Physiological status of bacteria used for environmental applications

Abstract: Several bacteria have properties of interest for biotechnological applications, such as bioremediation of pollutants and biocontrol of plant pathogens. In order to perform their intended tasks in the environment the cells need to remain viable and active. Therefore, the aim of this thesis was to use a combination of molecular approaches to determine the physiological status of specific bacterial populations in soil. Complementary experiments were done in pure cultures to gain a better understanding of specific physiological states, such as bacterial dormancy. In some studies, the bacteria were tagged with the following marker genes to enable them to be specifically detected in soil: gfp (encoding the green fluorescent protein, GFP), luxAB (encoding bacterial luciferase) or luc (encoding eukaryotic luciferase). Viability stains, 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), were used to stain active and dead cells, respectively. The marker-gene tagged cells were incubated in soil under different conditions and the number of GFP fluorescent and stained cells was enumerated by flow cytometry at specified sampling periods. Luciferase activity was used to monitor metabolic activity of the population. In addition, the number of culturable cells was determined by selective plate counting and compared to the results obtained by flow cytometry. Finally, in one study, proteomics was used to elucidate which proteins were expressed under different nutrient conditions. The physiological status of Arthrobacter chlorophenolicus A6 (a chlorophenol degrading bacterium) was investigated after introduction into soil incubated at different temperatures, 5 and 28 °C. The majority of the A6 population remained metabolically active after 20 days of incubation in soil at 5 °C. However, there was a fraction of the GFP-fluorescent A6 population that was not stained with CTC or PI, presumably indicating a subfraction of dormant cells that were alive but inactive. By contrast, after the same period of incubation at 28 °C, the majority of the cells died. The ability of A. chlorophenolicus A6 to enter a state of dormancy during incubation at cold temperatures, makes this strain a good candidate for treating chlorophenol contaminated soil in temperate climates. Two Pseudomonas fluorescens strains, proposed for improving crop yields, were also studied. Pseudomonas fluoresens A506 is used to reduce frost damage to plants and Pseudomonas fluorescens SBW25 is a plant growth promoting bacterium. First, a GFPtagged variant of the A506 strain was studied to determine whether GFP could be used to detect the cells when they were viable but non-culturable (VBNC). The results showed that GFP tagged cells could be detected even in a V13NC state as long as the cell membrane was intact. The SBW25 strain was studied in pure cultures and in soil to determine the physiological status of the cells under different nutritional conditions, using many of the approaches described above for A6. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI, indicating that most of the cells were presumably dormant. In soil, a subpopulation of the SBW25 cell population died. However, approximately 60% of the population in soil apparently entered a state of dormancy, similar to that observed under starvation conditions in pure cultures. Several differences were found in the proteins that were expressed when SBW25 was incubated under nutrient rich conditions compared to starvation conditions. These differences provide a clue as to what proteins enable SBW25 to survive starvation and dormant states.