ligand astaxanthin (C40H52O4), and for which only a structure determined using NMR is currently available. The engineered variants, designated NeuroAstaP, are 51 amino acid shorter than the 22 kDa parent protein, have 38%-42% sequence identity to it, exhibit good yields, are expressed in a soluble, mostly monomeric form, and demonstrate efficient binding of carotenoids in vitro and in cells. Altogether, our work further tests the limits of using machine learning for protein engineering and paves the way for MPNN-based modification of proteins based on NMR-derived structures.

Phylogenetic placement of Mt Bamboutos endemic skink, Trachylepis mekuana (Chirio and Ineich, 2001)

African skinks of the genus Trachylepis is one of the most diverse genera of lizards in Africa. Although, many species have not been validated phylogenetically in recent years. In this study we evaluate the phylogenetic status of the Cameroon Volcanic Line endemic montane skink, Trachylepis mekuana. We recover this species as part of the larger Trachylepis varia group, with sister relationship to the morphological similar skinks from Eastern Africa Trachylepis megalura and central Africa Trachylepis raymondlaurenti. Low sequence divergence (<1.6% 16S) have been observed among the three species. Based on some morphological and colouration differences, as well as mostly allopatric distribution we regard them as good species.

Geo-classification of drug-resistant travel-associated Plasmodium falciparum using Pfs47 and Pfcpmp gene sequences (USA, 2018-2021).

Travel-related malaria is regularly encountered in the United States, and the U.S. Centers for Disease Control and Prevention (CDC) characterizes Plasmodium falciparum drug-resistance genotypes routinely for travel-related cases. An important aspect of antimalarial drug resistance is understanding its geographic distribution. However, specimens submitted to CDC laboratories may have missing, incomplete, or inaccurate travel data. To complement genotyping for drug-resistance markers Pfcrt, Pfmdr1, Pfk13, Pfdhps, Pfdhfr, and PfcytB at CDC, amplicons of Pfs47 and Pfcpmp are also sequenced as markers of geographic origin. Here, a bi-allele likelihood (BALK) classifier was trained using Pfs47 and Pfcpmp sequences from published P. falciparum genomes of known geographic origin to classify clinical genotypes to a continent. Among P. falciparum-positive blood samples received at CDC for drug-resistance genotyping from 2018 to 2021 (n = 380), 240 included a travel history with the submission materials, though 6 were excluded due to low sequence quality. Classifications obtained for the remaining 234 were compared to their travel histories. Classification results were over 96% congruent with reported travel for clinical samples, and with collection sites for field isolates. Among travel-related samples, only two incongruent results occurred; a specimen submitted citing Costa Rican travel classified to Africa, and a specimen with travel referencing Sierra Leone classified to Asia. Subsequently, the classifier was applied to specimens with unreported travel histories (n = 140; 5 were excluded due to low sequence quality). For the remaining 135 samples, geographic classification data were paired with results generated using CDC's Malaria Resistance Surveillance (MaRS) protocol, which detects single-nucleotide polymorphisms in and generates haplotypes for Pfcrt, Pfmdr1, Pfk13, Pfdhps, Pfdhfr, and PfcytB. Given the importance of understanding the geographic distribution of antimalarial drug resistance, this work will complement domestic surveillance efforts by expanding knowledge on the geographic origin of drug-resistant P. falciparum entering the USA.

Repurposing Tolfenamic Acid to Anchor the Uncharacterized Pocket of the PUB Domain for Proteolysis of the Atypical E3 Ligase HOIP.

The E3 ligase HOIP is vital for the NF-κB pathway and is implicated in cancer and immunity. However, it remains challenging to achieve high selectivity by directly targeting the conserved catalytic RBR domain of HOIP. Herein, we identified four low-molecular-weight compounds that bind to an uncharacterized pocket of the HOIP PUB domain (HOIPPUB). The complex structure facilitated the discovery of the first single-digit micromolar ligand of HOIPPUB, tolfenamic acid, which exhibited over 30-fold selectivity due to the low sequence identity of the uncharacterized pocket of HOIPPUB. Although tolfenamic acid did not block the substrate recognition and linear ubiquitination activity of HOIP, a ligand of the uncharacterized PUB pocket of HOIP (LUPH), by chemical linking pomalidomide with tolfenamic acid, degraded HOIP, reduced NEMO ubiquitination and p65 phosphorylation, and eventually inhibited NF-κB activation and breast cancer cell proliferation. Our work proposes an alternative strategy to target the nonfunctional pocket of the PUB domain with high sequence diversity to promote HOIP degradation, rather than targeting the conserved RBR domain to block the catalytic function of HOIP.

Transcription factor networks drive perforin activity in the anti-bacterial immune response of tilapia

Perforin, produced by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), is one of the effectors of cell-mediated cytotoxicity (CMC) in vertebrates, playing a paramount role in killing target cells. However, whether and how perforin is involved in adaptive immune responses in early vertebrates remains unclear. Using Nile tilapia (Oreochromis niloticus) as a model, we investigated the characteristics of perforin in early vertebrates. Oreochromis niloticus perforin (OnPRF) possesses 2 conserved functional domains, membrane attack complex/perforin (MACPF) and protein kinase C conserved region 2 (C2) domains, although they share low amino acid sequence similarity with other homologs. OnPRF was widely expressed in various immune tissues and could respond to lymphocyte activation and T-cell activation in vitro at both the transcriptional and protein levels, indicating that it may be involved in adaptive immune responses. Furthermore, after infection with Edwardsiella piscicida and Aeromonas hydrophila, the mRNA and protein levels of OnPRF were significantly up-regulated within the adaptive immune response period. Additionally, we revealed that many transcription factors were involved in the transcriptional regulation of OnPRF, including p65, c-Fos, c-Jun, STAT1 and STAT4, and there was a synergy among these transcription factors. Overall, these findings demonstrate the involvement of OnPRF in T-cell activation and adaptive immune response in tilapia, thus providing new evidence for comprehending the evolution of immune response in early vertebrates.

Crystal structure of the alternative complex III from the phototrophic bacterium Chloroflexus aurantiacus.

Alternative complex III (ACIII) is a multi-subunit quinol:electron acceptor oxidoreductase that couples quinol oxidation with transmembrane proton translocation in bacterial respiratory and photosynthetic electron transport chains. Four ACIII cryoelectron microscopy (cryo-EM) structures are known. However, the effects of cryo-EM versus X-ray crystallography structure determination on ACIII structure are unclear. Here, we report a 3.25Å crystal structure of photosynthetic ACIII from Chloroflexus aurantiacus (CaACIIIp), revealing eight subunits (ActA-G and I) with four iron-sulfur clusters and six c-type hemes, a menaquinol-binding site, and two proton translocation passages. Structural comparisons with the previously reported cryo-EM structures reveal slight local conformational changes in the solvent-exposed regions of ActB, ActD, ActG, and the transmembrane (TM) helix of subunit I. The regions conferring structural flexibility possess low sequence conservation across species. However, the core functional modules containing the menaquinol-binding pocket, redox centers, and proton translocation passages remain unchanged, preserving the enzyme's activity.

Angiostrongylus vasorum in golden jackals (Canis aureus) and red foxes (Vulpes vulpes) from Northern Bosnia and Herzegovina.

In this study, we conducted post-mortem examinations of golden jackals (Canis aureus) and red foxes (Vulpes vulpes) from the northern region of the Republic of Srpska (Bosnia and Herzegovina) to detect the presence of Angiostrongylus vasorum. An epidemiological survey was conducted in the densest golden jackal population in the country. Over two time intervals, we examined a total of 30 jackals and 16 foxes. The presence of A. vasorum was confirmed in two jackals (6.6%) and one fox (6.25%). These findings strongly suggest that the spread of A. vasorum can be facilitated by the active expansion and migration of golden jackals in Southeastern Europe. The morphological and molecular detection of A. vasorum in jackals and foxes confirmed its presence and active circulation in wildlife, while the phylogenetic analysis of the ITS-2 gene indicated a low sequence distance from European isolates.

Emergence and development of Chihuahuan Desert grasses under simulated wet-dry sequences

We evaluated the response (emergence, speed of germination, survival) of nine grasses (six native, three introduced) commonly occurring in the State of Chihuahua, Mexico to four irrigation “wet-dry” sequences that simulate sequences of days with and without rainfall, under greenhouse conditions. The sequences provide a total of 120, 100, 80 and 60 mm (high, medium, low, and very low) of water for 40 days. The native species were Bouteloua curtipendula, B. dactyloides, B. eriopoda, B. gracilis, Digitaria californica, and Leptochloa dubia. The introduced grasses were Cynodon dactylon, Eragrostis curvula, and E. superba. A decrease in water, resulting from the drier sequences, delayed the emergence from 4 to 7 days in most species. The native B. dactyloides displayed the best potential for growth with high emergence (18%) and high survival (44%) under low or very low water sequences. In general, survival for native species was higher than for introduced species under water restrictions. The native species showed high survival and high root:shoot ratio (RSR) under drought, which was not observed in introduced species. As expected, we found that plant species responded differently to modeled rainfall sequences, providing insight into plant adaptations to drought and potential for survival under environmental variation.

Barcode 100K Specimens: In a Single Nanopore Run.

It is a global priority to better manage the biosphere, but action must be informed by comprehensive data on the abundance and distribution of species. The acquisition of such information is currently constrained by high costs. DNA barcoding can speed the registration of unknown animal species, the most diverse kingdom of eukaryotes, as the BIN system automates their recognition. However, inexpensive sequencing protocols are critical as the census of all animal species is likely to require the analysis of a billion or more specimens. Barcoding involves DNA extraction followed by PCR and sequencing with the last step dominating costs until 2017. By enabling the sequencing of highly multiplexed samples, the Sequel platforms from Pacific BioSciences slashed costs by 90%, but these instruments are only deployed in core facilities because of their expense. Sequencers from Oxford Nanopore Technologies provide an escape from high capital and service costs, but their low sequence fidelity has, until recently, constrained adoption. However, the improved performance of its latest flow cells (R10.4.1) erases this barrier. This study demonstrates that a MinION flow cell can characterise an amplicon pool derived from 100,000 specimens while a Flongle flow cell can process one derived from several thousand. At $0.01 per specimen, DNA sequencing is now the least expensive step in the barcode workflow.

RNA transcription assisted universal CRISPR/Cas12a system for programmable analysis of multiple colorectal cancer-associated microRNAs

Accurate analysis of multiple microRNA (miRNA) levels is significantly valuable for early diagnosis of colorectal cancer noninvasively considering the miRNA expression is highly relevant to the occurrence and progression of cancer. However, the low abundance and high sequence homology of miRNAs make their precise determination extremely challenging. Here, we developed a universal and programmable diagnostic strategy allowing for analyzing multiple colorectal cancer-associated miRNAs. The system combined sequentially programmable rolling circle transcription (RCT) and the CRISPR/Cas12a system with high trans-cleavage activity to achieve highly sensitive and specific detection of four target miRNAs. Owing to the remarkable performance of universal RCT-Cas12a strategy, this biosensor could detect miR-21, miR-17, miR-31 and miR-92a with a LOD of 2.1, 1.6, 3.7 and 1.0 pM, respectively. This strategy had a unique advantage in distinguishing human normal colon epithelial cells lines (NCM460) from human colon cancer cells (HT29). In particular, the designed system exhibited superior analytical capability in distinguishing paracancerous and colorectal cancer tissues from patients undergoing colorectal cancer surgery. This arbitrarily programmable, scalable, fast and specific strategy potentially offered an attractive alternative to handle varied challenges encountered with CRISPR-based systems, and held immense promise in scientific research and clinical applications.

Electrostatic Preorganization in Three Distinct Heterogeneous Proteasome β-Subunits.

The origin of the enzyme's powerful role in accelerating chemical reactions is one of the most critical and still widely discussed questions. It is already accepted that enzymes impose an electrostatic field onto their substrates by adopting complex three-dimensional structures; therefore, the preorganization of electric fields inside protein active sites has been proposed as a crucial contributor to catalytic mechanisms and rate constant enhancement. In this work, we focus on three catalytically active β-subunits of 20S proteasomes with low sequence identity (∼30%) whose active sites, although situated in an electrostatically miscellaneous environment, catalyze the same chemical reaction with similar catalytic efficiency. Our in silico experiments reproduce the experimentally observed equivalent reactivity of the three sites and show that obliteration of the electrostatic potential in all active sites would deprive the enzymes of their catalytic power by slowing down the chemical process by a factor of 1035. To regain enzymatic efficiency, besides catalytic Thr1 and Lys33 residues, the presence of aspartic acid in position 17 and an aqueous solvent is required, proving that the electrostatic potential generated by the remaining residues is insignificant for catalysis. Moreover, it was found that the gradual decay of atomic charges on Asp17 strongly correlates with the enzyme's catalytic rate deterioration as well as with a change in the charge distributions due to introduced mutations. The computational procedure used and described here may help identify key residues for catalysis in other biomolecular systems and consequently may contribute to the process of designing enzyme-like synthetic catalysts.

Shotgun metagenomic analysis reveals the diversity of PHA producer bacterial community and PHA synthase gene in Addis Ababa municipal solid waste disposal area ‘Qoshe’

BackgroundPolyhydroxyalkanoates (PHAs) are naturally produced biopolymers with significant scientific and biotechnological potential. This study aimed to investigate the diversity of the PHA-producing bacterial community and PhaC genes in soil samples collected from a municipal solid waste disposal site known as “Qoshe” in Addis Ababa, Ethiopia, using a shotgun metagenomics approach. The SqueezeMeta pipeline was used to analyze the microbial community in the waste samples. A CD search against the TIGRFAM protein family database was performed to identify the complete-length multidomain sequences of PhaC genes and classify them into their respective classes. Statistical analysis and data visualization were performed using RStudio with R version 4.2.3.ResultsThe findings of this study suggest that known and unknown taxa likely contribute to the phaC genes of municipal solid waste. Taxonomic profiling of the metagenomic data revealed that the majority of the PHA-producing taxa belonged to the phylum Proteobacteria (80%), followed by Actinomycetota (16.5%). Furthermore, this study identified Thiomonas and unclassified Mycobacterium as the main contributors to class I PhaC genes. Class II PhaC genes are predominantly associated with the Pseudomonadaceae family, followed by unclassified Hyphomicrobials and Acidimicrobiales. Class III PhaC genes are abundantly related to the Methylococcaceae family, specifically the Methylocaldum genus. The analysis of PhaC gene sequences revealed high level of diversity, with a significant proportion of putative PhaC genes exhibiting low sequence identity with each other and PhaC gene in the database. Notably, the sequence variation observed within the same PhaC gene classes suggests the potential presence of previously unidentified PhaC gene variants.ConclusionsOverall, this research improves our understanding of the diversity of PHA-producing taxa and PhaC genes in municipal solid waste environments, providing opportunities for sustainable PHA production and waste management strategies. However, additional studies, including the isolation and characterization of specific strains, are necessary to confirm the PHA production capabilities of these strains and explore their biotechnological potential.

Characterization of a Novel Acid-Stable Chitosanase from Lentinula edodes Suitable for Chitooligosaccharide Preparation

As high-value chitosan derivatives, chitooligosaccharides (COSs) with biodegradable, biocompatible, nontoxic, antimicrobial, and antioxidant activities have been widely applied in food-related fields. Chitosanases can hydrolyze chitosan to produce COSs. Herein, a chitosanase (LeCho1) from Lentinula edodes was successfully expressed in Escherichia coli and was then purified and characterized. LeCho1 had a low sequence identity with other chitosanases reported from the GH75 family. The recombinant protein showed a molecular mass of 27 kDa on SDS-PAGE. LeCho1 preferentially hydrolyzed chitosan with a high degree of deacetylation (DDA) and exhibited maximal activity (71.88 U/mg) towards 95% DDA chitosan at pH 3.0 and 50 °C. It possessed good stability at pH 2.0–6.0 and temperatures below 45 °C. Its hydrolytic activity was remarkably enhanced by the metal ion Mn2+ at 1 mM, while it was totally inhibited by 1 mM Fe3+ or 10 mM EDTA. Its Km and Vmax values were 0.04 μM and 76.81 μmol·min−1·mg−1, respectively, indicating good substrate affinity. LeCho1 degraded chitosan into COSs with degrees of polymerization (DPs) of 2–5, while it had no action on COSs with DPs of less than 5, revealing its endo-chitosanase activity. This study proved that chitosanase LeCho1 is a promising candidate in the industrial preparation of COSs due to its excellent properties.

Deep mutational scanning of CYP2C19 in human cells reveals a substrate specificity-abundance tradeoff.

The Cytochrome P450s (CYPs) enzyme family metabolizes ∼80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, leading to improper dosing and severe adverse drug reactions. Due to low sequence conservation, predicting variant effects across CYPs is challenging. Even closely related CYPs like CYP2C9 and CYP2C19, which share 92% amino acid sequence identity, display distinct phenotypic properties. Using Variant Abundance by Massively Parallel sequencing (VAMP-seq), we measured the steady-state protein abundance of 7,660 single amino acid variants in CYP2C19 expressed in cultured human cells. Our findings confirmed critical positions and structural features essential for CYP function and revealed how variants at conserved positions influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. Double and triple mutants showed distinct interactions, highlighting a region that points to differing thermodynamic properties between the two homologs. These positions are known contributors to substrate specificity, suggesting an evolutionary tradeoff between stability and enzymatic function. Finally, we analyzed 368 previously unannotated human variants, finding that 43% had decreased abundance. By comparing variant effects between these homologs, we uncovered regions underlying their functional differences, advancing our understanding of this versatile family of enzymes.

FamLink2 – A comprehensive tool for likelihood computations in pedigrees analyses involving linked DNA markers accounting for genotype uncertainties

There is an increasing demand for software that can handle an arbitrary number of linked markers in forensic genetics; primarily with application to inference of relationships and direct matching but also in applications such as ancestry inference and mixture interpretation. With the emergence of sequencing technologies, denser sets of SNP markers are generated and analyzed. Additionally, sequence data of low quality and quantity DNA generate uncertainty about the underlying true genotype. We provide an efficient implementation of a general model for pedigree likelihood computations with genetic marker data using a three-layered approach. The top and first layer is the population model where allele frequencies and population substructure are accounted for. The second layer is the inheritance model which efficiently handles linked markers using an IBD model. The third and bottom layer is the observational level where we model the likelihood of the true genotype given underlying reads as well as parameters for errors. We exemplify the utility of our implementation as well as provide validation according to guidelines established by the ISFG using a combination of two published SNP panels. We demonstrate that computations are feasible for panels encompassing 10,000 markers and we argue that, due to the properties of the underlying algorithm, extending the number of markers will result in a linear increase in computation time. In addition we study the impact of parameters used in our model and suggest some guidelines pertaining to their values. The results demonstrate that a probabilistic model for low coverage sequence read data is needed instead of relying on an a threshold based genotype and applying our general model for inference of relationships on a real case can be superior, i.e. higher information content, to other methods relying on either fixed genotypes with low quality sequence data or simple pair wise relationship tests. In summary, the implementation, FamLink2 (freely available at https://famlink.se), can jointly handle genetic linkage, genotype uncertainty and population substructure for an arbitrary pedigree with data for any number of individuals. Whereas the current study will focus on calculations disregarding mutations, FamLink2 has the ability to model mutations for certain built-in pedigrees.

A study on the development of O2O operation service platform using augmented reality

An attempt of sequence selection is made for multiple alignments of transmembrane proteins in order to detect the structural similarity in the protein families. Treatment for the sequence selection, which is based on the pairwise sequence identities, is applied to ten sequence data sets of functional group of transmembrane proteins extracted from SWISS-PROT. The treatment excludes the sequences with low sequence identity from the divergent data sets effectively. Obtained multiple alignments are evaluated using two newly developed indices. The selected sequences, which are well aligned with few inserted gaps, seem to contain enough information for extracting the structural features of transmembrane functional groups.

Streptococcus suis 5’-nucleotidases contribute to adenosine-mediated immune evasion and virulence in a mouse model

ABSTRACT Streptococcus suis (S. suis) is an important swine bacterial pathogen and causes human infections, leading to a wide range of diseases. However, the role of 5’-nucleotidases in its virulence remains to be fully elucidated. Herein, we identified four cell wall-anchored 5’-nucleotidases (Snts) within S. suis, named SntA, SntB, SntC, and SntD, each displaying similar domains yet exhibiting low sequence homology. The malachite green reagent and HPLC assays demonstrated that these recombinant enzymes are capable of hydrolysing ATP, ADP, and AMP into adenosine (Ado), with the hierarchy of catalytic efficiency being SntC>SntB>SntA>SntD. Moreover, comprehensive enzymatic activity assays illustrated slight variances in substrate specificity, pH tolerance, and metal ion requirements, yet highlighted a conserved substrate-binding pocket, His–Asp catalytic dyad, metal, and phosphate-binding sites across Snts, with the exception of SntA. Through bactericidal assays and murine infection assays involving in site-mutagenesis strains, it was demonstrated that SntB and SntC collaboratively enhance bacterial survivability within whole blood and polymorphonuclear leukocytes (PMNs) via the Ado-A2aR pathway in vitro, and within murine blood and organs in vivo. This suggests a direct correlation between enzymatic activity and enhancement of bacterial survival and virulence. Collectively, S. suis 5’-nucleotidases additively contribute to the generation of adenosine, influencing susceptibility within blood and PMNs, and enhancing survival within blood and organs in vivo. This elucidation of their integral functions in the pathogenic process of S. suis not only enhances our comprehension of bacterial virulence mechanisms, but also illuminates new avenues for therapeutic intervention aimed at curbing S. suis infections.

DNA palette code for time-series archival data storage

Abstract The long-term preservation of large volumes of infrequently accessed cold data poses challenges to the storage community. Deoxyribonucleic Acid (DNA) is considered a promising solution due to its inherent physical stability and significant storage density. The information density and decoding sequence coverage are two important metrics that influence the efficiency of DNA data storage. In this study, we propose a novel coding scheme called DNA Palette code, which is suitable for cold data, especially time-series archival datasets. These datasets are not frequently accessed but necessitate reliable long-term storage for retrospective research. The DNA Palette code employs unordered combinations of index-free oligonucleotides (oligos) to represent binary information. It can achieve high net information density encoding and lossless decoding with low sequencing coverage. When sequencing reads are corrupted, it can still effectively recover partial information, preventing the complete failure of file retrieval. The in vitro testing of clinical brain magnetic resonance imaging (MRI) data storage, as well as simulation validations using large-scale public MRI datasets (10 GB), planetary science datasets, and meteorological datasets, demonstrate the advantages of our coding scheme, including high information density, low decoding sequence coverage, and wide applicability.

Genome Analysis of a Potential Novel Vibrio Species Secreting pH- and Thermo-Stable Alginate Lyase and Its Application in Producing Alginate Oligosaccharides.

Alginate lyase is an attractive biocatalyst that can specifically degrade alginate to produce oligosaccharides, showing great potential for industrial and medicinal applications. Herein, an alginate-degrading strain HB236076 was isolated from Sargassum sp. in Qionghai, Hainan, China. The low 16S rRNA gene sequence identity (<98.4%), ANI value (<71.9%), and dDDH value (<23.9%) clearly indicated that the isolate represented a potential novel species of the genus Vibrio. The genome contained two chromosomes with lengths of 3,007,948 bp and 874,895 bp, respectively, totaling 3,882,843 bp with a G+C content of 46.5%. Among 3482 genes, 3332 protein-coding genes, 116 tRNA, and 34 rRNA sequences were predicted. Analysis of the amino acid sequences showed that the strain encoded 73 carbohydrate-active enzymes (CAZymes), predicting seven PL7 (Alg1-7) and two PL17 family (Alg8, 9) alginate lyases. The extracellular alginate lyase from strain HB236076 showed the maximum activity at 50 °C and pH 7.0, with over 90% activity measured in the range of 30-60 °C and pH 6.0-10.0, exhibiting a wide range of temperature and pH activities. The enzyme also remained at more than 90% of the original activity at a wide pH range (3.0-9.0) and temperature below 50 °C for more than 2 h, demonstrating significant thermal and pH stabilities. Fe2+ had a good promoting effect on the alginate lyase activity at 10 mM, increasing by 3.5 times. Thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI-MS) analyses suggested that alginate lyase in fermentation broth could catalyze sodium alginate to produce disaccharides and trisaccharides, which showed antimicrobial activity against Shigella dysenteriae, Aeromonas hydrophila, Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. This research provided extended insights into the production mechanism of alginate lyase from Vibrio sp. HB236076, which was beneficial for further application in the preparation of pH-stable and thermo-stable alginate lyase and alginate oligosaccharides.

Modelling soil prokaryotic traits across environments with the trait sequence database ampliconTraits and the R package MicEnvMod

We present a comprehensive, customizable workflow for inferring prokaryotic phenotypic traits from marker gene sequences and modelling the relationships between these traits and environmental factors, thus overcoming the limited ecological interpretability of marker gene sequencing data. We created the trait sequence database ampliconTraits, constructed by cross-mapping species from a phenotypic trait database to the SILVA sequence database and formatted to enable seamless classification of environmental sequences using the SINAPS algorithm. The R package MicEnvMod enables modelling of trait – environment relationships, combining the strengths of different model types and integrating an approach to evaluate the models' predictive performance in a single framework. Traits could be accurately predicted even for sequences with low sequence identity (80 %) with the reference sequences, indicating that our approach is suitable to classify a wide range of environmental sequences. Validating our approach in a large trans-continental soil dataset, we showed that trait distributions were robust to classification settings such as the bootstrap cutoff for classification and the number of discrete intervals for continuous traits. Using functions from MicEnvMod, we revealed precipitation seasonality and land cover as the most important predictors of genome size. We found Pearson correlation coefficients between observed and predicted values up to 0.70 using repeated split sampling cross validation, corroborating the predictive ability of our models beyond the training data. Predicting genome size across the Iberian Peninsula, we found the largest genomes in the northern part. Potential limitations of our trait inference approach include dependence on the phylogenetic conservation of traits and limited database coverage of environmental prokaryotes. Overall, our approach enables robust inference of ecologically interpretable traits combined with environmental modelling allowing to harness traits as bioindicators of soil ecosystem functioning.