Microbial Biotechnology最新文献

{"title":"A New Method for Constructing High-Resolution Phylogenomic Topologies Using Core Gene-Associated MNP Markers: A Case Study From Agaricus bisporus","authors":"Fei Liu,&nbsp;Zhi-Xin Cai,&nbsp;Wen-Yi Kang,&nbsp;Wen-Zhi Chen,&nbsp;Yuan-Ping Lu,&nbsp;Mei-Yuan Chen,&nbsp;Rui-Lin Zhao","doi":"10.1111/1751-7915.70070","DOIUrl":"https://doi.org/10.1111/1751-7915.70070","url":null,"abstract":"<p>Accurate strain identification is essential for economically significant fungi, as it aids in understanding their diverse agronomic traits, pathogenicity, and other important characteristics. However, traditional methods often face challenges related to limited accuracy, high workloads, and reproducibility issues. Recently, multiple nucleotide polymorphism (MNP) markers have been employed in mushroom strain identification, demonstrating significantly improved accuracy and reproducibility. Nevertheless, the identification of strains across different species still heavily depends on specific and often overly complex MNP markers. In this study, we address these challenges by developing a novel method for constructing high-resolution phylogenomic topologies using core gene-associated multiple nucleotide polymorphism (cgMNP) markers, focusing on <i>Agaricus bisporus</i> (button mushroom). Utilising resequencing data from 213 cultivated and wild strains of <i>A. bisporus</i>, we identified 84 cgMNP markers within 83 core genes from 1011 MNP markers. Phylogenetic analysis based on cgMNP sequences and the genetic distance between strain pairs allowed for precise identification of all strains. Moreover, the successful transferability of these cgMNP markers to an additional 385 <i>A. bisporus</i> strains and other fungal species, including <i>Flammulina filiformis</i> (enoki mushroom) and <i>Saccharomyces cerevisiae</i> (yeast), highlights their cross-species applicability. The high resolution and strong congruence of cgMNP markers with whole-genome data provide a robust and reliable method for strain-level discrimination in fungi. The success of this approach in <i>A. bisporus</i> sets a promising precedent for its application to a broader range of fungal taxa.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70070","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143447041","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Initial Genome Editing Toolset for Caldimonas thermodepolymerans, the First Model of Thermophilic Polyhydroxyalkanoates Producer","authors":"Anastasiia Grybchuk-Ieremenko,&nbsp;Kristýna Lipovská,&nbsp;Xenie Kouřilová,&nbsp;Stanislav Obruča,&nbsp;Pavel Dvořák","doi":"10.1111/1751-7915.70103","DOIUrl":"https://doi.org/10.1111/1751-7915.70103","url":null,"abstract":"<p>The limited number of well-characterised model bacteria cannot address all the challenges in a circular bioeconomy. Therefore, there is a growing demand for new production strains with enhanced resistance to extreme conditions, versatile metabolic capabilities and the ability to utilise cost-effective renewable resources while efficiently generating attractive biobased products. Particular thermophilic microorganisms fulfil these requirements. Non-virulent Gram-negative <i>Caldimonas thermodepolymerans</i> DSM15344 is one such attractive thermophile that efficiently converts a spectrum of plant biomass sugars into high quantities of polyhydroxyalkanoates (PHA)—a fully biodegradable substitutes for synthetic plastics. However, to enhance its biotechnological potential, the bacterium needs to be ‘domesticated’. In this study, we established effective homologous recombination and transposon-based genome editing systems for <i>C. thermodepolymerans</i>. By optimising the electroporation protocol and refining counterselection methods, we achieved significant improvements in genetic manipulation and constructed the AI01 chassis strain with improved transformation efficiency and a Δ<i>phaC</i> mutant that will be used to study the importance of PHA synthesis in <i>Caldimonas</i>. The advances described herein highlight the need for tailored approaches when working with thermophilic bacteria and provide a springboard for further genetic and metabolic engineering of <i>C. thermodepolymerans</i>, which can be considered the first model of thermophilic PHA producer.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70103","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143455777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Neglected Human Risk Factors Determining Sustainable Health by Microbial Causes: Individual Versus Social Conducts, Scientific Versus Stultified Behaviour","authors":"Fernando Baquero","doi":"10.1111/1751-7915.70097","DOIUrl":"https://doi.org/10.1111/1751-7915.70097","url":null,"abstract":"<p>Microbes constitute a ubiquitous warp, a highly sensitive skin of the biosphere that can be scratched and damaged by all human activities. However, the existence of life in general and the human species, in particular, depends on the intelligent preservation of such a biological microbiological cement linking our health with the health of Earth. We are responsible for maintaining sustainable health by managing our damaging individual and social behaviour, and we are also charged with the duty of correcting the microbial disequilibrium we are provoking. The harmful secondary effects resulting from the nature of the species <i>Homo sapiens</i> are frequently neglected. However, sustainable health by microbial causes depends on our individual and social psychology. The role of individual psychology, social behaviour (including the ‘tragedy of the commons’), based on collective psychology, culture, values and social norms, and the influence on sustainable health of the methodology of research and management of interventions are briefly analysed. As a general antidote to our unavoidable natural stultified behaviour, education in science is the only possibility to counteract mistakes and restore human dignity.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70097","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143446721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genomic, Transcriptomic and Suspect/Non-Target Screening Analyses Reveal the Role of CYP450s in the Degradation of Imazalil and Delineate Its Transformation Pathway by Cladosporium herbarum","authors":"Christina V. Papazlatani,&nbsp;Sotirios Vasileiadis,&nbsp;Eleni I. Panagopoulou,&nbsp;Dimitrios E. Damalas,&nbsp;Panagiotis A. Karas,&nbsp;Evdoxia Gerovasileiou,&nbsp;Nikolaos S. Thomaidis,&nbsp;Dimitrios G. Karpouzas","doi":"10.1111/1751-7915.70102","DOIUrl":"https://doi.org/10.1111/1751-7915.70102","url":null,"abstract":"<p>Imazalil (IMZ), a major surface water contaminant characterised by high environmental recalcitrance and toxicity, is used in fruit-packaging plants to control fungal infestations during storage. This leads to the production of wastewaters which should be treated on site before their environmental release. We previously isolated a <i>Cladosporium herbarum</i> strain, the first microorganism that could degrade IMZ. Here we describe the genetic network utilised by the fungus to degrade IMZ and its detailed transformation. Genomic and transcriptomic analysis of <i>C. herbarum</i> pointed to the involvement of strongly upregulated CYP450s in IMZ degradation, as further verified by cessation of its biodegradation by CYP450 inhibitors. LC-QTOF-HRMS analysis and suspect/non-target screening identified nine transformation products (TPs) of IMZ. IMZ biotransformation mainly proceeded through O-dealkylation, while other less important paths, most probably controlled by upregulated oxidases, were operative involving successive hydroxylation reactions. These lead to the formation of TPs like IMZ_313 and IMZ_331, with the former being further transformed through imidazole ring scission to IMZ_288, a TP reported for the first time. We provide first evidence for the transformation mechanism of IMZ by <i>C. herbarum</i> and the potential genes/enzymes involved, paving the way for the use of <i>C. herbarum</i> in the biodepuration of agro-industrial effluents.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143446722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel Gene Clusters for Secondary Metabolite Synthesis in Mesophotic Sponge-Associated Bacteria","authors":"Nuo Chen,&nbsp;Liwei Liu,&nbsp;Jingxuan Wang,&nbsp;Deqiang Mao,&nbsp;Hongmei Lu,&nbsp;Tânia Keiko Shishido,&nbsp;Shuai Zhi,&nbsp;Hua Chen,&nbsp;Shan He","doi":"10.1111/1751-7915.70107","DOIUrl":"https://doi.org/10.1111/1751-7915.70107","url":null,"abstract":"<p>Mesophotic coral ecosystems (MCEs) host a diverse array of sponge species, which represent a promising source of bioactive compounds. Increasing evidence suggests that sponge-associated bacteria may be the primary producers of these compounds. However, cultivating these bacteria under laboratory conditions remains a significant challenge. To investigate the rich resource of bioactive compounds synthesised by mesophotic sponge-associated bacteria, we retrieved 429 metagenome-assembled genomes (MAGs) from 15 mesophotic sponges, revealing a strong correlation between bacterial diversity and sponge species. Furthermore, we identified 1637 secondary metabolite biosynthetic gene clusters (BGCs) within these MAGs. Among the identified BGCs, terpenes were the most abundant (495), followed by 369 polyketide synthases (PKSs), 293 ribosomally synthesised and post-translationally modified peptides (RiPPs) and 135 nonribosomal peptide synthetases (NRPSs). The BGCs were classified into 1086 gene cluster families (GCFs) based on sequence similarity. Notably, only five GCFs included experimentally validated reference BGCs from the Minimum Information about a Biosynthetic Gene cluster database (MIBiG). Additionally, an unusual abundance of BGCs was detected in <i>Entotheonella</i> sp. (s191209.Bin93) from the Tectomicrobia phylum. In contrast, members of Proteobacteria and Acidobacteriota harboured fewer BGCs (6–7 on average), yet their high abundance in MCE sponges suggests a potentially rich reservoir of BGCs. Analysis of the BGC distribution patterns revealed that a subset of BGCs, including terpene GCFs (FAM_00447 and FAM_01046), PKS GCF (FAM_00235), and RiPPs GCF (FAM_01143), were widespread across mesophotic sponges. Furthermore, 32 GCFs were consistently present in the same MAGs across different sponges, highlighting their potential key biological roles and capacity to yield novel bioactive compounds. This study not only underscores the untapped potential of mesophotic sponge-associated bacteria as a source of bioactive compounds but also provides valuable insights into the intricate interactions between sponges and their symbiotic microbial communities.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143431133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of Oxygen Availability on the Organelle-Specific Redox Potentials and Stress in Recombinant Protein Producing Komagataella phaffii","authors":"Aliki Kostopoulou,&nbsp;Corinna Rebnegger,&nbsp;Borja Ferrero-Bordera,&nbsp;Matthias Mattanovich,&nbsp;Sandra Maaß,&nbsp;Dörte Becher,&nbsp;Brigitte Gasser,&nbsp;Diethard Mattanovich","doi":"10.1111/1751-7915.70106","DOIUrl":"https://doi.org/10.1111/1751-7915.70106","url":null,"abstract":"<p>The yeast <i>Komagataella phaffii</i> (syn. <i>Pichia pastoris</i>) is a highly effective and well-established host for the production of recombinant proteins. The redox balance of its secretory pathway, which is multi-organelle dependent, is of high importance for producing secretory proteins. Redox imbalance and oxidative stress can significantly influence protein folding and secretion. Glutathione serves as the main redox buffer of the cell and cellular redox conditions can be assessed through the status of the glutathione redox couple (GSH-GSSG). Previous research often focused on the redox potential of the endoplasmic reticulum (ER), where oxidative protein folding and disulphide bond formation occur. In this study, in vivo measurements of the glutathione redox potential were extended to different subcellular compartments by targeting genetically encoded redox sensitive fluorescent proteins (roGFPs) to the cytosol, ER, mitochondria and peroxisomes. Using these biosensors, the impact of oxygen availability on the redox potentials of the different organelles was investigated in non-producing and producing <i>K. phaffii</i> strains in glucose-limited chemostat cultures. It was found that the transition from normoxic to hypoxic conditions affected the redox potential of all investigated organelles, while the exposure to hyperoxic conditions did not impact them. Also, as reported previously, hypoxic conditions led to increased recombinant protein secretion. Finally, transcriptome and proteome analyses provided novel insights into the short-term response of the cells from normoxic to hypoxic conditions.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143396989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Production of the Sesquiterpene Bisabolene From One- and Two-Carbon Compounds in Engineered Methanosarcina acetivorans","authors":"Andrea Mentrup,&nbsp;Luca V. Scheitz,&nbsp;Theo Wallenfang,&nbsp;Michael Rother","doi":"10.1111/1751-7915.70105","DOIUrl":"10.1111/1751-7915.70105","url":null,"abstract":"<p>The isoprenoid bisabolene, one of the simplest monocyclic sesquiterpenes, is a natural plant product that, in addition to its biological function, serves as a precursor for many industrial products. Due to the low concentration of bisabolene and the long harvest cycle, industrial production of this isoprenoid in plants is economically challenging. Chemical synthesis of bisabolene also suffers from significant disadvantages, such as low yields, toxic side products and high costs. Archaea appear suitable producers of isoprenoids, as their membrane lipids consist of isoprenoid ethers, which are synthesised via a variant of the mevalonate (MVA) pathway. Archaeal model species have versatile metabolic capacities, which makes them potential candidates for biotechnological applications. Here, we engineered <i>Methanosarcina acetivorans</i> for production of α-bisabolene from one-carbon substrates by introducing a bisabolene synthase from <i>Abies grandis</i>. Expression of a codon-optimised bisabolene synthase gene in <i>M. acetivorans</i> resulted in 10.6 mg bisabolene/L of culture. Overexpressing genes of the MVA pathway only slightly increased bisabolene yields, which, however, were reached much earlier during incubations than in the corresponding parent strain. The data presented argue for the suitability of <i>M. acetivorans</i> for the biotechnical production of certain isoprenoids.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70105","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143389054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multifaceted Antipathogenic Activity of Two Novel Natural Products, Chermesiterpenoid B and Chermesiterpenoid B Seco Acid Methyl Ester, Against Pseudomonas aeruginosa","authors":"Dan-Dan Li,&nbsp;Ying Wang,&nbsp;Huiyan Li,&nbsp;Wen-Xin Niu,&nbsp;Jongki Hong,&nbsp;Jee H. Jung,&nbsp;Joon-Hee Lee","doi":"10.1111/1751-7915.70101","DOIUrl":"https://doi.org/10.1111/1751-7915.70101","url":null,"abstract":"<p><i>Pseudomonas aeruginosa</i> is an opportunistic human pathogen that causes both acute and chronic infections due to its virulence factors, biofilm formation and the ability to suppress the host immune system. Quorum sensing (QS) plays a key role in regulating these pathogenic traits and also downregulates the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in host cells. In this study, we isolated two novel natural products from the jellyfish-derived fungus <i>Penicillium chermesinum</i>, chermesiterpenoid B (Che B) seco acid methyl ester (Che B ester) and Che B. Both compounds act as partial agonists of PPAR-γ and exhibit anti-QS activity. Che B ester and Che B were found to inhibit biofilm formation, reduce the production of proteases and decrease the infectivity of <i>P. aeruginosa</i>, all without affecting bacterial growth. In host cells, Che B ester and Che B reduced <i>P. aeruginosa</i>-induced inflammation by activating PPAR-γ. This multifaceted function makes these compounds promising candidates for developing new antipathogenic agents against bacterial infections with few side effects.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70101","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143389052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microbiome Literacy: Enhancing Public and Academic Understanding Through the ‘Microbiome & Health’ Online Course","authors":"Matthias Schweitzer,&nbsp;Birgit Wassermann,&nbsp;Ahmed Abdelfattah,&nbsp;Tomislav Cernava,&nbsp;Gabriele Berg","doi":"10.1111/1751-7915.70094","DOIUrl":"https://doi.org/10.1111/1751-7915.70094","url":null,"abstract":"<p>Microorganisms are fundamental to life on Earth, influencing biogeochemical processes, soil fertility, and the health of humans, animals and plants. Human activities have left a remarkable footprint on the environment, including global microbiomes. Enhancing awareness and improving education about microbiome functions can contribute to a sustainable economy and resilient systems. However, public understanding of microbiome science is hindered by misinformation and limited accessible educational resources. To address this, we developed the massive open online course (MOOC) ‘Microbiome &amp; Health’, available on iMooX.at and YouTube. The course, structured into six units, covers microbiome concepts, methodologies, human and plant microbiomes, antibiotic resistance, and environmental impacts, aligning with the <i>One Health</i> concept and the Sustainable Development Goals of the United Nations. Initial results show that the MOOC provides the means to increase microbiology literacy, with 73.2% external participation and above-average completion rates. Integration as a mandatory component in university courses has improved student performance, halting declining grades and pass rates. This highlights the MOOC's potential to enhance public and academic understanding of microbiome science, fostering informed decisions for sustainable health and environmental stewardship as well as paving the way for new microbiome-based solutions in biotechnology.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70094","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143389053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multiple Chaperone DnaK–FliC Flagellin Interactions are Required for Pseudomonas aeruginosa Flagellum Assembly and Indicate a New Function for DnaK","authors":"Gabriella Molinari,&nbsp;Sara S. Ribeiro,&nbsp;Katrin Müller,&nbsp;Benjamin E. Mayer,&nbsp;Manfred Rohde,&nbsp;Alejandro Arce-Rodriguez,&nbsp;Juan José Vargas-Guerrero,&nbsp;Albert Avetisyan,&nbsp;Josef Wissing,&nbsp;Werner Tegge,&nbsp;Lothar Jänsch,&nbsp;Mark Brönstrup,&nbsp;Antoine Danchin,&nbsp;Martina Jahn,&nbsp;Kenneth N. Timmis,&nbsp;Simon Ebbinghaus,&nbsp;Dieter Jahn,&nbsp;José Manuel Borrero-de Acuña","doi":"10.1111/1751-7915.70096","DOIUrl":"https://doi.org/10.1111/1751-7915.70096","url":null,"abstract":"<p>The DnaK (Hsp70) protein is an essential ATP-dependent chaperone foldase and holdase found in most organisms. In this study, combining multiple experimental approaches we determined FliC as major interaction partner of DnaK in the opportunistic bacterial pathogen <i>Pseudomonas aeruginosa</i>. Implementing immunofluorescence microscopy and electron microscopy techniques DnaK was found extracellularly associated to the assembled filament in a regular pattern. <i>dnaK</i> repression led to intracellular FliC accumulation and motility impairment, highlighting DnaK essentiality for FliC export and flagellum assembly. SPOT–membrane peptide arrays coupled with artificial intelligence analyses suggested a highly dynamic DnaK–FliC interaction landscape involving multiple domains and transient complexes formation. Remarkably, in vitro fast relaxation imaging (FReI) experiments mimicking ATP-deprived extracellular environment conditions exhibited DnaK ATP-independent holdase activity, regardless of its co-chaperone DnaJ and its nucleotide exchange factor GrpE. We present a model for the DnaK-FliC interactions involving dynamic states throughout the flagellum assembly stages. These results expand the classical view of DnaK chaperone functioning and introduce a new participant in the <i>Pseudomonas</i> flagellar system, an important trait for bacterial colonisation and virulence.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 2","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70096","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143397065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}