Genome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1

Genome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1 Michele Castro, Graciela Maria Dias, Tiago Salles, Núbia Cabral, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-154442/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jan, 2022 Read the published version in Discover Genetics and Evolution → Version 1 posted You are reading this latest preprint version Abstract Background : Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role on biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids’ modulation of those group behaviors. Results : Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type P. aeruginosa PAO1 and a rhlA -mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the non-motile phenotype of the rhlA -minus strain. Swarming motility was restored with the addition of exogenous rhamnolipids obtained from the wild-type strain. Conclusions : Our results show significant evidence that rhamnolipids and/or their precursors, 3-(3-hydroxyalkanoyloxy) alkanoic acids, the major biosynthetic products of rhlABC pathway, seem to modulate gene expression in P. aeruginosa . Swarming motility assays support this hypothesis, since the non-motile rhlA -mutant strain had its swarming ability restored by the addition of exogenous rhamnolipids. Epigenetics & Genomics Pseudomonas aeruginosa Rhamnolipids Swarming Motility Flagella Transcriptomic Profile Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations No competing interests reported. Supplementary Files Castroetal2021SupplementaryFiguresandTables.pdf Cite Share Download PDF Status: Published Journal Publication published 29 Jan, 2022 Read the published version in Discover Genetics and Evolution → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-154442","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":10450539,"identity":"2d47dfe6-84e9-489d-b826-75c6ce39899d","order_by":0,"name":"Michele Castro","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Castro","suffix":""},{"id":10450540,"identity":"97f9a896-d908-403c-8fc0-89fd685d0bb3","order_by":1,"name":"Graciela Maria Dias","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Graciela","middleName":"Maria","lastName":"Dias","suffix":""},{"id":10450541,"identity":"08ecc793-0eee-4d13-b9f0-9efc5d1bba42","order_by":2,"name":"Tiago Salles","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tiago","middleName":"","lastName":"Salles","suffix":""},{"id":10450542,"identity":"b09c836c-fda7-4df3-b56a-9d585fd7f918","order_by":3,"name":"Núbia Cabral","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Núbia","middleName":"","lastName":"Cabral","suffix":""},{"id":10450543,"identity":"84672fec-7f00-4cf5-b8a5-a28891040d9a","order_by":4,"name":"Danielly Mariano","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Danielly","middleName":"","lastName":"Mariano","suffix":""},{"id":10450544,"identity":"4f007e23-1c00-4979-b4c3-3eb1d34f80e5","order_by":5,"name":"Hadassa Oliveira","email":"","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hadassa","middleName":"","lastName":"Oliveira","suffix":""},{"id":10450545,"identity":"6e5eaa2c-97ea-4bc5-b073-beb8cde81826","order_by":6,"name":"Eliana Abdelhay","email":"","orcid":"","institution":"Instituto Nacional do Câncer","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eliana","middleName":"","lastName":"Abdelhay","suffix":""},{"id":10450546,"identity":"f1b5a079-1fbf-444e-8784-b84752d91d11","order_by":7,"name":"Renata Gomes","email":"","orcid":"","institution":"Instituto Nacional do Câncer","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Renata","middleName":"","lastName":"Gomes","suffix":""},{"id":10450547,"identity":"e3652972-169d-46d1-b187-55e0156f3f67","order_by":8,"name":"Bianca Neves","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACAygtxwYiKxjY4CIEtRiDtZwhRUtiA0QLA2Et5uxnDz7mqahL72NPYJM4uIePwVz6AH4tlj15ycY8Zw7ntvE8YJM48IyNwbIvgYDDbvCYSc5sO5DbJpHAJv3hANAvZwj55QaP+c+Z/+rS2YBaJA4QqcWM4WMDcwLxWix7cowlPhw7bNjG87DZAqiFx7KHgBZz9jOGHxJq6uTl25MP3jhw4JicOQ8BLUgAHDXHSNDAwJAAImpI0TEKRsEoGAUjBAAA73g+OJLL5lkAAAAASUVORK5CYII=","orcid":"","institution":"Federal University of Rio de Janeiro","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bianca","middleName":"","lastName":"Neves","suffix":""}],"badges":[],"createdAt":"2021-01-25 18:32:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-154442/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-154442/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00294-021-01225-9","type":"published","date":"2022-01-30T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":5745069,"identity":"324f4f99-112c-46b2-939a-9a406a9db53e","added_by":"auto","created_at":"2021-02-08 17:26:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1187574,"visible":true,"origin":"","legend":"Differentially expressed genes. Hierarchical clustered heatmaps of the datasets show a total of 53 genes, with a fold-change ≤ -1.5 or ≥ 1.5 and p-value \u003c 0.05 between samples of P. aeruginosa PAO1 (PAO1 replicas 1, 2 and 3) and rhlA-mutant (rhlA replicas 1, 2 and 3). The relative differences in gene expression are proportional to the intensity of the coloration (heatmap), with blue referring to decreased and red to increased expression.","description":"","filename":"Figure1lateralcladogram15012021.png","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1/48324910ff746ffb8d72455c.png"},{"id":5745323,"identity":"7eb4dffa-9a6f-47fc-af68-8395ffd79428","added_by":"auto","created_at":"2021-02-08 17:29:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87822,"visible":true,"origin":"","legend":"Functional classification of P. aeruginosa genes differentially expressed in the microarrays. rhlA-knockout mutant compared to wild-type parental strain PAO1, with a fold change ≤ -1.5 or ≥ 1.5. The analyses were performed using the COG database.","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1/1624005636acb207694c7733.png"},{"id":5745320,"identity":"132397d4-9bdf-4519-ae62-28bc26a8c55a","added_by":"auto","created_at":"2021-02-08 17:29:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58998,"visible":true,"origin":"","legend":"Expression of flagellar genes fliC, flgL, fliD and fliT in P. aeruginosa PAO1 and rhlA-mutant strain. a) The graph represents the quantification of mRNA of genes fliC, flgL, fliD and fliT by RT-qPCR, normalized by the 16S rRNA gene, used as endogenous control. Bars indicate ± standard error. (*) indicate significantly different values, p \u003c 0.05 with the paired t-test. b) Representation of operon organization (nucleotide positions 1164275–1197833 of the PAO1 chromosome) and hierarchical regulation of flagellar genes: Class III (flgL), Class II (fliD and fliT) and Class IV (fliC). Tested genes are represented in blue, black arrows indicate operons and direction of transcription.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1/4d6f8b996c7c790770eb890d.jpg"},{"id":5745321,"identity":"4171c9b1-4645-4d88-98ae-c47ea9cf2382","added_by":"auto","created_at":"2021-02-08 17:29:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":231393,"visible":true,"origin":"","legend":"Expression of rhlA and rhlB in P. aeruginosa PAO1 and rhlA-mutant strain. The picture shows the quantification of mRNA of a) rhlA and b) rhlB by RT-qPCR, with 16S rRNA gene as endogenous control. Expression values of rhlA-mutant strain are relative to the control (PAO1), normalized as 1.0. Bars indicate ± standard error. Relative expression was calculated using the comparative Cq method.","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1/126c89e2cf253f4569149851.png"},{"id":5745064,"identity":"4aa7bbad-59a0-4217-aa70-377009543a63","added_by":"auto","created_at":"2021-02-08 17:26:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47511,"visible":true,"origin":"","legend":"Swarming motility assay. a) P. aeruginosa PAO1, b) rhlA-knockout mutant and c) rhlA-knockout mutant supplemented with exogenous rhamnolipids.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1/3d371331da27434aea334abf.jpg"},{"id":29392520,"identity":"fdefdced-2a24-4107-aab6-0b63defeb5a6","added_by":"auto","created_at":"2022-11-22 15:01:00","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1117951,"visible":true,"origin":"","legend":"","description":"","filename":"Castroetal04022021.pdf","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1_covered.pdf"},{"id":13582281,"identity":"9ed3dbd2-cd57-4092-9ed3-7b22eaec1aaf","added_by":"auto","created_at":"2021-09-17 04:30:33","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1106378,"visible":true,"origin":"","legend":"","description":"","filename":"Castroetal04022021.pdf","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1_covered.pdf"},{"id":5745743,"identity":"cab49057-1945-40de-92ba-48b511f396cc","added_by":"auto","created_at":"2021-02-08 17:36:02","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":986006,"visible":true,"origin":"","legend":"","description":"","filename":"Castroetal04022021.pdf","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1_stamped.pdf"},{"id":5745553,"identity":"bc068052-eeab-45de-92f3-3a018f9b9974","added_by":"auto","created_at":"2021-02-08 17:32:44","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":367951,"visible":true,"origin":"","legend":"","description":"","filename":"Castroetal2021SupplementaryFiguresandTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-154442/v1/be3b83846c99f0141c87c226.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGenome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in \u003cem\u003ePseudomonas Aeruginosa\u003c/em\u003e PAO1\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThis preprint is available for \u003ca href='/article/rs-154442/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pseudomonas aeruginosa, Rhamnolipids, Swarming Motility, Flagella, Transcriptomic Profile","lastPublishedDoi":"10.21203/rs.3.rs-154442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-154442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role on biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids’ modulation of those group behaviors. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 and a \u003cem\u003erhlA\u003c/em\u003e-mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the non-motile phenotype of the \u003cem\u003erhlA\u003c/em\u003e-minus strain. Swarming motility was restored with the addition of exogenous rhamnolipids obtained from the wild-type strain. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Our results show significant evidence that rhamnolipids and/or their precursors, 3-(3-hydroxyalkanoyloxy) alkanoic acids, the major biosynthetic products of \u003cem\u003erhlABC \u003c/em\u003epathway, seem to modulate gene expression in \u003cem\u003eP. aeruginosa\u003c/em\u003e. Swarming motility assays support this hypothesis, since the non-motile \u003cem\u003erhlA\u003c/em\u003e-mutant strain had its swarming ability restored by the addition of exogenous rhamnolipids.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-08 17:26:42","doi":"10.21203/rs.3.rs-154442/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6dd66442-ca78-4a73-9aa6-5e05936568e4","owner":[],"postedDate":"February 8th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":2287847,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2022-11-22T15:00:47+00:00","versionOfRecord":{"articleIdentity":"rs-154442","link":"https://doi.org/10.1007/s00294-021-01225-9","journal":{"identity":"discover-genetics-and-evolution","isVorOnly":false,"title":"Discover Genetics and Evolution"},"publishedOn":"2022-01-30 00:00:00","publishedOnDateReadable":"January 30th, 2022"},"versionCreatedAt":"2021-02-08 17:26:42","video":"","vorDoi":"10.1007/s00294-021-01225-9","vorDoiUrl":"https://doi.org/10.1007/s00294-021-01225-9","workflowStages":[]},"version":"v1","identity":"rs-154442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-154442","identity":"rs-154442","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}