BMC Microbiology | |
Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria | |
Methodology Article | |
María Trinidad Gallegos1  Lorena Romero-Jiménez1  Juan Sanjuán1  David Rodríguez-Carbonell1  Daniel Pérez-Mendoza1  | |
[1] Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain; | |
关键词: c-di-GMP; 7; Signal transduction; Biofilms; Exopolysaccharide production; Bacterial motility; Plasmid stability; | |
DOI : 10.1186/s12866-015-0521-6 | |
received in 2015-07-17, accepted in 2015-09-18, 发布年份 2015 | |
来源: Springer | |
【 摘 要 】
BackgroundThe cyclic diguanylate (c-di-GMP) is currently considered an ubiquitous second messenger in bacteria that influences a wide range of cellular processes. One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes.ResultsWe have constructed mini-Tn7 delivery vectors for the integration and stable expression of the pleD* gene encoding a highly active DGC, which can be used to artificially increase the intracellular levels of c-di-GMP in Gram negative bacteria. The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria. Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility. However, the mini-Tn7pleD* constructs resulted far more stable in the absence of antibiotics than the plasmid-based pleD* constructs. Furthermore, we have also implemented an inducible system to modulate pleD* expression and intracellular c-di-GMP rises “on demand”.Conclusionsmini-Tn7pleD* constructs are very stable and are maintained during bacterial free-living growth as well as during interaction with eukaryotic hosts, in the absence of selective pressure. This high stability ensures experimental homogeneity in time and space with regard to enhancing c-di-GMP intracellular levels in bacteria of interest.
【 授权许可】
CC BY
© Romero-Jiménez et al. 2015
【 预 览 】
Files | Size | Format | View |
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RO202311090767813ZK.pdf | 863KB | download |
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