Marker genes : molecular tools for in situ monitoring of bacterial distribution and activity in environmental samples

Abstract: Many different microorganisms carry out environmental functions, such as nutrient cycling, plant growth enhancement, or biodegradation. Some microorganisms also negatively affect their environment by causing disease. In order to gain a better understanding of the fate and behavior of specific microorganisms in nature, specific monitoring methods are required.This work focuses on the development of marker genes to monitor bacteria in environmental samples. The gfp gene, encoding the Green Fluorescent Protein (GFP), and the luxAB genes, encoding bacterial luciferase, were developed and applied as tools to track specific bacteria. To enable a strong expression of the eukaryotic gfp gene in bacteria, an expression cassette was assembled by fusing gfp to a ribosome binding site under control of a constitutive promoter. A variant cassette containing two copies of gfp was also constructed for higher expression of GFP. The gfp cassettes were inserted into Tn5 based transposon vectors for stable integration of gfp into the chromosomes of different bacterial strains. The Tn5-gfp cassettes were used to tag both Gram- and Gram+ strains with gfp. The intensity of fluorescence was dependent on the copy number of gfp. However, one copy of gfp was enough for visualisation of single bacterial cells by epifluorescence microscopy and confocal laser microscopy as well as for quantification by flow cytometry. GFP-fluorescent cells could also easily be distinguished from the indigenous bacterial population in soil samples and on plant surfaces by their fluorescence phenotype.In order to simultaneously quantify bacterial numbers and activity, a dual marker cassette containing both gfp and luxAB genes was constructed. Expression of the luminescence phenotype conferred by the luxAB genes is dependent on cellular energy reserves. The luciferase activity was highly correlated to the cell number when cells were actively growing, but decreased relative to cell number under starvation conditions. On the other hand, the GFP phenotype was insensitive to starvation conditions allowing gfp-tagged cells to be monitored independently of nutrient availability. Also, cells were detected by their GFP fluorescence when they were viable but nonculturable. The GFP fluorescence phenotype was found to be dependent on cell membrane integrity, as GFP was lost from dead cells with permeabilised membranes.The dual gfp-luxAB marker was useful for in situ studies of the plant growth promoting bacterium, Pseudomonas fluorescens SBW25, during colonisation of wheat plants starting from seed inoculum. The highest bacterial concentrations were found on the wheat seed, compared to the roots and leaves. By stereomicroscopy the in situ distribution of P. fluorescens SBW25 cells and the regions where they were metabolically active, were simultaneously monitored on the same wheat seeds. The P. fluorescens SBW25 cells showed a preference for specific regions on the wheat seed, such as the groove formed between the scutellum and the coleoptile. Interestingly, the P. fluorescens SBW25 cells were metabolically active on all plant parts where they were localised.In conclusion, the methods developed in this study are optimal tools for detection and quantification of bacteria and have wide applications for monitoring specific bacterial populations of interest in environmental samples.