JUNIATA STUDENT RESEARCH SCHOLARSHIP APPLICATION
(Please submit electronically to any member of the Student Research & Fellowship Committee)
Name: XXXX
Date: XXXX
Names of Your Advisors: XXXX
Year of Graduation: XXXX
Title of Your POE: Biology
Faculty Sponsor of the Project: XXXX
Dates of the Project: January 2006-present
Is the project a student-initiated independent study? Yes If so, for how many credit hours? 1
Do you have other sources of funding through the College? If so, give details.
No
TITLE OF THE PROJECT:
Identification of a Serine Protease in the Model Actinobacterium Streptomyces coelicolor
PROJECT DESCRIPTION: (1) Define the nature and significance of your research project. Give special
attention to the research goals of the project and the methodology you will use to pursue those goals. Include
enough detail in your methods to make it clear that the project has been carefully planned and that the methods
address the goals. Include the scholarly significance of the specific work proposed and/or broader significance
of the work to society. Write for a broadly trained, non-expert audience, avoiding jargon. In addition, mention
(2) the ways that you believe the project will contribute to your own educational development, and (3) the
tangible outcome of the project, such as a written paper, artistic production or artifact, or presentation before an
audience at Juniata or a professional meeting. If you have written other documents concerning your research
plans you may wish to include them with this application. This section of the proposal should not exceed two
single spaced pages below, including figures and references. Note that attending a workshop or conference is
not considered research.
INTRODUCTION
Streptomyces are a well-studied group of gram positive, high G+C bacteria. Their production of a wide range of
secondary metabolites, and ubiquitous presence in soil environments makes them invaluable research models (5, 10).
Secondary metabolites produced by Streptomyces encompass a variety of pharmacologically active biomolecules,
including over 2/3 commercially available antibiotics and several chemotherapeutic agents (10). Ecologically,
Streptomyces plays an undeniable role in the soil and successfully exploits a variety of carbon sources from fungi, plants
and insects (5, 10, 16). Most intensely studied of this distinctive genus is Streptomyces coelicolor, whose complete
genome was sequenced from an ordered cosmid library in 2002 (2, 15). The wealth of bioinformatics data available for S.
coelicolor coupled with its pharmaceutical importance and morphological novelties makes it an ideal model to study
bacterial development.
The production of pharmaceutically important biomolecules and environmental success are intimately tied with
the filamentous lifestyle of S. coelicolor (11). The complex lifestyle of morphological differentiation observed in S.
coelicolor is a novel feature among bacteria (12); and affords unique research opportunities to explore prokaryotic
development.The filamentous life cycle begins with a single mononucleoid spore, which germinates to form a branching
network of multinucleoid hyphae termed vegetative mycelium.
Differentiation commences with the formation of aerial hyphae, which
grow skyward and away from the surface of the newly-formed colony.
Later, these aerial hyphae septate to become uninucleoid compartments,
which metamorphose into chains of gray-pigmented spores (11). These
mature spores disperse into the environment, completing the cycle (9). The
process is summarized in fig. 1. Morphological differentiation is the
coordinated effort of numerous biomolecular processes (5, 6,7, 11); and the
adaptive powers it confers to S. coelicolor (including the synthesis of
antibiotics and other secondary metabolites), is just beginning to be well
understood. (9).
For most bacteria, cell division is absolutely essential to colony formation
and viability; this is not the case in Streptomycetes (8, 12). Developmental
mutants are often viable and may be identified with some ease
morphologically and then isolated for study (7). Furthermore, the role that
newly identified developmental genes play in cell division and other critical
roles can be more fully understood in a broad molecular context because the
S. coelicolor genome project has been completed (2).
METHODS
Using a highly efficient Tn5 based mini-transposon (3, 4, 15) I have identified numerous S. coelicolor mutants. I
screened these insertion mutants using an assortment of media and visual screens to identify
putative developmental mutants. Simultaneously, I chose one mutant of particular interest isolated
by another lab colleague to examine phenotypically, sequence using inverse PCR, and begin
protein characterization procedures. The mutation occurred in SCO2637, a gene encoding a
putative secreted serine protease that shares a conserved domain with the subtilase family of
serine proteases. The subtilisin family is the second largest serine protease family characterized to
date (14) and is typified by highly conserved domain containing an Asp/Ser/His catalytic triad and
an alpha/beta fold containing a 7-stranded parallel beta sheet (14). This super family of serine
proteases is considerably diverse in function; however, based upon amino acid sequence
SCO2637 has a 26.0% identity with stetterlysin, a thermally stable excreted serine protease from
Thermococcus stetteri (9, 24) and a 23.8% identity with a characterized extracellular protease in
FIG.2. Phase contrast
Bacillus subtilis (12, 16, 14).
micrograph of wild-type
RESULTS/DISCUSSION
S. coelicolor MT1110
Upon examination of BPK 12 using several characterization techniques, I found several
abnormalities. Visually, colonies are deficient in gray spore pigment. Phase contrast micrographs (WT) and developmental
mutant BPK 12 after five
(fig. 2) show the distinctly mutant phenotype of BPK 12: unusually short spore chains and
days growth. A deviantelongated spore compartments. Fluorescent confocal micrographs demonstrate that BPK 12
sized spore is highlighted
progresses normally through chromosome segregation, although condensation of the chromosome by the arrow in the figure.
may be abnormal (fig. 3).
FIG. 1. Morphological differentiation: the
developmental life cycle of Streptomyces
coelicolor.. Modified from Flärdh and Buttner
(11).
From a bioinformatic analysis, I found that the transposon insterted itself in SCO2637, a putative secreted serine
protease. From these data, and phenotypic analyses I infer that SCO2637 plays an indirect role in sporulation; the mutant
gene results in lower levels of gray spore pigment and abnormal spores, but chromosome
segregation proceeds. SCO2637 does not seem compulsory for strain viability.
The Subtilase family of serine proteases is strikingly diverse and performs a
wide variety of roles in prokaryotes (1, 13). To complicate matters further, Siezen and
Leunissen (14) found that high sequence variability (with the exception of the Asp/His/Ser
residues of the catalytic triad) is widespread. The complexities inherent to this family of
serine proteases make it difficult and often speculative to determine function based on amino
acid sequence alone. The protein encoded by SCO2637 is not exceptional in this regard.
There is considerable variation between both its sequence and the subtilase consensus
sequence and the sequences of the most similar published proteins: stetterlysin and Vpr.
Sloma and colleagues found that Vpr is not compulsory for development in B.
subtilis. Furthermore, knock-out mutants lacking Vpr and seven other secreted serine
proteases were completely viable and able to secrete sufficient amounts of extracellular
protein (14). These extracellular proteins included one or more proteases, as protease
activity was observed in the supernatant of cultures (14). Like B. subtilis, S. coelicolor
depends upon secreted molecules to colonize soil environments. It seems likely that S. coelicolor relies upon a similarly
complex system to coordinate the secretion of proteins into the environment regulate their modification. I propose that
sco2637 plays some role in this system, perhaps modifying a secreted protein that serves as part of signal transduction. In
the future, I would like to confirm that the transposon insertion is indeed the cause of the observed phenotype. Several
attempts to perform linkage procedures to accomplish this have been unsuccessful. To this end, I would like to utilize a
commercially available insertion mutation as a more efficient method of obtaining linkage. Once a gene of interest is
identified, a plasmid with a mutation in that gene can be ordered. I will use this plasmid to rapidly construct a mutant for
my gene of interest and compare this new mutant to the one that was originally identified in our laboratory. Once linkage
has been ascertained, a bevy of molecular techniques including protease assays can be used to further characterize BPK
12. These techniques, in coordination with those I have used thus far in this research project (inverse PCR, phase
contrast/confocal microscopy) will improve my chances to pursue molecular research at the graduate level, my primary
goal upon graduation in May XXXX.
REFERENCES
FIG.3. Confocal fluorescent
micrographs of MT1110
(WT) and BPK 12. Nucleic
acids are stained by
propidium iodide.
1.
Barrett, A.J., and N.D. Rawlings. 1995. Arch. Biochem. Biophys. 318: 247-250.
2.
Bentley, S. D., K. F. Chater, A. M. Cerdeno-Tarraga, et al. 2002. Nature 417:141-147.
3.
XXX, J.A. 2006. Ph.D. Dissertation. Duquesne University, Pittsburgh. PA.
4.
XXX, J. A. and J. R. McCormick. Manuscript in final revision prior to submission to J. Bacteriol.
5.
Champness, W. 2000. Coordinating ed., Y.V. Brunn, and L.J. Shimkets. ASM Press, Washington DC.
6.
Chater, K.F. 2000. Coordinating ed., Y.V. Brunn, and L.J. Shimkets. ASM Press, Washington DC.
7.
Chater, K.F. 1998. Microbiol. 144: 1465-1478.
8.
Errington, J., R.A. Daniel, and D.J. Scheffers. 2003. Microbiol. Mol. Biol. Rev. 67: 52-65.
9.
Flärdh, K., and M.J. Buttner. 2009. Nat. Rev. Microbiol. 7: 36-49.
10. Hopwood, D.A. 1999. Microbiol. 145: 2183-2202.
11. Kelemen, G.H. M.J. Buttner. 1998. Current Opinion in Microbiol. 1: 656-662.
12. Margolin, M. 2000. FEMS Microbiol. Rev. 24: 513-548.
13. Siezen, R.J., and J.A.M. Leunissen. Protein Sci. 6: 501-523.
14. Sloma, A., G.A. Rufo Jr., K.A. Theriault, M. Dwyer, et al. 1991. J. Bacteriol. 173: 6889-6895.
15. Redenbach, M., H.M. Kieser, D. Denapaite, et al. 1996. 21: 77-96.
16. Voorhortst, W.G.B., A. Warner, W.M de Vos, and R.J. Seizen. 1997. Protein Eng. Des. Sel. 10: 905-914.
JUSTIFICATION FOR FINANCIAL REQUEST: Why do you need financial assistance to complete your
project? Provide a carefully itemized list of your anticipated expenditures for such things as supplies
(chemicals, photocopying, artist’s materials, etc.), audiovisual aids, or travel funds for library or museum
research, conducting interviews, or visiting field sites. The maximum support for excellent projects is $500.
Do not list travel to a conference to give a presentation. There is a separate application form for defraying travel
expenses to give a presentation that you may wish to fill out later if your research is going well.
A major component of my research project is determining whether the mutant phenotype I observe in
BPK 12 is linked (i.e. caused by) the transposon insertion rather than a random mutation elsewhere on the
chromosome. I have previously performed linkage procedures to ascertain this. However, despite repeated
attempts and careful troubleshooting I have not been able to successfully complete the necessary procedure. I
would like to apply for extra funding in the amount of 240.00 USD (80 USD each) to purchase a Tn5062
insertion plasmid for my gene of interest (SCO2637) as well as two additional transposon mutagenized plasmids
that contain mutations in genes flanking my gene of interest. This molecular tool will allow me to determine if
the transposon insertion event is the cause of the mutant phenotype, allowing me to rapidly progress in my
research. I will also be able to subclone the genetic region for future genetic manipulation and create two
additional mutants of interest.
SPONSOR’S STATEMENT: Speak to the potential value of the research experience to the student,
appropriateness of methods, and potential of the student to perform the research. The SRFC encourages faculty
sponsors to offer useful additional information for interpreting and adding context to the project
Rationale #1: Student X is an excellent student
Student X is a senior research student who has worked in my microbial genetics laboratory for about 1 year.
Student X is an excellent student who is particularly interested in a career that couples his interests in molecular
microbiology and archaeology. He has been awarded a von Liebig Summer Undergraduate Research
Fellowship and an I99 Regional Networking Scholarship to work in my laboratory during the summer 2008
before leaving to study abroad at the University of Aberdeen funded by a Saint Andrew’s Scholarship. Student
X returned in the Fall of 2009 and presented his research this past November at the Annual Biomedical
Research Conference for Minority Students as a selected speaker, winning an Outstanding Presentation Award
at this conference. He also presented his research at the Allegheny Branch Meeting of the American Society for
Microbiology, the Swarthmore Microbial Educators Undergraduate Research Symposium and the Juniata
Summer and Liberal Arts Symposia. Student X has been approved by the United States for his Fulbright
Scholarship application and is awaiting the approval for the host country of Iceland where he has already spent
last summer conducting independent research.
Rationale #2: The proposed research is able to be finished in the short time frame available.
Rationale #3: The proposed research will greatly enhance the Senior Honors Thesis quality of a
deserving student.
Rationale #4: The requested materials will serve more than one purpose in the proposed research.
Rationale #5: The requested plasmids are perpetuated in E. coli strains for eternal use that extends far
beyond this project.
The above proposal requests three large plasmid clones. These clones are pieces of circular DNA containing
part of the Streptomyces chromosome with an insertion mutation that inactivates a particular gene of interest.
One of these clones will allow Student X to construct a mutant inactivated for his gene of interest. This will
enable Student X to compare his original mutant to the newly constructed one, confirming that the gene is
causing the observed defect and is of interest for continued characterization. This is a critical step for
determining whether the gene is interesting for publication. Similar mutant constructions have been performed
in our laboratory for other genes within a few weeks time. This will allow Student X to have a complete story
for his Senior Honors Thesis, enabling Student X to graduate with distinction in his POE. The other two clones
will allow Student X to simultaneously construct two additional mutants with insertions in related genes of
unknown function, creating a more multi-dimensional thesis and future publication. Additionally, because the
genes are adjacent to each other the plasmids can be used for subcloning to create other useful plasmids that
may be used in this project if time permits as well as in future projects.
Rationale #6: I have many students conducting research in my laboratory to gain hands-on experience in
preparation for future careers in science and medicine. The biology department is willing to share the costs by
buying additional plasmids.