www.postersession.com D. radiodurans was first discovered growing on irradiated meat cans in 1956 (Slade, 2011). This bacteria belongs to a family of known as Deinococcaceae. This family has the the unique capability to withstand prolonged periods of desiccation, extreme heat, ionizing radiation and ultraviolet radiation (Anja, 2011). D. radiodurans is non-motile, non-spore forming, gram positive cocci, which usually appear in characteristic tetrads (Anja, 2011). The full mechanism by which D. radiodurans is able to withstand such intense deviation from a normal environment is largely unknown. Desiccation and radiation have both been previously shown to lead to DNA double stranded breaks, and ultraviolet radiation readily forms thymine dimers (Makarova, 2001). These changes to DNA are deleterious to most other bacteria. Additionally, oxidative stress through desiccation quickly leads to oxidative damage through free radicals (Slade, 2011). D. radiodurans overexpresses a multitude of DNA double stranded break proteins in the homologous recombination and single stranded annealing repair mechanisms. D. radiodurans also has an upregulated amount of repeated sequences and transposases (Makarova, 2011). Additionally, this bacteria has a unique nucleoid structure, uncommon lipid membrane arrangement, and an unusually high ratio of Manganese to Iron ratio (Zimmerman, 2005). Understanding how D. radiodurans acquired these changes are important for deciphering the exact role some of these mechanisms may play. Ultimately, by subjecting this bacteria to a combination of stressors, one may begin to unravel how D. radiodurans was granted its evolutionary gifts. Methods Discussion Attenuation of Deinococcus radiodurans’ resistance to Ultraviolet Radiation, Heat, and Desiccation Brian Covello, Dr. Brittany Gasper Acknowledgements Bacterial Culture D. radiodurans were a gift of Dr. Brittany Gasper. Bacteria were originally grown in TSB. For experimentation purposes, the bacteria was quadrant streaked onto a TSA plate. Growth of all bacteria was carried out in a 37 degree Celsius incubator. TSA plates were wrapped with parafilm to prevent desiccation. S. aureus was utilized as a control. Heat Aliquots of TSB containing the bacteria were placed in a test tube. This test tube was then placed in water at 85 degrees Celsius. After initial data indicated death of D. radiodurans, the temperature was lowered to 75 degrees Celsius, where the bacteria was able to grow. Ultraviolet Radiation Aliquots of TSB containing the bacteria were placed onto a slide and the slide was placed into an empty petri dish. The petri dish was syran wrapped to avoid contamination with Bacillus spores. The dish was then placed under a UV hood for 45 minutes. A thin layer of syran wrap was found to allow ultraviolet to pass through. Desiccation Aliquots of TSB containing the bacteria were placed in a petri dish. The dish was then placed in the incubator for 24 hours periods. Serial Dilutions For the purpose of counting survival rates, serial dilutions were made after the bacteria had been exposed to either individual treatments or the combined treatments, spread plated and titer calculations were recorded. Thank you… Florida Southern College for providing the funding, Dr. Brittany Gasper for providing the Deinococcus radiodurans and laboratory space along with much needed guidance and direction. Thank you fellow students and colleagues for support. Introduction Results Abstract Sources Question Hypothesis D. radiodurans acquired the ability to withstand heat and radiation as a byproduct of exposure to desiccation. D. radiodurans will be best suited to tolerate desiccation. Under which conditions of heat, dessication, and UV radiation is Deinococcus radiodurans best able to survive? What has allowed D. radiodurans to acquire such a unique ability to thrive under conditions of stress? Figure 1 Figure 2 Figure 4 Figure 3 Figure 1 shows an electron microscopy image of D. radiodurans. Figure 2 shows D. radiodurans quadrant streaked onto a TSA plate for the purpose of isolating colonies. Figure 4 shows D. radiodurans under a 100X objective in oil immersion. Figure 3 shows an unknown bacillus- like bacteria, which caused contamination in the UV hood. Heat Ultraviolet Radiation Desiccation Combined Stresses 0 10 20 30 40 50 60 70 80 90 100 55 60 65 70 75 80 85 % Survival Degrees Celsius Survival at 15 Minutes Heat D. radiodurans S. aureus 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12 14 16 % Survival Minutes Survival at 85 Degrees Celsius D. radiodurans S. aureus 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 % Survival Minutes Survival for UV Radiation D. radiodurans S. aureus Figure 5 Figure 6 Figure 7 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 % Survival Days Survival under Desiccation D. radiodurans S. aureus 0 10 20 30 40 50 60 70 80 90 100 D, H, U H, U D, U U D, U, H U, H D, H H 68 66 94 89 52 41 98 75 % Survival Treatment Combinations Survival v. Stress Combinations Figure 8 Figure 9 Figure 9 depicts treatment combinations. For the combined stressors the following abbreviations were utilized: D represents desiccation for a period of 24 hours, H represents heat of 70 degrees Celsius for 15 minutes, and U represents ultraviolet radiation for 45 minutes. Per the figures above, D. radiodurans’ survival rate is 100% for a desiccation of 24 hours, 75% for 70 degrees Celsius at 15 minutes, and 88% for UV exposure for 45 minutes. Fig. 5 shows D. radiodurans ability to survive at temperatures of up to 85 degrees for 15 minutes, while S. aureus, the control, was only able to survive up to 70 degrees Celsius. Fig. 6 depicts survival rates at a constant 85 degrees Celsius with varying amounts of time. S. aureus had a 0% survival rate at 10 minutes, while D. radiodurans was surviving past 15 minutes. Fig. 7 shows D. radiodurans maintaining its ability to grow up to the two hour mark under constant UV exposure. This is remarkable when compared to the control, which was nearly killed at 15 minutes UV exposure. Two areas of relative stability can also be noted for D. radiodurans. Fig. 8 indicates that the researchers were unable to kill D. radiodurans even at 7 days desiccation, while S. aureus was quickly killed around 1.5 days. Fig. 9 shows that across the board, any treatments that began with desiccation had a better survival rate. D. radiodurans clearly has installed mechanisms for survival under conditions of heat, UV, and desiccation when compared to S. aureus. D. radiodurans did not become affected by 15 minutes of heat until 70 degrees Celsius. More surprisingly however, was D. radiodurans’ ability to thrive in up to two hours of ultraviolet radiation. D. radiodurans seems to contain buffer regions of survival stability between 0-45 minutes and 75-105 minutes UV exposure. This may be due to an interplay of two or more mechanisms. Possibly, one mechanism may be utilized under short periods of ultraviolet radiation, and a second mechanism utilized under sustained periods of ultraviolet radiation. D. radiodurans was unaffected by desiccation up to 1 week, and it seems that D. radiodurans was best adapted to handle desiccation better than either heat or ultraviolet radiation. This may provide support that this bacteria’s resistance to ultraviolet radiation and heat is simply a byproduct of evolutionary pressures from desiccation. Support for the hypothesis is also given in the combination treatment. D. radioduran’s has a better survival rate when it is first exposed to desiccation. Future research may concentrate on examining how long D. radioduran’s can survive under desiccation. Deinococcus radiodurans is a unique gram positive, non-spore forming cocci bacteria that has acquired the ability to withstand extreme environmental stress such as heat, ultraviolet radiation, and desiccation. Although these stressors cause various types of breaks in the DNA and mutations to occur, D. radiodurans has unique proteins, nucleoid associations, and lipid composition which help it survive (Witte, 2005). This study tested heat, UV, and desiccation stressors on D. radiodurans. This bacteria was found to be able to withstand desiccation best. In addition, survival rate was increased when the desiccation treatment was first. This may signify that D. radiodurans’ ability to withstand stressors is a byproduct of its ability to withstand desiccation. Ultimately, further research is warranted to unravel the mechanism by which desiccation affects D. radiodurans. Slade, DS. (2011). Oxidative stress resistance in deinococcus radiodurans . Microbiology and Molecular Biology Reviews, 75(1), 133-191. Anja, B. (2011). Effect of relative humidity on deinococcus radiodurans' resistance to prolonged desiccation, heat, ionizing germicidal, and environmentally relevant uv radiation. Microbial Ecology, (16), 715-722. Makarova, KS. (2001). Genome of the extremely radiation-resistant Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev, (65), 44-79. Witte G. (2005). Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization. Nucleic Acids Res, (33), 1662-1670. Zimmerman, JM. (2005). A ring like nucleoid is not necessary for radioresistance in the Deinococcaceae. BMC Microbiol, (5), 17. www.seti.cli
Brian Covello: Microbiology Research On Bacteria's Ability to Withstand Stressors
Jun 14, 2015
Brian Covello's Microbiology Research On Bacteria's Ability to Withstand Stressors
Taken from www.wikipedia.org
Ionizing radiation resistance mechanisms[edit]
Deinococcus accomplishes its resistance to radiation by having multiple copies of its genome and rapid DNA repair mechanisms. It usually repairs breaks in its chromosomes within 12–24 hours through a 2-step process. First, D. radiodurans reconnects some chromosome fragments through a process called single-stranded annealing. In the second step, multiple proteins mend double-strand breaks through homologous recombination. This process does not introduce any more mutations than a normal round of replication would.
A persistent question regarding D. radiodurans is how such a high degree of radioresistance could evolve. Natural background radiation levels are very low—in most places, on the order of 0.4 mGy per year, and the highest known background radiation, near Ramsar, Iran is only 260 mGy per year. With naturally occurring background radiation levels so low, organisms evolving mechanisms specifically to ward off the effects of high radiation are unlikely.
Valerie Mattimore of Louisiana State University has suggested the radioresistance of D. radiodurans is simply a side effect of a mechanism for dealing with prolonged cellular desiccation (dryness). To support this hypothesis, she performed an experiment in which she demonstrated that mutant strains of D. radiodurans which are highly susceptible to damage from ionizing radiation are also highly susceptible to damage from prolonged desiccation, while the wild-type strain is resistant to both.[14] In addition to DNA repair, D. radiodurans use LEA proteins (Late Embryogenesis Abundant proteins)[15] expression to protect against desiccation.[16]
Scanning electron microscopy analysis has shown that DNA in D. radiodurans is organized into tightly packed toroids, which may facilitate DNA repair.[17]
A team of Croatian and French researchers led by Miroslav Radman have bombarded D. radiodurans to study the mechanism of DNA repair. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step, there is crossover by means of RecA-dependent homologous recombination.[18]
D. radiodurans is capable of genetic transformation, a process by which DNA derived from one cell can be taken up by another cell and integrated into the recipient genome by homologous recombination.[19] When DNA damages (e.g. pyrimidine dimers) are introduced into donor DNA by UV irradiation, the recipient cells efficiently repair the damages in the transforming DNA as they do in cellular DNA when the cells themselves are irradiated.
Taken from www.wikipedia.org
Ionizing radiation resistance mechanisms[edit]
Deinococcus accomplishes its resistance to radiation by having multiple copies of its genome and rapid DNA repair mechanisms. It usually repairs breaks in its chromosomes within 12–24 hours through a 2-step process. First, D. radiodurans reconnects some chromosome fragments through a process called single-stranded annealing. In the second step, multiple proteins mend double-strand breaks through homologous recombination. This process does not introduce any more mutations than a normal round of replication would.
A persistent question regarding D. radiodurans is how such a high degree of radioresistance could evolve. Natural background radiation levels are very low—in most places, on the order of 0.4 mGy per year, and the highest known background radiation, near Ramsar, Iran is only 260 mGy per year. With naturally occurring background radiation levels so low, organisms evolving mechanisms specifically to ward off the effects of high radiation are unlikely.
Valerie Mattimore of Louisiana State University has suggested the radioresistance of D. radiodurans is simply a side effect of a mechanism for dealing with prolonged cellular desiccation (dryness). To support this hypothesis, she performed an experiment in which she demonstrated that mutant strains of D. radiodurans which are highly susceptible to damage from ionizing radiation are also highly susceptible to damage from prolonged desiccation, while the wild-type strain is resistant to both.[14] In addition to DNA repair, D. radiodurans use LEA proteins (Late Embryogenesis Abundant proteins)[15] expression to protect against desiccation.[16]
Scanning electron microscopy analysis has shown that DNA in D. radiodurans is organized into tightly packed toroids, which may facilitate DNA repair.[17]
A team of Croatian and French researchers led by Miroslav Radman have bombarded D. radiodurans to study the mechanism of DNA repair. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step, there is crossover by means of RecA-dependent homologous recombination.[18]
D. radiodurans is capable of genetic transformation, a process by which DNA derived from one cell can be taken up by another cell and integrated into the recipient genome by homologous recombination.[19] When DNA damages (e.g. pyrimidine dimers) are introduced into donor DNA by UV irradiation, the recipient cells efficiently repair the damages in the transforming DNA as they do in cellular DNA when the cells themselves are irradiated.
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D. radiodurans was first discovered growing on irradiated meat cans in 1956 (Slade, 2011). This bacteria belongs to a family of known as Deinococcaceae. This family has the the unique capability to withstand prolonged periods of desiccation, extreme heat, ionizing radiation and ultraviolet radiation (Anja, 2011). D. radiodurans is non-motile, non-spore forming, gram positive cocci, which usually appear in characteristic tetrads (Anja, 2011). The full mechanism by which D. radiodurans is able to withstand such intense deviation from a normal environment is largely unknown. Desiccation and radiation have both been previously shown to lead to DNA double stranded breaks, and ultraviolet radiation readily forms thymine dimers (Makarova, 2001). These changes to DNA are deleterious to most other bacteria. Additionally, oxidative stress through desiccation quickly leads to oxidative damage through free radicals (Slade, 2011). D. radiodurans overexpresses a multitude of DNA double stranded break proteins in the homologous recombination and single stranded annealing repair mechanisms. D. radiodurans also has an upregulated amount of repeated sequences and transposases (Makarova, 2011). Additionally, this bacteria has a unique nucleoid structure, uncommon lipid membrane arrangement, and an unusually high ratio of Manganese to Iron ratio (Zimmerman, 2005). Understanding how D. radiodurans acquired these changes are important for deciphering the exact role some of these mechanisms may play. Ultimately, by subjecting this bacteria to a combination of stressors, one may begin to unravel how D. radiodurans was granted its evolutionary gifts.
Methods
Discussion
Attenuation of Deinococcus radiodurans’ resistance to Ultraviolet Radiation, Heat, and Desiccation
Brian Covello, Dr. Brittany Gasper
Acknowledgements
Bacterial Culture D. radiodurans were a gift of Dr. Brittany Gasper. Bacteria were originally grown in TSB. For experimentation purposes, the bacteria was quadrant streaked onto a TSA plate. Growth of all bacteria was carried out in a 37 degree Celsius incubator. TSA plates were wrapped with parafilm to prevent desiccation. S. aureus was utilized as a control. Heat Aliquots of TSB containing the bacteria were placed in a test tube. This test tube was then placed in water at 85 degrees Celsius. After initial data indicated death of D. radiodurans, the temperature was lowered to 75 degrees Celsius, where the bacteria was able to grow. Ultraviolet Radiation Aliquots of TSB containing the bacteria were placed onto a slide and the slide was placed into an empty petri dish. The petri dish was syran wrapped to avoid contamination with Bacillus spores. The dish was then placed under a UV hood for 45 minutes. A thin layer of syran wrap was found to allow ultraviolet to pass through. Desiccation Aliquots of TSB containing the bacteria were placed in a petri dish. The dish was then placed in the incubator for 24 hours periods. Serial Dilutions For the purpose of counting survival rates, serial dilutions were made after the bacteria had been exposed to either individual treatments or the combined treatments, spread plated and titer calculations were recorded.
Thank you… Florida Southern College for providing the funding, Dr. Brittany Gasper for providing the Deinococcus radiodurans and laboratory space along with much needed guidance and direction. Thank you fellow students and colleagues for support.
Introduction
Results Abstract
Sources
Question
Hypothesis D. radiodurans acquired the ability to withstand heat and radiation as a byproduct of exposure to desiccation. D. radiodurans will be best suited to tolerate desiccation.
Under which conditions of heat, dessication, and UV radiation is Deinococcus radiodurans best able to survive? What has allowed D. radiodurans to acquire such a unique ability to thrive under conditions of stress?
Figure 1 Figure 2
Figure 4 Figure 3 Figure 1 shows an electron microscopy image of D. radiodurans. Figure 2 shows D. radiodurans quadrant streaked onto a TSA plate for the purpose of isolating colonies. Figure 4 shows D. radiodurans under a 100X objective in oil immersion. Figure 3 shows an unknown bacillus-like bacteria, which caused contamination in the UV hood.
Heat
Ultraviolet Radiation
Desiccation
Combined Stresses
0 10 20 30 40 50 60 70 80 90
100
55 60 65 70 75 80 85
% S
urvi
val
Degrees Celsius
Survival at 15 Minutes Heat
D. radiodurans S. aureus
0 10 20 30 40 50 60 70 80 90
100
0 2 4 6 8 10 12 14 16
% S
urvi
val
Minutes
Survival at 85 Degrees Celsius
D. radiodurans S. aureus
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120
% S
urvi
val
Minutes
Survival for UV Radiation
D. radiodurans
S. aureus
Figure 5 Figure 6
Figure 7
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7
% S
urvi
val
Days
Survival under Desiccation
D. radiodurans
S. aureus
0
10
20
30
40
50
60
70
80
90
100
D, H, U H, U D, U U D, U, H U, H D, H H
68 66
94 89
52
41
98
75 % S
urvi
val
Treatment Combinations
Survival v. Stress Combinations
Figure 8
Figure 9
Figure 9 depicts treatment combinations. For the combined stressors the following abbreviations were utilized: D represents desiccation for a period of 24 hours, H represents heat of 70 degrees Celsius for 15 minutes, and U represents ultraviolet radiation for 45 minutes. Per the figures above, D. radiodurans’ survival rate is 100% for a desiccation of 24 hours, 75% for 70 degrees Celsius at 15 minutes, and 88% for UV exposure for 45 minutes.
Fig. 5 shows D. radiodurans ability to survive at temperatures of up to 85 degrees for 15 minutes, while S. aureus, the control, was only able to survive up to 70 degrees Celsius. Fig. 6 depicts survival rates at a constant 85 degrees Celsius with varying amounts of time. S. aureus had a 0% survival rate at 10 minutes, while D. radiodurans was surviving past 15 minutes. Fig. 7 shows D. radiodurans maintaining its ability to grow up to the two hour mark under constant UV exposure. This is remarkable when compared to the control, which was nearly killed at 15 minutes UV exposure. Two areas of relative stability can also be noted for D. radiodurans. Fig. 8 indicates that the researchers were unable to kill D. radiodurans even at 7 days desiccation, while S. aureus was quickly killed around 1.5 days. Fig. 9 shows that across the board, any treatments that began with desiccation had a better survival rate.
D. radiodurans clearly has installed mechanisms for survival under conditions of heat, UV, and desiccation when compared to S. aureus. D. radiodurans did not become affected by 15 minutes of heat until 70 degrees Celsius. More surprisingly however, was D. radiodurans’ ability to thrive in up to two hours of ultraviolet radiation. D. radiodurans seems to contain buffer regions of survival stability between 0-45 minutes and 75-105 minutes UV exposure. This may be due to an interplay of two or more mechanisms. Possibly, one mechanism may be utilized under short periods of ultraviolet radiation, and a second mechanism utilized under sustained periods of ultraviolet radiation. D. radiodurans was unaffected by desiccation up to 1 week, and it seems that D. radiodurans was best adapted to handle desiccation better than either heat or ultraviolet radiation. This may provide support that this bacteria’s resistance to ultraviolet radiation and heat is simply a byproduct of evolutionary pressures from desiccation. Support for the hypothesis is also given in the combination treatment. D. radioduran’s has a better survival rate when it is first exposed to desiccation. Future research may concentrate on examining how long D. radioduran’s can survive under desiccation.
Deinococcus radiodurans is a unique gram positive, non-spore forming cocci bacteria that has acquired the ability to withstand extreme environmental stress such as heat, ultraviolet radiation, and desiccation. Although these stressors cause various types of breaks in the DNA and mutations to occur, D. radiodurans has unique proteins, nucleoid associations, and lipid composition which help it survive (Witte, 2005). This study tested heat, UV, and desiccation stressors on D. radiodurans. This bacteria was found to be able to withstand desiccation best. In addition, survival rate was increased when the desiccation treatment was first. This may signify that D. radiodurans’ ability to withstand stressors is a byproduct of its ability to withstand desiccation. Ultimately, further research is warranted to unravel the mechanism by which desiccation affects D. radiodurans.
Slade, DS. (2011). Oxidative stress resistance in deinococcus radiodurans . Microbiology and Molecular Biology Reviews, 75(1), 133-191.
Anja, B. (2011). Effect of relative humidity on deinococcus radiodurans' resistance to prolonged desiccation, heat, ionizing germicidal, and environmentally relevant uv radiation. Microbial Ecology, (16), 715-722.
Makarova, KS. (2001). Genome of the extremely radiation-resistant Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev, (65), 44-79.
Witte G. (2005). Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization. Nucleic Acids Res, (33), 1662-1670.
Zimmerman, JM. (2005). A ring like nucleoid is not necessary for radioresistance in the Deinococcaceae. BMC Microbiol, (5), 17.
www.seti.cli
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