Progress 06/21/17 to 05/15/21
Outputs
Target Audience:The project has a global target audience since the project addresses a problem (U contamination ) which is of global concern. We have been able to reach a large part of the scientific community by presenting our work at several conferences and locally to the general community by presenting the data at an area high school. We are also in the process of submitting a manuscript which will serve to share our findings with the global scientific community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided opportunities for underrepresented minority students to conduct independent research in the area on molecular environmental science. In addition, they have received experience in presenting their findings by participating in conferences. They have also contributed to the preparation of a manuscript for publication. How have the results been disseminated to communities of interest?During the course of the project, results have been disseminated by giving oral presentations (PI and undergraduate students) at various conferences. In 2019, the PI also disseminated the project's results by giving an oral presentation to high school science students at Dutch Fork High School in Columbia, SC. Without this presentation, this community would have no knowledge of the findings generated in the laboratory and how they contribute to and impact our understanding of environmental contaminant utilization. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In order to identify genes involved in biosurfactant production, we initially used transpositional mutagenesis to produce ~ 1800 A. piechaudii and 400 Flexibacter cf. sancti SRS random mutants. We needed to employ a method to screen the mutants for any increase/decrease in biosurfactant production. An atomized overspray technique proved unsuccessful in screening for biosurfactant activity. Previously, we reported on the performance of the methylene blue active substances (MBAS) assay to detect biosurfactant activity in the P. pituda, A. piechaudii and Flexibacter strains. Biosurfactant production is indicated by a blue coloring in/around the bacteria in the well, and/or a blue halo around the well. A. piechaudii and P. pituda exhibited biosurfactant production, while Flexibacter exhibited less biosurfactant production as compared to these strains. We also subjected a strain used in a previous phenanthrene utilization study, Sphingomonas BPH, to the MBAS assay. This strain is capable of degrading various polycyclic aromatic hydrocarbons. Three phenanthrene utilization mutants of this strain (mutants 257, 1778 and 1782) were made previously using Tn5 transpositional mutagenesis. Two of these mutants, 1778 and 1782, were selected and subjected to the MBAS assay. Our results revealed significant biosurfactant production by the wildtype strain BPH, but decreased biosurfactant production by the 1778 and 1782 mutants. Sphingomonas strains 1782 and 1778 were subjected to nucleotide sequencing to identify gene into which the transposon had inserted. When examining the insertion site in strain 1782, we found that the Tn5 transposon had inserted into the large subunit of the naph/bph dioxygenase gene bphA1. The bphA1 gene initiates the catabolism of biphenyl via a dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing deoxygenation of three and four-ring polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene. Thus, the disruption of the PAH biodegradation pathway in 1782 explains the lack of phenanthrene utilization, but not the decreased level of biosurfactant production. When examining the insertion site in strain 1778, we found the Tn5 transposon had inserted into a gene encoding for a protein bearing 97% identity to the lasso peptide B protein from Sphingopyxis bauzanensis. Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold and are typically between 15-24 amino acids in length and are grouped in 4 classes. This unique lariat topology involves the action of at least 2 enzymes: B (required to bind and cleave the precursor peptide) and C (required for cyclization/knot formation). Lasso peptides have a wide range of activities including antimicrobial, enzyme inhibitory and receptor antagonistic activities, but have not been shown to be involved in PAH utilization or biosurfactant production. This finding provided an interesting development in our research which must be fully examined. This resulted in a change to our goals to incorporate experiments designed to quantitate biosurfactant production in these strains and to determine if Sphingomonas BPH is capable of reducing U levels. These experiments are currently being performed at Savannah River National Laboratory (Aiken, SC). Our results revealed significant biosurfactant production by the wildtype strain BPH, but decreased biosurfactant production by the 1778 and 1782 mutants. The lasso peptide of Sphingomonas BPH was designated SpLp1. This finding resulted in a change to our goals to incorporate experiments designed to quantitate biosurfactant production in these strains and to determine if Sphingomonas BPH is capable of reducing U levels. These experiments are currently being performed at Savannah River National Laboratory (Aiken, SC). In addition to the proposal, during this period Drs. Simpson and Brigmon wrote a paper that will soon be submitted entitled "Roles(s) of Lasso Peptide in Polycyclic Aromatic Hydrocarbon Utilization in Sphingomonas". As a result of this finding, we generated a separate objectiveto analyze the abilities of the wildtype Sphingomonas strain and the 1778 and 1782 mutants to utilize certain metals. We tested heavy metal contaminated soils from SRS Steed Pond with varying U, chromium (Cr), and Nickel (Ni) concentrations with select microorganisms. One soil, #2300, had higher Ni but lower Cr and U concentrations than soil #2303. Soil #2303 had higher U and Cr concentrations. The soil pH was typical of SRS being acidic round pH 5. Bioremediation of contaminants of heavy metals in soils, water, and sediment is limited by bioavailability. Microorganisms have various mechanisms that have been used to remediate heavy metals including reduction or change to less mobile state, sorption, complexation, and metabolic byproducts that can influence bioavailability. Previous work had demonstrated that one biosurfactant producing bacteria, BPH (Sphingomonas SP. S37), could impact U bioavailability. In this study we tested three bacteria, BPH and two mutants generated from BPH in this study, M1 (Sphingomonas 1778) and M2 (Sphingomonas 1782), for their ability to impact heavy metal bioavailability on these two different soils. Desorption properties were not consistent for both soils. Soil #2303 (high OM, loamy texture, very high [U] and lower [Ni]) had increased Cr, Ni and U concentration in the aqueous phase at the end of the experiment, compared to when no microbes were added. Soil #2300 (little OM/clayey, very high [Ni] and low [U]): Cr and U had no response. Ni showed microbes bound released Ni. The pH of soil suspension was not influenced by microbial additions. We did seek to continue and expand our study on the role of lasso peptides in PAH degradation by submitting a grant to the National Institute of Environmental Health Science (NIEHS). The grant, entitled "Role(s) of Lasso Peptides in Natural and Microbial Attenuation of Polycyclic Aromatic Hydrocarbon Contaminated Soils", was written with Dr. Simpson as PI and Drs. Robin Brigmon (Savannah River National Laboratory) and Armand Dichosa (Los Alamos National Laboratory). Submitted in May of 2020, in the proposal we hypothesized that our prior results suggested a role for lasso peptides in PAH utilization by a yet to be elucidated mechanism. We proposed studies that would provide a better, mechanistic understanding of the possible involvement of SpLp1 in the utilization of environmental contaminants, such as phenanthrene and metals, identify the range of substances with which Sphingomonas SpLp1 may associate, demonstrate any ability of the expression of this lasso peptide to enhance natural attenuation of PAH-(and metal) contaminated soils/sediments, and identify novel environmental microorganisms that may also produce lasso peptides which may be employed to natural attenuation procedures.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
1. Waltena Simpson. 2019. Bioremediation of Uranium (U)-Contaminated Soils . Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
2. Waltena Simpson. 2019. Possible Role of Lasso Peptides in Biosurfactant Production and Polycyclic Aromatic Hydrocarbon Utilization . Dutch Fork High School. Columbia, SC. June 4, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
3. Makaela Jackson, Victoria Brown, Shakeela Shiggs, Jasmine Fox and Waltena Simpson. 2019. Efficient Delivery of Tn5 for the Production of Biosurfactant Deficient Mutants of Alcaligenes piechaudii . Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019. Makaela Jackson, won 1st Place in the Renewable Energy, Natural Resources and Environment Division. She successfully competed against 24 students from 12 different universities.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
4. Victoria Brown, Makaela Jackson and Waltena Simpson. 2019. Tn5 Transpositional Mutagenesis of Flexibacter cf. sancti SRS to Identify Genes Involved in Biosurfactant Production . Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
5. Makaela Jackson, Victoria Brown, and Waltena Simpson. 2018. LS-SCAMP symposium, Orangeburg, SC. November 9, 2018. She won first place in her area of competition (Biology).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
6. Makaela Jackson, Victoria Brown and Waltena Simpson. 2018. Annual Biomedical Research Conference for Minority Students (ABRCMS), Indianapolis, Indiana. November 13-17, 2018.
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audience is a global one since the project addresses a problem (U contamination ) which is of global concern. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Due to Covid 19, conferences that may have offered professional development were cancelled. We did, however, have our students expand their training by giving presentations of articles read during the summer. How have the results been disseminated to communities of interest?We have shared our new results with the scientific community by writing a grant for review. A manuscript is in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
South Carolina State University researchers have: Created and screened transconjugant mutants of P. putida and A. piechaudii. Both microorganisms are capable of reducing U levels in soil/groundwater. MBAS screening of P. putida and A. piechaudii transconjugants did not detect any that were deficient in biosurfactant production. Increased the number of underrepresented minorities conducting research in the area of environmental molecular biology by allowing SCSU students to perform independent studies. These students have participated in and presented their research at several conferences, and have won at least one prestigious award. Identified a protein that may be involved in biosurfactant production/activity and degradation of other environmental contaminants. Attempted sustainability of the proposal by submitting a grant to the National Institute of Environmental Health Sciences to further explore the role of lasso peptides in the utilization of various soil contaminants. This project would have been a collaboration between South Carolina State University, Savannah River National Laboratory (SRNL) and Los Alamos National Laboratory (LANL). Prepared a manuscript on the possible role of lasso peptides in phenanthrene utilization. During this year, we achieved results related to major objective above. Previously, while conducting studies targeted at producing biosurfactant deficient strains of A. piechaudii and P. putida, we performed a methylene blue active substance (MBAS) assay on these strains and another (Sphingomonas BPH) in the laboratory. Similar to P. putida, Sphingomonas BPH is capable of degrading various polycyclic aromatic hydrocarbons (PAHs) but had not be examined for an ability to degrade U. Sphingomonas 1778 and 1782 are both phenanthrene utilization mutants of Sphingomonas BPH generated by transpositional mutagenesis. Our results revealed significant biosurfactant production by the wildtype strain BPH, but decreased biosurfactant production by the 1778 and 1782 mutants. Examination of the insertion site of the pUTminiTn5-km luxAB transposon into Sphingomonas 1782 revealed that the transposon had inserted into the large subunit of the naph/bph dioxygenase gene bphA1. In Sphingomonas 1778 the transposon had inserted into a gene encoding for a protein bearing 97% identity to the lasso peptide B protein from Sphingopyxis bauzanensis. Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold and are typically between 15-24 amino acids in length and are grouped in 4 classes. This unique lariat topology involves the action of at least 2 enzymes: B (required to bind and cleave the precursor peptide) and C (required for cyclization/knot formation). Lasso peptides have a wide range of activities but have not been shown to be involved in PAH utilization or biosurfactant production. This finding provided an interesting development in our research which must be fully examined. The lasso peptide of Sphingomonas BPH was designated SpLp1. This finding resulted in a change to our goals to incorporate experiments designed to quantitate biosurfactant production in these strains and to determine if Sphingomonas BPH is capable of reducing U levels. These experiments are currently being performed at Savannah River National Laboratory (Aiken, SC). As part of this work, we tested heavy metal contaminated soils from SRS Steed Pond with varying U, chromium (Cr), and Nickel (Ni) concentrations with select microorganisms. One soil, #2300, had higher Ni but lower Cr and U concentrations than soil #2303. Soil #2303 had higher U and Cr concentrations. The soil pH was typical of SRS being acidic round pH 5. Bioremediation of contaminants of heavy metals in soils, water, and sediment is limited by bioavailability. Microorganisms have various mechanisms that have been used to remediate heavy metals including reduction or change to less mobile state, sorption, complexation, and metabolic byproducts that can influence bioavailability. Previous work had demonstrated that one biosurfactant producing bacteria, BPH (Sphingomonas SP. S37), could impact U bioavailability. In this study we tested three bacteria, BPH and two mutants generated from BPH in this study, M1 (Sphingomonas 1778) and M2 (Sphingomonas 1782), for their ability to impact heavy metal bioavailability on these two different soils. Desorption properties were not consistent for both soils. Soil #2303 (high OM, loamy texture, very high [U] and lower [Ni]) had increased Cr, Ni and U concentration in the aqueous phase at the end of the experiment, compared to when no microbes were added. Soil #2300 (little OM/clayey, very high [Ni] and low [U]): Cr and U had no response. Ni showed microbes bound released Ni. The pH of soil suspension was not influenced by microbial additions. This work and testing for biosurfactant quantification is ongoing. During the 2020 year, we initiated several more studies. Specifically, we performed additional transpositional mutagenesis of P. putida in an attempt to identify biosurfactant deficient mutants and to identify genes involved in biosurfactant production. While we successfully created additional P. putida transconjugants, the shut down of the University due to Covid 19 restrictions resulted in our being unable to screen them. We did, however, seek to continue and expand our study on the role of lasso peptides in PAH degradation by submitting a grant to the National Institute of Environmental Health Science (NIEHS). The grant, entitled "Role(s) of Lasso Peptides in Natural and Microbial Attenuation of Polycyclic Aromatic Hydrocarbon Contaminated Soils", was written with Dr. Simpson as PI and Drs. Robin Brigmon (Savannah River National Laboratory) and Armand Dichosa (Los Alamos National Laboratory). Submitted in May of 2020, in the proposal we hypothesized that our prior results suggested a role for lasso peptides in PAH utilization by a yet to be elucidated mechanism. We proposed studies that would provide a better, mechanistic understanding of the possible involvement of SpLp1 in the utilization of environmental contaminants, such as phenanthrene and metals, identify the range of substances with which Sphingomonas SpLp1 may associate, demonstrate any ability of the expression of this lasso peptide to enhance natural attenuation of PAH-(and metal) contaminated soils/sediments, and identify novel environmental microorganisms that may also produce lasso peptides which may be employed to natural attenuation procedures. The identification of these novel environmental microorganisms would be accomplished used gel microdroplets (GMD) as a potential materials delivery system. In addition to the proposal, during this period Drs. Simpson and Brigmon wrote on a paper that will soon be submitted entitled "Roles(s) of Lasso Peptide in Polycyclic Aromatic Hydrocarbon Utilization in Sphingomonas". These results and activities relate to objective 3. Regarding objective 4, training of undergraduate students in the area of environmental molecular biology Undergraduate students were still trained in environmental molecular biology even though the University was closed during our most productive times. Two undergraduate students, Victoria Brown and Makaela Jackson, worked remotely on the project during the summer of 2020 and for half of the Spring 2020 semester. These students were required to read, provide written summaries, and give power point presentations of relevant articles during these periods. These activities provided excellent preparation for the students, particularly as one, Makaela Jackson, is currently in graduate school
Publications
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience: The target audience is a global one since the project addresses a problem (U contamination) which is of global concern. During the 2019 year, we were able to reach a large part of the scientific community by presenting our work at a national conference. Results generated by the project also reach local audiences by means of presenting the data at an area high school. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided the undergraduate students (and the PI) the opportunity to attend and participate in professional conferences. Both the PI and her 2 undergraduate students participated in the 2019 1890 Association of Research Directors Symposium in Jacksonville, Florida. This conference has excellent professional development seminars for students targeting at preparing them for post-graduate endeavors, i.e. joining the workforce and graduate/professional school(s). At this conference, one student, Makaela Jackson, won 1st Place in the Renewable Energy, Natural Resources and Environment Division. She successfully competed against 24 students from 12 different universities. How have the results been disseminated to communities of interest? During the 2019 year, the PI and her undergraduate students disseminated the results of their studies by making a total of 4 presentations. Two were oral presentations and 2 were poster presentations. Three (3) of the presentations were given at the 1890 Association of Research Directors Symposium in Jacksonville, Fl, while one was given at an area high school. These presentations allow for members of the scientific community as well as members of the local community to become familiar with the research conducted by this laboratory. PI's Oral Presentations: Waltena Simpson. 2019. "Bioremediation of Uranium (U)-Contaminated Soils". Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019. Waltena Simpson. 2019. "Possible Role of Lasso Peptides in Biosurfactant Production and Polycyclic Aromatic Hydrocarbon Utilization". Dutch Fork High School. Columbia, SC. June 4, 2019. Students' Poster Presentations: Makaela Jackson, Victoria Brown, Shakeela Shiggs, Jasmine Fox and Waltena Simpson. 2019. "Efficient Delivery of Tn5 for the Production of Biosurfactanat Deficient Mutants of Alcaligenes piechaudii". Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019. Victoria Brown, Makaela Jackson and Waltena Simpson. 2019. "Tn5 Transpositional Mutagenesis of Flexibacter cf. sancti SRS to Identify Genes Involved in Biosurfactant Production". Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we anticipate concentrating more on Objectives 1 and 2.
Impacts
What was accomplished under these goals?
During this year, we achieved new results related to major objective #3 (above). In order to identify genes involved in biosurfactant production, we initially used transpositional mutagenesis to produce random A. piechaudii mutants. We needed to employ a method to screen the mutants for any increase/decrease in biosurfactant production. Previously, we reported on the performance of the methylene blue active substances (MBAS) assay to detect biosurfactant activity in the P. pituda, A. piechaudii and Flexibacter strains. Biosurfactant production is indicated by a blue coloring in/around the bacteria in the well, and/or a blue halo around the well. A. piechaudii and P. pituda exhibited biosurfactant production, while Flexibacter exhibited less biosurfactant production as compared to these strains. While we were conducting these MBAS assay on these strains, we also subjected a strain used in a previous phenanthrene utilization study, Sphingomonas BPH, to the same test. This strain is capable of degrading various polycyclic aromatic hydrocarbons. Three phenanthrene utilization mutants of this strain (mutants 257, 1778 and 1782) were made previously using Tn5 transpositional mutagenesis. Two of these mutants, 1778 and 1782, were selected and subjected to the MBAS assay. Our results revealed significant biosurfactant production by the wildtype strain BPH, but decreased biosurfactant production by the 1778 and 1782 mutants. Sphingomonas strains 1782 and 1778 were subjected to nucleotide sequencing to identify gene into which the transposon had inserted. When examining the insertion site in strain 1782, we found that the Tn5 transposon had inserted into the large subunit of the naph/bph dioxygenase gene bphA1. The bphA1 gene initiates the catabolism of biphenyl via a dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing deoxygenation of three and four-ring polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene. Thus, the disruption of the PAH biodegradation pathway in 1782 explains the lack of phenanthrene utilization by this mutants, but now the decreased level of biosurfactant production. We are currently examining the insertion site more extensively to understand this occurrence. When examining the insertion site in strain 1778, we found the Tn5 transposon had inserted into a gene encoding for a protein bearing 97% identity to the lasso peptide B protein from Sphingopyxis bauzanensis. Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold and are typically between 15-24 amino acids in length and are grouped in 4 classes. This unique lariat topology involves the action of at least 2 enzymes: B (required to bind and cleave the precursor peptide) and C (required for cyclization/knot formation). Lasso peptides have a wide range of activities including antimicrobial, enzyme inhibitory and receptor antagonistic activities, but have not been shown to be involved in PAH utilization or biosurfactant production. This finding provided an interesting development in our research which must be fully examined. This resulted in a change to our goals to incorporate experiments designed to quantitate biosurfactant production in these strains and to determine if Sphingomonas BPH is capable of reducing U levels. These experiments are currently being performed at Savannah River National Laboratory (Aiken, SC). Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold and are typically between 15-24 amino acids in length. The lariat topology of the lasso peptides involves the action of at least 2 enzymes: B (required to bind and cleave the precursor peptide) and C (required for cyclization, or lariat formation). Lasso peptides have a wide range of activities, including antimicrobial, enzyme inhibitory, and receptor antagonistic activities, but have not been shown to be involved in PAH utilization or biosurfactant production. Lasso peptides have been identified in several microorganisms and tend to be genomically organized into operons that consist of 3-4 genes: 1) the precursor peptide (A); 2) the protease (B); 3) the cyclase (C); and a transporter (D). It has been observed that the transporter may not always be present on lasso peptide encoding operons. We then determined the organization of the lasso peptide operon in Sphingomonas BPH. Our results indicate that the Sphingomonas lasso peptide operon consists of 3 genes, A, B and C. A D gene encoding for a transporter is not associated with this operon. Our results also indicated that a divergently transcribed set of genes is located adjacent to the lasso peptide operon in Sphingomonas BPH. This set of genes consists of an isopeptidase (E) and a TonB-dependent receptor. This specific organization has been identified in the microorganism A. excentricus . In this organism, the isopeptidase has been shown to hydrolyze the lasso peptide and convert it into a linear form. We hypothesize that in Sphingomonas BPH, the isopeptidase gene may serve the same function. These findings were unexpected and caused us to contribute significant time to the investigation of these strains and the lasso peptide cluster. It is possible that the lasso peptide may play a role in biosurfactant production. As identifying such genes is a major objective of the project, this change is a worthwhile endeavor. During this reporting period, we also screened ~400 of the previously generated A. piechaudii transconjugants using the MBAS assay. The original number of transconjugants produced was approximately 1800, however a significant number of them were no longer viable at the time of testing. Several variables associated with the MBAS assay required reworking, and during this times many transconjugants were lost. None of the A. piechaudii transconjugants tested were biosurfactant deficient. We are currently performing additional transpositional mutagenesis studies to generate more transconjugants (both P. pituda and A. piechaudii) for screening.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
1. Waltena Simpson. 2019. Bioremediation of Uranium (U)-Contaminated Soils . Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
2. Waltena Simpson. 2019. Possible Role of Lasso Peptides in Biosurfactant Production and Polycyclic Aromatic Hydrocarbon Utilization . Dutch Fork High School. Columbia, SC. June 4, 2019.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
3. Makaela Jackson, Victoria Brown, Shakeela Shiggs, Jasmine Fox and Waltena Simpson. 2019. Efficient Delivery of Tn5 for the Production of Biosurfactanat Deficient Mutants of Alcaligenes piechaudii . Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
4. Victoria Brown, Makaela Jackson and Waltena Simpson. 2019. Tn5 Transpositional Mutagenesis of Flexibacter cf. sancti SRS to Identify Genes Involved in Biosurfactant Production . Association of Research Directors Symposium. Jacksonville, Fl. March 30-April 3, 2019.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience: The target audience for this project is global as it encompasses areas with U contaminated soils/sediments. The project may result in the production of a cost-effective means of reducing the amount of U in sediments and soils. This will reduce a global health hazard to individuals who grow and/or maintain crops as well as those who consume them as U can be absorbed into certain agricultural products. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has provided opportunities for professional development as one of the undergraduate student participants has presented her data at both local and state conferences. Her preparation of her poster and power point presentations allowed for her to expand in a way not offered by traditional coursework, but by independent research. How have the results been disseminated to communities of interest?The results have been disseminated by student participation in conferences. Ms. Makaela Jackson has given both oral and poster presentations here at SCSU and at the ABRCMS conference in Indiana this year. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we anticipate that SRNL will have received funding and will have performed objectives 1 and 2.
Impacts
What was accomplished under these goals?
During the 2018 year, objectives 3 and 4 were initiated. The focus of this objective is the identification of genes involved in biosurfactant production in A. piechaudii and P. pituda. Identifying these genes may provide new insight not only into how these microorganism produce biosurfactants but also a means to enhance biosurfactant production. We chose to employ a system of random transpositional mutagenesis using the miniTn5 transposon, as it has been successful in the previous creation of stable transconjugants of other soil microorganisms. The PANC-1 Plus Mini-Tn5 package was purchased (BioMedal, Spain). This package contains 4 conjugal plasmids: pUTminiTn5-Km (confers resistance to kanamycin), pUTminiTn5-Sm (confers resistance to streptomycin) , pUTminiTn5-Cm (confers resistance to chlorampenical), and pUTminiTn5-Tc (confers resistance to tetracycline). To determine the best conjugal plasmid to utilize, we assessed the abilities of the bacterial strains to survive in the presence of (or be killed by) the antibiotics kanamycin, streptomycin, chloramphenicol and tetracycline. Unsurprisingly, both A. piechaudii and P. pituda exhibited multiantibiotic resistance. We selected the conjugal plasmid pUTminiTn5-Tc because this experiment demonstrated both strains to be resistant to the antibiotics kanamycin and streptomycin, but sensitive to tetracycline. Several techniques were utilized to produce tranconjugants of A. piechaudii and P. pituda in an attempt to produce random mutations in genes involved in biosurfactant production. These techniques were electroporation, chemical transformation, dot conjugation and line streak conjugation. Electroporation was performed using the following paramerters to introduce pUTminiTn5-Tc (30 ng) into the bacteria: 25mF, 200? , and 2500mV. For chemical transformation, the bacteria were made competent using MgCl2 and 100ng of pUTminiTn5-Tc. In dot transformation, 300 ml was taken from overnight cultures of A. piechaudii and mixed in a microcentrifuge tube with 300 ml of E. coli S17-lpir/pUTminiTn5-Tc. From this mixture, 100 ml was removed and "dotted" in 3 separate locations onto and LB plate to allow conjugation to occur. To perform line streak conjugation, cultures of A. piechaudii and E. coli S17-lpir/pUTminiTn5-Tc were grown to an OD600 of approx. 0.4. The cultures were then centrifuged and concentrated in 1/10 the original culture's volume. Wire loops were then used to transfer bacteria from the cultures and produce adjacent line streaks on LB plates. The streaks were allowed to grow for 1 day and were then mixed with a steel spreader and the mixed cultures allowed to conjugate overnight. For line and dot conjugations, after overnight mating the mixed bacteria was removed from the LB plate, resuspended in 1x PBS and plated on LB plates containing tetracycline (20 mg/ml) and kanamycin (100 mg/ml). For all techniques, the LB-tet-kan plates were allowed to incubate at 28oC for 10-12 days. Our results demonstrated that the line streak conjuation method was most efficient at producing tranconjugants. Specifically, this method produced 1800 transconjugants, while electroporation and chemical transformation each generated 0 transconjugants , and dot conjugation produced only 37. We then attempted to screen the transconjugants to detect any that are deficient in biosurfactant production using an atomized assay. This atomized assay, which involved an overspray of light parafilm oil, was developed as a way to screen large numbers of bacteria for biosurfactant production.As a positive control for this experiment, we requested and were provided with the biosurfactant producing strain P. syringae B728a (courtesy of Dr. Steve Lindow, UC Berkeley). Our studies indicated that this assay could not be used to detect biosurfactant production in the wildtype strains of A. piechaudii or P. pituda provided to us by SRNL. After consultation with Dr. Brigmon and further research, we determined that the ability of microorganisms to lyse red blood cells (i.e hemolysis) serves as an indication of biosurfactant production. We therefore plated the wildtype strains onto 5% TSA blood agar plates. Both strains failed to demonstrate hemolytic activity. However, two previously generated phenanthrene utilization mutants of Sphingomonas strain BPH demonstrated hemolytic activity and will be further investigated to determine if they produce biosurfactants and if the gene disrupted in them is involved in biosurfactant production as well. We met and conferenced with SRNL for a solution to this problem. We have determined that the methylene blue active substances (MBAS) assay will need to be performed in order to screen the 1,800 A. piechaudii transconjugants generated. The MBAS assay was initially employed to detect biosurfactant -producing strains at SRNL. This procedure requires the production of indicator plates containing mineral salts medium, trace elements, methylene blue and the catatonic detergent cetyltrimethylammonium bromide (CTAB). To perform the MBAS assay, individual shallow wells must be cut into the agar of the indicator plate and bacterial cultures to be analyzed placed within. Alternatively, we have been supplied by SRNL with several (6) different strains which exhibit better biosurfactant activity that those originally provided should the situation arise where we must change strains. Studies are currently underway to perform MBAS analysis of all of the wildtype strains in our possession. Once optimal parameters are set, this assay will be performed to screen all of the A. piechaudii transconjugants.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
1. Ms. Jackson gave an oral presentation at the Annual Biomedical Research Conference for Minority Students (ABRCMS) in Indianapolis, Indiana. November 13-17, 2018.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
2. Ms. Jackson gave a poster presentation at the annual LS-SCAMP symposium held at South Carolina State University. She won first place in her area of competition (Biology),Louis Stokes South Carolina Alliance for Minority Participation (LS-SCAMP) Program in celebration of Research Scholars at the 2018 Undergraduate Research Symposium and Showcase on Friday, November 9, 2018 SCSU Orangeburg, SC.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
3. M. Jackson*, V. Brown, S. Shiggs, J. Fox, W. Simpson, South Carolina State University, Orangeburg, SC 29117; and R. Brigmon, Savannah River National Laboratory, Aiken, SC 29808. Efficient Delivery of Tn5 for the Production of Biosurfactant Deficient Mutants of Alcaligenes piechaudii. Presentation at the 2019 ARD Research Symposium, Jacksonville, FL. March 30 - April 3, 2019.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
4. V. Brown*, M. Jackson, W. Simpson, South Carolina State University, Orangeburg, SC 29117; and R. Brigmon, Savannah River National Laboratory, Aiken, SC 29808. Tn5 Transpositional Mutagenesis of Flexibacter cf. sancti SRS to Identify Genes Involved in Biosurfactant Production. Presentation at the 2019 ARD Research Symposium, Jacksonville, FL. March 30 - April 3, 2019.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
5. W. Simpson*, M. Durant, South Carolina State University, Orangeburg, SC, 29117; and R. Brigmon, Savannah River National Laboratory, Aiken, SC, 29808. Bioremediation of Uranium (U) Contaminated Soil. Presentation at the 2019 ARD Research Symposium, Jacksonville, FL. March 30 - April 3, 2019.
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Progress 06/21/17 to 09/30/17
Outputs
Target Audience: The target audience for this project is global as it encompasses areas with U contaminated soils/sediments. The project may result in the production of a cost-effective means of reducing the amount of U in sediments and soils. This will reduce a global health hazard to individuals who grow and/or maintain crops as well as those who consume them as U can be absorbed into certain agricultural products. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided an opportunity for a student, Shakeela Shiggs, to be selected as an undergraduate researcher. This will allow her to be trained in molecular methods and environmental research. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, the transpositional mutagenesis studies will be performed in order to produce biosurfactant deficient mutants of P. pituda and A. piechaudii and assess their roles in U attenuation. Plasmids containing variations of the Tn-5 minitransposon have been procured from Biomedal (Spain) and are ready to be used in mutagenesis experiments. Biosurfactant deficient mutants will be identified by overspraying all transconjugants produced with atomized mineral oil drops. Mutants displaying reduced halos compared to the wildtype will be categorized as biosurfactant deficient and selected for further analysis. These mutants will also be utilized in experiments to determine the effect of their biosurfactants in U remediation. In addition, using U-containing soil from the SRNL's Mixed Waste Management Facility (MWMF), experiments to determine /quantify the optimal amounts of amendments needed to immobilize U under aerobic conditions will be conducted. Accomplishing these goals will require 1) the hiring of additional undergraduate students at SCSU, 2) sorption batch experiments to be conducted at SRNL, and 3) the internship of SCSU students in the lab of Dr. Brigmon of SRNL during the 2018 summer.
Impacts
What was accomplished under these goals?
To accomplish the goals listed above, viable cultures of P. pituda and A. piechaudii must be possessed. During this period, both bacterial strains were procured from the Savannah River National Laboratory (SRNL), subcultured and stored. Accomplishment of the objective of examination of microbial amendments and their sequestering capacity for U under aerobic conditions and anaerobic conditions is a task to be performed by SRNL.To accomplish the objective of identifying genes involved in biosurfactant production in P. pituda and A. piechaudii using transpositional mutagenesis, a Tn-5 package containing the following conjugation plasmids was ordered: pUT mini-Tn5 Km, pUT mini-Tn5 Cm, pUT mini-Tn5 Sm/Sp, and pUT mini-Tn5 Tc. These plasmids confer resistance to the antibiotics kanamycin, chloramphenicol, streptomycin and tetracycline, respectively. The plasmids were recently received and are now ready to be used in conjugation experiments to yield biosurfactanct deficient mutants. The objective of examination of the effect of amendments on microbial activity and its implications for U sequestering will be accomplished in year 2 of the project.
Publications
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