Present address: Guido V. Bloemberg, Institute of Medical Microbiology, University of Zurich, Gloriastr. 32, Postfach, CH-8006 Zurich, Switzerland.
Genetic tools for tagging Gram-negative bacteria with mCherry for visualization in vitro and in natural habitats, biofilm and pathogenicity studies
Article first published online: 28 JAN 2010
© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Letters
Volume 305, Issue 1, pages 81–90, April 2010
How to Cite
Lagendijk, E. L., Validov, S., Lamers, G. E.M., De Weert, S. and Bloemberg, G. V. (2010), Genetic tools for tagging Gram-negative bacteria with mCherry for visualization in vitro and in natural habitats, biofilm and pathogenicity studies. FEMS Microbiology Letters, 305: 81–90. doi: 10.1111/j.1574-6968.2010.01916.x
Editor: Jeff Cole
- Issue published online: 4 MAR 2010
- Article first published online: 28 JAN 2010
- Received 11 December 2009; revised 14 January 2010; accepted 14 January 2010.Final version published online 18 February 2010.
- autofluorescent proteins;
Live-cell imaging techniques are essential to gain a better understanding of microbial functioning in natural systems, for example in biofilms. Autofluorescent proteins, such as the green fluorescent protein (GFP) and the red fluorescent protein (DsRed), are valuable tools for studying microbial communities in their natural environment. Because of the functional limitations of DsRed such as slow maturation and low photostability, new and improved variants were created such as mCherry. In this study, we developed genetic tools for labeling Gram-negative bacteria in order to visualize them in vitro and in their natural environment without the necessity of antibiotic pressure for maintenance. mcherry was cloned into two broad host-range cloning vectors and a pBK-miniTn7 transposon under the constitutive expression of the tac promoter. The applicability of the different constructs was shown in Escherichia coli, various Pseudomonas spp. and Edwardsiella tarda. The expression of mcherry was qualitatively analyzed by fluorescence microscopy and quantified by fluorometry. The suitability of the constructs for visualizing microbial communities was shown for biofilms formed on glass and tomato roots. In addition, it is shown that mCherry in combination with GFP is a suitable marker for studying mixed microbial communities.