Progress 08/15/02 to 06/30/07
Outputs
OUTPUTS: Large volume (9-20L) fed-batch bioreactors have been developed for the cultivation of Propionibacterium jensenii. Semi-purification methods using combination of ammonium sulfate, boiling, and ion exchange have been developed to isolate and purify jenseniin P. Further fractionation and purification of jenseniin P using C-18 silica gel and acetonitrile elusion has been developed. Analytical isoelectric focusing determination and 2D analysis of jenseniin P polypeptide have been conducted and preparatory amino acid sequencing of jenseniin P is near completion.
PARTICIPANTS: Tzuen-Rong Tzeng, assistant professor, served as the PI on this project and was responsible for overseeing the research activities, research designs, data analysis and communication. John Abercrombie, lecturer, served as an investigator responsible for designing and carrying out designed experiments for the cultivation of Propionibacterium jensenii, isolation, purification, and characterization of jenseniin P. Gaoyan Wang, graduate student, was responsible for carrying out experiments for the genetic analysis of Propionibacterium jensenii gene(s) and creation of mutants. Hong Luo, associate professor, served as a collaborator in the development of transgenic plants. Protein sequence analysis and the related activities were conducted in collaboration with the Proteomic Center of the Clemson University Genomics Institute.
TARGET AUDIENCES: Undergraduate student education through Creative Inquiry projects aiming to educate students on the importance of emerging drug-resistant microorganisms and possible approaches to minimize the use of antibiotics or to develop antibiotic displacement techonolgies.
Impacts
The completion of the amino acid sequence analysis will allow for cloning of jenseniin P gene(s) for large scale production in bioreactors or transgenic plants. A collaborative effort utilizing transgenic turf grass for large production of jenseniin P has been established. Large scale production of jenseniin P utilizing transgenic tobacco and the existing large number of tobacco growers in the State could have great positive impacts on the economy of the State. Data generated from this report period will be published and used to secure additional federal funding.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Variants of Propionibacterium jensenii yielding higher concentrations of jenseniin P proteins have been isolated for use in the production of jenseniin P. Variants of P. jensenii tolerating higher or lower concentrations of jenseniin P have been isolated to facilitate the gene analysis of genes involved in jenseniin P production and immunity. New methods utilizing low concentration of penicillin to facilitate the lysis of P. jensenii for extraction of DNA has been developed and published in the 2006 ASM General Meeting Proceedings. Further characterization of jenseniin P has been performed in this 2006 report period and published in the 2006 ASM General Meeting Proceedings. Analytical isoelectric focusing determination, 2D analysis, and preparatory amino acid sequencing service agreement is in place with the Clemson University Genomics Institute to complete the characterization of jenseniin P in the 2007 report period.
Impacts
Data generated from this report period will be published and used to secure additional federal funding through the STTR programs. A commercial partner has been identified and mutual non-disclosure agreement has been signed. Other methods, including transgenic plants, for large scale production of jenseniin P have been explored to utilize the existing large number of plant growers in the State.
Publications
- Partial Characterization of the Propionibacterium Bacteriocin Jenseniin P which has Potential for Anti-Acne Applications, ASM General Meeting, 2006, J.G. Abercrombie, C. Durham, S.F. Barefoot, T-R. J. Tzeng
- Non-Mechanical and Non-Enzymatic Lysis of Bacteriocin Producing Gram(+) Propionibacterium jensenii B1264, ASM General Meeting, 2006, T-R. J. Tzeng, R.M. Holland, A.M. Gude, S.F. Barefoot
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Progress 01/01/05 to 12/31/05
Outputs
Of the 12 variants isolated, variant no. 10 produces significantly higher concentration of jensenii P than the original strain and the other variants. Variants no. 3 and 7 exhibit tolerance to higher concentration of jensenii P that they produce. This data suggests that the gene(s) for production of jensenii P and the immunity gene might be located on separate polycistrons and under the control of different regulatory system. An effort to localize the production and immunity genes via plasmid curing is currently underway. Jenseniin P is inhibited by proteinase K and protease. Jenseniin P is stable in the pH range of 2 to 12 for at least 1 hour. Jenseniin P retains its activity at temperatures of 45, 80, and 100 degree C for at least 2 hours.
Impacts
The completion of jensenii P peptide sequence will allow us to manually synthesize the peptide and overcome the low yield limitation for clinical trial. Cloning of jensenii P cDNA could also allow us to mass produce the peptide in GRAS microorganisms.
Publications
- DNA Profile of Jenseniin P Producing Propionibacterium jensenii Variants. Adam Gude and Jeremy Tzeng. Howard Hughes Medical Institute Undergraduate Research Award Colloquium, 2005
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Progress 01/01/04 to 12/31/04
Outputs
Twelve variants of the original antimicrobial peptide jenseniin P producer P. jensenii B1264 that tolerated higher concentration of jenseniin P have been isolated. Of the twelve variants, one expressed 4x and two expressed 2x amount of jenseniin P of the original strain and two expressed no detectible jenseniin P. Jenseniin P is a protein with a molecular wt. of less than 6 KDa. Amino acid sequence of this protein has been partially sequenced. Progresses are continued to be made to further purify the protein and to improve the cultivation, recovery and detection methods, and to increase the yield. Resistance of P. jensenii B1264 to jenseniin P has been determined to be not linked directly to the production of jenseniin P. Genetic analysis of the producer strain and variants is also undertaken to localize the gene(s) ending jenseniin P. A new DNA extraction protocol has been established to effectively lyse P. jensenii without the use of lytic enzymes or mechanical
force. Genome library of P. jensenii is currently in progress
Impacts
Developing reliable methods for producing jenseniin P remains a critical step in evaluating its effectiveness against the agents that are implicated in acne. Genetic methods to de-regulate production or cloning of jensenii P encoding genes may be more effective.
Publications
- Anti-Acne Property of Bacteriocin Produced by Propionibacterium jensenii B1264. Carolyn Durham, John Abercrombie, and Jeremy Tzeng Department of Biological Sciences, Clemson University, REU - NSF, 2004
- A Genetic Profile of the jenseniin P. Gene from Propionibacterium jensenii B1264. Adam Larkins, Chapman High School, and Tzuen-Rong J. Tzeng, Department of Biological Sciences, Clemson University, SPRI, 2004
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Progress 01/01/03 to 12/31/03
Outputs
Work continued with ImmuCell. Additional jenseniin P was produced at Clemson University in large-scale (13-L) fed-batch producer cultures and shipped on ice to ImmuCell. They separated fluids from producer cells using large-scale filtration techniques. They then ammonium sulfate precipitated (80%) activity from cell-free fluids and applied various techniques for isolation of jenseniin P. In one experiment, an estimated 72,000 activity units (AU) of jenseniin P were dialyzed, filtered, resuspended in TRIS buffer and applied to a Sephadex G-200 liquid chromatography column. Fractions were collected, lyophilized separately and shipped to Clemson for bioassay. Activity was eluted in two broad bands: one near the functional exclusion limit of the column (>200 Da) and one approximating the column void volume (<20 Da). Only two fractions in the second broad peak were active against the producer culture and presumably contain the most concentrated activity. These eluted
lyophilized fractions currently are being examined for impurities by SDS-PAGE to determine suitability for protein sequencing. Jenseniin P can be produced reproducibly but at extremely low concentrations (likely <1 ng per mL). The process of isolating producer variants that tolerate higher concentration of jenseniin P has been initiated as a way to increase bacteriocin yield. Data on protein size and sequence are required to develop genetic tools for future monitoring of regulation and synthesis of jenseniin P.
Impacts
Developing reliable methods for producing jenseniin P remains a critical step in evaluating its effectiveness against the agents that are implicated in acne. Genetic methods to de-regulate production may be more effective.
Publications
- Stefanie H. Baker, F. Yesim Ekinci Kitis, R. Glen Quattlebaum, and S. F. Barefoot. 2004. Sensitization of gram-negative and gram-positive bacteria to jenseniin G by sublethal injury. J. Food Protect. (In press).
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Progress 01/01/02 to 12/31/02
Outputs
For the project's first six months, we worked with ImmuCell to produce sufficient jenseniin P for their purification procedures. Larger-scale (13-L) fed-batch producer cultures reproducibly yielded directly detectable bacteriocin activity; this finding contrasts sharply with results from earlier batch culture methods where activity was detected only after concentration. Activity was detected at day 5 in 13-L cultures and is stable; these results allow for earlier harvest of jenseniin P. Detectable activity was not eluted from producer cells at pH 2.5, 4.0, 8.0 or 10; activity was recovered only in culture supernates. ImmuCell has better facilities to remove cells from large volumes of spent cultures. To permit shipping, we assessed the stability of bacteriocin activity in cultures to shipping conditions. Activity was stable at -20C, 4C and 20C for up to 4 days. Some bacteriocin-resistant producers lack susceptibility to feed-back inhibition and produce higher
concentrations of bacteriocins so we are currently attempting to select for jenseniin P-resistant producer variants.
Impacts
Developing reliable methods for producing jenseniin P is a critical first step in evaluating its effectiveness against the agents that are implicated in acne.
Publications
- Baker, S.H., F.Y Ekinci and S.F. Barefoot. 2001. Injury-enhanced spectrum of jenseniin G, a heat-stable, peptide bacteriocin produced by Propionibacterium thoenii P126. Symposium abstract. 3nd International Symposium on Propionibacteria, Zurich, Switzerland. July 8-11.
- Ekinci, F.Y., S.H. Baker and S.F. Barefoot. 2001. Mode of action of jenseniin G, a heat-stable, peptide bacteriocin produced by Propionibacterium thoenii P126. Symposium abstract. 3nd International Symposium on Propionibacteria, Zurich, Switzerland. July 8-11.
- Pittman, J.A., J. Hatch, D. Delozier, L. Oliver and S. Barefoot. 2002. Global food safety education campaign. Presented at Thinking Globally- Working Locally: A Conference for Food Safety Education. Orlando, FL. Sept. 20-22, 2002.
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