Welcome to our lab
In our lab, we are mainly interested how bacterial species grow on plant leaves (the phyllosphere), which growth pattern they exhibit and how they interact with other species that share the leaf with them (figure 1). To do so, we mostly rely on fluorescence microscopy techniques such as confocal laser scanning microscopy and widefield epifluorescence microscopy. This is in combination with bacteria that are either tagged with fluorescent protein genes, stained using fluorescence in situ hybridisation (FISH), or stained using fluorescent live/dead stains that allow to determine if individual bacterial cells are either alive or dead. To analyse our data we employ image cytometry and spatial statistic tools which allow us to assess pair aggregation patterns of leaf colonising bacteria (figure 2).
Figure 1 schematic of bacterial colonisation on the phyllosphere of Arabidopsis.
Figure 2 fluorescence micrograph of bacteria that were recovered from environmentally grown Arabidopsis plants and were labelled using FISH. Random dipoles were used to analyse spatial correlation resulting in pair correlation plots.
Since many leaf colonising bacteria cannot be used with the molecular toolbox available for E. coli and other model bacteria, a lot of research time in our lab is dedicated to adapt existing tools to make environmental bacteria more tractable to genetic manipulation (figure 3-7) and to effectively "make them shine". More information about "Chromatic bacteria toolset" can be found here.
Widefield microscopy of environmental bacteria expressing fluorescent proteins. (A) E. coli DH5α (pMRE145). (B) E. coli DH5α::MRE-Tn5-145. (C) E. coli DH5α::MRE-Tn7-145. (D) Bradyrhizobium sp. Leaf396::MRE-Tn5-165. (E) Methylobacterium sp. Leaf92::MRE-Tn5-165. (F) Sphingomonas melonis FR1 (pMRE145). (G) S. melonis FR1::MRE-Tn5-145. (H) S. melonis FR1::MRE-Tn7-145. (I) Sphingomonas phyllosphaerae FA2 (pMRE135). (J) S. phyllosphaerae FA2::MRE-Tn5-145. (K) Erwinia amylovoraCFBP1430S (pMRE135). (L) E. amylovora CFBP1430S::MRE-Tn5-145. (M) E. amylovoraCFBP1430S::MRE-Tn7-145. (N) Pantoea agglomerans 299R (pMRE135). (O) P. agglomerans 299R::MRE-Tn5-145. (P) P. agglomerans 299R::MRE-Tn7-145. (Q) Pseudomonas citronellolis P3B5 (pMRE145). (R) P. citronellolis P3B5::MRE-Tn5-145. (S) Pseudomonas syringae pv. syringae B728a (pMRE145). (T) P. syringae B728a::MRE-Tn5-145. Exposure times used during image acquisition are depicted in the corresponding images. Scale bars represent 5 μm.
Normalized (A) absorption and (B) emission spectra of the fluorescent proteins used in this work.
Microscopy images of fluorescent bacteria. (A) Widefield epifluorescence micrograph of E. coli expressing either mTB2 (blue), mCl3 (green), or mSc (red). (B) Widefield epifluorescence micrograph of E. coli expressing either mTB2 (blue), mTq2 (cyan), sYFP2 (green), or mSc (red). (C) Confocal microscopy of a mixed E. coli expressing either mTB2 (blue), mTq2 (magenta), mCl3 (yellow), mO2 (green), mSc (white), or mCa (red). (D) Confocal microscopy of a mixed E. coli expressing either mTB2 (cyan), mTq2 (magenta), sGFP2 (yellow), sYFP2 (gray), mO2 (red), mSc (green), or mCa (blue). In all cases, E. coli harboring pMRE14X plasmids series were used. Scale bar: 10 μm.
Figure 3 several phyllosphere isolates tagged with different constitutively expressed fluorescent proteins excited with UV light.
Figure 4 Soybean root nodule colonized by sYFP2 expressing Bradyrhizobium japonicum.
Figure 5 Arabidopsis leaf colonized by Pantoea agglomerans (in cyan) and Pseudomonas syringae DC3000 (in red)
Next to this main theme, we are currently working on:
Environmental studies to screen for antibiotic resistant bacteria,
the characterisation of oil degrading bacteria from plant leaves,
biocontrol of Erwinia amylovora on apple blossoms,
and the phyllosphere microbiota of kunzea in close vicinity of hydrothermal springs.