Putative Enzyme Research Articles

The Soilborne Fungus Verticillium longisporum and Its Interactions with the Brassicaceous Hosts.

Verticillium longisporum, a soilborne fungal species, is the causative agent of Verticillium stripe disease in Brassica species and represents a notable threat to agricultural production, particularly in regions where oilseed rape is a major crop, including Europe, North America, and Asia. The microsclerotia of this pathogen can persist in the soil for extended periods, with a potential lifespan of up to a decade, thereby posing a substantial challenge for the complete eradication of the pathogen from infested soil. The genome of V. longisporum is amphidiploid and resulted from the hybridization of V. dahliae (D genotypes) and an unidentified species (A1 genotype). At least three independent hybridization events are estimated to have occurred, resulting in three distinct lineages: A1/D1, A1/D2, and A1/D3. Genome sequence analysis revealed the presence of mating-type idiomorphs, putative cell wall-degrading enzymes, and effectors. However, due to the complexity of the genome, there is a paucity of research on the molecular interactions between V. longisporum and Brassica crops. This review summarizes the extant knowledge regarding the pathogenicity factors that V. longisporum deploys upon infection and the host immune responses against this attack, highlighting aspects that remain to be elucidated and the molecular tools available for studying this interaction. A better understanding of the molecular interactions in this pathosystem will contribute to developing more effective control measures against this disease in Brassica oilseed and cabbage crops.

Development of a CRISPR-based cytosine base editor for restriction-modification system inactivation to enhance transformation efficiency in Vibrio Sp. dhg

BackgroundVibrio sp. dhg is a fast-growing, alginate-utilizing, marine bacterium being developed as a platform host for macroalgae biorefinery. To maximize its potential in the production of various value-added products, there is a need to expand genetic engineering tools for versatile editing.ResultsThe CRISPR-based cytosine base editing (CBE) system was established in Vibrio sp. dhg, enabling C: G-to-T: A point mutations in multiple genomic loci. This CBE system displayed high editing efficiencies for single and multiple targets, reaching up to 100%. The CBE system efficiently introduced premature stop codons, inactivating seven genes encoding putative restriction enzymes of the restriction-modification system in two rounds. A resulting engineered strain displayed significantly enhanced transformation efficiency by up to 55.5-fold.ConclusionsDeveloping a highly efficient CBE system and improving transformation efficiency enable versatile genetic manipulation of Vibrio sp. dhg for diverse engineering in brown macroalgae bioconversion.

Long-read, multi-amplicon sequencing to explore genetic diversity associated with starch degrading phenotypes in amylolytic Lactobacillaceae.

Characterizing starch-degrading Lactobacillaceae and associated enzymes remains relevant as various industries seek to harness their activity to produce valuable by-products, develop novel food applications, and to aid the sustainable bioconversion of starch-rich resources. To support this, we developed a targeted methodological and analysis framework utilizing complimentary phenomic and genomic assays informative of the starch degrading potential of Lactobacillaceae. Adapted starch agar plate assays incorporating diversified starch sources and states facilitated the rating of extracellular amylolytic activity by starch-processing-line isolates [Lactobacillus amylovorus (n = 3), Lactobacillus amylolyticus (n = 2), and Limosilactobacillus reuteri (n = 2)] as weak to moderate based on the complete or partial hydrolysis of retrograded soluble (SS), or potato and wheat (WS), starches, respectively, and the partial hydrolysis of raw SS. In contrast, the known raw starch degrader, L. amylovorus NRRL B4540, was rated as strong, with complete hydrolysis of all retrograded starch sources and raw WS. To explore genetic diversity and the putative enzymes associated with phenotypic diversity amongst L. amylovorus and L. amylolyticus, a multi-amplicon sequencing approach using MinION™ was used to simultaneously sequence starch-degradation-associated genes identified from them. Gene and deduced amino acid sequence analysis suggested raw starch hydrolysis by L. amylovorus NRRL B4540 was largely attributed to amyA encoding a rare α-amylase with unique starch binding domain (targeting α-1,4 linkages), but which was predicted to also require the starch debranching activity (targeting α-1,6 linkages) associated with (putative) pul-encoded pullulanase (Pul) for complete hydrolysis. Without amyA, Pul was hypothesized necessary for observed starch degradation by L. amylovorus and L. amylolyticus test isolates; as a previously undescribed amylopullulanase with dual activity, or as a pullulanase requiring complimentary α-1,4 activity from an additional enzyme, potentially Gly2 (a putative maltogenic α-amylase). Whilst further work is required to characterize these enzymes, including those encoded by gene variants, the experimental approach described here provided the necessary evidence to warrant this. Further, this framework is likely adaptable for the direct analysis of Lactobacillaceae-rich microbiomes for amylolytic potential and for the targeted screening of various other functions across different taxa.

Gene expression and enzyme activity analysis of carbohydrate digestion in Strongylocentrotus purpuratus larvae.

Carbohydrates play multifaceted roles in marine ecosystems, serving as structural components in algae, energy storage molecules, and vital nutrients for marine organisms. The purple sea urchin Strongylocentrotus purpuratus undergoes metamorphosis during ontogeny, transitioning its feeding strategy from microalgae to macroalgae as the primary food source. However, the digestive mechanisms underlying carbohydrate digestion in sea urchin larvae remain poorly understood. We investigated the carbohydrate digestion capabilities of S. purpuratus larvae, using expression level analysis of candidate genes putatively involved in carbohydrate digestion, quantification of enzyme activity and characterization (pH and temperature optima) for the digestion of starch, laminarin, cellulose, xylan, and trehalose. Transcriptomic analyses revealed the expression of genes encoding putative carbohydrate-degrading enzymes during early larval development. RT-qPCR demonstrated age- and/or feeding-dependent expression patterns of glycosidase candidate genes β-1,3-glucanase (laminarinase), α-amylase, endo-ß-1,4-glucanase D-like (cellulase), xylanase/β-glucanase-like and trehalase. Furthermore, enzymatic assays elucidated differential temporal patterns, and thermal and pH optima of associated carbohydrate-degrading enzymes. A comparison of the enzymatic degradation of five substrates demonstrated that laminarinase activity is five times higher than the enzyme activities involved in digesting starch, cellulose, xylan, and trehalose, opening up a hypothesis regarding the importance of laminarin for larval growth.

Identification and Analysis of Cuticular Wax Biosynthesis Related Genes in Salicornia europaea Under NaCl Treatment.

Salinity is a major environmental factor that adversely affects plant growth and production. Cuticular wax protects plants against external environmental stress. The relationship between cuticular wax biosynthesis and salt tolerance remains unclear in Salicornia europaea. This study examined the cuticle thickness, wax load, morphology, composition, and the expression of cuticular wax biosynthesis gene identification and expression. The results showed that 600 mM NaCl treatment enhanced the cuticle thickness and total wax load; crystal wax structures were also observed after NaCl treatment. The cuticular wax was mainly composed of fatty acids, alcohols, alkenes, and esters. The alcohol class accounted for the largest proportion, with docosanol (C25H54OSi) being the main specific alcohol compound, followed by fatty acids and alkanes. After a sequence database search, six fatty acyl-CoA reductases (FARs), sixteen wax synthase/diacylglycerol acyltransferases (WS/DGATs), three fatty alcohol oxidases (FAOs), five eceriferums (CERs), and eight mid-chain alkanes (MAHs) were identified as the putative wax biosynthesis enzymes. Their expression analysis revealed a differential response to 100 and 600 mM NaCl treatment and reached the highest level at 12 h or 48 h. The genes that were evidently upregulated with higher fold changes under salinity, such as SeFAR1, SeFAR2, and SeFAR3 are implied to synthesize primary alcohols, and SeWSs convert the primary alcohols to wax esters; SeCER1 and SeCER3 are also supposed to catalyze the conversion of aldehydes to alkanes while SeMAH7 catalyze alkanes to secondary alcohols in S. europaea in response to NaCl treatment. This study demonstrated that both the decarbonylation and acyl-reduction wax biosynthesis pathways may not be independent from each other.

Insights into putative alginate lyases from epipelagic and mesopelagic communities of the global ocean

Alginate lyases and oligoalginate lyases catalyze the cleavage of the glycosidic bonds of alginate, an acidic polysaccharide synthesized by brown algae and other organisms. These enzymes are highly diverse, currently classified into 15 families of the Carbohydrate-Active Enzyme (CAZy) database. We explored the structural and taxonomic diversity, the biogeographic distribution of the genes and transcripts, and the potential environmental drivers of putative alginate-degrading enzymes from picoplanktonic communities of the upper layers of the global ocean. The identified sequences were first analyzed using sequence similarity networks to assess their relationship with CAZy members. Sequences related to the PL5, PL6, PL7, PL17, and PL38 families had higher gene and transcript abundances, with temperature being a key driver of the structuring of the community members carrying putative alginate lyase genes. PL5 homologs included variants in a key residue of the active site, and sequences assigned to ‘Candidatus Pelagibacter’ showed high gene and transcript abundances that negatively correlated with inorganic phosphorus concentrations. Sequences assigned to Flavobacteriia and/or Gammaproteobacteria classes dominated the PL6, PL7, and PL17 families, in particular those closely related to sequences from uncultured Polaribacter and Alteromonas australica. In the PL38 family, while sequences assigned to taxa from the Planctomycetota, Verrucomicrobiota, and Bacteroidota phyla showed the highest relative gene abundance at most regions and depths, high expression levels were observed at high latitudes in sequences assigned to Eukaryota (e.g., Phaeocystis antarctica). Overall, the putative enzymes uncovered in this study could be involved in various physiological processes, including alginate assimilation and biosynthesis.

Haloalkane dehalogenases other than LinB can contribute to the γ-hexachlorocyclohexane utilization.

Haloalkane dehalogenases (HLDs) convert halogenated compounds to corresponding alcohols by a simple hydrolytic mechanism. Although many bacterial strains possess HLDs or HLD homologues, LinB is the only HLD known to be involved in the utilization of γ-hexachlorocyclohexane (γ-HCH), a man-made chlorinated pesticide. In this study, to gain insight into the functional evolution of HLDs toward the γ-HCH utilization, the linB gene in γ-HCH-degrading Sphingobium japonicum strain UT26 was replaced by 7 other HLD or HLD homologous genes, including 3 putative ancestral enzymes. Interestingly, strains carrying genes for DmmA from marine metagenome or Rluc_anc, an ancestor of Renilla-luciferin 2-monooxygenase (Rluc) and LinB, produced 2,5-dichlorophenol and 2,5-dichlorohydroquinone from γ-HCH, which are indicators of LinB activity, and grew in minimal medium supplied with γ-HCH as a sole carbon source. These results indicated that other HLDs in addition to LinB can play roles in the γ-HCH utilization.

OXA β-lactamases from Acinetobacter spp. are membrane bound and secreted into outer membrane vesicles.

β-lactamases from Gram-negative bacteria are generally regarded as soluble, periplasmic enzymes. NDMs have been exceptionally characterized as lipoproteins anchored to the outer membrane. A bioinformatics study on all sequenced β-lactamases was performed that revealed a predominance of putative lipidated enzymes in the Class D OXAs. Namely, 60% of the OXA Class D enzymes contain a lipobox sequence in their signal peptide, that is expected to trigger lipidation and membrane anchoring. This contrasts with β-lactamases from other classes, which are predicted to be mostly soluble proteins. Almost all (>99%) putative lipidated OXAs are present in Acinetobacter spp. Importantly, we further demonstrate that OXA-23 and OXA-24/40 are lipidated, membrane-bound proteins in Acinetobacter baumannii. In contrast, OXA-48 (commonly produced by Enterobacterales) lacks a lipobox and is a soluble protein. Outer membrane vesicles (OMVs) from A. baumannii cells expressing OXA-23 and OXA-24/40 contain these enzymes in their active form. Moreover, OXA-loaded OMVs were able to protect A. baumannii, Escherichia coli, and Pseudomonas aeruginosa cells susceptible to piperacillin and imipenem. These results permit us to conclude that membrane binding is a bacterial host-specific phenomenon in OXA enzymes. These findings reveal that membrane-bound β-lactamases are more common than expected and support the hypothesis that OMVs loaded with lipidated β-lactamases are vehicles for antimicrobial resistance and its dissemination. This advantage could be crucial in polymicrobial infections, in which Acinetobacter spp. are usually involved, and underscore the relevance of identifying the cellular localization of lactamases to better understand their physiology and target them.IMPORTANCEβ-lactamases represent the main mechanism of antimicrobial resistance in Gram-negative pathogens. Their catalytic function (cleaving β-lactam antibiotics) occurs in the bacterial periplasm, where they are commonly reported as soluble proteins. A bioinformatic analysis reveals a significant number of putative lipidated β-lactamases, expected to be attached to the outer bacterial membrane. Notably, 60% of Class D OXA β-lactamases (all from Acinetobacter spp.) are predicted as membrane-anchored proteins. We demonstrate that two clinically relevant carbapenemases, OXA-23 and OXA-24/40, are membrane-bound proteins in A. baumannii. This cellular localization favors the secretion of these enzymes into outer membrane vesicles that transport them outside the boundaries of the cell. β-lactamase-loaded vesicles can protect populations of antibiotic-susceptible bacteria, enabling them to thrive in the presence of β-lactam antibiotics. The ubiquity of this phenomenon suggests that it may have influenced the dissemination of resistance mediated by Acinetobacter spp., particularly in polymicrobial infections, being a potent evolutionary advantage.

The Assembly of Fish Gut Microbiomes Through Habitat Variation Provides Insight Into Redbelly Tilapia Invading a Large Subtropical River.

The environment in which fish reside markedly shapes the composition of their gut microbiome. However, the precise mechanisms by which the assembly process of fish gut microbiota adapts to diverse habitat conditions remain largely uncharted, especially in the case of invasive tilapia, renowned for its remarkable adaptability to environmental changes. In this study, we employed high-throughput 16S rRNA gene sequencing to explore the gut microbiome of redbelly tilapia from three distinct habitats. Our results showed substantial disparities in both the composition and diversity (alpha and beta) of the gut microbiome between wild and pond-cultured redbelly tilapia. Notably, stochastic processes emerged as the dominant forces governing the assembly of the gut microbial community in redbelly tilapia. As the habitat shifted from pond-cultured to wild, the influence of undominated processes in gut microbial community assembly waned, while the effect of dispersal limitation intensified. Co-occurrence network analysis suggested that habitat variation contributed to the enhanced complexity of the gut microbial network in invasive tilapia during their transition from pond to wild environments. Across the three distinct habitats, variations were observed in the influence of environmental factors on the gut microbiota of tilapia. Moreover, our findings demonstrated that the gut microbiome of wild tilapia possessed unique characteristics, such as higher alpha diversity and a relatively greater abundance of genes encoding putative cellulolytic enzymes crucial for digesting the preferred food source of tilapia (hydrophytes), particularly in fragmented habitats with well-developed cascade dams. Additionally, we identified that OTU8895 (Clostridum_sensu_stricto_1) and OTU11387 (unclassified Bacilli) were core biomarkers of the gut bacterial community in wild redbelly tilapia, offering valuable insights for the monitoring and management of invasive tilapia populations.

Genomic insights into the probiotic potential and genes linked to gallic acid metabolism in Pediococcus pentosaceus MBBL6 isolated from healthy cow milk.

Pediococcus pentosaceus is well known for its probiotic properties, including roles in improving health, antimicrobial production, and enhancing fermented food quality. This study aimed to comprehensively analyze the whole genome of P. pentosaceus MBBL6, isolated from healthy cow milk, to assess its probiotic and antimicrobial potentials. P. pentosaceus MBBL6, isolated from a healthy cow milk at BSMRAU dairy farm, Gazipur, Bangladesh, underwent comprehensive genomic analysis, including whole genome sequencing, assembly, annotation, phylogenetic comparison, and assessment of metabolic pathways and secondary metabolites. Antimicrobial efficacy was evaluated through in-vitro and in-vivo studies, alongside in-silico exploration for potential mastitis therapy. We predicted 1,906 genes and 204 SEED sub-systems involved in carbohydrate metabolism and vitamin B complex biosynthesis, with a focus on lactose metabolism in MMBL6. Notably, 43 putative carbohydrate-active enzyme genes, including lysozymes, suggest the ability of MBBL6 for carbohydrate biotransformation and antimicrobial activity. The genome also revealed primary metabolic pathways for arginine and gallic acid metabolism and secondary metabolite gene clusters, including T3PKS and RiPP-like regions. Importantly, two bacteriocin biosynthesis gene clusters namely bovicin_255_variant and penocin_A, were identified in MBBL6. The safety assessment of MBBL6 genome revealed no virulence genes and a low pathogenicity score (0.196 out of 1.0). Several genes related to survival in gastrointestinal tract and colonization were also identified. Furthermore, MBBL6 exhibited susceptibility to a wide range of antibiotics in-vitro, and effectively suppressed mastitis pathogens in an in-vivo mouse mastitis model trial. The observed bacteriocin, particularly bovicin, demonstrated the ability to disrupt the function of an essential protein, Rho factor of mastitis pathogens by blocking transcription termination process. Taken together, our in-depth genomic analysis underscores the metabolic versatility, safety profile, and antimicrobial potential of P. pentosaceus MBBL6, suggesting its promise for applications in therapeutics, bioremediation, and biopreservation.

Antidiabetic potency of glimepiride and naringin: an in silico and in vitro investigation

Glimepiride (GLM) is one of the potential antidiabetic drugs used in clinics for a long time. It is currently used in combination with metformin along with other drugs, but has shown various complications in patients from long-term use. Thus, the hypothesis is to use a lower dose of GLM with a non-toxic class of flavonoid, naringin (NARN), for better therapy with minimal side-effects. Initially, we assessed the binding efficacy of GLM and NARN against nine putative target enzymes using AutoDock 4.2 software. We also analysed the drug chemistry, drug-ability, and cytotoxicity, as well as performed molecular dynamic (MD) simulation at 100 ns with individual and combination states using GROMACS-2022 software. Both candidates showed higher binding efficacy, especially against the AKT-serine/threonine kinase-1 (AKT1) target enzyme (−11.85 kcal/mol), and demonstrated higher stability and compatibility with AKT1 from MD-simulation (based on RMSD, Rg, RMSF, and H-bond plots) in combination than individual form. The in vitro cytotoxicity with human embryonic kidney (HEK-293) cells suggested 100 µg/mL (observed 80% of the cell viability) as a non-toxic dose for further study. Alpha-amylase, alpha-glucosidase, and DPP-IV inhibition assays revealed that both GLM and NARN inhibited up to 60% at 100 µg/mL in a concentration-dependent manner. At the end, selecting a lower dose of GLM and a higher dose of NARN (2:8 v/v ratio) showed up to 87% inhibition at 100 µg/mL. Both in silico and in vitro studies suggest that the investigated formulation could be a potential and non-toxic dose for diabetics.

Abstract 4141772: “SETDB2 regulates the Innate Immune Memory response and influences Inflammatory Immune landscape in Atherosclerosis”

Background: Several pro-inflammatory signals, including microbial cell components such as β-glucan or metabolites produced during western-diet feeding, function as initial “triggers” that train macrophages to respond more robustly to a later secondary inflammatory challenge. This crude “memory” response in innate immune cells is termed as “trained immunity." This memory response is mediated, at least in part, through epigenomic changes to the chromatin landscape. Aim: Role of SETDB2 in trained immunity and atherosclerosis. Results: Our preliminary findings suggest that SETDB2, a putative epigenomic enzyme that is a member of the KMT1 family of lysine methyltransferases, is induced by β-glucan training in macrophages, and knocking out SETDB2 in myeloid cells (SETDB2mKO) altered the magnitude of the secondary pro-inflammatory response in the two-step training protocol described in Background. RNA sequencing revealed a dynamic profile of transcripts that were altered in SETDB2mKO with different gene clusters, consistent with SETDB2 positively regulating some genes while negatively regulating others. We also developed SETDB2 knock-in (SETDB2KI) mice that crippled its potential enzymatic function. Interestingly, SETDB2KI data suggested that SETDB2’s enzyme activity is essential for its role in negatively regulating genes, while positively regulated genes require SETDB2 but not its enzyme activity. Several metabolic and inflammatory pathway genes were regulated by SETDB2, as were several genes known to be involved in immune cell infiltration. Bone marrow transplantation from global SETDB2-deficient mice into LDLRKO resulted in more severe atherosclerotic plaques following western diet feeding, and single-cell RNA sequencing revealed increased neutrophil and reduced CD4+ T-cell populations in lesions from the SETDB2-deficient plaques. When combined together, the positive and negative regulation of different gene clusters by SETDB2 in the in-vitro training study and the more severe atherosclerosis with altered immune profiles in the SETDB2-deficient plaques in vivo suggest that SETDB2 positively and negatively regulates CD4+ T cell and neutrophil recruitment to atherosclerotic plaques, respectively. Conclusion: Our preliminary findings show that SETDB2 regulates immuno-metabolic pathway genes via two different mechanisms, which might also influence the immune cell inflammatory landscape and severity of atherosclerosis.

Virulence Determinants Are Unevenly Distributed Within Streptomyces Species and Strains Causing Potato Common Scab in the Province of Quebec, Canada.

Potato common scab is an important bacterial disease afflicting potatoes around the world. Better knowledge of the local Streptomyces spp. populations causing this disease is key to developing durable control strategies. In this study, we isolated 230 Streptomyces strains from scab-infected potato tubers harvested from commercial potato fields located across the province of Quebec, Canada. The genetic diversity of this collection was first studied using repetitive element-based PCR fingerprinting, and the genomes of 36 representative strains were sequenced using PacBio's sequencing technology. This enabled us to identify the strains to the species level, to study the distribution of previously characterized virulence-associated genes and clusters, and to explore the repertoires of putative plant cell wall-degrading enzymes. In parallel, the virulence of the 36 strains was evaluated using a potato tuber slice assay. The diversity was higher than previously reported, as 11 phytopathogenic species were found across the province. Among them, S. scabiei and S. acidiscabies were the most abundant as well as the most virulent. Strains belonging to these two species harbored numerous virulence determinants, including the thaxtomin biosynthetic gene cluster. By contrast, most weakly virulent strains lacked this cluster but harbored at least one known virulence determinant. The results obtained suggest that a higher number of virulence-associated genes and clusters in the genome of phytopathogenic Streptomyces spp. are associated with greater virulence. This study contributes to increasing the publicly available genomic resources of scab-causing Streptomyces spp. and expands our knowledge on the diversity and virulence of this important bacterial pathogen.

Biodegradation of dimethachlon by Arthrobacter sp. K5: Mechanistic insights and ecological implications

Biodegradation of dimethachlon by Arthrobacter sp. K5: Mechanistic insights and ecological implications

Integrated metabolite profiling and transcriptome analysis reveal candidate genes involved in the biosynthesis of benzylisoquinoline alkaloids in Corydalis solida

Structurally diverse benzylisoquinoline alkaloids (BIAs) are found in specific plant families, some of which are desirable for their efficient production because of their strong biological activities. Corydalis plants (e.g., Corydalis yanhusuo) of the family Papaveraceae also produce various BIAs; thus, they have been used in traditional Chinese medicine. Because metabolic engineering and synthetic biology using microorganisms are promising technologies for the effective production of useful metabolites, elucidation of the biosynthetic pathway of each BIA is indispensable. Although several enzyme genes involved in the biosynthesis of Corydalis BIAs have recently been isolated, many remain unknown, such as the protoberberine alkaloid C-methyltransferase involved in the biosynthesis of corydaline, one of the main BIAs found in the tubers of Corydalis plants. In this study, we performed transcriptome analysis combined with metabolite profiling of different tissues of Corydalis solida. Based on the high accumulation of several BIAs, including protopine, allocryptopine, and corydaline, genes encoding putative biosynthetic enzymes, including cytochrome P450, methyltransferase, and oxidase proteins, that were highly expressed in the tubers were screened. Two OMT genes, CsOMT1 and CsOMT2, were highly expressed in the tuber, and further characterization using crude enzyme preparations demonstrated that CsOMT1 showed 7-O-methylation activity against reticuline, whereas CsOMT2 catalyzed 9-O-methylation of scoulerine, followed by 2-O-methylation of tetrahydrocolumbamine. Our findings provide valuable information for the isolation of novel biosynthetic enzyme genes in Corydalis species.

Analysis of Plant and Fungal Transcripts from Resistant and Susceptible Phenotypes of Leptospermum scoparium Challenged by Austropuccinia psidii.

Austropuccinia psidii is the causal pathogen of myrtle rust disease of Myrtaceae. To gain understanding of the initial infection process, gene expression in germinating A. psidii urediniospores and in Leptospermum scoparium-inoculated leaves were investigated via analyses of RNA sequencing samples taken 24 and 48 h postinoculation (hpi). Principal component analyses of transformed transcript count data revealed differential gene expression between the uninoculated L. scoparium control plants that correlated with the three plant leaf resistance phenotypes (immunity, hypersensitive response, and susceptibility). Gene expression in the immune resistant plants did not significantly change in response to fungal inoculation, whereas susceptible plants showed differential expression of genes in response to fungal challenge. A putative disease resistance gene, jg24539.t1, was identified in the L. scoparium hypersensitive response phenotype family. Expression of this gene may be associated with the phenotype and could be important for further understanding the plant hypersensitive response to A. psidii challenge. Differential expression of pathogen genes was found between samples taken 24 and 48 hpi, but there were no significant differences in pathogen gene expression that were associated with the three different plant leaf resistance phenotypes. There was a significant decrease in the abundance of fungal transcripts encoding three putative effectors and a putative carbohydrate-active enzyme between 24 and 48 hpi, suggesting that the encoded proteins are important during the initial phase of infection. These transcripts, or their translated proteins, may be potential targets to impede the early phases of fungal infection by this wide-host-range obligate biotrophic basidiomycete.

Streptomyces castrisilvae sp. nov. and Streptomyces glycanivorans sp. nov., novel soil streptomycetes metabolizing mutan and alternan.

Six bacterial strains, Mut1T, Mut2, Alt1, Alt2, Alt3T, and Alt4, were isolated from soil samples collected in parks in Gothenburg, Sweden, based on their ability to utilize the insoluble polysaccharides α-1,3-glucan (mutan; Mut strains) or the mixed-linkage α-1,3/α-1,6-glucan (alternan; Alt strains). Analysis of 16S rRNA gene sequences identified all strains as members of the genus Streptomyces. The genomes of the strains were sequenced and subsequent phylogenetic analyses identified Mut2 as a strain of Streptomyces laculatispora and Alt1, Alt2 and Alt4 as strains of Streptomyces poriferorum, while Mut1T and Alt3T were most closely related to the type strains Streptomyces drozdowiczii NBRC 101007T and Streptomyces atroolivaceus NRRL ISP-5137T, respectively. Comprehensive genomic and biochemical characterizations were conducted, highlighting typical features of Streptomyces, such as large genomes (8.0-9.6 Mb) with high G+C content (70.5-72.0%). All six strains also encode a wide repertoire of putative carbohydrate-active enzymes, indicating a capability to utilize various complex polysaccharides as carbon sources such as starch, mutan, and cellulose, which was confirmed experimentally. Based on phylogenetic and phenotypic characterization, our study suggests that strains Mut1T and Alt3T represent novel species in the genus Streptomyces for which the names Streptomyces castrisilvae sp. nov. and Streptomyces glycanivorans sp. nov. are proposed, with strains Mut1T (=DSM 117248T=CCUG 77596T) and Alt3T (=DSM 117252T=CCUG 77600T) representing the respective type strains.

Evolutionary History of the DD41D Family of Tc1/Mariner Transposons in Two Mayetiola Species.

Tc1/mariner elements are ubiquitous in eukaryotic genomes including insects. They are diverse and divided into families and sub-families. The DD34D family including mauritiana and irritans subfamilies have already been identified in two closely related species of Cecidomyiids M. destructor and M. hordei. In the current study the de novo and similarity-based methods allowed the identification for the first time of seven consensuses in M. destructor and two consensuses in M. hordei belonging to DD41D family whereas the in vitro method allowed the amplification of two and three elements in these two species respectively. Most of identified elements accumulated different mutations and long deletions spanning the N-terminal region of the transposase. Phylogenetic analyses showed that the DD41D elements were clustered in two groups belonging to rosa and Long-TIR subfamilies. The age estimation of the last transposition events of the identified Tc1/mariner elements in M. destructor showed different evolutionary histories. Indeed, irritans elements have oscillated between periods of silencing and reappearance while rosa and mauritiana elements have shown regular activity with large recent bursts. The study of insertion sites showed that they are mostly intronic and that some recently transposed elements occurred in genes linked to putative DNA-binding domains and enzymes involved in metabolic chains. Thus, this study gave evidence of the existence of DD41D family in two Mayetiola species and an insight on their evolutionary history.

Characterization of Potential Plastic-Degradation Enzymes from Marine Bacteria.

Polyethylene terephthalate (PET) and polyethylene (PE) are prominent polymer materials that comprise a significant portion of commercial plastic waste. Their durability and slow degradation rate have resulted in significant accumulation of plastic on Earth. In a recent study, macrotranscriptomic profiling of a reconstituted marine bacterial community identified 10 putative enzymes capable of directly acting on PE or PET (PEases or PETases). Among these enzymes, three recombinant proteins were reported to possess PE degradation activity. To select potential plastic degrading enzyme candidates for protein engineering efforts, we expressed and purified eight out of the 10 candidates, excluding two due to poor expression and/or solubility. Notably, several candidate proteins displayed significant esterase activity on p-nitrophenyl butyrate and exhibited unexpected thermostability despite their marine origin. Additionally, we observed dose- and time-dependent hydrolytic activity on the PET trimer substrate. Structural analysis and mutagenesis of a candidate protein confirmed the presence of catalytic triad residues, classifying it as an esterase. Furthermore, we elucidated the structural importance of the two disulfide bonds. Through point mutation experiments, we observed an enhanced hydrolytic activity of a selected enzyme candidate on PET nanoparticles. Our findings challenge the classification of the enzymes directly acting on PE and highlight the significance and complexity of validating PE degradation enzymes identified through metagenomic analysis.

The Response of the Gut Physiological Function and Microbiome of a Wild Freshwater Fish (Megalobrama terminalis) to Alterations in Reproductive Behavior.

The fish gut microbiome is well known for its role in degrading nutrients to improve the host's digestion and absorption efficiency. In this study, we focused on the core physiological adaptability during the various reproductive stages of the black Amur bream (Megalobrama terminalis) to explore the interaction mechanisms among the fish host gut mucosal structure, gut enzyme activity, and gut microbial metabolism in the course of the host's reproductive cycle. Our findings showed that M. terminalis exhibited locomotion metabolic type (aids in sporting) in the reproductive stage, and a change to visceral metabolic type (aids in digestion) during non-reproductive and post-reproductive stage phases. The impact of metabolic type selection and energy demand during various reproductive stages on fish nutrition strategy and digestive function was substantial. Our resulted showed that mitochondria in intestinal epithelial cells of reproductive M. terminalis appeared autophagy phenomenon, and the digestive enzyme activities in the intestines of reproductive M. terminalis were lower than those in the non-reproductive and post-reproductive individuals. Moreover, these differences in nutrition strategy have a prominent impact on the gut microbiome of reproductive M. terminalis, compared to non-reproductive and post-reproductive samples. Our findings showed that reproductive females had lower levels of alpha diversity compared to non-reproductive and post-reproductive females. Our results also showed a greater functional variety and an increase in functional genes related to carbohydrate, lipid, amino acid, cofactors, and vitamin metabolic pathways in the NRS and PRS group. It is noteworthy that an enrichment of genes encoding putative enzymes implicated in the metabolism of taurine and hypotaurine was observed in the RS samples. Our findings illustrated that the stability and resilience of the gut bacterial community could be shaped in the wild fish host-microbiome interactions during reproductive life history.