cfb040ca315e9484401f79c9d0511d47 pmc_10304964.pdf 729380271d2869d35e16a433df07d2de9a090b01 pmc_10304964.pdf 0134332a19bb1a706b8fac2a7784f1e490f39b00f9fe7029c258fbe2ce5b5a96 pmc_10304964.pdf Title: Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia Subject: Methods Keywords: Stenotrophomonas maltophilia; fluorescent labeling; transposon; mini-Tn7; biofilm Author: Uwe Mamat;Manuel Hein;Dörte Grella;Claire S. Taylor;Thomas Scholzen;Ifey Alio;Wolfgang R. Streit;Pol Huedo;Xavier Coves;Oscar Conchillo-Solé;Andromeda-Celeste Gómez;Isidre Gibert;Daniel Yero;Ulrich E. Schaible Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Fri Jun 16 05:52:08 2023 CEST ModDate: Mon Jun 19 15:54:05 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 20 Encrypted: no Page size: 584.957 x 782.986 pts Page rot: 0 File size: 3739325 bytes Optimized: no PDF version: 1.4 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- BHOILF+AdvOTce3d9a73 Type 1C WinAnsi yes yes no 181 0 BHOIMG+AdvOT56309c18.I Type 1C WinAnsi yes yes no 691 0 BHOJCF+AdvOT07517017 Type 1C WinAnsi yes yes no 692 0 BHOJCG+AdvOT3b30f6db.B Type 1C WinAnsi yes yes no 48 0 BHOJCH+AdvOT46dcae81 Type 1C WinAnsi yes yes no 49 0 BHOJCI+AdvOT46dcae81+fb Type 1C Custom yes yes yes 50 0 BHOJCJ+AdvOT65f8a23b.I Type 1C WinAnsi yes yes no 51 0 BHOJDJ+AdvOT8817665d Type 1C WinAnsi yes yes no 83 0 BHOJDK+AdvOTd0125be5.BI Type 1C WinAnsi yes yes no 84 0 BHOJDL+AdvP7DA6 Type 1C WinAnsi yes yes no 85 0 BHOJDM+AdvOT46dcae81+20 Type 1C WinAnsi yes yes no 52 0 BHOJDN+AdvOT07517017+fb Type 1C Custom yes yes yes 693 0 BHOJHN+AdvOT9d186844 Type 1C WinAnsi yes yes no 80 0 BHOJJN+AdvOT65f8a23b.I+fb Type 1C Custom yes yes yes 105 0 BHOJJO+AdvOT3b30f6db.B+fb Type 1C Custom yes yes yes 106 0 BHOKOG+AdvP80675 Type 1C WinAnsi yes yes no 162 0 BHOLAH+AdvMPI4IT Type 1C WinAnsi yes yes no 178 0 BHOLMD+AdvP4C4E74 Type 1C Custom yes yes no 219 0 BHOMFA+AdvPSMP13 Type 1C WinAnsi yes yes no 266 0 BHOMGB+AdvPSUnvGMP Type 1C WinAnsi yes yes no 269 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-10-26 02:25:11 CEST RepresentationInformation: pmc_10304964.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-10-18 02:19:07 CEST Size: 3739325 Format: PDF Version: 1.4 Status: Well-Formed, but not valid SignatureMatches: PDF-hul ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - 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referenced object 'lBB95' cannot be found Offset: 3484018 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'lBB38' cannot be found Offset: 3484018 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'lBB96' cannot be found Offset: 3484018 MIMEtype: application/pdf PDFMetadata: Objects: 753 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: true NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: RESULTS AND DISCUSSION Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Children: Item: Title: The 3′-end of the glmS gene in S. maltophilia. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Construction of mini-Tn7 delivery plasmids for S. maltophilia. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Chromosomal insertion of mini-Tn7 elements and fluorescent labeling of S. maltophilia. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: Stability of mini-Tn7 insertions. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Growth properties of fluorescently labeled S. maltophilia strains. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Biofilm formation of fluorescently labeled S. maltophilia strains. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: Virulence of fluorescently labeled S. maltophilia strains. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Item: Title: MATERIALS AND METHODS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Children: Item: Title: Bacterial strains, plasmids and growth conditions. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@69b0fd6f Item: Title: Cell culture. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@757942a1 Item: Title: Construction of mini-Tn7 delivery plasmids for fluorescent labeling of S. maltophilia. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4a87761d Item: Title: Delivery of mini-Tn7 plasmids into S. maltophilia. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@66d1af89 Item: Title: Confocal laser-scanning microscopy of bacterial cells and biofilms. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@8646db9 Item: Title: Stability testing. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@37374a5e Item: Title: Growth of strains with mini-Tn7 insertions. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4671e53b Item: Title: Antibiotic susceptibility testing. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@2db7a79b Item: Title: Biofilm formation assay. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6950e31 Item: Title: Virulence in Galleria mellonella. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@b7dd107 Item: Title: Data availability. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@42eca56e Item: Title: SUPPLEMENTAL MATERIAL Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@52f759d7 Item: Title: ACKNOWLEDGMENT Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7cbd213e Item: Title: REFERENCES Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@192d3247 Info: Title: Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia Author: Uwe Mamat;Manuel Hein;Dörte Grella;Claire S. Taylor;Thomas Scholzen;Ifey Alio;Wolfgang R. Streit;Pol Huedo;Xavier Coves;Oscar Conchillo-Solé;Andromeda-Celeste Gómez;Isidre Gibert;Daniel Yero;Ulrich E. Schaible Subject: Methods Keywords: Stenotrophomonas maltophilia; fluorescent labeling; transposon; mini-Tn7; biofilm Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Fri Jun 16 06:22:08 CEST 2023 ModDate: Mon Jun 19 16:24:05 CEST 2023 ID: 0xb0312765b584de2fd5a8bfa3d8226d37, 0x08d4ca0c6b1ab70658c0ac4ffa34172d Filters: FilterPipeline: FlateDecode Images: Image: NisoImageMetadata: FormatName: image/png ImageWidth: 67 ImageHeight: 67 BitsPerSample: 8 BitsPerSampleUnit: integer Filter: FlateDecode Fonts: Type1: Font: BaseFont: BHOKOG+AdvP80675 FontSubset: true FirstChar: 46 LastChar: 54 FontDescriptor: FontName: BHOKOG+AdvP80675 Flags: Nonsymbolic FontBBox: -20, -229, 1010, 812 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: BHOJJN+AdvOT65f8a23b.I+fb FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: BHOJJN+AdvOT65f8a23b.I+fb 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LastChar: 1 FontDescriptor: FontName: BHOLMD+AdvP4C4E74 Flags: Symbolic FontBBox: -20, -958, 1114, 770 FontFile3: true EncodingDictionary: Differences: true XMP: Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia 10.1128/aem.00317-23 Uwe Mamat Manuel Hein Dörte Grella Claire S. Taylor Thomas Scholzen Ifey Alio Wolfgang R. Streit Pol Huedo Xavier Coves Oscar Conchillo-Solé Andromeda-Celeste Gómez Isidre Gibert Daniel Yero Ulrich E. Schaible Methods Methods Stenotrophomonas maltophilia fluorescent labeling transposon mini-Tn7 biofilm American Society for Microbiology Mamat et al. Copyright © 2023 Mamat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. 20230517 2023 ABSTRACT Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as Stenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7 delivery plasmids for labeling of S. maltophilia with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3′-end of the conserved glmS gene of different S. maltophilia wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in Galleria mellonella. It is also shown that the mini-Tn7 elements remained stably integrated in the genome of S. maltophilia over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7 delivery plasmids are valuable tools for generating fluorescently labeled S. maltophilia strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium S. maltophilia is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of S. maltophilia infections worldwide. One important virulence-related trait of S. maltophilia is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system for S. maltophilia to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions. 20230223 20230421 1098-5336 Pages: Page: Label: 1 Annotations: Annotation: Subtype: Link Rect: 50, 585, 97, 594 Annotation: Subtype: Link Rect: 364, 585, 396, 594 Annotation: Subtype: Link Rect: 411, 585, 482, 594 Annotation: Subtype: Link Rect: 324, 574, 372, 583 Annotation: Subtype: Link Rect: 393, 574, 438, 583 Annotation: Subtype: Link Rect: 195, 43, 200, 52 Destination: 302 Annotation: Subtype: Link Rect: 204, 43, 209, 52 Destination: 302 Annotation: Subtype: Link Rect: 417, 124, 513, 131 Annotation: Subtype: Link Rect: 400, 115, 456, 122 Annotation: Subtype: Link Rect: 459, 24, 526, 31 Annotation: Subtype: Link Rect: 508, 683, 544, 719 Page: Label: 2 Annotations: Annotation: Subtype: Link Rect: 283, 677, 287, 686 Destination: 302 Annotation: Subtype: Link Rect: 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Rect: 455, 24, 522, 31 Checksum: 9684fa8e Type: CRC32 Checksum: cfb040ca315e9484401f79c9d0511d47 Type: MD5 Checksum: 729380271d2869d35e16a433df07d2de9a090b01 Type: SHA-1