Physiological status of bacteria used for environmental applications
Author: Maraha, Ninwe
Date: 2007-01-12
Location: Moas Båge, MA636, Södertörns Högskola, Alfred Nobels Allé 7
Department: Institutionen för laboratoriemedicin / Department of Laboratory Medicine
View/ Open:
thesis.pdf (2.575Mb)
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.
List of papers:
I. Lowder M, Unge A, Maraha N, Jansson JK, Swiggett J, Oliver JD (2000). "Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506." Appl Environ Microbiol 66(8): 3160-5
Pubmed
II. Backman A, Maraha N, Jansson JK (2004). "Impact of temperature on the physiological status of a potential bioremediation inoculant, Arthrobacter chlorophenolicus A6." Appl Environ Microbiol 70(5): 2952-8
Pubmed
III. Maraha N, Backman A, Jansson JK (2004). "Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry." FEMS Microbiol Ecol 51(1): 123-32
Pubmed
IV. Maraha N, VerBerkmoes NC, Spiers A, Shah M, Timms-Wilson T, Goodall T, Jansson JK (2006). "Use of proteomics to study impact of nutrient status on Pseudomonas fluorescens SBW25." (Manuscript)
I. Lowder M, Unge A, Maraha N, Jansson JK, Swiggett J, Oliver JD (2000). "Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506." Appl Environ Microbiol 66(8): 3160-5
Pubmed
II. Backman A, Maraha N, Jansson JK (2004). "Impact of temperature on the physiological status of a potential bioremediation inoculant, Arthrobacter chlorophenolicus A6." Appl Environ Microbiol 70(5): 2952-8
Pubmed
III. Maraha N, Backman A, Jansson JK (2004). "Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry." FEMS Microbiol Ecol 51(1): 123-32
Pubmed
IV. Maraha N, VerBerkmoes NC, Spiers A, Shah M, Timms-Wilson T, Goodall T, Jansson JK (2006). "Use of proteomics to study impact of nutrient status on Pseudomonas fluorescens SBW25." (Manuscript)
Issue date: 2006-12-22
Rights:
Publication year: 2007
ISBN: 91-7357-063-X
Statistics
Total Visits
Views | |
---|---|
Physiological ...(legacy) | 787 |
Physiological ... | 162 |
Total Visits Per Month
September 2023 | October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | |
---|---|---|---|---|---|---|---|
Physiological ... | 1 | 3 | 11 | 0 | 1 | 0 | 0 |
File Visits
Views | |
---|---|
thesis.pdf(legacy) | 558 |
thesis.pdf | 145 |
thesis.pdf.txt(legacy) | 2 |
Top country views
Views | |
---|---|
United States | 379 |
China | 86 |
Sweden | 60 |
Germany | 55 |
South Korea | 39 |
Denmark | 16 |
United Kingdom | 16 |
Ireland | 16 |
Finland | 13 |
Russia | 13 |
Top cities views
Views | |
---|---|
Beijing | 44 |
Tempe | 43 |
Seoul | 32 |
Sunnyvale | 31 |
Romeo | 27 |
Kiez | 18 |
Stockholm | 13 |
Ballerup | 12 |
Dublin | 9 |
Woodbridge | 7 |