Applied and Environmental Microbiology最新文献

{"title":"Unraveling the relationship between population composition and metabolite induction in high-precision cellulosic bacterial-fungal co-cultivations.","authors":"Ana M Palacio-Barrera, Jethro L Hemmann, Ivan Schlembach, Maurice Finger, Jochen Büchs, Gerald Lackner, Miriam A Rosenbaum","doi":"10.1128/aem.01397-26","DOIUrl":"https://doi.org/10.1128/aem.01397-26","url":null,"abstract":"<p><p>Co-cultures of filamentous microorganisms are a tool to unlock the biosynthetic machinery encoded in silent gene clusters. However, if bioactive compounds are to be produced biotechnologically, it is essential to understand the dynamics of microbial partnership. In this context, we have previously introduced a co-cultivation platform based on low-cost crystalline cellulose as a substrate. Here, the fungus <i>Trichoderma reesei</i> RUT-C30 degrades α-cellulose into soluble sugars to support the growth of the natural product-producing bacterium <i>Streptomyces coelicolor</i> A3(2), which is unable to utilize cellulose. This co-cultivation approach mimics a carbon-limited interaction scenario of natural habitats. We investigated how the population dynamics of both strains affect metabolite production by combining high-throughput online fluorescence monitoring with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. We then determined the metabolic profile of the cultures and identified metabolites specifically induced during co-cultivation. The coupled analysis of population ratios and metabolomic data indicated that the highest number of induced metabolites compared to axenic cultures is observed in co-cultures with a balanced ratio of both strains. This analysis further led to deciphering a biotransformation of dimerumic acid, a hydroxamate siderophore produced by <i>T. reesei</i>, into the dehydroxylated derivatives eleutherazine B and talarazine A by <i>S. coelicolor</i>. Additionally, a small cluster of unique metabolites strongly induced in co-cultures was found to be structurally similar to steroidal lactones of the withanolide class, thus highlighting the potential for the discovery of natural products. Overall, the platform described combines natural product prospecting with a biotechnological production strategy that can be extended also to other co-cultures.IMPORTANCEThis work provides a comprehensive analysis of population dynamics and metabolite production in a complex microbial co-culture. This approach integrates three key aspects. First, high-throughput online monitoring enables biomass tracking through fluorescence labeling in a system containing two filamentous microorganisms with distinct macro- and micromorphologies and a non-soluble fibrous substrate. Second, concomitant metabolomics analysis during co-cultivation settings helps to resolve metabolic interaction patterns. Interestingly, a metal-acquisition compound produced by the first microbe is chemically modified by the second, suggesting a competition strategy for an essential micronutrient. Third, from a biotechnological perspective, substrate solubilization resembles a fed-batch strategy that minimizes carbon catabolite repression during secondary metabolite production. Overall, this platform advances our understanding of microbial interactions and culture conditions that can be transferred to other culture platforms of relev","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0139726"},"PeriodicalIF":4.2,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149340395","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":"Hydrogen production and fermentation in heliobacteria.","authors":"Tatum B Vilaboy, Christopher J Gisriel, Mitchell Gibson, Jabreel M Hania, Savanah D Gisriel, Julie A Turk, Bridget Krause, Andrey Kanygin, Patricia Baker, Kevin E Redding","doi":"10.1128/aem.01061-26","DOIUrl":"https://doi.org/10.1128/aem.01061-26","url":null,"abstract":"<p><p>Heliobacteria are the only phototrophic members of the gram-positive bacteria. <i>Heliomicrobium modesticaldum</i> can grow chemotrophically in the dark using pyruvate as a carbon source. It can grow phototrophically only on acetate (plus CO₂). We found that dihydrogen production occurred only when fixing N₂ and increased ~5-fold upon replacing N₂ with argon. Introduction of genes encoding a [FeFe] hydrogenase and maturases (<i>hydAEFG</i>) from <i>Acetivibrio thermocellus</i> conferred the ability to produce H₂ at much higher levels. The modified cells made the most H₂ when grown chemotrophically, less when grown mixotrophically, and none when grown phototrophically, suggesting that only excess electrons were available for H₂ production. We tested several molecules as potential reduced fermentation products by this species. Only propanoate was observed, and its extracellular concentration could reach several millimolar. There was an inverse correlation between light intensity and propanoate production when the cells were grown on pyruvate, and phototrophic cultures produced no detectable propanoate. We conclude that propanoate is the major reduced fermentation product made by <i>Hmb. modesticaldum,</i> and we propose a pathway to produce it that is essentially a mini-cycle within the reductive TCA (rTCA) cycle, combining the first four reactions of the rTCA cycle with methylmalonyl-CoA mutase and epimerase, and methylmalonyl-CoA:pyruvate carboxytransferase. This pathway would, in effect, dispose of four reducing equivalents while driving the conversion of pyruvate to propanoate. The genes for the last six enzymes of this pathway seem to be part of an operon; the genes and potential operon are conserved in other heliobacteria, suggesting that this pathway exists in all or most heliobacteria.</p><p><strong>Importance: </strong>This study establishes that heliobacteria, the only phototrophic members of the <i>Bacillota</i>, produce propanoic acid as the reduced fermentation product when growing chemotrophically. The pathway used to make it resembles that of the <i>Propionibacteria</i>, except in the context of a mini-cycle within the reductive TCA (rTCA) cycle. This work confirms that the <i>Heliomicrobium</i> genus lacks a functional hydrogenase to make hydrogen as a way to dispose of excess electrons. Finally, it is shown that these organisms can be engineered to make dihydrogen as a reduced fermentation product if the genes for a clostridial hydrogenase and assembly proteins are expressed in <i>Heliomicrobium modesticaldum</i>.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0106126"},"PeriodicalIF":4.2,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149340394","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":"Cultivation of halophilic archaea in shallow subsurface Martian conditions has implications for extant life on Mars.","authors":"Adam Robinson, Shannon McQuaig-Ulrich, Tyler Dondero, Aaron Celestian, Scott M Perl","doi":"10.1128/aem.01749-26","DOIUrl":"https://doi.org/10.1128/aem.01749-26","url":null,"abstract":"&lt;p&gt;&lt;p&gt;The present-day Martian surface is generally considered inhospitable to life because of low atmospheric pressure, intense surface radiation, global desiccation, and oxidizing chemistry, which has been increasing since the late Noachian. However, shallow Martian subsurface regions where mineralogy has shown groundwater movement may include localized hypersaline environments capable of retaining liquid water and supporting microbial metabolism. &lt;i&gt;Haloferax volcanii&lt;/i&gt;, a model halophilic archaeon, has previously been shown to survive under low-pressure Martian conditions (24 mbar) and to grow anaerobically supported by the Mars-relevant oxyanions nitrate and perchlorate under high-salinity conditions. Here, we investigated whether &lt;i&gt;H. volcanii&lt;/i&gt; could actively grow under a combination of environmental and chemical conditions relevant to potentially habitable shallow subsurface Martian lacustrine settings. Cultures were incubated for 160 days under anoxic, CO₂-enriched, low-pressure conditions (24 mbar) in hypersaline liquid media supplemented with nitrate or perchlorate. Growth was observed in all low-pressure treatments and was confirmed by increases in optical density and biological reduction of nitrate and perchlorate. Scanning electron microscopy revealed extensive biofilm formation in low-pressure cultures, and Raman spectroscopy demonstrated the persistence of carotenoid biosignatures after prolonged incubation under Martian conditions. Liquid loss remained below 4% across all treatments, indicating long-term stability of hypersaline brines throughout the experiment. These results demonstrate for the first time that a halophilic archaeon is capable of active growth and metabolism under a Mars-relevant combination of low pressure, high salinity, anoxia, and oxidizing chemistry, providing experimental support for the potential habitability of localized shallow subsurface Martian environments.IMPORTANCEThe search for cellular life is a major objective of future Mars exploration. While many studies have examined whether microorganisms can survive under Martian conditions, far fewer have demonstrated active growth and metabolism. Here, we document &lt;i&gt;Haloferax volcanii&lt;/i&gt; as the first halophilic archaeon capable of active growth under a defined combination of Mars-relevant low atmospheric pressure, high salinity, anoxia, and oxidizing chemical conditions. These findings expand the current understanding of the environmental limits of microbial growth and provide experimental evidence that localized brine environments in the shallow Martian subsurface could support active microbial metabolism if suitable organics and liquid water are present. These results are also relevant to planetary protection efforts, as they help constrain the potential for terrestrial halophilic microorganisms introduced during robotic or human exploration to persist and remain metabolically active in Martian environments. Furthermore, this study establishes a pr","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0174926"},"PeriodicalIF":4.2,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149332165","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":"Correction for Lubin et al., \"Characterization of chemotaxis in soybean symbiont <i>Bradyrhizobium diazoefficiens</i>\".","authors":"Matthew B Lubin, Daniel H Teixeira, Brittany J Belin","doi":"10.1128/aem.02050-26","DOIUrl":"https://doi.org/10.1128/aem.02050-26","url":null,"abstract":"","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0205026"},"PeriodicalIF":4.2,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149332164","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":"Novel electron transport powers archaeal methane metabolism across varied substrates and environments.","authors":"Anupkumar Rai, Shikha Singh Chauhan, Divya Prakash","doi":"10.1128/aem.01816-26","DOIUrl":"https://doi.org/10.1128/aem.01816-26","url":null,"abstract":"<p><p>Methanogens are responsible for producing approximately one billion metric tons of methane each year within Earth's anaerobic environments as a byproduct of their energy metabolism. They play a vital role in the global carbon cycle. There are three main pathways for methanogenesis: hydrogenotrophic, which uses H₂ and CO₂ to produce methane; methylotrophic, which utilizes methylated compounds like methanol and amines; and aceticlastic, which converts acetate into methane and CO₂. Instead of substrate-level ATP production, methanogens conserve energy through a membrane-bound electron transport chain and flavin-based electron bifurcation (FBEB). Beyond the canonical modes of growth in methanogens, <i>Methanosarcina acetivorans</i>, a model acetotrophic methanogen, can conserve energy via direct interspecies electron transfer (DIET). Here, we review the metabolic pathways and electron transport processes involved in energy conservation during fermentative, respiratory, and DIET modes. Additionally, we discuss the role of FBEB in energy conservation in gut-associated <i>Methanomassiliicoccus luminyensis</i>. Finally, we discuss the potential roles of heterodisulfide reductase (Hdr) and the process of electron transfer during the reaction mechanism of electron-bifurcating HdrA2B2C2 complex in cytochrome-containing methanogens. Understanding the complex redox control mechanisms in these organisms has broader implications for sustainable energy and environmental management.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0181626"},"PeriodicalIF":4.2,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149332095","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":"An integrated cell culture-nanopore sequencing (ICC-NanoporeSeq) method for the simultaneous detection of multiple enterovirus types and quantification of infectious Enterovirus B.","authors":"Aina Astorch-Cardona, Simon Rouault, Tamar Kohn","doi":"10.1128/aem.01830-26","DOIUrl":"https://doi.org/10.1128/aem.01830-26","url":null,"abstract":"<p><p>Enteroviruses (EVs) are ubiquitous contaminants of surface waters, where they can remain infectious for long periods of time. Most methods used for EV monitoring are unable to distinguish between infectious and non-infectious particles, or between EV types. Because different types exhibit both distinct environmental persistence and health implications, there is a need for type-resolved infectivity measurements. Here, we developed integrated cell culture-nanopore sequencing (ICC-NanoporeSeq), a method combining short-term cell culture amplification with nanopore sequencing of the VP1 gene. The ICC approach was adapted from a previously described ICC-reverse transcriptase quantitative PCR (ICC-RTqPCR) protocol, while the NanoporeSeq workflow was derived from a clinical EV typing protocol and optimized for environmentally circulating EV types. Using samples containing known concentrations of 10 EV types, the NanoporeSeq method accurately and reproducibly recovered the original proportions of all EV types after correction of biases. When applied to wastewater extracts, the method detected 45 EV types across five species. Furthermore, type-specific calibration curves generated with ICC-NanoporeSeq enabled quantification of the infectious concentrations of six EV types of the Enterovirus B species, allowing a simultaneous and type-resolved assessment of infectivity in mixed samples. Overall, ICC-NanoporeSeq provides a scalable approach for the parallel analysis of multiple EV types. Compared with the predecessor ICC-RTqPCR method, it eliminates the need for multiple type-specific PCR primers, and can therefore be readily expanded to include additional EV types.IMPORTANCECurrent methods used to detect enteroviruses (EVs) in environmental samples generally measure viral genome copies without determining whether viruses remain infectious, limiting their use in public health risk assessment or water quality monitoring. At the same time, available infectivity assays are often labor-intensive and cannot distinguish between different EV types. Here, we developed integrated cell culture-nanopore sequencing (ICC-NanoporeSeq), a method combining cell culture and nanopore sequencing to simultaneously quantify the infectious concentrations of multiple EV types in samples containing mixed EV populations. The method provides an efficient and scalable approach for studying EVs in complex environmental matrices. ICC-NanoporeSeq has potential applications in wastewater-based epidemiology, environmental surveillance, and disinfection studies, where understanding the persistence of different EV types simultaneously is crucial.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0183026"},"PeriodicalIF":4.2,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149332163","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":"Harnessing phage defense systems to develop a <i>Burkholderia</i> bacterial host.","authors":"Stephanie C Heard, Galen M Ptacek, Adjo E Kadjo, Alessandra S Eustáquio","doi":"10.1128/aem.01706-26","DOIUrl":"https://doi.org/10.1128/aem.01706-26","url":null,"abstract":"<p><p>Heterologous production of metabolites and proteins requires the transfer of foreign DNA into a host. One common barrier to the genetic engineering of bacteria is their plethora of phage defense systems. Targeted deletion of defense system components can improve DNA transfer for downstream applications. We sought to improve the transformation efficiency of <i>Burkholderia</i> sp. FERM BP-3421, a β-proteobacterial host that has great potential for the high-titer production of bioactive small molecules. We investigated restriction-modification systems and the recently described Kiwa phage defense system. Through deletion of three restriction enzyme genes, transformation efficiency was improved nearly 7-fold. The electroporation protocol was also optimized with respect to temperature, cell density, and buffer composition, which resulted in an additional 40-fold improvement in transformation efficiency, reaching 1 × 10⁶ CFU/µg plasmid DNA. Interestingly, a mimicry by methylation approach, where DNA is pre-methylated using two host DNA methyltransferases, outperformed the transformation efficiency obtained with restriction enzyme deletion mutants nearly 3-fold. Moreover, contrary to previous reports in <i>Escherichia coli</i>, deletion of the Kiwa system did not improve transformation efficiency into <i>Burkholderia</i> sp. FERM BP-3421. Understanding the impact of bacterial innate immune systems on genetic tractability is vital to improving non-model host strains to produce high-value molecules of interest.</p><p><strong>Importance: </strong>The ability to transfer DNA into bacteria is key to genetic engineering. However, bacteria have evolved immune defenses to recognize and protect themselves from foreign DNA, thwarting genetic manipulation. Here, we identified putative defense systems in a β-proteobacterial strain of interest and characterized their impact on DNA transfer. The defense systems studied belong to the well-known class of restriction-modification systems and to the recently described Kiwa system. Intriguingly, we observed higher DNA transfer efficiency by avoiding restriction-modification systems via pre-methylation rather than by restriction gene deletion. Future studies should investigate the underlying mechanism for this difference. Moreover, contrary to what was reported for γ-proteobacteria <i>Escherichia coli</i>, we showed that the Kiwa system does not impair DNA transfer into the β-proteobacteria <i>Burkholderia</i> sp. strain. There has been significant interest in the discovery of novel defense systems. Learning more about their complexity is important to enable genetic manipulation of previously intractable strains.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0170626"},"PeriodicalIF":4.2,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149332094","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":"The attachment rate of <i>Vibrio anguillarum</i> to microplastics strongly varies with strain identity, particle characteristics, and environmental conditions.","authors":"Méril Massot, Lukas Wimmer, Mhamad Aly Moussawi, Jeanne Hamet, Tatjana Parac-Vogt, Andreja Rajkovic, Lea-Ann Dailey, Martijn Callens, Stéphanie Bedhomme","doi":"10.1128/aem.01687-26","DOIUrl":"https://doi.org/10.1128/aem.01687-26","url":null,"abstract":"<p><p>Microplastics (MPs), resulting from plastic objects and waste degradation, are increasingly abundant, particularly in marine environments. They exhibit a hydrophobic surface on which biofilms form easily. Metagenomic analyses of these biofilms have revealed that they often contain bacterial species potentially pathogenic to humans or animals. For this reason, MPs are suspected to present a risk to public health by acting as a vector for pathogenic bacterial species. To better understand this hazard, we studied different factors potentially affecting the bacterial attachment rate to MPs. Focusing on the fish pathogen <i>Vibrio anguillarum</i>, a collection of 16 GFP-labeled strains was assembled, including isolates from infected fish. Their attachment rates were measured using fluorescence microscopy on three types of MPs (milled polypropylene and polyethylene terephthalate particles, and commercially available polystyrene beads). A strong effect of the particle type was found, with higher attachment rates on milled particles, whose surface appeared qualitatively rougher based on visual examination of scanning electron microscopy images. Our results also revealed an intraspecific variation in attachment rate, highlighting the need for testing several strains of the same species to assess attachment rate and related hazards. Finally, it was observed that when a biofilm had formed by field incubation of the MPs, the differences in attachment rates between particle types were erased. The attachment of <i>V. anguillarum</i> depends on a combination of strain-related factors, particle characteristics, and environmental conditions, highlighting the need to combine microbiology, materials science, and environmental sciences to achieve a comprehensive risk assessment associated with MPs.IMPORTANCEMicroplastics (MPs) are increasingly abundant, particularly in marine environments. They are suspected to present a risk to public health by acting as a vector for pathogenic bacterial species. Focusing on the fish pathogen <i>Vibrio anguillarum</i>, we measured the attachment rates of 16 strains on three types of microplastics (milled polypropylene and polyethylene terephthalate particles, and commercially available polystyrene beads). This study highlights a strong effect of the particle type, with higher attachment rates on irregular particles, as well as a strong intraspecific variation. In a second experiment, it was observed that when a natural marine biofilm had already formed on the microplastic particles, differences in attachment rates between particle types were erased. Our findings emphasize the importance of accounting for multiple factors when assessing the risks of MPs as carriers of pathogenic bacteria, with our study addressing the first step of the conceptual pathway linking attachment, persistence, transport, exposure, and ultimately infection.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0168726"},"PeriodicalIF":4.2,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149332097","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 switch-rheostat circuit governs quorum sensing homeostasis in a phytopathogen.","authors":"Nemanja Ristović, Iris Bertani, Gianluca Triolo, Michael P Myers, Cristina Bez, Vittorio Venturi","doi":"10.1128/aem.01488-26","DOIUrl":"https://doi.org/10.1128/aem.01488-26","url":null,"abstract":"<p><p><i>Pseudomonas fuscovaginae</i>, a wide-host-range plant pathogen of several cereal and grass species, possesses two canonical <i>N</i>-acyl homoserine lactone (AHL)-based quorum sensing (QS) systems called PfsI/R and PfvI/R, both of which are inactive under laboratory conditions but active <i>in planta</i>. The <i>pfsI-pfsR</i> intergenic region encodes RsaM, a putative protein that has since been hypothesized to act as a repressor switch of this QS circuit. In the present study, we demonstrate that the stringent repression exerted on the PfsI/R system depends entirely on the divergent promoter/intergenic <i>pfsR-rsaM</i> region rather than RsaM itself. Remarkably, this regulatory switch element stringently represses the expression of both the <i>pfsR</i> and <i>rsaM</i> genes. We further show for the first time that RsaM is endogenously expressed and functions as a negative regulator, modulating the PfsI/R circuit instead of preventing its activation. Taken together, our results evidence a unique two-tiered/hierarchical repression of a QS system, provided by a master repressor switch and a repressor modulator.</p><p><strong>Importance: </strong><i>Pseudomonas fuscovaginae</i> is a globally occurring plant pathogen that employs <i>N</i>-acyl homoserine lactone (AHL)-based quorum sensing (QS) to regulate virulence. In this bacterium, QS signaling circuits display a rather unusual feature: the lack of activation at high cell densities under standard laboratory conditions. A hypothetical regulator named RsaM was previously linked to this phenomenon as a repressor switch acting on the PfsI/R AHL QS system in the absence of an unknown signal or stimulus. In this study, we show that a regulatory element within the <i>pfsR</i>-<i>rsaM</i> intergenic region acts as the primary switch of the PfsI/R QS system, independently of RsaM. Conversely, RsaM functions as a post-activation modulator that fine-tunes the QS response. This study advances our understanding of the regulatory configurations of unconventional QS systems by revealing a two-tier control mechanism in which a master regulatory switch governs circuit activation, while the previously uncharacterized protein RsaM controls signaling output once the system is engaged.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0148826"},"PeriodicalIF":4.2,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149332162","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":"Sustainable 2-keto-L-gulonic acid biosynthesis via single-strain one-step fermentation.","authors":"Fang Wang, Qian Zhou, Yupu Wang, Zhiwen Wang, Dong Li","doi":"10.1128/aem.01631-26","DOIUrl":"https://doi.org/10.1128/aem.01631-26","url":null,"abstract":"<p><p>L-ascorbic acid (vitamin C) is an essential antioxidant molecule that maintains normal physiological functions in humans. Currently, industrial production relies primarily on a three-strain, two-step fermentation process to produce 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. 2-KLG is then converted to vitamin C through chemical lactonization. However, this process is limited by prolonged fermentation cycles, poor stability of the mixed-culture system, and complex process control. In recent years, engineered single-strain platforms capable of one-step 2-KLG biosynthesis have emerged as a promising strategy for more sustainable vitamin C production. Nevertheless, the efficiency of L-sorbose-to-2-KLG conversion in this system remains low, making it a major bottleneck to further improving 2-KLG production. This review summarizes recent advances on single-strain, one-step fermentation systems using <i>Gluconobacter oxydans</i> as the chassis strain. It focuses on strategies including genetic tool development, metabolic engineering, and key enzyme optimization. The review aims to provide a theoretical basis and technical reference for optimizing and industrializing green biomanufacturing systems for vitamin C.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0163126"},"PeriodicalIF":4.2,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149332096","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}