{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T09:49:55Z","timestamp":1765792195644,"version":"3.48.0"},"reference-count":52,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T00:00:00Z","timestamp":1765756800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Komagataeibacter species are the best producers of bacterial nanocellulose membranes (BNC)\u2014amazing biomaterial with unique properties and applications in the medical and food industries. The molecular mechanisms of BNC synthesis control remain poorly understood and the need for BNC production and structure improvement is growing. Looking for the genes significant for biosynthesis, we studied the unexplored effect of ClpP proteolytic subunit inactivation on Komagataeibacter xylinus E25 cell morphology and production of BNC. A mutant with a disrupted clpP gene and a complemented strain were obtained. The colonies of the mutant cells, in contrast to the wild-type and complemented ones, were smaller, irregular, and were surrounded by a polymeric noncellulosic envelope. Additionally, the mutant cells were longer and organized in chains, showing different growth and production dynamics of BNC when grown under standard conditions. We also observed worse growth and production of BNC at 5 \u00b0C above optimal temperature and in the presence of increased levels of ethanol. E25 mutant cells were characterized by lower viability under stress conditions. The 3D microstructure of BNC displayed thicker fibers and denser packing and contained more hard-to-extract exopolysaccharides (HE-EPSs). Based on the outcomes, we conclude that the effect of ClpP on K. xylinus decreased resistance to stress and lowered the BNC production level.<\/jats:p>","DOI":"10.3390\/ijms262412047","type":"journal-article","created":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T09:36:39Z","timestamp":1765791399000},"page":"12047","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Effect of clpP Gene Disruption on Cell Morphology, Growth, and the Ability to Synthesize Cellulose of Komagataeibacter\u00a0xylinus E25"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2525-9751","authenticated-orcid":false,"given":"Marzena","family":"J\u0119drzejczak-Krzepkowska","sequence":"first","affiliation":[{"name":"Institute of Molecular and Industrial Biotechnology, Lodz University of Technology, Stefanowskiego Street 2\/22, 90-537 Lodz, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5403-7640","authenticated-orcid":false,"given":"Karolina","family":"Ludwicka","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Industrial Biotechnology, Lodz University of Technology, Stefanowskiego Street 2\/22, 90-537 Lodz, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stanislaw","family":"Bielecki","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Industrial Biotechnology, Lodz University of Technology, Stefanowskiego Street 2\/22, 90-537 Lodz, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1989","DOI":"10.1007\/s10570-012-9777-2","article-title":"Cellulose Production by Enterobacter sp. CJF-002 and Identification of Genes for Cellulose Biosynthesis","volume":"19","author":"Sunagawa","year":"2012","journal-title":"Cellulose"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e05075","DOI":"10.1002\/advs.202505075","article-title":"Genetic Impacts on the Structure and Mechanics of Cellulose Made by Bacteria","volume":"12","author":"Laurent","year":"2025","journal-title":"Adv. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Kaczmarek, M., J\u0119drzejczak-Krzepkowska, M., and Ludwicka, K. (2022). Comparative Analysis of Bacterial Cellulose Membranes Synthesized by Chosen Komagataeibacter Strains and Their Application Potential. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23063391"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.ijfoodmicro.2010.01.006","article-title":"Population Dynamics of Acetic Acid Bacteria During Traditional Wine Vinegar Production","volume":"138","author":"Vegas","year":"2010","journal-title":"Int. J. Food Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Suresh, S. (2018). Biosynthesis and Assemblage of Extracellular Cellulose by Bacteria. Handbook of Environmental Materials Management, Springer.","DOI":"10.1007\/978-3-319-58538-3_71-1"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1128\/mr.55.1.35-58.1991","article-title":"Cellulose Biosynthesis and Function in Bacteria","volume":"55","author":"Ross","year":"1991","journal-title":"Microbiol. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2448","DOI":"10.1128\/aem.55.10.2448-2452.1989","article-title":"Alternative Environmental Roles for Cellulose Produced by Acetobacter xylinum","volume":"55","author":"Cannon","year":"1989","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Tr\u010dek, J., Dogsa, I., Accetto, T., and Stopar, D. (2021). Acetan and Acetan-Like Polysaccharides: Genetics, Biosynthesis, Structure, and Viscoelasticity. Polymers, 13.","DOI":"10.3390\/polym13050815"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3965","DOI":"10.1007\/s10570-014-0443-8","article-title":"Characterization of Water-Soluble Exopolysaccharides from Gluconacetobacter xylinus and Their Impacts on Bacterial Cellulose Crystallization and Ribbon Assembly","volume":"21","author":"Fang","year":"2014","journal-title":"Cellulose"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1016\/j.carbpol.2014.09.028","article-title":"Characterization of Cellulose and Other Exopolysaccharides Produced from Gluconacetobacter Strains","volume":"115","author":"Fang","year":"2015","journal-title":"Carbohydr. Polym."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"978","DOI":"10.1002\/jbm.b.34191","article-title":"Stable Composite of Bacterial Nanocellulose and Perforated Polypropylene Mesh for Biomedical Applications","volume":"107","author":"Ludwicka","year":"2019","journal-title":"J. Biomed. Mater. Res. B Appl. Biomater."},{"key":"ref_12","first-page":"527","article-title":"Modified Bacterial Cellulose Tubes for Regeneration of Damaged Peripheral Nerves","volume":"9","author":"Cala","year":"2013","journal-title":"Arch. Med. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"120252","DOI":"10.1016\/j.carbpol.2022.120252","article-title":"The State-of-the-Art Application of Functional Bacterial Cellulose-Based Materials in Biomedical Fields","volume":"300","author":"Qian","year":"2023","journal-title":"Carbohydr. Polym."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Ludwicka, K., Kaczmarek, M., and Bia\u0142kowska, A. (2020). Bacterial Nanocellulose\u2014A Biobased Polymer for Active and Intelligent Food Packaging Applications: Recent Advances and Developments. Polymers, 12.","DOI":"10.3390\/polym12102209"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"20494","DOI":"10.1038\/s41598-024-71648-0","article-title":"How Carbon Sources Drive Cellulose Synthesis in Two Komagataeibacter xylinus Strains","volume":"14","author":"Lasagni","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Anguluri, K., La China, S., Brugnoli, M., Cassanelli, S., and Gullo, M. (2022). Better under Stress: Improving Bacterial Cellulose Production by Komagataeibacter xylinus K2G30 (UMCC 2756) Using Adaptive Laboratory Evolution. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.994097"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Cielecka, I., Ryngaj\u0142\u0142o, M., and Bielecki, S. (2020). BNC Biosynthesis with Increased Productivity in a Newly Designed Surface Air-Flow Bioreactor. Appl. Sci., 10.","DOI":"10.3390\/app10113850"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"6565","DOI":"10.1007\/s00253-020-10671-3","article-title":"Towards Control of Cellulose Biosynthesis by Komagataeibacter Using Systems-Level and Strain Engineering Strategies: Current Progress and Perspectives","volume":"104","author":"Kubiak","year":"2020","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"6997","DOI":"10.1128\/JB.06158-11","article-title":"Complete Genome Sequence of NBRC 3288, a Unique Cellulose-Nonproducing Strain of Gluconacetobacter xylinus Isolated from Vinegar","volume":"193","author":"Ogino","year":"2011","journal-title":"J. Bacteriol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1007\/s12010-019-03003-3","article-title":"In Vitro Thermal and Ethanol Adaptations to Improve Vinegar Fermentation at High Temperature of Komagataeibacter oboediens MSKU 3","volume":"189","author":"Taweecheep","year":"2019","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"115243","DOI":"10.1016\/j.carbpol.2019.115243","article-title":"Superfine Bacterial Nanocellulose Produced by Reverse Mutations in the BcsC Gene during Adaptive Breeding of Komagataeibacter oboediens","volume":"226","author":"Taweecheep","year":"2019","journal-title":"Carbohydr. Polym."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5723","DOI":"10.1128\/jb.173.18.5723-5731.1991","article-title":"An Acetobacter xylinum Insertion Sequence Element Associated with Inactivation of Cellulose Production","volume":"173","author":"Coucheron","year":"1991","journal-title":"J. Bacteriol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Jedrzejczak-Krzepkowska, M., Kubiak, K., Ludwicka, K., and Bielecki, S. (2016). Bacterial NanoCellulose Synthesis, Recent Findings. Bacterial Nanocellulose: From Biotechnology to Bio-Economy, Elsevier Inc.","DOI":"10.1016\/B978-0-444-63458-0.00002-0"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e00606","DOI":"10.1016\/j.btre.2021.e00606","article-title":"Functional and Genomic Characterization of Komagataeibacter uvaceti FXV3, a Multiple Stress Resistant Bacterium Producing Increased Levels of Cellulose","volume":"30","author":"Nascimento","year":"2021","journal-title":"Biotechnol. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Deng, Y., Nagachar, N., Fang, L., Luan, X., Catchmark, J.M., Tien, M., and Kao, T.H. (2015). Isolation and Characterization of Two Cellulose Morphology Mutants of Gluconacetobacter hansenii ATCC23769 Producing Cellulose with Lower Crystallinity. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0119504"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Zheng, J., Wu, Y., Lin, Z., Wang, G., Jiang, S., Sun, X., Tu, H., Yu, Z., and Qu, D. (2020). ClpP Participates in Stress Tolerance, Biofilm Formation, Antimicrobial Tolerance, and Virulence of Enterococcus Faecalis. BMC Microbiol., 20.","DOI":"10.1186\/s12866-020-1719-9"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Moreno-Cinos, C., Goossens, K., Salado, I.G., Van Der Veken, P., De Winter, H., and Augustyns, K. (2019). ClpP Protease, a Promising Antimicrobial Target. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20092232"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1222","DOI":"10.1002\/jobm.201300747","article-title":"Contribution of ClpP to Stress Tolerance and Virulence Properties of Streptococcus Mutans","volume":"54","author":"Hou","year":"2014","journal-title":"J. Basic. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Xie, F., Zhang, Y., Li, G., Zhou, L., Liu, S., and Wang, C. (2013). Correction: The ClpP Protease Is Required for the Stress Tolerance and Biofilm Formation in Actinobacillus pleuropneumoniae. PLoS ONE, 8.","DOI":"10.1371\/annotation\/fbe62755-866e-4a4e-baf8-4bb40019a8fb"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.vetmic.2015.11.020","article-title":"ClpP Participates in Stress Tolerance and Negatively Regulates Biofilm Formation in Haemophilus parasuis","volume":"182","author":"Huang","year":"2016","journal-title":"Vet. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1016\/j.bbrc.2018.12.071","article-title":"The Regulation of Porphyromonas gingivalis Biofilm Formation by ClpP","volume":"509","author":"He","year":"2019","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1376","DOI":"10.1016\/j.micinf.2007.06.012","article-title":"Role of ClpP in Biofilm Formation and Virulence of Staphylococcus Epidermidis","volume":"9","author":"Wang","year":"2007","journal-title":"Microbes Infect."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5041","DOI":"10.1128\/JB.00505-13","article-title":"Regulation of Host Hemoglobin Binding by the Staphylococcus aureus Clp Proteolytic System","volume":"195","author":"Farrand","year":"2013","journal-title":"J. Bacteriol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1021\/acs.jproteome.0c00668","article-title":"Global Inventory of ClpP- and ClpX-Regulated Proteins in Staphylococcus aureus","volume":"20","author":"Kirsch","year":"2020","journal-title":"J. Proteome Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1139\/cjm-2012-0681","article-title":"Armand-Frappier Outstanding Student Award\u2014Role of ATPdependent Proteases in Antibiotic Resistance and Virulence","volume":"59","author":"Breidenstein","year":"2013","journal-title":"Can. J. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"5658","DOI":"10.1093\/emboj\/17.19.5658","article-title":"An Essential Protease Involved in Bacterial Cell-Cycle Control","volume":"17","author":"Jenal","year":"1998","journal-title":"EMBO J."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1497","DOI":"10.1099\/mic.0.065797-0","article-title":"ClpP Deletion Causes Attenuation of Salmonella Typhimurium Virulence through Mis-Regulation of RpoS and Indirect Control of CsrA and the SPI Genes","volume":"159","author":"Knudsen","year":"2013","journal-title":"Microbiology"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1046\/j.1365-2958.1998.00797.x","article-title":"Initiation of Biofilm Formation in Pseudomonas Fluorescens WCS365 Proceeds via Multiple, Convergent Signalling Pathways: A Genetic Analysis","volume":"28","author":"Kolter","year":"1998","journal-title":"Mol. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"6357","DOI":"10.1128\/JB.184.22.6357-6366.2002","article-title":"Regulation and Physiological Significance of ClpC and ClpP in Streptococcus Mutans","volume":"184","author":"Lemos","year":"2002","journal-title":"J. Bacteriol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1445","DOI":"10.1111\/j.1365-2958.2004.04368.x","article-title":"Clp ATPases Are Required for Stress Tolerance, Intracellular Replication and Biofilm Formation in Staphylococcus aureus","volume":"54","author":"Frees","year":"2004","journal-title":"Mol. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"5783","DOI":"10.1128\/JB.00074-06","article-title":"Global Regulatory Impact of ClpP Protease of Staphylococcus aureus on Regulons Involved in Virulence, Oxidative Stress Response, Autolysis, and DNA Repair","volume":"188","author":"Michel","year":"2006","journal-title":"J. Bacteriol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1074\/mcp.M112.017004","article-title":"Life and Death of Proteins: A Case Study of Glucose-Starved Staphylococcus aureus","volume":"11","author":"Michalik","year":"2012","journal-title":"Mol. Cell. Proteom."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/MMBR.00043-12","article-title":"Cyclic Di-GMP: The First 25 Years of a Universal Bacterial Second Messenger","volume":"77","author":"Galperin","year":"2013","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1038\/sj.jim.7000303","article-title":"Factors Affecting the Yield and Properties of Bacterial Cellulose","volume":"29","author":"Krystynowicz","year":"2002","journal-title":"J. Ind. Microbiol. Biotechnol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2727","DOI":"10.1099\/00221287-148-9-2727","article-title":"ClpP Is Involved in the Stress Response and Degradation of Misfolded Proteins in Salmonella enterica Serovar Typhimurium","volume":"148","author":"Thomsen","year":"2002","journal-title":"Microbiology"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Gonz\u00e1lez-Garc\u00eda, Y., Meza-Contreras, J.C., Guti\u00e9rrez-Ortega, J.A., and Manr\u00edquez-Gonz\u00e1lez, R. (2022). In Vivo Modification of Microporous Structure in Bacterial Cellulose by Exposing Komagataeibacter xylinus Culture to Physical and Chemical Stimuli. Polymers, 14.","DOI":"10.3390\/polym14204388"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"6673","DOI":"10.1007\/s00253-019-09904-x","article-title":"Effect of Ethanol Supplementation on the Transcriptional Landscape of Bionanocellulose Producer Komagataeibacter xylinus E25","volume":"103","author":"Jacek","year":"2019","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1677","DOI":"10.1271\/bbb.66.1677","article-title":"Effects of Acetan on Production of Bacterial Cellulose by Acetobacter xylinum","volume":"66","author":"Ishida","year":"2002","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.nbt.2024.03.004","article-title":"Complete Genome Sequence and Transcriptome Response to Vitamin C Supplementation of Novacetimonas hansenii SI1\u2014Producer of Highly-Stretchable Cellulose","volume":"81","author":"Cielecka","year":"2024","journal-title":"New Biotechnol."},{"key":"ref_50","unstructured":"Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. [3rd ed.]."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1007\/s12257-014-0316-x","article-title":"Knock-out of Glucose Dehydrogenase Gene in Gluconacetobacter xylinus for Bacterial Cellulose Production Enhancement","volume":"20","author":"Kuo","year":"2015","journal-title":"Biotechnol. Bioprocess. Eng."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"2733","DOI":"10.1099\/mic.0.051003-0","article-title":"The Dickeya dadantii Biofilm Matrix Consists of Cellulose Nanofibres, and Is an Emergent Property Dependent upon the Type Iii Secretion System and the Cellulose Synthesis Operon","volume":"157","author":"Jahn","year":"2011","journal-title":"Microbiology"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/24\/12047\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T09:47:51Z","timestamp":1765792071000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/24\/12047"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,15]]},"references-count":52,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["ijms262412047"],"URL":"https:\/\/doi.org\/10.3390\/ijms262412047","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,15]]}}}