Progress 10/01/17 to 09/30/20
Outputs
Target Audience: During the award period, we primarily presented this work to researchers and undergraduates interested in microbial genetics, molecular biology, and biochemistry. Each year, the graduate student presented a talk at a weekly seminar attended by several research groups on campus. He also presented a poster at a national conference, the Molecular Genetics of Bacteria and Phages. The PI gave a talks to graduate program students and to freshman interested in the microbiology major on campus and highlighted this work. The graduate student mentored two undergraduates, a microbiology major and a biochemistry major, for their respective independent research project credits. The undergraduates presented posters are university-wide poster sessions attended by hundreds of undergraduates. Due to COVID, the two international meetings were cancelled (BACELL in Kobe, Japan, in April 2020 and Molecular Genetics of Bacteria and Phages in Madison, WI in August 2020) at which we had anticipated presenting work associated with this award. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the course of this award, the project has provided opportunities for training graduate, two undergraduates, and a high school student. The primary contributor to this project began as a graduate student in the UW-Madison Bacteriology Master's degree program. Midway through this project, the graduate student successfully defended a Master's thesis focused mostly on results from Objective 1 of this project. He matriculated into the Microbiology Doctoral Training Program here at UW-Madison and continued this work while preparing for this PhD candidacy exam. Through regular meetings with the PI and interactions with senior graduate students and laboratory scientists, the graduate student made significant advances in his ability to conduct independent research. In addition to daily function in the laboratory, this project has provided the student with professional development such as, coordinating resource distribution with multiple laboratories, writing skills from preparing graduate funding and program applications, and presentation skills from research presentations, committee meetings, and an oral preparation at cross-departmental seminar. In the fall of 2018, the graduate student began mentoring two UW-Madison undergraduates. With guidance from the PI, and attendance at campus mentor training workshops. The graduate student developed skills in teaching and overseeing his mentees in sub-aims of his projects. He gained experience editing and providing constructive feedback when his mentees developed posters for undergraduate symposium presentations. The graduate student also mentored a high school student at UW-Madison through the summer PEOPLE Program. He taught the student fundamentals of laboratory techniques and basics of the laboratory research process. The high school student had the opportunity to present a ~5min description of the summer work associated with this project to her summer cohort. How have the results been disseminated to communities of interest?Each year, the graduate student presented a talk at a weekly seminar attended by several research groups on campus. He also presented a poster at a national conference, the Molceular Genetics of Bacteria and Phages. The PI gave a talks to gradaute program students and to freshman interested in the microbiology major on campus and highlighted this work. The graduate student mentored two undergraduates, a microbiology major and a biochemistry major, for their respective independent research project credits. The undergraduates presented posters are university-wide poster sessions attended by hundreds of undergraduates. Due to COVID, the two international meetings were cancelled (BACELL in Kobe, Japan, in April 2020 and Molecular Genetics of Bacteria and Phages in Madison, WI in August 2020) at which we had anticiapted presenting work associated with this award. Although additional experiments are still needed, we anticipate that results from this work will be included in forthcoming manuscripts from the lab. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Associated with Objective 1, in which the goal was to identify genetic determinants for the ESX secretion system: (1) We conducted two genetic screens. In the first screen, we examined ~4000 strains of B. subtilis, each of which is missing a different gene from the genome. This provided a list of 345 so-called candidate genes that might be involved in ESX secretion. In the second screen, we examined over 250,000 colonies of B. subtilis, each of which has a unique genetic disruption called a transposon insertion somewhere within the genome. This yielded a list of 102 candidates. (2) Using secondary confirmation, bioinformatic analysis, and literature searches, we grouped the candidates from the two screens and identified a core set of gene candidates for follow-up experiments. In particular, we focused on one candidate gene that was identified in both of the screens. This gene is linked to the ability of B. subtilis cells to switch between a motile and a non-motile state. (3) We conducted a series of biofilm and motility assays to test for effects of this mutant in the context of the secretion system background. The results of these experiments did not suggest a link between this gene and the secretion phenotype found in the original screen. Simultaneously, we were remaking clean genetic background strains for the top candidates. In this process, we found that the blue/white phenotypes from the screen were not stable, including our top candidate. (4) That led us to test whether the quantity of ESX substrate made by the cell was influencing the secretion result. The major result from this work is that genetic elements that control the quantities of ESX substrate contribute to this otherwise un-regulated export of substrate into the media. Associated with Objective 2, in which the goal was to determine the range of proteins that can be accommodated by the ESX system: (1) We designed and produced a panel of secretion fusions with a range of sizes and biochemical properties. The fusion partners we used were chosen based on their use and utility in industrial applications. (2) We tested for protein secretion of the fusions by immunoblotting for the presence of the protein in the extracellular medium. All of the fusions were made and present in the cells. Only two of the fusions were detected as secreted. The other two were not. Secretion status did not correlate strictly with size or surface charge properties, so additional study will be required to determine characteristics of secretion-prone vs secretion-inhibited substrates. (3) We produced a secretion fusion that can be locked into its three-dimensional structure when in the presence of a complementary peptide. (4) We tested for protein secretion of this "locked" protein. This fusion was made and present in the cells, but not detected in the secreted sample. We found that presence of the fusion appeared to inhibit secretion of other protein substrates for the secretion apparatus. (5) We retested the original fusions that we had made. For a couple of the fusions, we found a similar result of apparent inhibition to secretion of other substrates by the fusions. The major results from these observations are a. some fusions are viable for secretion, and b. some fusions may be able to functions as "stoppers" for the secretion apparatus.
Publications
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2020
Citation:
Genetic and Molecular Characterization of the Type VII Secretion System in Bacillus subtilis
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience: We primarily targeted other researchers and undergraduates interested in microbial genetics, molecular biology, and biochemistry. Mr. Finn presented a talk at a weekly seminar attended by several research groups on campus. The PI gave a talk to freshman interested in the microbiology major on campus and highlighted this work. Mr. Finn mentored two undergraduates, a microbiology major and a biochemistry major, in their Biology 152 mentored research project on campus. Both of the students had the opportunity to present posters on their work at university-sponsored poster sessions at the end of their respective mentored research semesters. In August 2019, Mr. Finn presented the work completed to-date for his defense of his Masters thesis. This presentation was attended by faculty from the UW-Madison Bacteriology Masters program. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, the project has provided opportunities for training graduate, undergraduate students. The primary contributor to this project is a graduate student in the UW-Madison Bacteriology Masters degree program, Mr. James Finn. Through regular meetings with the PI and interactions with senior graduate students and laboratory scientists, Mr. Finn has made significant advances in his ability to conduct independent research. In addition to daily function in the laboratory, this project provided Mr. Finn with professional development such as, coordinating resource distribution with multiple laboratories, writing skills from preparing graduate funding and program applications, and presentation skills from research presentations, committee meetings, and an oral preparation at cross-departmental seminar. In the fall of 2018, Mr. Finn began mentoring two UW-Madison undergraduates. With guidance from the PI, and attendance at campus mentor training workshops, Mr. Finn developed skills in teaching and overseeing his mentees in sub-aims of his projects. He gained experience editing and providing constructive feedback when his mentees developed posters for undergraduate symposium presentations. How have the results been disseminated to communities of interest?Mr. Finn presented a talk at a weekly seminar attended by several research groups on campus. The PI gave a talk to freshman interested in the microbiology major on campus and highlighted this work. Mr. Finn mentored two undergraduates, a microbiology major and a biochemistry major, in their Biology 152 mentored research project on campus. Both of the students had the opportunity to present posters on their work at university-sponsored poster sessions at the end of their respective mentored research semesters. In August 2019, Mr. Finn presented the work completed to-date for his defense of his Masters thesis. This presentation was attended by faculty from the UW-Madison Bacteriology Masters program. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue the functional validation of the hits from the genetic screens. We will also test the newly made protein fusions for secretion by the ESX system.
Impacts
What was accomplished under these goals?
During this reporting period, the two most significant accomplishments were the completion of the two genetic screens designed to identify previously unknown genes associated with the ESX protein secretion system in Bacillus subtilis. The output of these two labor-intensive screens allows us to now explore which conditions and genetic modifications can increase protein secretion yields by this system. In the first screen, we examined ~4000 strains of B. subtilis, each of which is missing a different gene from the genome. This provided a list of 345 so-called candidate genes that might be involved in ESX secretion. In the second screen, we examined over 250,000 colonies of B. subtilis, each of which has a unique disruption called a transposon insertion somewhere within the genome. This yielded a list of 102 candidates. Using secondary confirmation, bioinformatic analysis, and literature searches, we were able group the candidates from the two screens and identify a core set of gene candidates that are now undergoing follow-up experimentation for their possible contributions to ESX secretion. In particular, we have focused on one candidate gene that was identified in both of the screens. This gene is linked to the ability of B. subtilis cells to switch between a motile and a non-motile state. Armed with this knowledge, we can test, for example, whether forcing all of the cells to be either motile or non-motile will alter the protein secretion yields of a bacterial culture. The major accomplishment for Aim 2 was the successful construction of four new protein fusions for further testing.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:We primarily targeted other researchers in this field including microbial geneticists, molecular biologists, and biochemists. Mr. Finn presented a poster at the national conference, Molecular Genetics of Bacteria and Phages in 08/2018, which had over 400 attendees. The PI presented a seminar that included some of this work in the Distinguished Lectures in Microbiology seminar series at UW-Madison in 09/2018. The PI also gave talks to incoming UW-Madison freshman exploring the microbiology major, as well as to the UW-Madison Microbiology Club. A high school student from the PEOPLE program who trained on this project over the summerhad the opportunity to present a poster related to this work. Changes/Problems:During the first reporting period, we discovered that the twoB. subtilisstrains, previously demonstratedto differ in ESX secretion when using an immunoblotting method to analyze differences,exhibit indistinguishableESXsubstrate secretion when assessed using our blue/white method. Wetherefore modified the genetic screen to focus on genetic hits that reveal general secretion inhibitors or enhancers, as opposed to the strain-specific genetic hits we originally expected to identify(objective 1.1). This was a fortuitous adjustment, as finding suchuniversal genetic hits will be more valuable and transferrable across secretion-relevant strains. What opportunities for training and professional development has the project provided?The project has provided opportunities for training graduate,undergraduate, and high schoolstudents. The primary contributor to this project is a graduate student in the UW-Madison Bacteriology Masters degree program, Mr. James Finn. Through reguar meetings with the PI and interactions with senior graduate students and laboratory scientists,Mr. Finn has made significant advances in his ability to conduct independent research. In addition to daily function in the laboratory, this projecthas provided Mr. Finn withprofessional development such as, coordinating resource distribution with multiple laboratories, writing skills from preparing graduate funding and program applications, and presentation skills from research presentations,committee meetings,and a poster preparation at a national conference. Over the summer of 2018, Mr. Finn mentored a high school student at UW-Madison through the PEOPLE Program. Mr. Finn taught the student fundamentals of laboratory techniques and basics of the laboratory researchprocess. In the fall of 2018, Mr. Finn began mentoring two UW-Madison Microbiology major undergraduates, and began learning how to design and coordinate appropriate projects for students at this stage. How have the results been disseminated to communities of interest?Mr. Finn presented a poster at the national conference, Molecular Genetics of Bacteria and Phages in 08/2018 which had over 400 attendees. The PI presented a seminar that included some of this work in the Distinguished Lectures in Microbiology seminar series at UW-Madison in 09/2018. The PI also gave talks to incoming UW-Madison freshman exploring the microbiology major, as well as to the UW-Madison Microbiology Club. A high school student from the PEOPLE program who trained on this project over the summerhad the opportunity to present a poster related to this work. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will(1) verify the genetic hitsidentified by the KO library screen by first,constructing newB. subtilisstrains containing direct KO's, second,testing the new strains for ESX substrate secretion using our blue/white assay, and, third, directly followingESX substrate secretion with polyclonal antibodies to the ESX substrate, and(2) conduct the transposonscreen andvalidate genetic hits as described for the KO library hits.Additionally, we will complete the construction ofB. subtilisstrains containing ESX substrate protein fusions and test the strains for secretion of the fusions.
Impacts
What was accomplished under these goals?
During the first reporting period, we focused our efforts on Objective 1:Dissect genetic requirements of the ESX secretion system inBacillus subtilis. We accomplished the following crucial steps towards thisobjective: (1) we obtained and re-arrayed into a functional format for screeninga knock-out (KO) librarycontaining all ~4000 nonessentialBacillus subtilisgenes, (2) we screened the arrayed KO library for changes in ESX substrate export, and after secondary screeningof thehits, identified over three hundred hits to send for sequencing identification and further validation, (3) we built bacterial strains that will allow for screening of a transposon library with ~130,000 independent mutants in the second funding year. Towards Objective 2: Determine the range of proteins that can be accommodated by the ESX system,we have designed and begun molecular geneticconstruction ofB. subtilisstrains containing ESX substrate protein fusions. These protein constructs are fusions of the ESX proteins with additional proteinsof various sizes and folding state stability. Most fusions that we will testare for enzymes that are established, important industrial products.
Publications
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