Applied and Environmental Microbiology最新文献

{"title":"Structural and catalytic characterization of the 2,5-dihydroxypyridine ring-cleaving dioxygenase Hpo from <i>Pseudomonas putida</i> S16.","authors":"Zhenyu Cheng, Xu Han, Hongli Wei, Haiyang Hu, Ping Xu, Weidong Liu, Hongzhi Tang","doi":"10.1128/aem.01467-26","DOIUrl":"https://doi.org/10.1128/aem.01467-26","url":null,"abstract":"<p><p>2,5-Dihydroxypyridine (2,5-DHP) is a potentially carcinogenic intermediate in pyridine catabolism. Hpo, a non-heme Fe(II)-dependent dioxygenase from <i>Pseudomonas putida</i> S16, catalyzes ring cleavage of 2,5-DHP, yet its structure has remained uncharacterized. We determined the crystal structure of Hpo at 2.56 Å resolution, revealing a catalytic Fe center coordinated by His257, His310, and Asp312 in a conserved 2-His-1-carboxylate motif, with Ser294 positioned directly below the iron and essential for iron binding. The substrate-binding pocket, formed primarily by Phe94, Phe97, Phe186, Phe188, and Arg285, incorporates potential hydrogen-bonding interactions from Glu169 and His181. Hpo employs a distinctive Phe94-Leu95-Leu96-Phe97 motif for substrate recognition, contrasting with the Met102-Leu103-Leu104-His105 motif in the isoenzyme NicX. Molecular dynamics simulations spanning 200 ns demonstrated that Phe94 and Phe97 mutations destabilized substrate binding, confirming their role in substrate recognition, and explaining the corresponding loss of catalytic activity. These findings reveal the active-site architecture and substrate engagement mechanism of Hpo, advancing our understanding of pyridine ring-cleaving non-heme Fe(II)-dependent dioxygenases.IMPORTANCEPyridine compounds are common environmental contaminants that pose risks to human health. The microbial degradation of pyridine compounds generates toxic intermediates, including 2,5-dihydroxypyridine (2,5-DHP), which require enzymatic detoxification. Prior to this study, only one 2,5-DHP dioxygenase had been structurally characterized, leaving the structural basis of this rare enzyme subgroup unresolved. By determining the Hpo crystal structure and identifying residues critical for iron coordination and catalysis, this work addresses a significant knowledge gap and establishes a structural framework for understanding pyridine ring-cleaving non-heme Fe(II)-dependent dioxygenases and may also provide an enzymatic perspective on the removal of pyridine contaminants from the environment.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0146726"},"PeriodicalIF":4.2,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assembly mechanisms and successional patterns of sediment bacterial communities along a riverine wetland restoration chronosequence.","authors":"Yongjuan Chen, Chenyang Li, Shuyi Xie, Lingling Zhu, Mingxiang Liu, Hengpeng Li, Wangshou Zhang","doi":"10.1128/aem.01355-26","DOIUrl":"https://doi.org/10.1128/aem.01355-26","url":null,"abstract":"<p><p>Understanding the assembly mechanisms of bacterial communities in sediment is vital for assessing ecological outcomes of wetland restoration. This study examined these processes within sediment bacterial communities along a restoration chronosequence (2, 4, and 8 years) in riverine wetlands. Employing multi-seasonal sampling combined with 16S rRNA gene sequencing, null model frameworks, and co-occurrence network analyses, we observed distinct stage-dependent differences in community assembly. The 2-year wetland was characterized by a relatively high contribution of stochastic processes (notably dispersal limitation), although deterministic processes already dominated assembly. With increasing restoration age, the influence of deterministic processes, especially homogeneous selection, strengthened and remained dominant through the late stage (8 years). Community assembly exhibited significant spatiotemporal variation, with seasonal fluctuations strongly associated with this variation. Concurrently, bacterial co-occurrence networks demonstrated increased modularity, indicative of increasingly modular, functionally compartmentalized community structures. Network topology differed consistently along the restoration gradient, from simple and loosely connected to highly complex and modular configurations, reflecting ecosystem maturation. These findings reveal a pattern of increasing deterministic selection and network modularity with restoration age, thereby advancing understanding of microbial processes during wetland restoration and providing a scientific foundation for ecosystem recovery assessment.IMPORTANCEWetland restoration is widely regarded as a nature-based solution, yet the microbial recovery trajectories following restoration remain poorly characterized. Our study reveals a consistent pattern across restoration ages in sediment bacterial communities across 2-, 4-, and 8-year restored wetlands: from relatively high stochasticity alongside dominant deterministic selection, to strengthened deterministic dominance, and finally to modular, functionally compartmentalized networks. This pattern is associated with restoration duration itself, not with measured environmental factors, and occurs despite strong seasonal variation. Importantly, we show that co-occurrence network modularity and robustness can serve as quantitative, sensitive indicators of ecosystem maturation-more informative than traditional diversity indices. These findings provide a scientific basis for evaluating restoration success and suggest that allowing at least 8 years is necessary for functional stabilization. Our work offers microbial ecological criteria to guide adaptive management and engineering decisions in nature-based wetland restoration.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0135526"},"PeriodicalIF":4.2,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178751","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dinoflagellate blooms reshape bacterioplankton assembly and alter free-living and particle-attached community dynamics with implications for harmful algal bloom processes.","authors":"Huatao Yuan, Ling Li, Senjie Lin","doi":"10.1128/aem.00723-26","DOIUrl":"https://doi.org/10.1128/aem.00723-26","url":null,"abstract":"<p><p>Although shifts in bacterial communities during harmful algal blooms (HABs) have been studied extensively, the assembly processes of bacterioplankton communities across complete bloom cycles remain insufficiently resolved. In this study, based on 16S rRNA gene high-throughput sequencing, we characterized the succession and assembly of bacterioplankton communities across four stages of a coastal bloom caused by <i>Scrippsiella acuminata</i>. We found that the alpha diversity of free-living (FL) bacterioplankton decreased with bloom progression, whereas the alpha diversity of particle-attached (PA) bacterioplankton increased during bloom development. Among taxa that responded strongly to the bloom, Rhodobacteraceae, Flavobacteriaceae, and SAR86_clade showed marked changes in abundance. Null-model analysis suggests that stochastic processes, mainly drift and dispersal limitation, dominated the assembly of both FL and PA bacterial communities, but that the bloom increased the influence of deterministic processes, especially selection. Rhodobacteraceae showed the closest association with the bloom-forming dinoflagellate and may participate in infochemical exchange through compounds such as indole-3-acetic acid (IAA). Together, these results show that bloom progression restructures FL and PA bacterioplankton differently while shifting community assembly toward stronger environmental filtering.IMPORTANCEHarmful algal blooms (HABs) reshape microbial food webs and biogeochemical cycling in coastal waters. This study shows that free-living and particle-attached bacterioplankton respond differently to bloom progression, with contrasting diversity trajectories and distinct taxonomic shifts. It also demonstrates that stochastic assembly remains dominant throughout the bloom, whereas deterministic selection becomes more important during bloom stages. These findings provide a mechanistic framework for understanding how HABs restructure marine bacterioplankton and identify bacterial groups potentially involved in bloom-associated interactions.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0072326"},"PeriodicalIF":4.2,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cross-domain microbiome legacy of a natural <i>Phaeocystis globosa</i> bloom in eutrophic coastal waters: a framework for monitoring bloom onset, collapse, and post-bloom recovery.","authors":"Nianrong Yin, Satoshi Ishii, Yanrong Zhou, Jingming Hu, Xingchen Xia, Haiming Wang, Yue Wu, Milko A Jorquera, Zhe Du, Qian Zhang","doi":"10.1128/aem.01489-26","DOIUrl":"https://doi.org/10.1128/aem.01489-26","url":null,"abstract":"<p><p>When a harmful algal bloom collapses, does the coastal water quality recover? We addressed this question using the natural bloom of the haptophyte <i>Phaeocystis globosa</i> in coastal waters around Xiamen Island, China. Bacterial and microeukaryotic communities in the water samples (<i>n</i> = 105) monthly collected from five sites from September 2023 to March 2024 were analyzed using 16S and 18S rRNA amplicon sequencing, genotype-specific qPCR, iCAMP-based assembly inference, partial least squares path modeling, bloom stage-specific co-occurrence networks, and amplicon-based functional prediction. Bloom development drove coordinated physicochemical change. Both bacterial and microeukaryotic communities were displaced from their pre-bloom state but did not return to the original state within the 7-month series. However, their patterns of succession were different. Bacterial communities underwent coherent system-wide succession. They diverged spatially during the bloom. Their assembly shifted toward stochasticity, with the deterministic contribution falling from 46.3% pre-bloom to 27.8% during the bloom and recovering only to 30.3% post-bloom. Their co-occurrence network was most complex during the bloom period. In contrast, microeukaryotic communities retained stronger site-specific structure throughout succession, and their network connectivity peaked after the bloom collapsed. Water quality variables explained 45.9% of bacterial but only 13.1% of microeukaryotic community variation. Predicted functional profiles also showed stage-associated changes. The same bloom thus pushed bacterial and microeukaryotic communities toward different post-disturbance configurations on different timescales. We therefore propose a cross-domain monitoring framework that integrates stage-specific taxa, a community state index, and network complexity. This framework can diagnose bloom onset, collapse, and genuine post-bloom recovery in eutrophic coastal waters.IMPORTANCEHarmful algal blooms disturb coastal waters well beyond the period when the causative alga is abundant. This study shows that the effects of a <i>Phaeocystis globosa</i> bloom on the coastal microbiome lasted after the bloom itself ended and that bacteria and microeukaryotes recovered along different paths and on different timescales. Algal counts or nutrient concentrations alone therefore cannot adequately capture the recovery of coastal water quality. We suggest an alternative monitoring framework that combines stage-specific indicator taxa, a community state index, and network topology, so that coastal water quality can be assessed more reliably.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0148926"},"PeriodicalIF":4.2,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phosphonate degradation through the C-P lyase pathway by a dinoflagellate-associated <i>Mameliella alba</i> enhances host growth under phosphate limitation.","authors":"Yudong Cui, Jinghao Long, Wenhui Xiong","doi":"10.1128/aem.01846-26","DOIUrl":"https://doi.org/10.1128/aem.01846-26","url":null,"abstract":"<p><p>Phosphonates are distinguished by their chemically stable carbon-phosphorus (C-P) bonds and constitute approximately 25% of the dissolved organic P pool in the ocean. Most eukaryotic phytoplankton, including dinoflagellates, are unable to directly utilize extracellular phosphonates. However, their associated bacteria can facilitate indirect exploitation of this P source. This study presents the isolation and characterization of strain DH-1, a dinoflagellate-associated bacterium capable of phosphonate degradation. DH-1 was identified as <i>Mameliella alba</i>, and its genome contains a complete C-P lyase gene cluster. Growth assays demonstrated that DH-1 could maintain growth under conditions with 2-aminoethylphosphonate (2-AEP) as the sole P source, indicating that it can utilize this biogenic phosphonate. RT-qPCR analyses revealed that transcription levels of the C-P lyase subunits <i>phnJ</i> and <i>phnH</i>, as well as the phosphonate transport genes <i>phnC</i> and <i>phnD</i>, were significantly upregulated under phosphate-limited conditions. Furthermore, the expression of <i>phnJ</i> and <i>phnH</i> was also induced by 2-AEP, indicating that C-P lyase pathway in DH-1 is co-regulated by the Pho regulon and substrate induction. The growth of the dinoflagellate <i>Prorocentrum donghaiense</i> was significantly enhanced when both DH-1 and 2-AEP were added to P-limited cultures, whereas the alga did not grow in the presence of either 2-AEP or DH-1 alone. This study presents the first example of a specific dinoflagellate-associated bacterium that can promote algal growth by degrading phosphonates to release phosphate. These interactions are vital for the ecological success of dinoflagellates that live in P-limited environments, offering novel insights into the formation and dynamics of harmful algal blooms.IMPORTANCEPhosphonates are an important component of dissolved organic phosphorus in the ocean. They can serve as a potential phosphorus source when phosphate, which is directly bioavailable to phytoplankton, is scarce in marine environments. While eukaryotic phytoplankton typically lack the capacity to utilize phosphonates, certain algal-associated bacteria possess the ability to degrade these compounds. However, no specific strains have been thoroughly examined. Here, we report the discovery of a bacterial strain capable of phosphonate utilization through a specific metabolic pathway. This bacterium was isolated from the dinoflagellate <i>Prorocentrum donghaiense</i>, one of the most prevalent red tide-forming species in China. It can promote the growth of <i>P. donghaiense</i> by supplying phosphate derived from phosphonate degradation. These findings highlight the critical role of bacteria in facilitating phosphonate utilization by dinoflagellates. This mechanism may contribute to the development of harmful algal blooms, which underscores the ecological importance of the algae-bacteria interactions in marine ecosystems.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0184626"},"PeriodicalIF":4.2,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178864","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A high-throughput pipeline for isolation and screening of the citrus bacteriome identifies <i>Pseudomonas chlororaphis</i> with anti-<i>Candidatus</i> Liberibacter asiaticus activity.","authors":"Christopher Drozd, Kathryn Maebori, Carolina Ruiz Rivera, Kiana Callahan, Manikandan Ramasamy, Kranthi Mandadi, Katherine N Maloney, M Caroline Roper","doi":"10.1128/aem.01576-26","DOIUrl":"https://doi.org/10.1128/aem.01576-26","url":null,"abstract":"<p><p>Plant microbiomes are a rich source of secondary metabolites, often with pathogen- or disease-suppressive traits. Huanglongbing (HLB) is a serious disease of citrus for which there is currently no cure. Here, we tested the hypothesis that bacteria native to the citrus microbiome may possess inhibitory activity against the associated causal agent of HLB, <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las). We coupled a robotic microfluidic isolation platform with a high-throughput antimicrobial screening assay using <i>Liberibacter crescens</i>, a culturable surrogate for the unculturable <i>C</i>Las, to identify isolates with anti-<i>Liberibacter</i> activity. We identified 35 bacterial isolates with activity using this pipeline. One strongly inhibitory citrus root isolate, <i>Pseudomonas chlororaphis</i> (SRAM11), was selected for bioassay-guided fractionation. We determined a significant driver of <i>in vitro Liberibacter</i> inhibition was attributed to the production of phenazine-1-carboxamide (PCN) by <i>P. chlororaphis</i>. We also demonstrated significant anti-<i>C</i>Las activity of PCN, comparable to that of oxytetracycline, using an <i>ex vivo</i> citrus hairy root assay. <i>P. chlororaphis</i> and phenazines have been implicated in successful biocontrol of other diseases and represent a new tool with possible application to HLB mitigation.IMPORTANCEHuanglongbing (HLB) is a devastating citrus disease that causes millions of dollars in crop loss each year in the United States alone. In this study, we mined the citrus microbiome for bacteria that produce antimicrobials with suppressive activity against <i>Candidatus</i> Liberibacter asiaticus, the causal agent of HLB. We demonstrated that the citrus microbiome is a rich source of microorganisms that inhibit <i>Liberibacter</i> spp. We selected a <i>Pseudomonas chlororaphis</i> strain for further characterization and assigned anti-<i>Liberibacter</i> activity to the production of phenazine-1-carboxamide. Phenazine-producing <i>P. chlororaphis</i> strains are known biocontrols in other pathosystems, and this study highlights their potential for mitigation of HLB.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0157626"},"PeriodicalIF":4.2,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Waste oil substrates reshape the black soldier fly larval gut microbiome and biomass composition during bioconversion.","authors":"Reese Saho, Duy Trinh, Erin Kojima, Tong Wang, Charity Owings, Zachary Burcham","doi":"10.1128/aem.01082-26","DOIUrl":"https://doi.org/10.1128/aem.01082-26","url":null,"abstract":"<p><p>Black soldier fly larvae (BSFL) are generalist decomposers with promise for converting agricultural and food-processing by-products into value-added bioproducts, but BSFL performance on lipid-rich waste oil streams and the role of the gut microbiome in this process remain unclear. Here, we evaluated BSFL bioconversion of a standard chicken feed diet supplemented with three chemically distinct waste oils: acidulated vegetable oil (AVO), pork grease (PG), and used cooking oil (UCO). Larval performance, bioconversion rate, gut microbiome composition, total protein and fat content, and fatty-acid profiles were measured across bioconversion. Larval age was a major driver of gut microbiome structure, but waste oil supplementation further reshaped community membership and structure, with the strongest diet-associated effects occurring during early-to-intermediate bioconversion. Most differentially abundant taxa were members of the baseline core gut community, suggesting that oil supplementation primarily altered dominance patterns among resident taxa. PG and UCO supported larval growth, and bioconversion performance did not differ significantly from the chicken feed control, whereas AVO reduced bioconversion rate and showed weaker growth outcomes. Oil supplementation also increased larval fat content, reduced protein content, and shifted fatty-acid profiles toward the corresponding oil feedstocks, although larval biomass composition remained shaped by basal diet and host or microbial metabolism. These findings suggest that selected lipid-rich waste streams can support efficient BSFL bioconversion while restructuring resident gut microbiome members that may tolerate, metabolize, or indirectly respond to oil-associated conditions, contributing to substrate-dependent changes in larval lipid accumulation and fatty-acid composition.</p><p><strong>Importance: </strong>Agricultural and food-processing systems generate large amounts of lipid-rich by-products that are difficult to manage using conventional waste-valorization approaches. Black soldier fly larvae (BSFL) offer a biological route for recovering nutrients from these materials, but efficient conversion depends on interactions among substrate chemistry, larval physiology, and the gut microbiome. This study suggests that some waste oil streams can support larval growth while restructuring resident gut microbial communities and altering larval fatty-acid composition. These findings are important for agricultural biotechnology because they frame BSFL production as a host-microbiome bioconversion system rather than simply an insect-based waste-reduction process. Understanding how gut microbes respond to chemically distinct lipid wastes can guide substrate selection, pretreatment, and microbiome-informed optimization strategies for converting underutilized agricultural and food-processing residues into value-added bioproducts for circular agricultural systems.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0108226"},"PeriodicalIF":4.2,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149179016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Design of ancestral protein-glutaminase with improved thermal stability.","authors":"Takuto Ono, Hayato Araseki, Taichi Chisuga, Daisuke Fujinami, Isao Abe, Sohei Ito, Shogo Nakano","doi":"10.1128/aem.01736-26","DOIUrl":"https://doi.org/10.1128/aem.01736-26","url":null,"abstract":"<p><p>Protein-glutaminase (PG) from <i>Chryseobacterium proteolyticum</i> is an industrially valuable enzyme for protein deamidation; however, its application is limited because of insufficient thermal stability. Previous rational design studies have enhanced the thermostability of PG but often required labor-intensive, stepwise optimization. In this study, ancestral sequence reconstruction (ASR) was used to engineer thermostable PG variants in a single design step. Eight ancestral PGs were constructed, all of which exhibited enhanced thermal stability compared with that of the wild type. The most stable variant, AncPG1, had a melting temperature of 76.2°C, 13.6°C higher than that of the wild type. Mutational dissection identified five substitutions, N148G, N154D, L156Y, L168S, and D179S, that individually enhanced thermal stability and showed cumulative stabilizing effects when combined. These substitutions were predominantly located in surface-associated loop regions. Notably, although the ancestral variants showed reduced catalytic efficiency toward the small synthetic substrate Cbz-Gln-Gly, they retained reactivity toward macromolecular protein substrates when compared at equal activity units. Furthermore, AncPG1 showed greater absolute protein-substrate reactivity than WT CpPG under elevated temperature conditions. These findings provide further support for ASR as a strategy for generating thermostable enzyme variants with improved functional performance under elevated-temperature conditions.IMPORTANCEProtein-glutaminase from <i>Chryseobacterium proteolyticum</i> is used to improve functional properties of food proteins, but its thermal stability limits its use at elevated temperatures. In this study, ancestral sequence reconstruction generated PG variants with substantially increased melting temperatures while retaining reactivity toward protein substrates. AncPG1 also showed greater absolute reactivity than WT CpPG toward multiple protein substrates at 70°C when compared at the same protein concentration. Several substitutions that individually enhanced thermal stability were located in surface-associated loop regions. These results illustrate the utility of evolutionary sequence information for engineering thermostable protein-modifying enzymes.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0173626"},"PeriodicalIF":4.2,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"N_₂O reduction at low pH.","authors":"Lars R Bakken, Åsa Frostegård","doi":"10.1128/aem.01424-26","DOIUrl":"https://doi.org/10.1128/aem.01424-26","url":null,"abstract":"<p><p>Microbial N_₂O reduction in acid soils became a conundrum when NosZ maturation was found to be impaired at pH ≤6 in both model organisms and soil communities. Yet, evolution appears to have repeatedly circumvented this constraint. Examples include the identification of two small proteins that enable maturation of canonical NosZ at low pH, as well as the <i>Schinkia</i> sp. isolated by G. He, M. E. Davin, S. Kolodney, E. Stutzman, et al. (Appl Environ Microbiol 92:e00069-26, 2026, https://doi.org/10.1128/aem.00069-26), which carries a unique repertoire of <i>nosZ</i> genes and grows by N₂O respiration across pH 4.4-8.5. These discoveries help resolve the conundrum and may provide new options for mitigating N₂O emissions.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0142426"},"PeriodicalIF":4.2,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149178844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Epigenetics as a design variable in heterologous expression.","authors":"Adjo E Kadjo, Galen Ptacek, Barbara I Adaikpoh, Stephanie C Heard, Rasha M Bashatwah, Alessandra S Eustáquio","doi":"10.1128/aem.01024-26","DOIUrl":"https://doi.org/10.1128/aem.01024-26","url":null,"abstract":"<p><p>Heterologous expression-the expression of genes from one organism into another-is an important approach to produce value-added chemicals such as proteins and metabolites. The main challenge, however, is achieving high production yields that are commercially viable. Approaches used to improve productivity rely on editing the DNA sequence, including promoters, ribosome binding sites, and codon usage. Epigenetics, in the form of DNA methylation, may offer an additional layer of control to improve production yields but remains unexplored. Moreover, the choice of the host organism is crucial. We have been interested in developing the alternative bacterial host <i>Burkholderia</i> sp. FERM BP-3421. Four DNA methyltransferases were predicted to be encoded in the genome of this strain, one of which was silent under our growth conditions. After performing gene deletions or overexpression, we experimentally assigned the recognition motif of each DNA methyltransferase, one of which was shown to be essential. As expected, modulation of DNA methylation had major impacts on the transcriptome and metabolome of the host, varying depending on which DNA methyltransferase was modified. Two of the methyltransferases that methylate adenine had overlapping profiles, whereas the one methylating cytosine differed. Finally, we probed the role of DNA methylation during heterologous expression using green fluorescence protein and three different promoters. Expression levels varied in a context-dependent manner with mechanisms, in some cases, involving the presence of methylation motifs within promoter regions but also based on global host effects. This work establishes epigenetics as an important design variable to help improve heterologous expression yields.IMPORTANCEImproving heterologous expression currently relies on modifying the DNA sequence. Epigenetics offers an opportunity to improve outcomes but remains unexplored. We first investigated the effect of epigenetic DNA methylation on the growth, transcriptome, and metabolome of an alternative bacterial host we are interested in developing by generating three single, DNA methyltransferase mutants. Next, we showed that epigenetic DNA methylation or the lack thereof can affect heterologous expression outcomes. The effect was context-dependent, reaching 2.4-fold improvement in one instance. This work highlights the importance of being epigenetics-aware during host development for heterologous expression.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0102426"},"PeriodicalIF":4.2,"publicationDate":"2026-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149164129","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}