Genetic tools for tagging Gram-negative bacteria with mCherry for visualization in vitro and in natural habitats, biofilm and pathogenicity studies

Abstract

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.

Colonization Patterns of an mCherry-Tagged Pectobacterium carotovorum subsp. brasiliense Strain in Potato Plants

“…brasiliense strain. A plasmid (pMP7604) harboring mCherry (27) was used to generate an mCherry-tagged P. carotovorum subsp. brasiliense strain.…”

Bacterial adhesion onto nanofiltration and reverse osmosis membranes: Effect of permeate flux

“…Pseudomonas fluorescens PCL1701 (Lagendijk et al (2010)) and Pseudomonas putida PCL1480 (Lagendijk et al (2010)). Pseudomonas strains were stored at -80°C in King B broth (King et al (1954)) supplemented with 20% glycerol.…”

The effect of β-aminobutyric acid (BABA) on root knot nematode and soft rot pathogen disease complexes in Solanum tuberosum plants

“…A glycerol stock of Pectobacterium carotovorum subsp brasiliense (Pcb) transformed with a plasmid (pMP7604) harbouring the red fluorescent protein (mCherry) and tetracycline resistance genes was retrieved from the -70 C freezer (Lagendijk et al, 2010;Mongae et al, 2013). Single colonies of Pcb_mCherry were prepared from stocks by streaking on nutrient agar supplemented with 12.5 µg/ml tetracycline then incubated for two days at 37C.…”