Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria

BMC Microbiology

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