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Impact of temperature on the physiological status of a potential bioremediation inoculant, Arthrobacter chlorophenolicus A6
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. SLU.
2004 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 5, p. 2952-2958Article in journal (Refereed) Published
Abstract [en]

Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28degreesC. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system ("viable cellis") whereas propidium iodide (PI) was used to stain cells with damaged membranes ("dead cells"). Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28degreesC, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28degreesC, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5degreesC, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make A. chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.

Place, publisher, year, edition, pages
2004. Vol. 70, no 5, p. 2952-2958
National Category
Microbiology
Identifiers
URN: urn:nbn:se:sh:diva-15481DOI: 10.1128/AEM.70.5.2952-2958.2004ISI: 000221340400052PubMedID: 15128556Scopus ID: 2-s2.0-2442704506OAI: oai:DiVA.org:sh-15481DiVA, id: diva2:504653
Available from: 2012-02-21 Created: 2012-02-20 Last updated: 2017-07-19Bibliographically approved
In thesis
1. Physiological status of bacteria used for environmental applications
Open this publication in new window or tab >>Physiological status of bacteria used for environmental applications
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Karolinska Institutet, 2007. p. 49
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31242 (URN)91-7357-063-X (ISBN)
Public defence
2007-01-12, MA636, Alfred Nobels allé 7, Huddinge, 10:00 (English)
Opponent
Supervisors
Available from: 2016-11-30 Created: 2016-11-29 Last updated: 2016-11-30Bibliographically approved

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Backman, AgnetaMaraha, NinweJansson, Janet K

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