Progress 10/01/99 to 09/30/10
Outputs
OUTPUTS: ACTIVITIES - Created tools to be used in a new line of investigations for the lab. Our objective is to determine if the machinery used to transport plasmid ColIb-P9 during conjugation can be used to deliver toxic peptides and RNA. We obtained needed strains and examined their project-related phenotypes. Using PCR amplification and other cloning techniques we generates a sets of plasmids and examined their properties. We also continued our research aimed at identifying small compounds that lead to toxic levels of plasmid DNA replication. In addition, we recently began characterizing a unique resource at the University, the Raper Archive of soil-dwelling amoebae. The knowledge gained by these activities was disseminated in the following ways: 1) Networking by the PI while attending scientific a conference in New York; 2) The presentation of a poster at a conference in Argentina; 3) The presentation of a poster and talks at two Department of Bacteriology symposia. This project also contributed to the training/mentoring of two undergraduate students and five graduate students in the technical and analytical aspects of research science. PRODUCTS - Freezer stocks of host bacteria with confirmed phenotypes. Recombinant plasmid DNA with confirmed phenotypes. Laboratory procedures for reviving and characterizing an archive of soil amoebae. Preliminary data to establish a relationship between fluorescent output of biosensor plasmids and biosensor plasmid copy number. PARTICIPANTS: INDIVIDUALS - Principal Investigator: Marcin S. Filutowicz; Scientist: Peter J. McNamara; Senior Research Specialist: Sheryl A. Rakowski. PARTNER ORGANIZATIONS - National Institutes of Health, Department of Defense/Army, University of Wisconsin-Madison (Department of Bacteriology), Wisconsin Alumni Research Foundation. COLLABORATORS - N/A. TRAINING or PROFESSIONAL DEVELOPMENT - PhD Student: Abishek Muralimohan; Masters Student: Katarzyna Boryz; Masters Student: Leah Cronan; Masters Student: Musatif Hasan; Masters Student: Karolina Stojowska ; Undergraduate Student: Yue Hin Leung; Undergraduate Student: Nathaniel Shekem. TARGET AUDIENCES: 1) Students (lab members) - Provided laboratory training & experience, scientific communications training and experience, and procedural and analytical mentoring for seven students (see Participants). 2) Scientific Community - Shared insights into plasmid biology and antimicrobial development research in our lab through attendance and participation (posters and talks) in foreign and domestic conferences and symposia. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
CONJUGATION-BASED STUDIES - Small toxic peptides and sRNAs have enormous antimicrobial potential but this potential has been largely overlooked due to problems with extracellular stability and efficient uptake. We plan to determine if plasmid conjugation machinery can be used to deliver such unconventional antimicrobial agents. Our laboratory strains collection already contained one strain of E. coli we thought would be useful for these studies. However, we discovered it performed poorly in some experiments. This prompted us to obtain an alternate strain that performed better. We also acquired several strains of E. coli containing plasmid ColIb-P9, which encodes an enzyme that gets transferred from donor cells to recipients during conjugation. We examined the project-relevant phenotypes of the various strains and determined they were suitable for the research project. We began construction of plasmids with the properties needed for our project. The features of particular interest included the transferable enzyme, Sog, and sequences that allow plasmids to be mobilized during conjugation. The sog gene produces two variant enzymes, the longer SogL protein and a shorter SogS. We therefore expanded our cloning pursuit and used PCR amplification to generate constructs with the long sog and, additionally, constructs with the short sog. Plasmids that, for brevity's sake, we will refer to as SogL constructs contain the entire sog coding region and produce both forms of Sog. The ability of the constructs to express sogL was determined by transformation into a strain with a chromosomal mutant enzyme and identifying phenotypic rescue y the plasmid construct. The expression of short sog constructs was not examined. Further cloning experiments put sogL under control of an inducible lac promoter. In addition to the plasmid cloning work, we undertook a P1 transduction strategy to move the conjugation machinery into a strain we had previously engineered to have properties that are desirable for this project. Our attempts were unsuccessful, causing us to reevaluate the strain construction work. PLASMID OVERREPLICATION STUDIES - A new member of the lab has begun work on this project as part of her training for the Masters degree program. Techniques adopted as part of the high-throughput screening portion of the project are being modified and expanded upon in preparation for the evaluation of lead compounds. RAPER ARCHIVE STUDIES - Soil microorganisms have proven to be a rich source of bioactive compounds called polyketides. Recent work in the field of genomics indicates that soil dwelling amoebae may constitute the largest repository of polyketide synthase enzymes. The Raper Archive is a diverse collection of soil amoebae representing seventeen different species, and over a thousand unique isolates. This University resource has been sitting idle for many years. We have initiated a project to revive and characterize the amoebae in the archive. Procedures are being evaluated to revitalize, with minimal losses, stocks that have been stored as spores for over 4 decades. Additionally, we are designing a strategy for documenting archive-relevant data and materials.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: ACTIVITIES - Continued plasmid replication studies and efforts to develop protocols and resources for discovering novel, plasmid-based antibiotics. The knowledge gained by these activities was disseminated in the following ways: 1). The publication of one news article (Miller, N. 2009. The Drugs Start Here; In Grow Magazine; University of Wisconsin, Madison) and one review article (below), and 2) The production of laboratory recruitment materials (documents and a Powerpoint presentation). This project also contributed to the training/mentoring of three undergraduate students and five Masters students in the technical and analytical aspects of conducting scientific research. PRODUCTS - Generated tools, e.g. plasmid biosensors, from Gram-negative and Gram-positive bacteria to begin high-throughput screening for novel antibiotics. Conducted high throughput screens of a large commercial collection of small compounds (the Maybridge HitFinderTM library), which resulted in the identification of several preliminary candidates for antibiotic development. PARTICIPANTS: INDIVIDUALS - Principal Investigator: Marcin S. Filutowicz; Scientist: Peter J. McNamara; Senior Research Specialist: Sheryl A. Rakowski. PARTNER ORGANIZATIONS - National Institutes of Health, United States Army Research Office, University of Wisconsin-Madison (Department of Bacteriology), Wisconsin Alumni Research Foundation. COLLABORATORS - Dr. Jo Handelsman - University of Wisconsin-Madison. TRAINING or PROFESSIONAL DEVELOPMENT - Masters Student: Carly Campbell; Masters Student: Leah Cronan; Masters Student: David Danforth; Masters Student: Theresa Drake; Masters Student: Phoua Yang; Undergraduate Student: Brian Cole; Undergraduate Student: Yue Hin Leung; Undergraduate Student: Nathaniel Shekem. TARGET AUDIENCES: 1) Students (lab members) - Provided laboratory training & experience, scientific communications training and experience, and procedural and analytical mentoring for the eight students (see Participants). 2) Scientific Community - Shared insights into plasmid biology through published literature (see Outputs and Publications). 3) General Public (University of Wisconsin, CALS, students, alumni & staff) - Shared insights into antibiotics discovery research through published literature (see Outputs). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our research on plasmid-based antimicrobials relies on biosensors, which are specialized plasmids that signal changes that occur inside bacteria. These changes may influence the viability of the cells. Research was conducted to construct biosensors from Gram-positive bacteria and to study previously constructed biosensors from Gram-negative bacteria. GRAM-POSITIVE STUDIES - We began biosensor construction (in Escherichia coli) by cloning modules that harbor: 1) plasmid replication regions (from Bacillus plasmids), 2) a green fluorescent protein (gfp) gene variant, and 3) an E. coli general purpose phagemid. This methodology let us isolate more DNA for than could be directly obtained from Bacillus. Additionally, we adopted a gene expression system that can be used to manipulate the intracellular level of GFP within bacilli. One biosensor was constructed using a phagemid with a tetracycline resistance marker, however, the biosensor could not be used because KG1030 proved to be intrinsically resistant to tetracycline. A second biosensor with chloramphenicol resistance has been constructed. Biosensor-containing E. coli fluoresced brightly but initial attempts to transform the biosensors into B. subtilis were unsuccessful. This outcome prompted us to attempt transformation using other bacilli. This approach yielded limited success. We suspect the biosensors may not be stably maintained in these strains in the absence of selective pressure and are in the process of evaluating what impact this may have on planned high-throughput screening experiments. GRAM-NEGATIVE STUDIES - We conducted experiments to improve and characterize previously constructed biosensors from Gram-negative bacteria. Additionally, we were able to advance our studies to the point of using one of these biosensors for high-throughput screening for bioactive compounds. At the end of 2008 we switched our screening focus and began developing an assay for increases in GFP signal. In early 2009 we were able to use genetic manipulation to generate a suitable positive control for the screening assay. We then examined 14,400 HitFinderTM library compounds for their effects on fluorescence. To conduct the assay, liquid cultures of bacteria containing the biosensor were diluted then: 1) added to wells microtiter plates containing micromolar amounts of the compounds, 2) grown for several hours to allow the compounds to exert their effects, and 3) robotically processed for fluorescence and optical density (OD) measurements. Compounds that increased the fluorescence/OD ratio by 3 or more standard deviations above the background average were considered tentative hits. Promising compounds (92) were selected for a secondary "cherry-picking" screen. Repeat assays identified 8 compounds with a Relative Fluorescence value that was at least 3 times higher than cells not exposed to any compounds. Because GFP expression is an indirect indication of possible antibiotic activity, follow-up studies on "hit compounds" will be required to confirm their effect on the growth of plasmid-bearing bacteria.
Publications
- Filutowicz, M. 2009. Plasmids, Bacterial. In M. Schaechter (ed.), Encyclopedia of Microbiology. Academic Press, Oxford. p. 644-665.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: ACTIVITIES - Analyzed experiments on plasmid R6K replication to elucidate the role of cooperative nucleoprotein binding by a dimeric regulatory protein. Continued work to develop protocols and resources for discovering novel, plasmid-based antibiotics via high-throughput screening. The knowledge gained by these activities was disseminated in the following ways: 1) A poster presentation (Undergraduate Symposium 2008, University of Wisconsin - Madison), 2) Two PowerPoint presentations (International Plasmid Biology Conference 2008, Gdansk, Poland); 3) The publication of one research article and one review article, and 4) The production of laboratory recruitment materials. Trained/mentored three undergraduate students and two Masters students in the technical and analytical aspects of research science. PRODUCTS - Generated tools, e.g. plasmid biosensors, to begin high-throughput screening for novel antibiotics. Co-developed a research project for an undergraduate laboratory course at the University of Wisconsin - Madison (Microbiology 304). In conjunction with the Micro 304 class, generated a collection of Streptomyces via low-throughput screening that secrete compounds with interesting effects on bacterial growth and biosensor-encoded phenotypes. These compounds will be investigated for their potential to be developed into antimicrobial agents. PARTICIPANTS: INDIVIDUALS - Principal Investigator: Marcin Filutowicz; Senior Research Specialist: Sheryl A. Rakowski. PARTNER ORGANIZATIONS - University of Wisconsin-Madison (Department of Bacteriology), Wisconsin Alumni Research Foundation, US Army Research Office. COLLABORATORS - Dr. Jo Handelsman - University of Wisconsin-Madison. TRAINING or PROFESSIONAL DEVELOPMENT - PhD Student: Lisa Bowers; Masters Student: David Danforth; Masters Student: Leah Cronan; Undergraduate Student: Brian Cole; Undergraduate Student: Libby Rens; Undergraduate Student: Nathaniel Shekem. TARGET AUDIENCES: 1) Students (lab members) - Provided laboratory training & experience, scientific communications training and experience, and procedural and analytical mentoring for the six students listed above 2) Students (Micro 304) - Enhanced the curriculum of an undergraduate laboratory course in Microbiology at the University of Wisconsin - Madison (approximately 150 students). 3) Scientific Community - Shared insights into plasmid biology through published literature and scientific talks. 4) Biotechnology Community - Generated foundation for new technologies including a preliminary patent application, which spurred the creation of a new Biotech start-up, PlasmiGon, LLC. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Research reported last cycle demonstrated that cooperative binding by monomers of the replication protein pi helps fill iteron DNA sequences, a prerequisite for the replication of plasmid R6K. Although data suggested cooperativity by pi dimers might be negligible, that work was preliminary. In 2008, we published the results of our work examining the cooperative binding of pi dimers to iterons (Bowers & Filutowicz). Our results indicated that, although pi dimers do not bind cooperatively to adjacent iterons, they do bind cooperatively to alternate iterons. This characteristic of dimer binding interferes with saturating the iterons with replication protein. Thus, cooperativity not only enhances the initiator function of pi monomers, it likely enhances the inhibitor function of pi dimers as well. 2) The accumulation and spread of antibiotic resistance among pathogenic bacteria is a serious problem on its way to becoming a global crisis. Plasmid DNA is a major contributor to the problem because they can encode antibiotic resistance and can be spread by conjugation to plasmid-free bacteria. Our laboratory's basic research on plasmid biology prompted us to ask whether science can turn the tables and use plasmids against pathogenic bacteria. In Filutowicz et al., 2008, we described how one aspect of plasmid biology, the process of conjugation, might be co-opted to deliver toxins to bacterial pathogens and pests. We hope the review article will stimulate creative initiatives in plasmid biology research that address the antibiotic resistance problem. 3) Informal communication of our lab's work led to a collaborative research opportunity incorporated into the Microbiology curriculum of the University of Wisconsin - Madison. Using tools generated in our lab for a different but related purpose (described below), undergraduates were able to practice microbial isolation and culturing techniques as part of a low throughput screen for novel antibiotics. In this first year of collaboration, a group of 150 Microbiology majors was asked to collect soil samples from different locations. Streptomyces, a species renowned for producing valuable metabolites, were separated from other bacteria and characterized for their ability to alter the phenotypes of E. coli harboring a biosensor plasmid. Roughly 10% of the isolates yielded promising results in this preliminary screen. Students have been invited to conduct independent study projects (Microbiology 699) to purify and characterize the active compounds. 4) High-throughput screening for plasmid-based antimicrobial agents is in the process of being optimized. We identified stability issues with some of our biosensor constructs and different methods to enhance stability. We tested several compounds expected to elicit the desired response of our biosensor and confirmed the functionality of our biosensors. In trial runs under high-throughput conditions, however, our most advanced biosensor displays only modest sensitivity to the test compound. We are testing various parameters to improve sensitivity before initiating a full library screen.
Publications
- Bowers, L.M. and Filutowicz, M. 2008. Cooperative binding mode of the inhibitors of R6K replication, pi dimers. Journal of Molecular Biology, 377(3):609-615.
- Filutowicz, M., Burgess, R., Gamelli, R.L., Heinemann, J.A., Kurenbach, B., Rakowski, S.A. and Shankar, R. 2008. Bacterial conjugation-based antimicrobial agents. Plasmid, 60(1):38-44.
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Progress 01/01/07 to 12/31/07
Outputs
(N/A)
Impacts
(N/A)
Publications
- Bowers, L.M., Kruger, R., and Filutowicz, M. (2007) Mechanism of origin activation by monomers of R6K-encoded pi protein. Journal of Molecular Biology, 368(4):928-938.
- Kunnimalaiyaan, S., Rakowski, S.A., and Filutowicz, M. (2007) Structure-based functional analysis of the replication protein of plasmid R6K: Key amino acids at the pi/DNA Interface. Journal of Bacteriology, 189(13):4953-4956.
- Shankar, R., He, L.K., Szilagyi, A., Muthu, K., Gamelli, R.L., Filutowicz, M., Wendt, J.L., Suzuki, H., and Dominguez, M. (2007) A novel antibacterial gene transfer treatment for multidrug-resistant Acinetobacter baumannii-induced burn sepsis. Journal of Burn Care Research, 28(1):6-12.
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Progress 01/01/06 to 12/31/06
Outputs
One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called 'iterons'. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control, established by our laboratory. Dimers, the predominant form of pi in the cell, inhibit replication while monomers facilitate open complex formation and activate the ori. In the past reporting period we attempted to answer the following question: What mechanism or mechanisms allow pi monomers to out-compete dimers for iteron binding? A partial answer to this question was provided by (Kunnimalaiyaan et al., 2004, J. Biol. Chem.:279) in a series of surprising data which demonstrated that pi monomers contact a larger segment of iteron DNA than pi dimers. We
set up an in vivo plasmid incompatibility (pi titration) assay and found that pi monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but only slightly diminished by insertion of a full helical turn between two iterons. These studies also show that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. Pi monomer/iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. Pi dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem iterons.
Impacts
The NIH outlines its goals in funding extramural research, which are 'to advance our understanding of biological systems, improve the control of disease, and enhance health.' Our studies of the replication of a transmissible antibiotic-resistance plasmid have met the first of these goals and are ripe to produce technologies that address the mission of the NIH in its entirety. Bacteria cause a multitude of diseases in humans, plants and animals, the social and economic impacts of which can be devastating. Importantly, the fitnesses of clinically-isolated bacterial strains are frequently enhanced by the plasmids they carry. Moreover, many plasmids carry pathogenicity-related genes. If scientists and health practitioners had a greater capacity to manipulate the replication and transmission rates of plasmids, particularly outside the confines of the research laboratory, the benefits to human health would be immeasurable. The research being conducted in our laboratory is
uniquely positioned to be the launching pad for multiple and perhaps powerful health-related 'discoveries'. However, much still remains to be understood about the fundamental biochemical mechanisms controlling plasmid replication and transmission. Knowledge is power. In this case it could very likely bring with it the power to manipulate microbial landscapes and even plasmid ecology in ways to benefit human health and welfare.
Publications
- Peng, Y., Rakowski, S.A. and Filutowicz, M. 2006. Small deletionvariants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity. FEMS Microbiol. Lett. 261:245-252.
- Bowers, L.M., Kruger, R. and Filutowicz, M. 2007. Mechanism of origin activation by monomers of R6K-encoded pi protein. J. Mol. Biol. [in press]
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Progress 01/01/05 to 12/31/05
Outputs
Our model system for DNA replication studies is derived from plasmid R6K. Its 2 essential components are gamma origin (g-ori) and its cognate replication initiator (Rep) protein pi (encoded by pir.) Our focus is the multiple functions of pi and their underlying biochemical mechanisms Over the last period, our research has revealed the following: (1) Disruptions of cell growth can result from loss (presumptive) of pi's negative regulatory function. We observed that deletions of amino acids (aa's) affected by certain copy-up mutations retain copy-up phenotypes (Y Peng and M Filutowicz, unpublished data). This extended evidence that such mutations are due, primarily, to loss of pi's repressor function. Introducing several copy-up pir mutations in cis to g-ori appears to produce growth-inhibiting levels of plasmid replication. Supporting this analysis, an 'inhibitor-only' pi variant rescued cells containing the copy-up plasmids. Furthermore, different bacterial species
exhibited different levels of tolerance for the copy-up plasmids (Wild et al., 2004 Annals Microbiol., and unpublished data). Thus, in addition to providing insights into copy-up Rep, this work has practical ramifications regarding the ability to modify popular vectors for use in bacterial species beyond Escherichia coli. (2) In R6K, pi dimers bound to iterons (pi binding sites in g-ori) appear better able to couple DNA fragments than pi monomers (Kunnimalaiyaan. et al., 2005 J. Bacteriol.) Handcuffed structures produced by DNA pairing are thought to inhibit replication in Rep/iteron plasmids. Our experiments indicated that copy-up pi variants exhibiting a monomer-bias in binding DNA were less efficient than wt in handcuffing. This finding lets us make connections between pi dimers, replication inhibition and handcuffing that are simpler than the models invoked for other Rep proteins. Although pi was long believed to be the only Rep to bind iterons as a dimer, evidence is emerging
that other Reps can also bind iterons (weakly) in dimeric form. We expect that our handcuffing model may be the forerunner of simplified handcuffing models for other Reps. If not, our findings will have implications for analyzing the evolutionary divergence of the Rep/iteron plasmid family. (3) Our theoretical 3-dimensional structure of an iteron-bound pi monomer has predictive value in assigning the functional roles of aa's. We have used our recently published structural model (Kunnimalaiyaan et al., 2004 J. Biol. Chem.) to identify candidate aa's for structure/function relationships of pi. Initial experiments have focused on putative DNA contacting aa's and targeted mutagenesis has yielded several promising mutants. Preliminary data are highly encouraging; 5 of 6 pi variants show marked alterations in their DNA binding patterns. Moreover, the single variant exhibiting little to no alteration falls in an unstructured loop domain and thus was considered something of a 'wildcard' at
the outset. These experiments suggest that the 3D model will be a powerful tool in generating and testing hypotheses regarding the structure/function relationships of pi protein at the resolution of individual amino acids.
Impacts
The NIH outlines its goals in funding extramural research, which are 'to advance our understanding of biological systems, improve the control of disease, and enhance health.' Our studies of the replication of a transmissible antibiotic-resistance plasmid have met the first of these goals and are ripe to produce technologies that address the mission of the NIH in its entirety. Bacteria cause a multitude of diseases in humans, plants and animals, the social and economic impacts of which can be devastating. Importantly, the fitnesses of clinically-isolated bacterial strains are frequently enhanced by the plasmids they carry. Moreover, many plasmids carry pathogenicity-related genes. If scientists and health practitioners had a greater capacity to manipulate the replication and transmission rates of plasmids, particularly outside the confines of the research laboratory, the benefits to human health would be immeasurable. The research being conducted in our laboratory is
uniquely positioned to be the launching pad for multiple and perhaps powerful health-related 'discoveries'. However, much still remains to be understood about the fundamental biochemical mechanisms controlling plasmid replication and transmission. Knowledge is power. In this case it could very likely bring with it the power to manipulate microbial landscapes and even plasmid ecology in ways to benefit human health and welfare.
Publications
- Kunnimalaiyaan, S., Inman, R.B., Rakowski, S.A. and Filutowicz, M. 2005. Role of pi dimers in coupling ('handcuffing') of plasmid R6K's gamma ori iterons. J. Bacteriol. 187:3779-3785.
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Progress 01/01/04 to 12/31/04
Outputs
***** R6K is a self-transmissible antibiotic resistance plasmid that encodes the DNA-binding protein pi, a product of the pir gene. Pi protein plays multiple roles in plasmid replication and exhibits structural plasticity in its interactions with DNA target sites - the iterons and a non-iteron binding site. The latter target is located near the start sites for leading strand synthesis and we hypothesized that pi might negatively modulate priming by binding to this site. If true, copy-up mutants that stimulate replication more than wt pi might do so as a consequence of decreased binding to the non-iteron site. Indeed, we found that copy-up mutations weakened pi binding as would be expected for a dimer-dependent interaction. Furthermore, we showed that pi uses a single subunit of the dimer to contact the non-iteron DNA. ***** Although R6K is widely held to be a narrow host range plasmid, anecdotal evidence suggested that this might not be entirely accurate. Moreover,
single amino acid (aa) substitutions in initiator protein can alter/expand the plasmid host-range. Thus, we undertook experiments to explore the host range of R6K derivatives. Our findings indicate that the gamma ori plasmids can replicate in a variety of enteric bacteria as well as in some non-enteric species, and a copy-up mutation of pi protein can extend the plasmid host-range. Furthermore, in many genera gamma ori plasmids were able to achieve high copy numbers in contrast to the the low copy numbers of a minimal RK2 plasmid. ***** Studies on a select few (non-R6K) plasmids suggest that these replicons do not diffuse randomly in the cytoplasm. We initiated similar microscopy pilot experiments for R6K-derived gamma ori replicons containing binding site arrays for green fluorescence tagged proteins. Initial results showed that most cells manifest fluorescent foci at the cell poles. Additionally, over the course of 1.5 hours, some foci appeared to oscillate between the poles. *****
We recently published a homology-based 3D structural model of pi monomer bound to a single iteron. After examining our model for aa's likely to contact iteron nucleotides, 6 candidates were selected for further study: Ser71, Tyr74, Gly131, Gly211, Arg225 and Arg254. We have successfully conducted site-directed mutagenesis to change these aa's into alanines in preparation for contact probing experiments. Additionally, we have confirmed the inducible expression of pi protein from each mutant construct. To quickly survey the phenotypes of the mutants, we transformed the constructs into E. coli cells then attempted to establish the gamma ori suicide plasmid pFW25 into the pir mutant isolates. In preliminary data, pFW25 could be established in cells producing wt pi as well as cells producing pi*G211A whereas the other 5 targeted alanine mutants were unable to support gamma ori replication. The data are consistent with our hypotheses regarding the importance of at least 5 of 6 aa's that
were proposed for further study. This, in turn, substantiates our ability to successfully use the theoretical 3D structural model of pi protein to target key aa's for investigations of pi structure/function relationships.
Impacts
Plasmids have adopted numerous strategies to ensure their maintenance and spread, none of them more basic than the control of replication, and yet replication control remains poorly understood. Certainly the central importance of plasmids to the evolution of genomes is ample justification for studying plasmid replication. Beyond this, our studies past and ongoing, provide insight into the mechanisms of protein-protein and protein-DNA interactions, insight that may be harder to obtain when studying other systems.
Publications
- Bowers LM, LaPoint K, Anthony L, Pluciennik A, Filutowicz M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 2004; 340:11-18.
- Wild J, Czyz A, Rakowski SA, Filutowicz M. Gamma origin plasmids of R6K lineage replicate in diverse genera of Gram-negative bacteria. Annals Microbiol. 2004; 54:471-480.
- Kruger R, Rakowski SA, Filutowicz M. Participating elements in the replication of iteron-containing plasmids In: Plasmid Biology (eds. Funnell B, Phillips G.) ASM Press, Washington D. C. 2004; pp 25-45.
- Kunnimalaiyaan S, Kruger R, Ross W, Rakowski SA, Filutowicz M. Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K. J. Biol. Chem. 2004; 279(39):41058-41066.
- Kruger R, Rakowski SA, Filutowicz M. Isomerization and apparent DNA bending by pi, the replication protein of plasmid R6K. Biochem. Biophys. Res. Commun. 2004; 23:834-840.
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Progress 01/01/03 to 12/31/03
Outputs
One of our long-term goals is to elucidate how the pi protein of plasmid R6K exerts opposing functions in replication. Pi's activities rely, in part, on its binding seven 22-bp sequences called direct repeats (DRs). A role for two base pairs in pi/DR binding had been established by others, but comprehensive data were lacking. In particular, the relative importance of DR bases was not known with regard to binding the 2 forms of pi protein, monomers (activators) and dimers (inhibitors). To address this, we conducted pi/DR footprinting experiments using DMS-protection, methylation- and missing-base- interference, in conjunction with EMSA. The results showed that the DR binding specificities of monomers and dimers differ but their binding sites partially coincide. In complementary in vitro work, we utilized DR-mediated 'incompatibility' to isolate DR mutants that were pi-binding deficient. Two important classes of mutants were identified; one affects the binding of both
pi monomers and dimers while the other affects the binding of monomers, alone. Additional evidence for the different DR-binding interactions of pi monomers and dimers was generated by ligation enhancement experiments used to assess whether a protein can simultaneously bind 2 DNA sequences. Ligation occurred more efficiently with His-pi-wt than with a variant that is richer in pi monomers, suggesting that the complexes formed by pi dimers bound to DRs may form handcuffs more efficiently than monomer-bound complexes. We note that there was an increase in monomeric circles in reactions containing ligase and pi when compared to ligase-only reactions. This was likely the result of pi's known ability to bend DR-containing DNA, a property that we have recently explored in greater depth. As part of this work, the ability of pi to bend DRs was reexamined using techniques that discriminate between bending induced by its different isomers. We found that monomers and dimers bend a single DR to
similar degrees. Ongoing experiments looking at the cooperative binding of pi monomers and dimers seem poised to help explain how dimers can bend gamma origin (g-ori) DNA similarly to monomers yet not be active replication initiators. Additional experiments designed to provide insight on how pi functions as an initiator expanded upon earlier work by this lab (focused on open complex formation at g-ori). We examined the effects of ATP, Mg2+ and temperature on the DNA opening reaction by probing pi/g-ori complexes with KMnO4 as a means to identify DNA distortions. A hyperactive variant of pi was used which does not require host proteins (IHF/HU or DnaA) for open complex formation. Our data suggest that ATP hydrolysis may be unnecessary for open complex formation at g-ori. However, in the absence of ATP or Mg2+, DNA bending, rather than melting, appeared to occur. These and other results suggest, in part, that ATP probably effects an allosteric change in pi protein bound to g-ori.
Finally, as a matter of practical concern, we examined the effects of His-tagging on pi protein and determined that tagging does not change the replication (and other) properties of the pi variants commonly used in our work.
Impacts
Plasmids have adopted numerous strategies to ensure their maintenance and spread, none of them more basic than the control of replication, and yet replication control remains poorly understood. Certainly the central importance of plasmids to the evolution of genomes is ample justification for studying plasmid replication. Beyond this, our studies past and ongoing, provide insight into the mechanisms of protein-protein and protein-DNA interactions, insight that may be harder to obtain when studying other systems.
Publications
- Forest KT, Filutowicz MS. 2003. Remodeling of replication initiator proteins. Nat. Struct. Biol.; 10:496-498.
- Kruger R, Filutowicz M. 2003. Characterization of His-tagged, R6K-encoded pi protein variants. Plasmid; 50: 80-85.
- Kruger R, Filutowicz M. 2003. Pi protein- and ATP-dependent transitions from 'closed' to 'open' complexes at the gamma ori of plasmid R6K. Nucleic Acids Res.; 31: 5993-6003.
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Progress 01/01/02 to 12/31/02
Outputs
Recognition of the replication origin, ori, by a replication initiator protein, Rep protein, is the central event governing DNA synthesis in diverse biological systems. Our goal is to understand how Rep proteins control the frequency of the initiation of DNA replication. We investigate the mechanisms responsible for the complex regulation of an iteron-containing plasmid that can replicate in many species of Gram-negative bacteria. Our gammaori/pi system is derived from the antibiotic-resistance plasmid, R6K. pi is the Rep protein for R6K and it is encoded by the pir gene of the plasmid. Through oligomerization and DNA binding, pi acts as a multifaceted regulatory protein with different functions in replication control as well as pir transcription. The oligomerization of pi protein in conjunction with binding to seven, tandem 22-base-pair iterons in gamma ori leads to open complex, melted DNA, formation that is a prerequisite for replication initiation. This
interaction involves seven pi monomers that exhibit positive cooperativity when binding to the DNA. In contrast, the binding of three pi dimers in a negatively cooperative fashion leads to an inactive, replication inhibitory, complex. Only one pi dimer binds to the operator of the pir gene to repress its expression. Yet another category of nucleoprotein assembly in this system involves pi dimers bound to a non-iteron DNA binding site within the A+T-rich segment of gamma ori. The non-iteron binding site clearly contains a different nucleotide sequence and occurs three helical turns upstream of the DNA synthesis start sites mapped in vitro. Thus it is evident that pi exhibits remarkable structural and functional flexibility in complexes with DNA. We found that pir mRNA is translated into two polypeptides of different molecular masses. The short form, pi30, and the more abundant long form, pi35, are functionally distinct, acting as an inhibitor and an activator of replication,
respectively. Key to the success of our past and future work are pir mutants which alter the regulatory properties of these two forms of pi protein. We have, in our possession, powerful sets of pi variants that are hyperactive or dominant-negative in their replication phenotypes. Together, our studies are rapidly advancing our understanding of the regulatory sites and forms of pi protein bound to these sites. By investigating the relationships between the ATP-independent binding of pi to the iterons, closed complex, and the ATP-dependent remodeling of a closed to an open complex, we established the groundwork for studies of the priming mechanism of DNA synthesis.
Impacts
Plasmids have evolved numerous strategies to ensure their maintenance and spread, none of them more basic than the control of replication, and yet the mechanisms of copy number control remain poorly understood. Certainly the central importance of plasmids to the evolution of genomes is ample justification for studying plasmid replication. Beyond this, dissecting the details of replication initiation will provide valuable information about the mechanisms of protein-DNA and protein-protein interactions, information that may be less tractable when studying other systems.
Publications
- Kruger R, Filutowicz M. 2000 Dimers of pi protein bind the A+T-rich region of the R6K gamma origin near the leading strand start sites: Regulatory implications. J. Bacteriol.; 182:2461-2467.
- Kruger R, Konieczny, Filutowicz M. 2001 Monomer/dimer ratios of replication protein modulate the DNA strand-opening in a replication origin. J. Mol. Biol. 306:945-955.
- Kruger R, Konieczny I, Filutowicz M. 2001 Control of DNA strand opening at the gamma origin of the plasmid, R6K. Plasmid 45:135-137.
- Pluciennik A, Iyer RR, Napierala M, Larson JE, Filutowicz M, Wells RD.2002 Long CTG*CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination. J. Biol. Chem. 277:34074-34086.
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Progress 01/01/01 to 12/31/01
Outputs
Plasmid R6K encoded pi protein activates gamma origin of replication, represses transcription of its own gene, pir, and inhibits replication. These activities are achieved, in part, via interactions of pi with distinct, DNA targets, the Direct and Inverted Repeats (DR, IR), that share a common core sequence. We have previously proposed that an asymmetric isomer of pi dimer can bind to a single DR unit from the origin of replication while a symmetric isomer of pi dimer binds to the half-sites that occur in the pir gene operator (Urh et al., 1998; J. Mol. Biol., 283; 619-631). During the past granting period, we set out to test this model by examining the isomerization properties of purified, His tagged derivatives of pi protein (wt) and three variants, hyperactive in replication. Electrophoretic mobility shift assays, using specially designed DNA probes, revealed that pi dimers (all variants) display remarkable flexibility, being able to bind and bend all three DNA
molecules. Hyperactive pi variants, however, exhibit varying degrees of reduced binding (compared to wt.) These results, together with previously published data, suggest that dimers of wt pi differ, structurally, from dimers of copy-up variants. We also examined pi-dependent, intra-molecular and inter-molecular interactions between DNA probes containing 22-bp DRs. Such pi-mediated pairing (also known as handcuffing) is believed to require extensive and complex surface interactions between pi protomers, perhaps ultimately leading to the inhibition of replication. Surprisingly, our results suggest that handcuffing occurs with similar efficiencies in the presence of wt and copy up pi dimers. We are preparing a manuscript in which we discuss the implications of these findings.
Impacts
Plasmids replicate extra-chromosomally in bacteria. They provide the raw material for astonishingly rapid adaptation of bacterial populations faced with changing environments. Our studies help to understand this process by using strains of Escherichia coli that harbor antibiotic-resistance plasmid R6K.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
The origins of replication (oris) used by plasmids that replicate via the Cairns mode (frequently called theta replication) contain A+T-rich regions adjacent to multiple repeated sequences (iterons) to which Replication (Rep) initiator protein binds. This laboratory has been investigating initiation control by using a minimal replicon derived from the Escherichia coli plasmid, R6K. In this system, the cis-acting _ ori and the trans-acting pir gene which encodes the Rep protein pi are necessary and sufficient for the stable maintenance of this replicon. The replication functions of pi depend on protein-DNA and protein-protein interactions. Protein-DNA interactions include pi binding to seven 22 bp iterons (each containing TGAGNG motif). Monomers and dimers of pi_can bind to an iteron. Pi also binds to a non-iteron site (lacking TGAGNG motif), a site adjacent to the iterons that lies within the A+T-rich segment of _g ori. Only dimers but not monomers can bind this site.
Protein-protein interactions not only involve self-associations but pi interacts in solution with several, known replication proteins as well. * protein plays a direct role in copy number determination; it activates or inhibits replication depending on its intracellular levels. The regulatory role of pi_, is also evidenced by several-fold increases in g_ori replicon copy numbers in vivo when mutants of the pir gene, called copy-up_ replace the wild-type (wt) counterpart. Copy-up variants do not act in vivo by increasing the total level of pi_ [which is in great excess. Rather, they act by elevating the fractions of pi_species (monomers) that stimulate replication. Copy-up * variants activate a larger fraction of DNA template in vitro, when compared to wt *, but use the same start sites for leading strand synthesis. Thus, in comparative studies with wt pi_protein, these hyperactive variants are useful tools in dissecting the mechanisms controlling plasmid copy number. DNA
strand-separation within the A+T-rich segments occurs very early in the development of replication forks. In general, the events in initiation that follow it are likely to be very similar, biochemically, and therefore of little relevance to any replicon-specific control of initiation. It would seem reasonable then that ori-specific Rep proteins would exert their control over replication early in the process, at the open complex stage and/or at the prepriming complex formation. Our most recent paper was an extension of our previous work comparing the affects of wt and hyperactive variants of pi_ on g_ ori replication control. The elevated activity of copy-up variants begged the following question: Could their hyperactivity be directly related to a propensity to open the A+T-rich region present in _g ori? Indeed, we have reported this being the case. Taken together our data suggest that it is the monomer/dimer ratio of * which determines ability of the protein to open _ ori and in so
doing regulates plasmid copy number.
Impacts
Our data suggests that pi elicits its ability to control plasmid copy number at the DNA helix-opening step.
Publications
- Kruger, R., Konieczny, I. and Filutowicz, M. 2001 Monomer/dimer ratios of replication protein modulate the DNA strand opening in a replication origin. J. Mol. Biol. 306, 945-955.
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