Improved mini-Tn 7 Delivery Plasmids for Fluorescent Labeling of
Mamat, Uwe (Research Center Borstel)
Hein, Manuel (Research Center Borstel)
Grella, Dörte (Research Center Borstel)
Taylor, Claire S. (Research Center Borstel)
Scholzen, Thomas (Research Center Borstel)
Alio, Ifey (Universität Hamburg. Department of Microbiology and Biotechnology)
Streit, Wolfgang R. (Universität Hamburg. Department of Microbiology and Biotechnology)
Huedo Moreno, Pol (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Coves, Xavier (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Conchillo-Solé, Oscar (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Gómez Camacho, Andromeda Celeste (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Gibert, Isidre (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Yero, Daniel (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Schaible, Ulrich E. (Research Center Borstel)

Fecha:	2023
Resumen:	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 . Using a Tn 7 -based genomic integration system, we report the construction of improved mini-Tn 7 delivery plasmids for labeling of 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-Tn 7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different 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 . It is also shown that the mini-Tn 7 elements remained stably integrated in the genome of over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn 7 delivery plasmids are valuable tools for generating fluorescently labeled strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium 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 infections worldwide. One important virulence-related trait of 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-Tn 7 -based labeling system for to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.
Ayudas:	Agencia Estatal de Investigación PID2019-111364RB-I00 Agència de Gestió d'Ajuts Universitaris i de Recerca 2017/SGR-1062
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Materia:	Stenotrophomonas maltophilia ; Fluorescent labeling ; Transposon ; Mini-tn 7 ; Biofilm
Publicado en:	Applied and Environmental Microbiology, Vol. 89, Issue 6 (June 2023) , ISSN 1098-5336

DOI: 10.1128/aem.00317-23
PMID: 37195181

20 p, 3.6 MB

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Documentos de investigación > Documentos de los grupos de investigación de la UAB > Centros y grupos de investigación (producción científica) > Ciencias de la salud y biociencias > Instituto de Biotecnología y de Biomedicina (IBB)
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