Janet K. Jansson

Monitoring population size, activity, and distribution of gfp-luxAB-tagged Pseudomonas fluorescens SBW25 during colonization of wheat

Increasingly, focus has been directed towards the use of microorganisms as biological control agents to combat fungal disease, as an alternative to chemical fungicides. Pseudomonas fluorescens SBW25 is one bacterial strain that has been demonstrated to promote plant growth by biocontrol of pathogenic fungi. To understand the mode of action of this bacterium, information regarding its localization and metabolic activity on plants is important. In this study, a gfp/luxAB-tagged derivative of P. fluorescens SBW25, expressing the green fluorescent protein (GFP) and bacterial luciferase, was monitored during colonization of wheat starting from seed inoculation. Since bacterial luciferase is dependent on cellular energy reserves for phenotypic expression, metabolically active cells were detected using this marker. In contrast, the stable GFP fluorescence phenotype was used to detect the cells independently of their metabolic status. The combination of these two markers enabled P. fluorescens SBW25 cells to be monitored on wheat plants to determine their specific location and metabolic activity. Studies on homogenized wheat plant parts demonstrated that the seed was the preferred location of P. fluorescens SBW25 during the 65-day time period studied, but the leaves and roots were also colonized. Interestingly, the bacteria were also found to be metabolically active on all plant parts examined. In situ localization of P. fluorescens SBW25 using a combination of different microscopic techniques confirmed the preference for the cells to colonize specific regions of the seed. We speculate that the colonization pattern of P. fluorescens SBW25 can be linked to the mechanism of protection of plants from fungal infection.

Tracking genetically engineered microorganisms in nature

The past year has seen the continued development of methods for tracking genetically engineered microorganisms in nature, with an emphasis on increased sensitivity, specificity and quantitative ability. In addition, novel methods have been developed for tagging bacteria targeted for environmental release. Nevertheless, the limited number of field trials published to date have primarily relied on conventional monitoring methods, despite the availability of better and theoretically safer methods.

Quantification of the presence and activity of specific microorganisms in nature

Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.

Quantitative PCR of environmental samples

The polymerase chain reaction (PCR) is currently the most sensitive method for detection of specific DNA in environmental samples. Sensitivities in the range of 1–100 cells per gram of sediment or soil have been reported [24, 31, 32]. Ultimately, sensitivity of detection by PCR is dependent on several factors such as the target copy number and/or enhancement by use of nested primers or DNA probes.