Progress 11/01/15 to 10/31/20
Outputs
Target Audience:Target audience: The primary target audience is the scientific research community. Our general approach is to use model bacteria with good genetic tools and relatively complex adaptive processes to discover novel signaling and gene regulatory mechanisms, and characterize them at the molecular level. We expect many of the mechanisms to be conserved in many bacteria, so our work will establish new paradigms that will be used to investigate bacteria that are experimentally more difficult to work with, but have potential applications in agriculture, medicine, and industry. The secondary target audience is the general public. We engage in outreach activities to teach the public about bacteria and the scientific process. Efforts: Our team generates a diverse STEM workforce with strong interdisciplinary training by exposing postdocs, graduate students, and undergraduates to state-of-the-art microscopic, molecular, and computational approaches. Full participation of women and other individuals underrepresented in STEM is a high priority. To improve STEM education and increase public scientific literacy and engagement, we create interactive presentations for the Greer Community Center and outreach events at Michigan State University.? Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided research training and professional development for two postdocs, six graduate students, and thirteen undergraduates. All gained valuable research experience in microbial molecular biology and genetics. One senior research technician also worked on the project. The project provides a unique training environment with in-depth exposure to two well-studied model organisms. Kroos meets individually with members of his group at least weekly and makes himself available as needed at other times. These meetings include data analysis, discussion of relevant literature and of ongoing work in our group and in other groups, and design of future experiments. Kroos encourages all group members to present their research in a variety of venues, including weekly group meetings, a "Metabolism, Membranes, and Metalloenzymology Interest Group" that meets twice a month, weekly "summer research presentations" with about 15 groups in the "Gene Expression in Development and Disease Focus Group", and local, national, and international scientific meetings. When appropriate, my group meets to give "practice talks" prior to meetings and we offer constructive criticism to each other. Fifteen meetings were attended by one or more of six different members of the Kroos group in order to present a poster and/or talk. Kroos encourages postdocs, graduate students, and senior undergraduates to mentor less-experienced undergraduates in the lab. Typically, this involves a period of working jointly on experiments before the less-experienced individual transitions to an independent research project with the more-experienced individual continuing as mentor. Kroos engages postdocs, graduate students, and senior undergraduates in other activities that foster their professional development, including preparation of grant and fellowship applications, preparation of abstracts and posters for meeting presentations, preparation of manuscripts from our group, and reviews of manuscripts for journals. Kroos occasionally recommends postdocs to journal editors when he do not have time to review a manuscript. Kroos shares his experiences collaborating with computational, structural, and evolutionary biologists at MSU and other institutions. He encourages mentees to build a network of scientific colleagues by introducing them to scientists at MSU and at scientific meetings. Kroos hosts seminar speakers and encourages mentees to have lunch or dinner with the speaker. He makes students and postdocs aware of the abundant resources at MSU for professional development, which include career counseling and several seminar series about teaching. In compliance with MSU and federal regulations, all prospective group members receive safety trainingprior to working in the lab, and they complete annual refresher training. Also, all group members complete 1 h/yr of training in the responsible conduct of research presented by our chairperson or a senior faculty member. In addition, the Kroos group spends at least 2 h/yr discussing issues like 1) data acquisition and management, sharing, and ownership and 2) publication practices and responsible authorship, and peer review. How have the results been disseminated to communities of interest?In addition to seven research papers, a review of the gene regulatory network governing Myxococcus development including a comparison with Bacillus subtilis and Streptomyces was published (Trends in Genetics, 2117). Results of the project were also disseminated by presenting 25 talks or posters at 15 scientific meetings. Kroos also presented seminars about the work at the University of South Florida (2016) and the University of Wyoming (2017). Eight outreach educational activities were presented: 1. Educational activity presented at the Girl Scouts STEM Demo Day on 11/7/15, coordinated by the MSU Women in Engineering organization. Presentation entitled "Magnificent Motile Microbes" included demonstration of what microbes are and where we find them, how microbes use flagella to mediate movement in their environment, and provided activities to engage participants and assess understanding. The effort was co-led by graduate student Daniel Parrell and undergraduate researcher Fiona Buchanan, with help from postdoc Ramya Rajagopalan and undergraduate researcher Emily Titus during activities. Four groups of 10-15 girl scouts participated for 30 minutes each. 2. Educational activity presented at the Girl Scouts STEM Demo Day on 10/27/16, coordinated by the MSU Women in Engineering organization. Presentation entitled "Motile Microbes" included demonstration of what microbes are and where we find them, how microbes use flagella tomediate movement in their environment, and provided activities to engage participants and assess understanding. The effort was co-led by graduate student Daniel Parrell and postdoc Ramya Rajagopalan, with help from undergraduate researcher Elizabeth Lyons and visiting scholar Suchitha Raghunathanduring activities. Four groups of 10-15 girl scouts participated for 30 minutes each. 3. Educational activity presented at the Middle School Girls Math and Science Day on 3/4/17, coordinated by the MSU Graduate Women in Science organization. Presentation entitled "Marvelous Microbes" included demonstration of what microbes are, where we find them, what cellular structures microbes have that are similar and different from humans, and provided activities to engage participants and assess understanding. The effort was co-led by graduate student Daniel Parrell and undergraduate researcher Fiona Buchanan, with help from graduate student Y Hoang and undergraduate researcher Matthew Nelson during activities. Four groups of 10-15 middle school girls participated for 30 minutes each. 4. Educational activity presented at the Middle School Girls Math and Science Day on 3/3/18, coordinated by the MSU Graduate Women in Science organization. Presentation entitled "Microbes know how to work" discussed how yeast can be used tometabolize sugar. To engage participants and assess understanding, activities were conducted to demonstrate how yeast can be used to breakdown sugar pollutants in a local river, in order to save fish and other wildlife. The effort was co-led by graduate students Daniel Parrell, Y Hoang, and Shreya Saha. Four groups of 10-15 middle school girls participated for 30 minutes each. 5. Educational activity entitled "Myxo under the dissection scope" was part of the "Microbe Magic" exhibit at the MSU Science Festival on 4/7/18. A poster demonstrating morphological changes of Myxococcus xanthus during starvation-induced development was prepared for display. To engage participants and assess understanding, comparison of morphological changes between wild type and a sporulation-deficient mutant were viewed under a dissecting microscope and discussed. The effort was co-led by graduate students Daniel Parrell, Y Hoang, and Shreya Saha. Numerous people of all ages participated for variable amounts of time. 6. Educational activity presented at the Middle School Girls Math and Science Day on 3/2/19, coordinated by the MSU Graduate Women in Science organization. Presentation entitled "How do germs spread and how can we prevent it?"discussed germs and herd immunity. To engage participants, activities with glitter were conducted to illustrate how germs spread via direct or indirect contact. Participants also had an opportunity to view bacteria microscopically. Presenters demonstrated how herd immunity works to protect individuals that cannot be vaccinated such as babies or those with allergies. The effort was co-led by graduate students Shreya Saha and Y Hoang. Four groups of 7-10 middle school girls participated for 30 minutes each. 7. Educational activity entitled "Myxo motility and predation" was part of the "Microbe Magic" exhibit at the MSU Science Festival on 4/6/19. Exhibits of Myxococcus xanthus swarming on nutrient agar and eating Escherichia coli will be shown and explained, as well as a time-lapse movie of M. xanthus predation. Swarming vs. swimming motility will be contrasted and participants will be invited to play "pin the flagellum on the bacterium". The effort was co-led by graduate students Y Hoang, Shreya Saha, and Sandra Olenic, and undergraduate researchers Olivia Thomas and Tara Metcalfe also participated. Numerous people of all ages participated for variable amounts of time. 8. Educational activities entitled "Bacteria" and "DNA - the building block of life" at the Greer Community Center on 6/20/19 and 6/27/19, respectively. On the 20th, students were shown a PowerPoint presentation that served as an introduction to bacteria. Afterwards, students were instructed to swab various items in the classroom (i.e. notebooks, hands before and after using sanitizer, keyboards) and streak their samples onto agar plates. During this activity, students were asked "Which samples will grow the most? Why?" At the end of this session, students filled out a handout where they labeled the parts of a bacterial cell and wrote down their predictions for bacterial growth. On the 27th, the same group of students was shown the plates they had streaked. Students were divided into small groups to evaluate the bacterial growth. After this discussion, a brief PowerPoint presentation was given on DNA. The presentation emphasized that DNA is the building block for all life like bacteria, plants, and humans. Students then worked independently and extracted DNA from wheatgerm. Then a brief discussion was held to evaluate student learning. The effort was led by graduate student Sandra Olenic. Approximately 30 students, ages 9-12, participated in both sessions, each lasting 2 hours. 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 nature, bacteria exist as members of communities in which cells respond to signals from each other and the environment. Understanding how bacteria integrate signals and respond appropriately is a fundamental challenge of great practical significance. Microbial communities impact global processes like cycling of elements between soil, water, and air, and primary productivity of the oceans; they impact ecosystems and all the organisms that inhabit them. Limited understanding of how microbes control complex behaviors in response to each other and their environment impedes our ability to harness them for pollution and climate control, and for increased bioenergy and food production. Our research helps fill this crucial knowledge gap while training scientists. Our general approach is to use model bacteria to discover novel signaling and gene regulatory mechanisms, and characterize them at the molecular level. We expect many of the mechanisms to be conserved, so our work will establish new paradigms that will be used to investigate other bacteria that that impact the quality of life globally. Our basic research is intended to provide new knowledge. Application of this knowledge to problems in agriculture, medicine, and industry will result in economic and health benefits to mankind, and environmental benefits to the world. Our work focused on two bacteria that undergo sporulation in response to starvation. Sporulation is an adaptive response that allows bacteria to survive in the absence of food by becoming dormant. The two bacteria undergo very different sporulation processes. Bacillus subtilis is partitioned into two compartments, the mother cell and the forespore, each of which expresses distinct sets of genes under the control of different sigma subunits of RNA polymerase. One gene set expressed in the mother cell is under the control of sigmaK RNA polymerase. We discovered that sigmaK is first made as an inactive precursor (Pro-sigmaK) and that cleavage to the active form depends on SpoIVFB, an intramembrane metalloprotease (IMMP). Homologs of SpoIVFB are present in nearly all organisms studied. Bacterial IMMPs play important roles during stress responses and infections. Understanding how IMMPs function in bacteria could lead to the development of new antibiotics. In eukaryotes, IMMPs cleave transcription factors that regulate lipid metabolism in animals, chloroplast development in plants, and responses to stress in both animals and plants. Knowledge about bacterial IMMPs will facilitate studies of eukaryotic IMMPs, which could lead to the development of novel therapeutics and growth modulators. The objectives of ourB. subtiliswork are: 1. Determine how SpoIVFB recognizes Pro-sigmaK. We purified a complex of SpoIVFB and Pro-sigmaK, determined parts of each protein needed to form the complex, and characterized the subunit composition of the complex (Journal of Biological Chemistry, 2016). We extended the work by performing chemical cross-linking followed by trypsin digestion and mass spectrometry to identify residues of the two proteins that are in proximity in the complex. Using this data and homology modeling, we built a model of the complex (Proceedings of the National Academy of Science, 2017). 2. Test the model that the CBS domain of SpoIVFB senses the ATP level and regulates access of Pro-sigmaKto the active site. We used sequence alignment and homology modeling to identify nine residues in the SpoIVFB CBS domain as candidates for involvement in ATP binding. SpoIVFB variants with Ala substituted for each residue were coexpressed with Pro-sigmaK in E. coli. Three variants exhibited strongly impaired Pro-sigmaK cleavage. We plan to purify these variants and test for ATP binding. We also tested the model that the ATP level rises in the mother cell after completion of forespore engulfment. We found that the mother cell ATP concentration rises about twofold coincident with increasing cleavage of Pro-sigmaK, but the rise in ATP was not necessary for cleavage. The forespore ATP concentration did not decline, suggesting that low ATP does not explain the lack of post-engulfment forespore gene expression in channel mutants (Molecular Microbiology, 2020). 3. Determine how BofA and SpoIVFA inhibit SpoIVFB. We found that inhibition requires three conserved BofA residues in or near transmembrane segment (TMS) 2 and that BofA TMS2 occupies the SpoIVFB active site. BofA TMS1 helps hinder interaction between the Proregion of Pro-sigmaK and SpoIVFB. A structural model of the SpoIVFB-BofA-Pro-sigmaK complex was built (manuscript in preparation). 4. Test whether RasP cleaves the cell division protein FtsL without a prior cleavage and identify a novel substrate(s) of RasP. B. subtilis RasP is a type of IMMP even more broadly conserved than SpoIVFB. We found that RasP appears to cleave MBP-FtsL without a prior cleavage upon coexpression in E. coli, but purified RasP failed to cleave MBP-FtsL in detergent micelles, although it did cleave truncated MBP-RsiW (Journal of Bacteriology, 2017). The second bacterium we study, Myxococcus xanthus, undergoes multicellular development. When starved, cells change their gene expression and send signals to each other, move to construct multicellular mounds, and some of the rod-shaped cells differentiate into round spores. Previously, we investigated gene regulation in response to short-range C-signaling during development. We discovered that several C-signal-dependent promoters are controlled by two transcription factors, MrpC and FruA, including the promoter of the dev operon whose products are important for sporulation. Therefore, we focused on the regulatory network involving MrpC, FruA, and Dev (the MFD network). The objectives of ourM. xanthuswork are: 1. Measure dynamical changes in the MFD network components and output before and during commitment of wild type and mutants, and use the data to build a mathematical model of the MFD network. We established methods to systematically quantify transcript and protein levels, as well as the number of rods and spores. Characterization of a csgA mutant (defective in C-signaling) suggests that C-signaling activates FruA at least ninefold posttranslationally, which in turn activates dev transcription (Molecular Microbiology, 2019). We clarified functions of Dev proteins, showing that DevTRS negatively autoregulate dev transcript accumulation and prevent DevI overproduction that strongly inhibits sporulation. DevI transiently inhibits sporulation when regulated normally, perhaps by inhibiting MrpC translation (Journal of Bacteriology, 2017). 2. Use the model to explore whether the network can operate as a switch that becomes irreversible and test this by measuring MFD network dynamics after addition of nutrients to developing cells. We measured the network response at the boundaries of ultrasensitivity to nutrient addition to 18-h developing cells. We found that the MrpC protein level correlated best with the sporulation response. MrpC and FruA were subject to transcriptional and posttranscriptional control, respectively (Journal of Bacteriology, 2018). 3. Use the model to explore if the MFD network can achieve an ultrasensitive response and test this by measuring MFD network response as a function of added C-signal or nutrients. We mixed csgA mutant and wild-type cells at slightly different ratios. Over a narrow range of ratios, mound formation and sporulation of the csgA mutant was rescued, consistent with threshold behavior, which we plan to test further. 4. Construct synthetically rewired MFD networks, measure their dynamical responses, and analyze the results using mathematical models of rewired networks. We rewired the MFD network by engineering inducible expression of FruA and MrpC. The inducible FruA strain was used in (Molecular Microbiology, 2019) but the inducible MrpC strain failed to make as much MrpC as wild-type cells during sporulation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Parrell, D. and L. Kroos 2020. Channels modestly impact compartment-specific ATP levels during Bacillus subtilis sporulation and a rise in the mother cell ATP level is not necessary for Pro-?K cleavage. Mol. Microbiol. 00:1-19.
- Type:
Websites
Status:
Published
Year Published:
2020
Citation:
https://bmb.natsci.msu.edu/faculty/lee-r-kroos/
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Our basic research on the mechanisms of signaling, gene regulation, and control of emergent behaviors in bacteria is intended to provide new knowledge. Application of this knowledge to problems in agriculture, medicine, and industry will result in economic and health benefits to mankind, and environmental benefits to the world. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided research training and professional development for one postdoc, four graduate students, and four undergraduates. All gained valuable research experience in microbial molecular biology and genetics. One senior research technician also worked on the project. The project provides a unique training environment with in-depth exposure to two well-studied model organisms. I meet individually with members of the group at least weekly and I make myself available as needed at other times. These meetings include data analysis, discussion of relevant literature and of ongoing work in our group and in other groups, and design of future experiments. I encourage all group members to present their research in a variety of venues, including weekly group meetings, a "Metabolism, Membranes, and Metalloenzymology Interest Group" that meets twice a month, weekly "summer research presentations" with about 15 groups in the "Gene Expression in Development and Disease Focus Group", and local, national, and international scientific meetings. When appropriate, my group meets to give "practice talks" prior to meetings and we offer constructive criticism to each other. I encourage postdocs, graduate students, and senior undergraduates to mentor less-experienced undergraduates in the lab. Typically, this involves a period of working jointly on experiments before the less-experienced individual transitions to an independent research project with the more-experienced individual continuing as mentor. I engage postdocs, graduate students, and senior undergraduates in other activities that foster their professional development, including preparation of grant and fellowship applications, preparation of abstracts and posters for meeting presentations, preparation of manuscripts from our group, and reviews of manuscripts for journals. I occasionally recommend postdocs to journal editors when I do not have time to review a manuscript. I share my experiences collaborating with computational, structural, and evolutionary biologists both here at MSU and at other institutions. I encourage mentees to build a network of scientific colleagues by introducing them to scientists at MSU and at scientific meetings. I host seminar speakers, and I encourage postdocs and students in my group to have lunch or dinner with the speaker. I make students and postdocs aware of the abundant resources at MSU for professional development, which include career counseling and several seminar series about teaching. In compliance with MSU and federal regulations, all prospective group members receive safety training prior to working in the lab, and they complete annual refresher training. Also, all group members complete 1 h/yr of training in the responsible conduct of research presented by our chairperson or a senior faculty member. In addition, my group spends at least 2 h/yr discussing issues like 1) data acquisition and management, sharing, and ownership and 2) publication practices and responsible authorship, and peer review. How have the results been disseminated to communities of interest?In addition to one publication, results of the project were disseminated at six presentations at scientific meetings. Three outreach educational activities were presented. One was at the Middle School Girls Math and Science Day on 3/2/19, coordinated by the MSU Graduate Women in Science organization. Our presentation entitled "How do germs spread and how can we prevent it?"discussed germs and herd immunity. To engage participants, activities with glitter were conducted to illustrate how germs spread via direct or indirect contact. Participants also had an opportunity to view bacteria microscopically. Presenters demonstrated how herd immunity works to protect individuals that cannot be vaccinated such as babies or those with allergies. The effort was co-led by graduate students Shreya Saha and Y Hoang. Four groups of 7-10 middle school girls participated for 30 minutes each. Our second outreach educational activity, entitled "Myxo under the dissection scope", was a part of the "Microbe Magic" exhibit at the MSU Science Festival on 4/7/18. A poster demonstrating morphological changes of Myxococcus xanthus during starvation-induced development was prepared for display. To engage participants and assess understanding, comparison of morphological changes between wild type and a sporulation-deficient mutant were viewed under a dissecting microscope and discussed. Numerous people of all ages participated for variable amounts of time. Our third activity was presented in two parts entitled "Bacteria" and "DNA - the building block of life" at the Greer Community Center on 6/20/19 and 6/27/19, respectively. On the 20th, students were shown a PowerPoint presentation that served as an introduction to bacteria. Afterwards, students were instructed to swab various items in the classroom (i.e. notebooks, hands before and after using sanitizer, keyboards) and streak their samples onto agar plates. During this activity, students were asked "Which samples will grow the most? Why?" At the end of this session, students filled out a handout where they labeled the parts of a bacterial cell and wrote down their predictions for bacterial growth. On the 27th, the same group of students was shown the plates they had streaked. Students were divided into small groups to evaluate the bacterial growth. After this discussion, a brief PowerPoint presentation was given on DNA. The presentation emphasized that DNA is the building block for all life like bacteria, plants, and humans. Students then worked independently and extracted DNA from wheatgerm. Then a brief discussion was held to evaluate student learning. The effort was led by graduate student Sandra Olenic. Approximately 30 students, ages 9-12, participated in both sessions, each lasting 2 hours. What do you plan to do during the next reporting period to accomplish the goals?Related to the objectives of our B. subtilis work: 1. We will remove the pro-sequence from Pro-sigmaK and use size exclusion chromatography and negative stain electron microscopy to determine whether it appears to form a more homogeneous complex with SpoIVFB. If so, we will try to determine the structure of the purified complex using X-ray crystallography and cryo-electron microscopy. We will further investigate the function of a short loop that interrupts a transmembrane segment of SpoIVFB near its active site, which our preliminary data suggests interacts with the pro-sequence of Pro-sigmaK. 2. We will finish our efforts to address reviewer comments and resubmit the manuscript describing use of luciferase to measure mother cell and forespore ATP levels. We will improve purification of the complex formed between Pro-sigmaK and SpoIVFB variants and measure the effects of ATP and other nucleotides. Using this in vitro assay, we will determine the effects of substitutions in the SpoIVFB CBS domain that impair cleavage of Pro-sigmaK upon coexpression in E. coli and result in failure of the SpoIVFB variants to accumulate in sporulating B. subtilis. 3. We will finish our ongoing experiments aimed at determining the mechanism by which BofA and SpoIVFA inhibit SpoIVFB cleavage of Pro-sigmaK, and submit a manuscript for publication. This work provided several clues about subcomplexes and truncated proteins that may allow purification of protein complexes suitable for structure determination, which we will pursue. 4. If resources permit, we will investigate why RasP fails to cleave FtsL in vitro and we will try to identify novel substrates of RasP. Related to the objectives of our M. xanthus work: 1. We will finish experiments documenting our discovery that regulation of sporulation genes is complex and involves negative regulation by unactivated FruA, and submit the work for publication. If resources permit, we will test whether expression of C-signal-dependent genes increases during streaming and mound formation, and identify genes important for those emergent behaviors. 2. We will finish knocking out our other candidate gene and test for involvement in cellular shape change during development. If resources permit, we will test whether C-signaling triggers a new pattern of gene expression at the onset of the rod-to-spore transition and identify genes important for initiating cellular shape change. 3. We will determine the mechanisms of C-signal transmission and FruA activation that drive threshold-dependent gene expression and successive emergent behaviors. 4. We will finish our ongoing analysis of data showing that cells transition from rods to spores as they move upward in nascent fruiting bodies, and submit a manuscript for publication. We will also finish ongoing experiments designed to better document our discovery that cells transitioning from rods to spores segregate two copies of the genome, and submit a manuscript for publication.
Impacts
What was accomplished under these goals?
Related to the objectives of our B. subtilis work: 1. We purified a complex of SpoIVFB and Pro-sigmaK and tried to determine its structure using X-ray crystallography and cryo-electron microscopy. Crystals failed to form in the presence of detergent. Small crystals formed using the lipidic cubic phase method. The crystals were exposed to synchrotron radiation, but failed to yield useful data. The purified complex was examined by negative stain electron microscopy. Thousands of particles were identified, but the particle shapes were heterogeneous and did not lead to useful class averages. It was concluded that a more homogeneous preparation would be needed. We also attempted to reconstitute cleavage of Pro-sigmaK by SpoIVFB using proteins purified in detergent micelles. Unfortunately, a very encouraging initial result could not be reproduced, despite extensive effort, including reconstitution of the proteins in bicelles. 2. We hypothesized that the CBS domain of SpoIVFB senses a rise in the mother-cell level of ATP when channels that allow the mother cell to feed the forespore are degraded upon completion of engulfment. We fused luciferase to promoters expressed only in the mother cell or forespore and used these fusions to measure ATP levels in each compartment during sporulation of wild type and channel mutants. We found that channels modestly impact compartment-specific ATP levels and this does not regulate Pro-sigmaK cleavage, so our initial hypothesis was not supported. A manuscript describing the results was submitted for publication. We are attempting to address reviewer comments. We also tried to test the model that ATP induces a conformational change in the CBS domain of SpoIVFB that positions Pro-sigmaK for cleavage using an assay for crosslinking between the two proteins. Nucleotide effects were observed in preliminary experiments, but more highly purified complex is needed to improve the results. We had identified several substitutions in the SpoIVFB CBS domain that impair cleavage of Pro-sigmaK upon coexpression in E. coli, possibly by reducing the binding of ATP. We introduced the substitutions into B. subtilis. Surprisingly, most of the SpoIVFB variants failed to accumulate during sporulation. We selected suppressor mutants with restored sporulation and identified intragenic suppressor mutations in spoIVFB that restored accumulation of the SpoIVFB variant to a low level. Altogether, our results suggest that ATP binding to the CBS domain does induce a conformational change in SpoIVFB and that substitutions in the CBS domain can impact SpoIVFB activity and stability. 3. Using a system we established that allows us to observe nearly complete inhibition of SpoIVFB cleavage of Pro-sigmaK by coexpressed BofA and SpoIVFA in E. coli, we performed numerous additional disulfide crosslinking experiments to test our model that a transmembrane segment of BofA is near the SpoIVFB active site and blocks access of Pro-sigmaK. Indeed, crosslinking time course experiments indicate that BofA specifically interferes with positioning of the pro-sequence of Pro-sigmaK in the active site of SpoIVFB. Nevertheless, Pro-sigmaK appears to remain associated with the protein complex via other contacts with the linker and CBS domains of SpoIVFB. Pull-down experiments also suggest that Pro-sigmaK remains associated with the protein complex, even though it is not cleaved. A manuscript is in preparation. We also tested the model that the C-terminal ends of BofA and SpoIVFA interact, using a disulfide crosslinking approach, but we found no evidence for interaction. 4. Because the grant was not fully funded, we have not been able to pursue the proposed work on RasP. Related to the objectives of our M. xanthus work: 1. We published a paper describing our systematic approach to the gene regulatory network and our computational and experimental results supporting the model that C-signaling activates FruA, which in turn activates sporulation genes. Our systematic analysis of expression of genes involved in sporulation revealed unexpected complexity, including negative regulation by unactivated FruA. We have further characterized regulation of several sporulation genes and a manuscript is in preparation. 2. Related to our objective of exploring whether the regulatory network can operate as a switch that becomes irreversible, we pursued two approaches to identify genes involved in cellular shape change during development, because shape change correlates with irreversible commitment to sporulation. One approach involved suppressors of a devS mutant that overproduces DevI, an inhibitor of sporulation. Genomic sequencing of four suppressor mutants was insufficient to identify mutations that might be responsible for suppression. More mutants and/or greater sequencing coverage will be needed. The other approach involved RNA-seq analysis of wild type and mrpC, fruA, and csgA mutants. Two candidates emerged from this approach, which also involved qPCR of RNA isolated after addition of nutrient medium at different concentrations to developing cells. One was knocked out and there was no defect in spore formation. A knockout mutant of the other candidate gene is being constructed. 3. We hypothesized that extracellular rescue of a csgA mutant unable to produce C-signal would exhibit threshold behavior. We mixed csgA mutant and wild-type cells at slightly different ratios. Over a narrow range of ratios, mound formation and sporulation of the csgA mutant (marked by fluorescent protein expression) were rescued in the mixtures, in agreement with our hypothesis. We plan to further characterize the threshold behavior using single-cell fluorescene microscopy methods we have devised to measure gene expression and motility. 4. We rewired the regulatory network by engineering inducible expression of FruA and MrpC. The inducible FruA strain allowed us to address reviewer comments and finish the publication mentioned under objective 1 of our M. xanthus work. The inducible MrpC strain failed to make as much MrpC as wild-type cells during sporulation. Results described in the publication mentioned under objective 1 suggest that the mrpC promoter is extremely strong, which is difficult to match with the available inducible promoters. 5. Using fluorescence microscopy methods we have devised, we discovered that cells transition from rods to spores as they move upward in nascent fruiting bodies. A manuscript is in preparation. We also discovered that cells in transition segregate two copies of the genome using three methods: a functional mNeonGreen-FruA fusion that binds to many sites in the genome, the DNA stain DAPI, and TetR-YFP binding to a binding-site array located at a particular position in the genome. These methods allowed us to visualize nucleoids or operator arrays in individual cells within nascent fruiting bodies. A manuscript is in preparation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Saha, S., Patra, P., Igoshin, O., and L. Kroos 2019. Systematic analysis of the Myxococcus xanthus developmental gene regulatory network supports posttranslational regulation of FruA by C-signaling. Mol. Microbiol. 111:1732-1752.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Our basic research on the mechanisms of signaling, gene regulation, and cell fate determination in bacteria is intended to provide new knowledge. Application of this knowledge to problems in agriculture, medicine, and industry will result in economic and health benefits to mankind, and environmental benefits to the world. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided research training and professional development for one postdoc, four graduate students, and seven undergraduates. All gained valuable research experience in microbial molecular biology and genetics. The project provides a unique training environment with in-depth exposure to two well-studied model organisms. I meet individually with members of the group at least weekly and I make myself available as needed at other times. These meetings include data analysis, discussion of relevant literature and of ongoing work in our group and in other groups, and design of future experiments. I encourage all group members to present their research in a variety of venues, including weekly group meetings, a "Metabolism, Membranes, and Metalloenzymology Interest Group" that meets twice a month, weekly "summer research presentations" with about 15 groups in the "Gene Expression in Development and Disease Focus Group", and local, national, and international scientific meetings. When appropriate, my group meets to give "practice talks" prior to meetings and we offer constructive criticism to each other. I encourage postdocs, graduate students, and senior undergraduates to mentor less-experienced undergraduates in the lab. Typically, this involves a period of working jointly on experiments before the less-experienced individual transitions to an independent research project with the more-experienced individual continuing as mentor. I engage postdocs, graduate students, and senior undergraduates in other activities that foster their professional development, including preparation of grant and fellowship applications, preparation of abstracts and posters for meeting presentations, preparation of manuscripts from our group, and reviews of manuscripts for journals. I occasionally recommend postdocs to journal editors when I do not have time to review a manuscript. I share my experiences collaborating with computational, structural, and evolutionary biologists both here at MSU and at other institutions. I encourage mentees to build a network of scientific colleagues by introducing them to scientists at MSU and at scientific meetings. I host seminar speakers, and I encourage postdocs and students in my group to have lunch or dinner with the speaker. I make students and postdocs aware of the abundant resources at MSU for professional development, which include career counseling and several seminar series about teaching. In compliance with MSU and federal regulations, all prospective group members receive safety training prior to working in the lab, and they complete annual refresher training. Also, all group members complete 1 h/yr of training in the responsible conduct of research presented by our chairperson or a senior faculty member. In addition, my group spends at least 2 h/yr discussing issues like 1) data acquisition and management, sharing, and ownership and 2) publication practices and responsible authorship, and peer review. How have the results been disseminated to communities of interest?In addition to two publications, results of the project were disseminated at two presentations at scientific meetings. Two outreach educational activities were presented. One was at the Middle School Girls Math and Science Day on 3/3/18, coordinated by the MSU Graduate Women in Science organization. Our presentation entitled "Microbes know how to work" discussed how yeast can be used tometabolize sugar. To engage participants and assess understanding, activities were conducted to demonstrate how yeast can be used to breakdown sugar pollutants in a local river, in order to save fish and other wildlife. The effort was co-led by graduate students Daniel Parrell, Y Hoang, and Shreya Saha. Four groups of 10-15 middle school girls participated for 30 minutes each. The same three students co-led our second outreach educational activity, entitled "Myxo under the dissection scope", which was a part of the "Microbe Magic" exhibit at the MSU Science Festival on 4/7/18. A poster demonstrating morphological changes of Myxococcus xanthus during starvation-induced development was prepared for display. To engage participants and assess understanding, comparison of morphological changes between wild type and a sporulation-deficient mutant were viewed under a dissecting microscope and discussed. Numerous people of all ages participated for variable amounts of time. What do you plan to do during the next reporting period to accomplish the goals?Related to the objectives of our B. subtilis work: 1. We will test various aspects of our model of the complex containing SpoIVFB and Pro-sigmaK. We will try to determine the structure of the purified complex using X-ray crystallography and cryo-electron microscopy. 2. We will test the model that ATP induces a conformational change in the CBS domain of SpoIVFB that positions Pro-sigmaK for cleavage. We will use a crosslinking assay between SpoIVFB and Pro-sigmaK that appears to be sensitive to ATP-binding to the CBS domain. We will test substitutions in the SpoIVFB CBS domain that impair cleavage of Pro-sigmaK upon coexpression in E. coli. We will also test suppressor mutations that we selected in B. subtilis. 3. We will investigate the mechanism by which BofA and SpoIVFA inhibit SpoIVFB cleavage of Pro-sigmaK. Interactions between the four proteins will be investigated using pull-down experiments. Additional disulfide cross-linking experiments will be performed to test our model that a transmembrane segment of BofA is near the SpoIVFB active site and blocks access of Pro-sigmaK. 4. We will investigate why RasP fails to cleave FtsL in vitro. We will try to identify novel substrates of RasP. Related to the objectives of our M. xanthus work: 1. We will finish the manuscript under revision, which describes our systematic approach to the gene regulatory network and our computational and experimental results supporting the model that C-signaling activates FruA, which in turn activates sporulation genes. 2. We will continue pursuing two approaches to identify genes involved in cellular shape change during development, one involving suppressors of a devS mutant that overproduces DevI, an inhibitor of sporulation, and the other involving RNA-seq analysis of wild type and mrpC, fruA, and csgA mutants. Candidates from the second approach, which also involved qPCR of RNA isolated after addition of nutrient medium at different concentrations to developing cells, will be knocked out. 3. We will further characterize localization of mNeonGreen-FruA, DNA, and membranes during development using fluorescence microscopy. Our goal is to understand cell fate determination during the developmental process.
Impacts
What was accomplished under these goals?
Related to the objectives of our B. subtilis work: 1. We purified a complex of SpoIVFB and Pro-sigmaK, determined parts of each protein needed to form the complex, and performed chemical cross-linking followed by trypsin digestion and mass spectrometry to identify residues of the two proteins that are in proximity in the complex. Using this data and homology modeling, we have built a model of the complex in collaboration with Michael Feig. The results were published. 2. We fused luciferase to promoters expressed only in the mother cell or forespore and we used these fusions to measure ATP levels in each compartment during sporulation. A manuscript describing the results is in preparation. 3. We used protein sequence alignments to identify substitutions in the SpoIVFB CBS domain that impair cleavage of Pro-sigmaK upon coexpression in E. coli. The same substitutions impair accumulation of SpoIVFB in B. subtilis during sporulation, so spores fail to form. We have selected suppressor mutations that restore sporulation of these strains, which are being characterized. CBS domains typically bind ATP. We are developing a crosslinking assay between SpoIVFB and Pro-sigmaK that appears to be sensitive to ATP-binding to the CBS domain. 4. We established a system to observe nearly complete inhibition of SpoIVFB cleavage of Pro-sigmaK by coexpressed BofA and SpoIVFA in E. coli. Numerous deletions and substitutions in BofA and SpoIVFA have been tested in the system. Many partially relieve inhibition. Three substitutions have been shown to relieve inhibition of cleavage in B. subtilis. Disulfide cross-linking data show that a transmembrane segment of BofA is near the SpoIVFB active site, perhaps blocking access of the Pro-sequence of Pro-sigmaK. Yet, Pro-sigmaK is still bound to SpoIVFB and is near BofA, suggesting that BofA shifts the position of the Pro-sequence so that it is not cleaved. Related to the objectives of our M. xanthus work: 1. Systematic characterization of a csgA mutant (defective in C-signaling) showed a twofold lower level of FruA protein and a 20-fold lower level of dev transcript compared to wild type during the period leading up to and including the time that cells commit to spore formation. Computational modeling shows that cooperative binding of MrpC and FruA cannot account for the 20-fold decrease in dev transcripts. Neither does instability of dev transcripts in the csgA mutant account for the difference from wild type. Boosting the FruA level in the csgA mutant had no effect on the dev transcript level. Together, the results are consistent with the model that C-signaling activates FruA, which in turn activates dev transcription. A manuscript describing these results is under revision. Systematic analysis of expression of genes involved in sporulation revealed unexpected complexity, including negative regulation by unactivated FruA. 2. Several genes are known to impact spore maturation, but we found that these genes are not necessary for the initial cellular shape change that appears to coincide with commitment to spore formation. As described below, MrpC and FruA appear to regulate shape change, but a better understanding of this process would be an important advance. Therefore, we initiated two approaches to identify genes involved in cellular shape change during development. 3. We measured the network response at the boundaries of ultrasensitivity to nutrient addition to 18-h developing cells. We found that the MrpC protein level correlated best with the sporulation response (i.e., the level decreased after nutrient addition, but recovered to a higher level after 12.5% nutrient addition than after 25% addition). Most of the difference appears to be due to a higher level of mrpC transcript recovery after 12.5% addition. Interestingly, the fruA transcript level did not recover, but the FruA protein level did recover at 27 h PS to a higher level after 12.5% addition than after 25% addition. The results suggest that MrpC and FruA regulate shape change, and that MrpC and FruA are subject to transcriptional and posttranscriptional control, respectively. These results were published. 4. We created a functional mNeonGreen-FruA fusion and developed methods to visualize the fusion protein, DNA, and membranes of individual cells at the base of developing fruiting bodies using fluorescence microscopy. The results show that mNeonGreen-FruA changes its association with the two copies of the nucleoid present in cells during development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Halder, S., Parrell, D., Whitten, D., Fieg, M., and L. Kroos 2017. Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-?K. Proc. Natl. Acad. Sci. Plus USA 114:E10677-E10686.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2018
Citation:
Hoang, Y and L. Kroos 2018. Ultrasensitive response of developing Myxococcus xanthus to the addition of nutrient medium correlates with the level of MrpC. J. Bacteriol. Epub ahead of print.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Saha S, Patra P, Igoshin OA, Kroos L. 2018. Systematic analysis of the Myxococcus xanthus developmental gene regulatory network supports posttranslational regulation of FruA by C-signaling. Mol. Microbiol. under revision.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Our basic research on the mechanisms of signaling and gene regulation in bacteria is intended to provide new knowledge. Application of this knowledge to problems in agriculture, medicine, and industry will result in economic and health benefits to mankind, and environmental benefits to the world. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided research training and professional development for two postdocs, four graduate student, and four undergraduates. All gained valuable research experience in microbial molecular biology and genetics. The project provides a unique training environment with in-depth exposure to two well-studied model organisms. I meet individually with members of the group at least weekly and I make myself available as needed at other times. These meetings include data analysis, discussion of relevant literature and of ongoing work in our group and in other groups, and design of future experiments. I encourage all group members to present their research in a variety of venues, including weekly group meetings, a "Metabolism, Membranes, and Metalloenzymology Interest Group" that meets twice a month, weekly "summer research presentations" with about 15 groups in the "Gene Expression in Development and Disease Focus Group", and local, national, and international scientific meetings. When appropriate, my group meets to give "practice talks" prior to meetings and we offer constructive criticism to each other. I encourage postdocs, graduate students, and senior undergraduates to mentor less-experienced undergraduates in the lab. Typically, this involves a period of working jointly on experiments before the less-experienced individual transitions to an independent research project with the more-experienced individual continuing as mentor. I engage postdocs, graduate students, and senior undergraduates in other activities that foster their professional development, including preparation of grant and fellowship applications, preparation of abstracts and posters for meeting presentations, preparation of manuscripts from our group, and reviews of manuscripts for journals. I occasionally recommend postdocs to journal editors when I do not have time to review a manuscript. I share my experiences collaborating with computational, structural, and evolutionary biologists both here at MSU and at other institutions. I encourage mentees to build a network of scientific colleagues by introducing them to scientists at MSU and at scientific meetings. I host seminar speakers, and I always treat postdocs and students in my group to dinner with the speaker. I make students and postdocs aware of the abundant resources at MSU for professional development, which include career counseling and several seminar series about teaching. In compliance with MSU and federal regulations, all prospective group members receive safety training prior to working in the lab, and they complete annual refresher training. Also, all group members complete 1 h/yr of training in the responsible conduct of research presented by our chairperson or a senior faculty member. In addition, my group spends at least 2 h/yr discussing issues like 1) data acquisition and management, sharing, and ownership and 2) publication practices and responsible authorship, and peer review. How have the results been disseminated to communities of interest?In addition to three publications, results of the project were disseminated at 10 presentations at scientific meetings. Two outreach educational activities were presented. One was at the Girl Scouts STEM Demo Day on 10/27/16. This event was coordinated by the MSU Women in Engineering organization. Our presentation entitled "Motile Microbes" included demonstration of what microbes are and where we find them, how microbes use flagella to mediate movement in their environment, and provided activities to engage participants and assess understanding. Four groups of 10-15 girl scouts participated for 30 minutes each. The other outreach educational activity was at the Middle School Girls Math and Science Day on 3/4/17, coordinated by the MSU Graduate Women in Science organization. Our presentation entitled "Marvelous Microbes" included demonstration of what microbes are, where we find them, what cellular structures microbes have that are similar and different from humans, and provided activities to engage participants and assess understanding. Four groups of 10-15 middle school girls participated for 30 minutes each. What do you plan to do during the next reporting period to accomplish the goals?Related to the objectives of our B. subtilis work: 1. We will test various aspects of our model of the complex containing SpoIVFB and Pro-sigmaK. 2. We will test the model that ATP induces a conformational change in the CBS domain of SpoIVFB that positions Pro-sigmaK for cleavage. We will use luciferase fusions to measure ATP levels in the mother cell and forespore compartments during sporulation of wild type and mutants defective in forming channels between the two compartments. We will characterize suppressor mutations that restore sporulation of B. subtilis strains expressing SpoIVFB CBS domain variants. We will finish measuring the binding of a fluorescent ATP to wild-type and variant GST-CBS fusion proteins in vitro. 3. Deletions and more substitutions in BofA and SpoIVFA that partially relieve inhibition of SpoIVFB cleavage of Pro-sigmaK in E. coli will be tested for effects on cleavage in B. subtilis. Interactions between the four proteins will be further investigated. Additional disulfide cross-linking experiments will be performed to test our model that a transmembrane segment of BofA is near the SpoIVFB active site and blocks access of Pro-sigmaK. 4. We will investigate why RasP fails to cleave FtsL in vitro. We will try to identify novel substrates of RasP. Related to the objectives of our M. xanthus work: 1. We plan to finish a manuscript describing our systematic approach to the MFD network and our computational and experimental results supporting the model that C-signaling activates FruA, which in turn activates sporulation genes. 2. We will continue pursuing two approaches to identify genes involved in cellular shape change during development, one involving suppressors of a devS mutant that overproduces DevI, an inhibitor of sporulation, and the other involving RNA-seq analysis of wild type and mrpC, fruA, and csgA mutants. 3. We plan to finish a manuscript describing our systematic comparison of the MFD network response after addition of 12.5% versus 25% nutrients at 18 h poststarvation. We will further characterize localization of mNeonGreen-FruA, DNA, and membranes during development using fluorescence microscopy. 4. We plan to characterize the network response to premature accumulation of FruA due to induction of our vanillate-inducible strain during growth and at the onset of starvation. Effects of inducible FruA in mrpC and csgA mutants will also be measured.
Impacts
What was accomplished under these goals?
Related to the objectives of our B. subtilis work: 1. We purified a complex of SpoIVFB and Pro-sigmaK, determined parts of each protein needed to form the complex, and performed chemical cross-linking followed by trypsin digestion and mass spectrometry to identify residues of the two proteins that are in proximity in the complex. Using this data and homology modeling, we have built a model of the complex in collaboration with Michael Feig. A manuscript is under review. 2. We fused luciferase to promoters expressed only in the mother cell or forespore and we are attempting to use these fusions to measure ATP levels in each compartment during sporulation. We used protein sequence alignments to identify substitutions in the SpoIVFB CBS domain that impair cleavage of Pro-sigmaK upon coexpression in E. coli. The same substitutions impair accumulation of SpoIVFB in B. subtilis during sporulation, so spores fail to form. We have selected suppressor mutations that restore sporulation of these strains, which are being characterized. The substitutions also impair ATP binding in vitro, as measured by binding of a fluorescent ATP analog to purified GST-CBS domain fusion protein derivatives. 3. We established a system to observe nearly complete inhibition of SpoIVFB cleavage of Pro-sigmaK by coexpressed BofA and SpoIVFA in E. coli. Numerous deletions and substitutions in BofA and SpoIVFA have been tested in the system. Many partially relieve inhibition. Three substitutions have been shown to relieve inhibition of cleavage in B. subtilis. Disulfide cross-linking data show that a transmembrane segment of BofA is near the SpoIVFB active site, perhaps blocking access of Pro-sigmaK. 4. We discovered that expression of inactive RasP can inhibit expression of other membrane proteins in E. coli. We overcame this problem by adding extra transmembrane segments to RasP. We demonstrated that active RasP cleaves FtsL and RsiW upon coexpression in E. coli. However, the cleavage products are unstable, so we could not map the cleavage site. We purified RasP and found that it cleaved purified RsiW but not purified FtsL. We mapped the cleavage site in RsiW to its transmembrane segment, demonstrating that RasP is an intramembrane protease. The results were published. Related to the objectives of our M. xanthus work: 1. Systematic characterization of a csgA mutant (defective in C-signaling) showed a twofold lower level of FruA protein and a 20-fold lower level of dev transcript compared to wild type during the period leading up to and including the time that cells commit to spore formation. Computational modeling shows that cooperative binding of MrpC and FruA cannot account for the 20-fold decrease in dev transcripts. Neither does instability of dev transcripts in the csgA mutant account for the difference from wild type. Together, the results are consistent with the model that C-signaling activates FruA, which in turn activates dev transcription. A manuscript describing these results is in preparation. Systematic analysis of expression of genes involved in sporulation revealed unexpected complexity, including negative regulation by unactivated FruA. 2. We found that the dev operon is dispensable for sporulation and that DevI acts as a timer to delay sporulation by about 6 h. The results were published. Other genes are known to impact spore maturation, but not the initial cellular shape change that appears to coincide with commitment to spore formation. As described below, MrpC and FruA appear to regulate shape change, but a better understanding of this process is necessary to address irreversibility of the network using modeling or experiments. Therefore, we initiated two approaches to identify genes involved in cellular shape change during development. 3. We measured the network response at the boundaries of ultrasensitivity to nutrient addition to 18-h developing cells. We found that the MrpC protein level correlated best with the sporulation response (i.e., the level decreased after nutrient addition, but recovered to a higher level after 12.5% nutrient addition than after 25% addition). Most of the difference appears to be due to a higher level of mrpC transcript recovery after 12.5% addition. Interestingly, the fruA transcript level did not recover, but the FruA protein level did recover at 27 h PS to a higher level after 12.5% addition than after 25% addition. The results suggest that MrpC and FruA regulate shape change, and that MrpC and FruA are subject to transcriptional and posttranscriptional control, respectively. A manuscript describing these results is in preparation. We created a functional mNeonGreen-FruA fusion and developed methods to visualize the fusion protein, DNA, and membranes of individual cells at the base of developing fruiting bodies using fluorescence microscopy. The results show that mNeonGreen-FruA changes its association with the two copies of the nucleoid present in cells during development. 4. Our efforts to rewire the network governing sporulation centered on characterization of a fruA mutant with a vanillate-inducible promoter fused to fruA integrated ectopically. We discovered that addition of vanillate during exponential growth and at the beginning of development reproducibly resulted in complete rescue of sonication-resistant spore formation at 36 h PS. The FruA level is being measured in samples collected every 6 h until 30 h PS, for comparison with the FruA level in developing wild-type cells. If we can convincingly demonstrate earlier accumulation of FruA in the strain with the vanillate-inducible fusion, we will fully analyze the network response.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Kroos, L. 2017. Highly signal-responsive gene regulatory network governing Myxococcus development. Trends Genet. 33:3-15.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Rajagopalan, R. and L. Kroos 2017. The dev operon regulates the timing of sporulation during Myxococcus xanthus development. J. Bacteriol. 199:e00788-16.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Parrell, D., Zhang, Y., Olenic, S., and L. Kroos 2017. Bacillus subtilis intramembrane protease RasP activity in Escherichia coli and in vitro. J. Bacteriol. 199:e00381-17.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Halder, S., Parrell, D., Whitten, D., Feig, M., and L. Kroos Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-sigmaK. Proc. Natl. Acad. Sci. Plus USA. Under review.
|
Progress 11/01/15 to 09/30/16
Outputs
Target Audience:Our basic research on the mechanisms of signaling and gene regulation in bacteria is intended to provide new knowledge. Application of this knowledge to problems in agriculture, medicine, and industry will result in economic and health benefits to mankind, and environmental benefits to the world. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided research training and professional development for two postdocs, four graduate student, and six undergraduates. All gained valuable research experience in microbial molecular biology and genetics. The project provides a unique training environment with in-depth exposure to two well-studied model organisms. I meet individually with members of the group at least weekly and I make myself available as needed at other times. These meetings include data analysis, discussion of relevant literature and of ongoing work in our group and in other groups, and design of future experiments. I encourage all group members to present their research in a variety of venues, including weekly group meetings, a weekly "Transcription Journal Club" with several other groups, a "Metabolism, Membranes, and Metalloenzymology Interest Group" that meets twice a month, weekly "summer research presentations" with about 15 groups in the "Gene Expression in Development and Disease Focus Group", and local, national, and international scientific meetings. When appropriate, my group meets to give "practice talks" prior to meetings and we offer constructive criticism to each other. I encourage postdocs, graduate students, and senior undergraduates to mentor less-experienced undergraduates in the lab. Typically, this involves a period of working jointly on experiments before the less-experienced individual transitions to an independent research project with the more-experienced individual continuing as mentor. I engage postdocs, graduate students, and senior undergraduates in other activities that foster their professional development, including preparation of grant and fellowship applications, preparation of abstracts and posters for meeting presentations, preparation of manuscripts from our group, and reviews of manuscripts for journals. I occasionally recommend postdocs to journal editors when I do not have time to review a manuscript. I share my experiences collaborating with computational, structural, and evolutionary biologists both here at MSU and at other institutions. I encourage mentees to build a network of scientific colleagues by introducing them to scientists at MSU and at scientific meetings. I host seminar speakers, and I always treat postdocs and students in my group to dinner with the speaker. I make students and postdocs aware of the abundant resources at MSU for professional development, which include career counseling and several seminar series about teaching. In compliance with MSU and federal regulations, all prospective group members receive safety training prior to working in the lab, and they complete annual refresher training. Also, all group members complete 1 h/yr of training in the responsible conduct of research presented by our chairperson or a senior faculty member. In addition, my group spends at least 2 h/yr discussing issues like 1) data acquisition and management, sharing, and ownership and 2) publication practices and responsible authorship, and peer review. How have the results been disseminated to communities of interest?In addition to one publications, results of the project were disseminated at one seminar and at six presentations at scientific meetings. One outreach educational activity was presented at the Girl Scouts STEM Demo Day on 11/7/15. This event was coordinated by the MSU Women in Engineering organization. Our presentation entitled "Magnificent Motile Microbes" included demonstration of what microbes are and where we find them, how microbes use flagella to mediate movement in their environment, and provided activities to engage participants and assess understanding. The effort was co-led by graduate student Daniel Parrell (who works on another project in the lab) and undergraduate Fiona Buchanan, with help from postdoc Ramya Rajagopalan and undergraduate Emily Titus during activities. Four groups of 10-15 girl scouts participated for 30 minutes each. What do you plan to do during the next reporting period to accomplish the goals?Related to the objectives of our B. subtilis work: 1. We will complete the manuscript in preparation that describes our model of SpoIVFB in complex with Pro-sigmaK. Aspects of the model will be tested experimentally. 2. We will create luciferase fusions to measure ATP levels in the mother cell and forespore compartments during sporulation. We will measure binding of a fluorescent ATP to wild-type and variant GST-CBS fusion proteins in vitro. 3. Deletions and substitutions in BofA and SpoIVFA that partially relieve inhibition of SpoIVFB cleavage of Pro-sigmaK will be tested for effects on cleavage in B. subtilis. Interactions between the four proteins will be investigated. 4. We will complete the manuscript in preparation that describes RasP activity on FtsL and RsiW in E. coli and in vitro. We will test the effects of C-terminal deletions in RsiW on cleavage by RasP in E. coli. We will investigate why RasP fails to cleave FtsL in vitro. Related to the objectives of our M. xanthus work: 1. We plan to finish measuring transcripts, proteins, and the numbers of rod-shaped cells and spores in a fruA mutant. We will establish immunoblot assays for CsgA and ExoI. The co-PI and his postdoc will refine the mathematical model of the network. Graduate student Shreya Saha from the PI's lab will travel to the co-PI's lab to collaborate on building the model. A manuscript describing our systematic approach to the MFD network and our model will be prepared. 2. The co-PI and his postdoc will continue to use the model to explore whether the network can operate as a switch that becomes irreversible. The PI's group will test this by measuring network dynamics after addition of nutrients to developing cells. This is a natural extension of graduate student Y Hoang's current work on specific objective 3, which involves adding different amounts of nutrients at 18 h poststarvation. For this objective, the experiments will likely involve adding a high level of nutrients at 24 versus 30 h poststarvation (since most cells commit to spore formation during this period), and systematically comparing the response of the MFD network. 3. We will systematically compare the response of the MFD network after addition of 12.5% versus 25% nutrients at 18 h poststarvation. Graduate student Shreya Saha plans to make C-signal (i.e., CsgA capable of rescuing development of a csgA mutant) using a construct and protocol provided by Larry Shimkets (University of Georgia), then add different amounts of it to developing cells to test for an ultrasensitive response. 4. We plan to measure the MrpC level in the vanillate-inducible strain during the first six hours after vanillate addition at the onset of starvation. Depending on the outcome, we may rebuild the fusion to achieve higher expression. We will systematically measure the response of the MFD network to fruA induction at the onset of starvation.
Impacts
What was accomplished under these goals?
Related to the objectives of our B. subtilis work: 1. We purified a complex of SpoIVFB and Pro-sigmaK, determined parts of each protein needed to form the complex, and characterized the subunit composition of the complex. This work was published in the J. Biol. Chem. We further determined parts of SpoIVFB needed to form the complex and we performed chemical cross-linking followed by trypsin digestion and mass spectrometry to identify residues of the two proteins that are in proximity in the complex. Using this data and homology modeling, we have built a model of the complex in collaboration with Michael Feig. A manuscript is in preparation. 2. Our efforts to build a FRET-based ATP sensor were unsuccessful, so we are pursuing luciferase fusions to measure ATP levels in vivo. We fused the SpoIVFB CBS domain to GST and established an assay to measure binding of a fluorescent ATP analog to the CBS domain. We used protein sequence alignments to identify substitutions in the CBS domain important for cleavage of Pro-sigmaK. The same substitutions will be tested for ATP binding in vitro. 3. We established a system to observe nearly complete inhibition of SpoIVFB cleavage of Pro-sigmaK by coexpressed BofA and SpoIVFA in E. coli. Numerous deletions and substitutions in BofA and SpoIVFA have been tested in the system. Many partially relieve inhibition. Their effects on cleavage in B. subtilis will be tested next. 4. We discovered that expression of inactive RasP can inhibit expression of other membrane proteins in E. coli. We overcame this problem by adding extra transmembrane segments to RasP. We demonstrated that active RasP cleaves FtsL and RsiW upon coexpression in E. coli. However, the cleavage products are unstable, so we could not map the cleavage site. We purified RasP and found that it cleaved purified RsiW but not purified FtsL. We mapped the cleavage site in RsiW to its transmembrane segment, demonstrating that RasP is an intramembrane protease. A manuscript is in preparation. Related to the objectives of our M. xanthus work: 1. We established methods to measure dynamical changes in the MrpC, FruA, Dev (MFD) network components and output before and during commitment to sporulation. We measured six transcripts, two proteins, and the numbers of rods and spores for wild type and three mutants. Our collaborators Oleg Igoshin and Pintu Patra have used the data to build initial mathematical models of the MFD network. 2. We discovered a positive feedback loop from DevI to MrpC in the MFD network. This feedback loop can allow the network to operate as an irreversible switch based on modeling results. To follow-up on these findings, network dynamics after addition of nutrients to developing cells will be measured. 3. We found that the MFD network exhibits an ultrasensitive response to the addition of nutrients to developing cells. The network response at the boundaries of ultrasensitivity will be measured. 4. We found that copper inhibits M. xanthus development, so we could not use the copper-inducible system. Therefore, we built fusions of mrpC and fruA to a vanillate-inducible promoter. Induction of mrpC at the onset of starvation accelerates aggregation but spores fail to form, perhaps because the level of MrpC is lower than normal. Similarly induced fruA appears to have little impact on aggregation. Sporulation will be measured. FruA accumulates earlier than normal and perhaps to a slightly higher level than normal. Network dynamics after mrpC or fruA induction will be measured more carefully.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Zhang, Y., S. Halder, R.A. Kerr, D. Parrell, B. Ruotolo, and L. Kroos 2016. Complex formed between intramembrane metalloprotease SpoIVFB and its substrate, Pro-sigmaK. J. Biol. Chem. 291:10347-10362.
- Type:
Other
Status:
Under Review
Year Published:
2016
Citation:
Kroos, L. Highly signal-responsive gene regulatory network governing Myxococcus development. Trends in Genet.
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