The σ54 System Directly Regulates Bacterial Natural Product Genes

The σ54 System Directly Regulates Bacterial Natural Product Genes | 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 The σ54 System Directly Regulates Bacterial Natural Product Genes Muqing Ma, Roy Welch, Anthony Garza This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-110328/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Feb, 2021 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutic agents and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery that controls their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explore this idea by analyzing four putative σ54 promoters identified in the sequences of Myxococcus xanthus natural product gene clusters. We show that mutations in the putative σ54-RNA polymerase binding regions reduce in vivo promoter activities during growth and development. We also show that the EBP Nla28 is important for the in vivo activities of three natural product promoters, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the putative Nla28 binding sites are mutated. These results indicate that the natural product promoters are bona fide σ54 promoter elements and three are direct targets of Nla28. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product clusters are located within genes and not in intergenic sequences. General Microbiology Molecular Biology enhancer binding proteins (EBPs) Myxococcus Nla28 σ54 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Supplementary Files NPNla28papersupplementaryfigurestablesMM.pdf Cite Share Download PDF Status: Published Journal Publication published 26 Feb, 2021 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 31 Dec, 2020 Reviewers agreed at journal 02 Dec, 2020 Reviews received at journal 01 Dec, 2020 Reviewers agreed at journal 20 Nov, 2020 Reviewers invited by journal 20 Nov, 2020 Editor assigned by journal 20 Nov, 2020 Editor invited by journal 20 Nov, 2020 Submission checks completed at journal 20 Nov, 2020 First submitted to journal 17 Nov, 2020 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. 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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-110328","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":5099517,"identity":"2749ca5d-7702-4c11-b5de-1beb463c66f0","order_by":0,"name":"Muqing Ma","email":"","orcid":"","institution":"Syracuse University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muqing","middleName":"","lastName":"Ma","suffix":""},{"id":5099518,"identity":"382d0cef-82fa-47ba-b324-971fa9897df8","order_by":1,"name":"Roy Welch","email":"","orcid":"","institution":"Syracuse University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Roy","middleName":"","lastName":"Welch","suffix":""},{"id":5099519,"identity":"aac3527b-c6d9-43f3-845e-7db537d71dd0","order_by":2,"name":"Anthony Garza","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYPACGwjFQ4KWNNK1HCZBC3//4mcff9Sctze4fYDxwds2IrRI3HhmPEPi2O3EDecSmA3nEqOF4cYBYwYDttsJBmcY2KR5idEif+P4Z4aEf+fsgVrYfxOlxeB8jzHDwbYDjBuAtjATpcXwBk8xY2NfcuLMM4zNknPOEaFF7vzxzYw/vtnZ851hPvjhTRkRWhgkEiC0wgHGBmLUAwH/AQgtT6yGUTAKRsEoGHkAAD/kOHy/MCsDAAAAAElFTkSuQmCC","orcid":"","institution":"Syracuse University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"","lastName":"Garza","suffix":""}],"badges":[],"createdAt":"2020-11-17 15:59:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-110328/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-110328/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-021-84057-4","type":"published","date":"2021-02-26T19:02:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3799351,"identity":"6854575d-d1d2-43a2-abcb-0ae127907c7a","added_by":"auto","created_at":"2020-11-24 17:58:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96103,"visible":true,"origin":"","legend":"The promoter regions of the MXAN1286, MXAN1579, MXAN1603 and MXAN3778 natural product loci. Nucleotides the match those in the consensus Nla28 binding site or the consensus σ54 RNA polymerase binding site are relatively large. The conserved GC dinucleotide in -12 region and the conserved GG dinucleotide in -24 region of the putative σ54 RNA polymerase binding sites are in bold. The underlined nucleotides represent the spacers between the two half Nla28 binding sites or the spacers between -12 and -24 promoter regions. ","description":"","filename":"1.PNG","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1/ec5f698a3d13dbd030ba8485.PNG"},{"id":3799352,"identity":"735403e6-cf95-4118-bf65-f49a3e87b9ea","added_by":"auto","created_at":"2020-11-24 17:58:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85549,"visible":true,"origin":"","legend":" Location of putative PK/NRP σ54 promoters identified in the M. xanthus genome. Of the 83 putative PK/NRP σ54 promoters identified in M. xanthus genome sequence, 74 (89%) are located in protein coding sequences (intragenic promoters) and 9 (11%) are located in non-coding sequences (intergenic promoters). Of the 74 intragenic promoters, 43 are located within a protein coding sequence in an operon or within the protein coding sequence of a single gene (internal promoters), and 31 are located in the protein coding sequence of an upstream gene (upstream promoters). ","description":"","filename":"2.PNG","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1/0c9eb50b049d9548115b66cc.PNG"},{"id":3799353,"identity":"b8f2d0be-662b-4234-9a1b-e893882d09a7","added_by":"auto","created_at":"2020-11-24 17:58:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120031,"visible":true,"origin":"","legend":"In vivo activities of wild-type MXAN1286, MXAN1579 and MXAN3778 promoters and derivatives of the promoters carrying a mutation in the putative -12 region, -24 region or spacer region. Wild-type and mutant fragments of the MXAN1286, MXAN1579 and MXAN3778 promoters were cloned into a lacZ expression vector and transferred to the wild-type M. xanthus strain DK1622. At various cell densities during growth (A-C) and time points during development (D-F), β-galactosidase-specific activities (defined as nanomoles of ONP produced per minute per milligram of protein) in cells carrying a wild-type or a mutant promoter fragment were determined. Mean β-galactosidase specific activities derived from three biological replicates are shown. Error bars represent standard deviations of the means.","description":"","filename":"3.PNG","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1/f51d9da60ddb30dde77c3a54.PNG"},{"id":3799354,"identity":"b17c8e09-19a3-4b03-ae00-9dcd9075e08e","added_by":"auto","created_at":"2020-11-24 17:58:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75394,"visible":true,"origin":"","legend":"In vivo activities of the wild-type MXAN1603 promoter and derivatives of the promoter carrying a mutation in the putative -12 region, -24 region or spacer region. Wild-type and mutant fragments of the MXAN1603 promoter were cloned into a lacZ expression vector and transferred to the wild-type M. xanthus strain DK1622. At various cell densities during growth (A) and time points during development (B), β-galactosidase-specific activities in cells carrying a wild-type or a mutant promoter fragment were determined. Mean β-galactosidase specific activities derived from three biological replicates are shown. Error bars represent standard deviations of the means. ","description":"","filename":"4.PNG","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1/e33d686a00affffe26d45bc2.PNG"},{"id":3799355,"identity":"1788a853-aff1-4ea0-b1e0-cc2ff6cc476e","added_by":"auto","created_at":"2020-11-24 17:58:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176238,"visible":true,"origin":"","legend":"In vivo activities of the MXAN1286, MXAN1579 and MXAN3778 promoters in wild-type and nla28- cells. Fragments of the MXAN1286, MXAN1579 and MXAN3778 promoters were cloned into a lacZ expression vector and transferred to the wild-type M. xanthus strain DK1622 or to a derivative of strain DK1622 with an inactivated nla28 gene. At various cell densities during growth (A-C) and time points during development (D-F), β-galactosidase-specific activities in cells carrying a wild-type or a mutant promoter fragment were determined. Mean β-galactosidase specific activities derived from three biological replicates are shown. Error bars represent standard deviations of the means.","description":"","filename":"5.PNG","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1/e44f372080b1ee58889e9bde.PNG"},{"id":3799356,"identity":"30ac19bc-7479-4696-8c9e-eca30e5e0224","added_by":"auto","created_at":"2020-11-24 17:58:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":179841,"visible":true,"origin":"","legend":"In vivo activities of MXAN1286, MXAN1579 and MXAN3778 promoters containing a wild-type or mutated Nla28 binding site. Fragments of the MXAN1286, MXAN1579 and MXAN3778 promoters were cloned into a lacZ expression vector and transferred to the wild-type M. xanthus strain DK1622. At various cell densities during growth (A-C) and time points during development (D-F), β-galactosidase-specific activities in cells carrying a wild-type or a mutant promoter fragment were determined. Mean β-galactosidase specific activities derived from three biological replicates are shown. Error bars represent standard deviations of the means. ","description":"","filename":"6.PNG","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1/817ec93e672278550a99ed9e.PNG"},{"id":3799357,"identity":"8a051d5f-8c19-4780-a584-78aa3424bce2","added_by":"auto","created_at":"2020-11-24 17:58:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":102522,"visible":true,"origin":"","legend":"EMSAs performed with Nla28-DBD and a MXAN1286, MXAN1579 or MXAN3778 promoter fragment carrying a wild-type or mutated Nla28 binding site. Binding reactions were performed with (+) or without (-) 2 μM of purified Nla28-DBD and a Cy5 end-labeled promoter fragment containing a wild-type (WT) or mutated (Mut) Nla28 binding site. ","description":"","filename":"7.PNG","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1/bcbb8e8f6b01f42cde16e673.PNG"},{"id":13558184,"identity":"55d8452c-ed24-4460-93f8-233daddf18d0","added_by":"auto","created_at":"2021-09-17 02:55:48","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":678191,"visible":true,"origin":"","legend":"","description":"","filename":"NPNla28PapermanuscriptMM.pdf","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1_covered.pdf"},{"id":3799358,"identity":"1714252a-f2c6-46a0-80d7-a5880e79950b","added_by":"auto","created_at":"2020-11-24 17:58:16","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":279339,"visible":true,"origin":"","legend":"","description":"","filename":"NPNla28PapermanuscriptMM.pdf","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1_stamped.pdf"},{"id":3799350,"identity":"8b393d10-48fb-4e43-af26-416ee999a216","added_by":"auto","created_at":"2020-11-24 17:58:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":597110,"visible":true,"origin":"","legend":"","description":"","filename":"NPNla28papersupplementaryfigurestablesMM.pdf","url":"https://assets-eu.researchsquare.com/files/rs-110328/v1/f486d3b1b48a602a2752ca98.pdf"}],"financialInterests":"","formattedTitle":"The σ54 System Directly Regulates Bacterial Natural Product Genes","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThis preprint is available for \u003ca href='/article/rs-110328/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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"enhancer binding proteins (EBPs), Myxococcus, Nla28, σ54","lastPublishedDoi":"10.21203/rs.3.rs-110328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-110328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutic agents and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery that controls their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explore this idea by analyzing four putative σ54 promoters identified in the sequences of Myxococcus xanthus natural product gene clusters. We show that mutations in the putative σ54-RNA polymerase binding regions reduce in vivo promoter activities during growth and development. We also show that the EBP Nla28 is important for the in vivo activities of three natural product promoters, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the putative Nla28 binding sites are mutated. These results indicate that the natural product promoters are bona fide σ54 promoter elements and three are direct targets of Nla28. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product clusters are located within genes and not in intergenic sequences.","manuscriptTitle":"The σ54 System Directly Regulates Bacterial Natural Product Genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-24 17:58:11","doi":"10.21203/rs.3.rs-110328/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-01-01T04:24:34+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"d2ab934a-2c75-42c1-9fb7-703902d82ff8","date":"2020-12-02T14:27:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-01T19:14:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c36e6ead-6df6-47b3-802a-8942653e725e","date":"2020-11-20T20:49:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-11-20T19:55:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-11-20T19:42:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-11-20T19:33:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-11-20T16:04:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2020-11-17T15:44:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ee3c8f7-6056-4070-80ab-ad5b6c388029","owner":[],"postedDate":"November 24th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1189633,"name":"General Microbiology"},{"id":1189634,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2021-08-18T19:14:17+00:00","versionOfRecord":{"articleIdentity":"rs-110328","link":"https://doi.org/10.1038/s41598-021-84057-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2021-02-26 19:02:34","publishedOnDateReadable":"February 26th, 2021"},"versionCreatedAt":"2020-11-24 17:58:11","video":"","vorDoi":"10.1038/s41598-021-84057-4","vorDoiUrl":"https://doi.org/10.1038/s41598-021-84057-4","workflowStages":[]},"version":"v1","identity":"rs-110328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-110328","identity":"rs-110328","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}