{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T23:54:28Z","timestamp":1778802868878,"version":"3.51.4"},"reference-count":48,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,2,2]],"date-time":"2024-02-02T00:00:00Z","timestamp":1706832000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002341","name":"Academy of Finland","doi-asserted-by":"publisher","award":["343565, 308766"],"award-info":[{"award-number":["343565, 308766"]}],"id":[{"id":"10.13039\/501100002341","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Plant Sci."],"abstract":"<jats:p><jats:italic>Methylorubrum extorquens<\/jats:italic> DSM13060 is an endosymbiont that lives in the cells of shoot tip meristems. The bacterium is methylotrophic and consumes plant-derived methanol for the production of polyhydroxybutyrate (PHB). The PHB provides protection against oxidative stress for both host and endosymbiont cells through its fragments, methyl-esterified 3-hydroxybutyrate (ME-3HB) oligomers. We evaluated the role of the genes involved in the production of ME-3HB oligomers in the host colonization by the endosymbiont <jats:italic>M. extorquens<\/jats:italic> DSM13060 through targeted genetic mutations. The strains with deletions in PHB synthase (<jats:italic>phaC<\/jats:italic>), PHB depolymerase (<jats:italic>phaZ1<\/jats:italic>), and a transcription factor (<jats:italic>phaR<\/jats:italic>) showed altered PHB granule characteristics, as <jats:italic>\u0394phaC<\/jats:italic> had a significantly low number of granules, <jats:italic>\u0394phaR<\/jats:italic> had a significantly increased number of granules, and <jats:italic>\u0394phaZ1<\/jats:italic> had significantly large PHB granules in the bacterial cells. When the deletion strains were exposed to oxidative stress, the <jats:italic>\u0394phaC<\/jats:italic> strain was sensitive to 10 mM HO\u00b7 and 20 mM H<jats:sub>2<\/jats:sub>O<jats:sub>2<\/jats:sub>. The colonization of the host, Scots pine (<jats:italic>Pinus sylvestris<\/jats:italic> L.), by the deletion strains varied greatly. The deletion strain <jats:italic>\u0394phaR<\/jats:italic> colonized the host mainly intercellularly, whereas the <jats:italic>\u0394phaZ1<\/jats:italic> strain was a slightly poorer colonizer than the control. The deletion strain <jats:italic>\u0394phaC<\/jats:italic> lacked the colonization potential, living mainly on the surfaces of the epidermis of pine roots and shoots in contrast to the control, which intracellularly colonized all pine tissues within the study period. In earlier studies, deletions within the PHB metabolic pathway have had a minor effect on plant colonization by rhizobia. We have previously shown the association between ME-3HB oligomers, produced by PhaC and PhaZ1, and the ability to alleviate host-generated oxidative stress during plant infection by the endosymbiont <jats:italic>M. extorquens<\/jats:italic> DSM13060. Our current results show that the low capacity for PHB synthesis leads to poor tolerance of oxidative stress and loss of colonization potential by the endosymbiont. Altogether, our findings demonstrate that the metabolism of PHB in <jats:italic>M. extorquens<\/jats:italic> DSM13060 is an important trait in the non-rhizobial endosymbiosis.<\/jats:p>","DOI":"10.3389\/fpls.2024.1302705","type":"journal-article","created":{"date-parts":[[2024,2,2]],"date-time":"2024-02-02T04:50:37Z","timestamp":1706849437000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["Biosynthesis of polyhydroxybutyrate by Methylorubrum extorquens DSM13060 is essential for intracellular colonization in plant endosymbiosis"],"prefix":"10.3389","volume":"15","author":[{"given":"Namrata","family":"Baruah","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roosa","family":"Haajanen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohammad Tanvir","family":"Rahman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna Maria","family":"Pirttil\u00e4","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Janne J.","family":"Koskim\u00e4ki","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2024,2,2]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"e01265","DOI":"10.1128\/AEM.01265-20","article-title":"3-Hydroxybutyrate derived from poly-3-hydroxybutyrate mobilization alleviates protein aggregation in heat-stressed Herbaspirillum seropedicae SmR1","volume":"86","author":"Alves","year":"2020","journal-title":"Appl. Environ. Microbiol."},{"key":"B2","doi-asserted-by":"publisher","first-page":"599","DOI":"10.1139\/w05-042","article-title":"Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti PHB synthesis and degradation pathway mutants","volume":"51","author":"Aneja","year":"2005","journal-title":"Can. J. Microbiol."},{"key":"B3","doi-asserted-by":"publisher","first-page":"459","DOI":"10.1016\/0022-2836(69)90288-5","article-title":"A complementation analysis of the restriction and modification of DNA in Escherichia coli","volume":"41","author":"Boyer","year":"1969","journal-title":"J. Mol. Biol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/2191-0855-2-26","article-title":"Examination of PHB depolymerases in Ralstonia eutropha: further elucidation of the roles of enzymes in PHB homeostasis","volume":"2","author":"Brigham","year":"2012","journal-title":"AMB Express"},{"key":"B5","doi-asserted-by":"publisher","first-page":"2265","DOI":"10.1016\/j.biortech.2008.11.020","article-title":"Enhanced production of medium-chain-length polyhydroxyalkanoates (PHA) by PHA depolymerase knockout mutant of Pseudomonas putida KT2442","volume":"100","author":"Cai","year":"2009","journal-title":"Bioresour Technol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"753","DOI":"10.1128\/AEM.72.1.753-759.2006","article-title":"Multicopy integration and expression of heterologous genes in methylobacterium extorquens ATCC 55366","volume":"72","author":"Choi","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1685","DOI":"10.1128\/AEM.71.4.1685-1693.2005","article-title":"Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN","volume":"71","author":"Compant","year":"2005","journal-title":"Appl. Environ. Microbiol."},{"key":"B8","doi-asserted-by":"publisher","first-page":"1390","DOI":"10.1094\/MPMI-06-21-0138-R","article-title":"Role and regulation of poly-3-hydroxybutyrate in nitrogen fixation in azorhizobium caulinodans","volume":"34","author":"Crang","year":"2021","journal-title":"Mol. Plant-Microbe Interact."},{"key":"B9","doi-asserted-by":"crossref","DOI":"10.1007\/978-94-009-0213-8_45","article-title":"Cycling of methanol between plants, methylotrophs and the atmosphere","author":"Fall","year":"1996"},{"key":"B10","doi-asserted-by":"publisher","first-page":"1648","DOI":"10.1073\/pnas.76.4.1648","article-title":"Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans","volume":"76","author":"Figurski","year":"1979","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B11","doi-asserted-by":"publisher","first-page":"293","DOI":"10.1128\/MMBR.00050-14","article-title":"The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes","volume":"79","author":"Hardoim","year":"2015","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"B12","doi-asserted-by":"publisher","first-page":"56","DOI":"10.1186\/s12866-018-1198-4","article-title":"Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol","volume":"18","author":"Heinze","year":"2018","journal-title":"BMC Microbiol."},{"key":"B13","doi-asserted-by":"crossref","DOI":"10.1007\/978-94-007-1599-8_6","article-title":"Diversity of endophytic bacteria in forest trees","volume-title":"Endophytes of Forest Trees. Forestry Sciences","author":"Izumi","year":"2011"},{"key":"B14","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1080\/07352689891304195","article-title":"Infection and colonization of sugar cane and other graminaceous plants by endophytic diazotrophs","volume":"17","author":"James","year":"1998","journal-title":"CRC Crit. Rev. Plant Sci."},{"key":"B15","doi-asserted-by":"publisher","first-page":"757","DOI":"10.1093\/jxb\/45.6.757","article-title":"Infection of sugar cane by the nitrogen-fixing bacterium Acetobacter diazotrophicus","volume":"45","author":"James","year":"1994","journal-title":"J. Exp. Bot."},{"key":"B16","doi-asserted-by":"publisher","first-page":"3244","DOI":"10.1128\/AEM.69.6.3244-3250.2003","article-title":"Involvement of the reserve material poly-\u03b2-Hydroxybutyrate in azospirillum brasilense stress endurance and root colonization","volume":"69","author":"Kadouri","year":"2003","journal-title":"Appl. Environ. Microbiol."},{"key":"B17","doi-asserted-by":"publisher","first-page":"309","DOI":"10.1007\/s00203-003-0590-z","article-title":"Poly \u03b2-hydroxybutyrate depolymerase (PhaZ) in Azospirillum brasilense and characterization of a phaZ mutant","volume":"180","author":"Kadouri","year":"2003","journal-title":"Arch. Microbiol."},{"key":"B18","doi-asserted-by":"publisher","first-page":"2218","DOI":"10.1128\/AEM.02532-07","article-title":"Cultivation-independent characterization of Methylobacterium populations in the plant phyllosphere by automated ribosomal intergenic spacer analysis","volume":"74","author":"Knief","year":"2008","journal-title":"Appl. Environ. Microbiol."},{"key":"B19","doi-asserted-by":"publisher","first-page":"6174","DOI":"10.1128\/JB.184.22.6174-6181.2002","article-title":"Poly-\u03b2-hydroxybutyrate biosynthesis in the facultative methylotroph Methylobacterium extorquens AM1: identification and mutation of gap11, gap20, and phaR","volume":"184","author":"Korotkova","year":"2002","journal-title":"J. Bacteriol."},{"key":"B20","doi-asserted-by":"publisher","first-page":"332","DOI":"10.1038\/nchembio.2043","article-title":"Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals","volume":"12","author":"Koskim\u00e4ki","year":"2016","journal-title":"Nat. Chem. Biol."},{"key":"B21","doi-asserted-by":"publisher","first-page":"e00039","DOI":"10.1128\/mBio.00039-15","article-title":"The intracellular scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins","volume":"6","author":"Koskim\u00e4ki","year":"2015","journal-title":"mBio"},{"key":"B22","doi-asserted-by":"publisher","first-page":"391","DOI":"10.1093\/treephys\/tpab102","article-title":"The meristem-associated endosymbiont Methylorubrum extorquens DSM13060 reprograms development and stress responses of pine seedlings","volume":"42","author":"Koskim\u00e4ki","year":"2022","journal-title":"Tree Physiol."},{"key":"B23","doi-asserted-by":"publisher","first-page":"2177","DOI":"10.3390\/cells10092177","article-title":"A universal approach to analyzing transmission electron microscopy with imagej","volume":"10","author":"Lam","year":"2021","journal-title":"Cells"},{"key":"B24","first-page":"770","article-title":"Produit de d\u00e9shydratation et de polym\u00e9risation de l\u2019acide b-oxybutyrique","volume":"8","author":"Lemoigne","year":"1926","journal-title":"Bull. Soc. Chim. Biol. (Paris)"},{"key":"B25","doi-asserted-by":"publisher","first-page":"596","DOI":"10.1242\/jcs.001362","article-title":"Targeted alterations in iron homeostasis underlie plant defense responses","volume":"120","author":"Liu","year":"2007","journal-title":"J. Cell Sci."},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1186\/1756-0500-1-1","article-title":"Development of a broad-host-range sacB-based vector for unmarked allelic exchange","volume":"1","author":"Marx","year":"2008","journal-title":"BMC Res. Notes"},{"key":"B27","doi-asserted-by":"publisher","first-page":"617","DOI":"10.1021\/jf052555p","article-title":"Novel fluorometric assay for hydroxyl radical scavenging capacity (HOSC) estimation","volume":"54","author":"Moore","year":"2006","journal-title":"J. Agric. Food Chem."},{"key":"B28","doi-asserted-by":"publisher","first-page":"975","DOI":"10.1007\/s00425-012-1646-6","article-title":"Iron and reactive oxygen responses in Pinus sylvestris root cortical cells infected with different species of Heterobasidion annosum sensu lato","volume":"236","author":"Mucha","year":"2012","journal-title":"Planta"},{"key":"B29","doi-asserted-by":"publisher","first-page":"fuaa058","DOI":"10.1093\/femsre\/fuaa058","article-title":"The protective role of PHB and its degradation products against stress situations in bacteria","volume":"45","author":"M\u00fcller-Santos","year":"2021","journal-title":"FEMS Microbiol. Rev."},{"key":"B30","doi-asserted-by":"publisher","first-page":"1359","DOI":"10.1104\/pp.108.4.1359","article-title":"Methanol emission from leaves (enzymatic detection of gas-phase methanol and relation of methanol fluxes to stomatal conductance and leaf development)","volume":"108","author":"Nemecek-Marshall","year":"1995","journal-title":"Plant Physiol."},{"key":"B31","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12866-018-1317-2","article-title":"Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation","volume":"18","author":"Nishihata","year":"2018","journal-title":"BMC Microbiol."},{"key":"B32","doi-asserted-by":"publisher","first-page":"1261","DOI":"10.1007\/s11274-009-0296-8","article-title":"Effect of ethanol and hydrogen peroxide on poly(3-hydroxybutyrate) biosynthetic pathway in Cupriavidus necator H16","volume":"26","author":"Obruca","year":"2010","journal-title":"World J. Microbiol. Biotechnol."},{"key":"B33","doi-asserted-by":"publisher","first-page":"3073","DOI":"10.1128\/AEM.66.7.3073-3077.2000","article-title":"Detection of intracellular bacteria in the buds of Scotch pine (Pinus sylvestris L.) by in situ hybridization","volume":"66","author":"Pirttil\u00e4","year":"2000","journal-title":"Appl. Environ. Microbiol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"993","DOI":"10.1093\/treephys\/tpu062","article-title":"Interaction with ectomycorrhizal fungi and endophytic Methylobacterium affects nutrient uptake and growth of pine seedlings in vitro","volume":"34","author":"Pohjanen","year":"2014","journal-title":"Tree Physiol."},{"key":"B35","doi-asserted-by":"publisher","first-page":"4299","DOI":"10.1128\/AEM.00757-16","article-title":"Regulation of polyhydroxybutyrate synthesis in the soil bacterium bradyrhizobium diazoefficiens","volume":"82","author":"Quelas","year":"2016","journal-title":"Appl. Environ. Microbiol."},{"key":"B36","doi-asserted-by":"publisher","first-page":"3145","DOI":"10.1128\/JB.02203-12","article-title":"Analysis of two polyhydroxyalkanoate synthases in Bradyrhizobium japonicum USDA 110","volume":"195","author":"Quelas","year":"2013","journal-title":"J. Bacteriol."},{"key":"B37","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/j.1574-6968.2007.00918.x","article-title":"Bacterial endophytes: recent developments and applications","volume":"278","author":"Ryan","year":"2008","journal-title":"FEMS Microbiol. Lett."},{"key":"B38","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1016\/0378-1119(94)90324-7","article-title":"Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum","volume":"145","author":"Sch\u00e4fer","year":"1994","journal-title":"Gene"},{"key":"B39","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1007\/s00792-012-0445-0","article-title":"Hydroxybutyrate prevents protein aggregation in the halotolerant bacterium Pseudomonas sp. CT13 under abiotic stress","volume":"16","author":"Soto","year":"2012","journal-title":"Extremophiles"},{"key":"B40","doi-asserted-by":"publisher","first-page":"7245","DOI":"10.1128\/AEM.71.11.7245-7252.2005","article-title":"Methylotrophic metabolism is advantageous for Methylobacterium extorquens during colonization of Medicago truncatula under competitive conditions","volume":"71","author":"Sy","year":"2005","journal-title":"Appl. Environ. Microbiol."},{"key":"B41","doi-asserted-by":"publisher","first-page":"3825","DOI":"10.1128\/JB.187.11.3825-3832.2005","article-title":"Analysis of transient polyhydroxybutyrate production in Wautersia eutropha H16 by quantitative Western analysis and transmission electron microscopy","volume":"187","author":"Tian","year":"2005","journal-title":"J. Bacteriol."},{"key":"B42","doi-asserted-by":"publisher","first-page":"3814","DOI":"10.1128\/JB.187.11.3814-3824.2005","article-title":"Kinetic studies of polyhydroxybutyrate granule formation in Wautersia eutropha H16 by transmission electron microscopy","volume":"187","author":"Tian","year":"2005","journal-title":"J. Bacteriol."},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-10-92","article-title":"Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti","volume":"10","author":"Trainer","year":"2010","journal-title":"BMC Microbiol."},{"key":"B44","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1007\/s00253-006-0354-1","article-title":"The role of PHB metabolism in the symbiosis of rhizobia with legumes","volume":"71","author":"Trainer","year":"2006","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"B45","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1111\/cmi.12059","article-title":"Consequences of flagellin export through the type III secretion system of Pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant Nicotiana benthamiana","volume":"15","author":"Wei","year":"2013","journal-title":"Cell. Microbiol."},{"key":"B46","doi-asserted-by":"publisher","first-page":"566","DOI":"10.1002\/jemt.22375","article-title":"Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells","volume":"77","author":"White","year":"2014","journal-title":"Microsc Res. Tech"},{"key":"B47","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/S0167-7012(98)00103-1","article-title":"Bi-functional gfp-and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies","volume":"35","author":"Xi","year":"1999","journal-title":"J. Microbiol. Methods"},{"key":"B48","doi-asserted-by":"publisher","first-page":"3788","DOI":"10.1128\/JB.185.13.3788-3794.2003","article-title":"Ralstonia eutropha H16 Encodes Two and Possibly Three Intracellular Poly[ d -(\u2013)-3-Hydroxybutyrate] Depolymerase Genes","volume":"185","author":"York","year":"2003","journal-title":"J. Bacteriol."}],"container-title":["Frontiers in Plant Science"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fpls.2024.1302705\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,2,8]],"date-time":"2024-02-08T13:31:00Z","timestamp":1707399060000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fpls.2024.1302705\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,2]]},"references-count":48,"alternative-id":["10.3389\/fpls.2024.1302705"],"URL":"https:\/\/doi.org\/10.3389\/fpls.2024.1302705","relation":{},"ISSN":["1664-462X"],"issn-type":[{"value":"1664-462X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,2]]},"article-number":"1302705"}}