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Education

Ph.D.: Biochemistry, Microbiology, and Molecular Biology, 1994. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA

General Areas of Interest:

• Microbial physiology
• Photosynthesis
• Gene regulation
• Microbial biotechnology
• Microbial diversity

Cyanobacteria are a morphologically and developmentally diverse group of prokaryotes whose light harvesting structures called phycobilisomes (see Fig 1) are very similar to those found in red algal chloroplasts in structure and function, but quite distinct from higher plant light harvesting systems.  Cyanobacteria are important contributors to many ecosystems and have been used as a model system to study photosynthesis because we have developed genetic systems for many strains, they have fast growth rates, and the genome sequences of many cyanobacteria have been completed.  

Cyanobacteria alter the composition of their phycobilisomes in response to light intensity, light quality, and nutrient availability. Their phycobilisomes (PBS) are composed primarily of phycobiliproteins (PBP). Phycobiliproteins range in color from yellow to red to purple to blue, depending upon which of a combination of four possible chromophores called bilins are covalently attached to these proteins.  The structures and biosynthetic pathways of the two most common bilins, phycocyanobilin and phycoerythrobilin are shown below in Figure 2.  

The long-term goal of this research project is to understand how the cyanobacterial light-harvesting phycobiliproteins and their chromophores are synthesized, the order that these post-translational modifications occur, and how the phycobilisome is assembled.   There are four known classes of bilin attachment enzymes (called bilin lyases) which are distinct in their substrate specificity and in their amino acid sequences: the E/F type, the S/U type, the T type bilin lyases, and the autocatalytic type.  Our approach is to identify enzymes involved in the post-translational modification of phycobiliproteins and to characterize each gene’s involvement by cloning each gene thought to be involved in the biosynthesis of a particular phycobiliprotein in a plasmid, putting all of these plasmids inside E. coli and growing the cells to allow expression of all the recombinant cyanobacterial enzymes, and seeing if phycobiliproteins are produced.  We have successfully produced allophycocyanin inside E. coli (see Fig 3).

Figure 1: Structure of a phycobilisome containing allophycocyanin (AP) in the core portion and phycocyanin (PC) in the rods. Each phycobiliproteins is composed of alpha and beta subunits which assemble into trimers as shown. These trimers resemble a donut in structure and can further stack into hexamers. The rods in this phycobilisome are composed of 3 hexamers of phycocyanin. The colors shown are close to the colors of the isolated proteins.

Figure 2:  Biosynthetic pathway for the bilin chromophores attached to phycobiliproteins in cyanobacteria (Frankenberg, et al., Plant Cell 13:965-978; Dammeyer, et al, 2008. Current Biology 18:442-448)

Figure 3: Photographs of E. coli pellets or extracts after growth with the plasmids listed above each picture.

People:

Dr. Wendy Schluchter, Associate Professor

Ph.D.: The Pennsylvania State University, University Park, PA.  Biochemistry, Microbiology, and Molecular Biology, 1989-1994.  Department of Biochemistry and Molecular Biology

Grant funding:

April 2009-Mar 2012. “Biosynthesis and Assembly of Phycobiliproteins in Cyanobacteria” NSF. Wendy Schluchter, PI; $411,000.

Aug 2004- Aug 2009 “UMEB: Mentoring Undergraduate Research in Conservation Biology and Ecology” NSF. Steve Johnson, PI;  Co-PIs: Wendy Schluchter, Nicola Anthony, Carla Penz.  Funds awarded: $ 438,902

June 2002-Aug, 2009 “CAREER:  Elucidation of the biosynthetic and degradative pathways for phycobiliproteins in cyanobacteria”. NSF.  Wendy Schluchter, PI. Funds awarded: $523,147. (no cost extension)

Graduate students, current: 

Avijit Biswas:  PhD student, Department of Chemistry, 2006-present

Tierna Dragomani:  M.S. Student, Department of Biological Sciences,

Kali Hicks:  M.S. student, Department of Biological Sciences, 2003-present

Tierna, Avi and Yasmin.

Graduate students, past:

Crystal Miller:  Identification and Characterization of Enzymes involved in post-translational modifications of phycobiliproteins in the Cyanobacterium Synechocystis sp. PCC 6803. MS Summer 2007

Shervonda Williams: Characterization of a new family of phycobiliprotein lyases in cyanobacteria:  Bilin attachment to allophycocyanin. MS Fall 2007

Nicolle Saunée:  Identification and characterization of a new class of bilin lyases in Synechococcus sp. PCC 7002. MS Fall 2006

 Amy Theriot Pierce: M.S. 2003.   Characterization of a quintuple phycobiliprotein lyase mutant.

Heidi Shoup. M.S. 2002. Generation of quadruple phycobiliprotein lyase mutant and characterization of a putative methyl transferase.

Ruth Waterman: M.S. 2002. Application of the yeast two-hybrid system to identify protein-protein interactions among putative lyases and phycocyanin subunits from the cyanobacterium Synechocystis sp. PCC 6803.

Padmanabhan Mahadevan.  M.S.  2001. Characterization of phycobiliprotein lyase mutants in the cyanobacterium Synechocystis sp. PCC 6803.

Nicolle, Crystal, Wendy, Vonda, Leon, Alan (back row) and Ariane (front row).

Undergraduates doing research in my lab over the last 5 years

UMEB students:

Yasmin Vasquez: (Aug 2007-present) Cloning and expression of the hox-1 gene involved in the conversion of Heme to Biliverdin IXα

Brandy Turner (June 2006-May 2008).  Cloning of apcA with apcF for co-expression of the recombinant proteins in E. coli. 

Alan Williams: (June 2005-Fall 2005) Cloning and overexpression of recombinant histidine-tagged cpcBA from Synechococcus sp. PCC 7002.

Ariane Fletcher: (June 2005-Fall 2005) Cloning and expression of recombinant histidine tagged apcF from Synechococcus sp. PCC 7002.

Bios  4091: Senior Honor’s Thesis Research:
Eduardo Gallo (6 cr, honor’s thesis: Fall 2003-Spring 2004) Characterization of a family of proteins for bilin addition activity and interaction with the beta subunit of phycocyanin from Synechococcus sp. PCC 7002. 

BIOS3092 Independent Research

Christopher Benjamin (1 cr, Spring 2009)
Lauren Ruffino (1 cr, Spring 2008): 
Angela Hernandez (1 cr, Spring 2007): 
Jesse Allen (1 cr, Spring 2007): 
Bukola Folorunsho (1 cr Fall 2006):
Latira Haynes (2 cr, Fall 2006):
Angela Hernandez (3 cr, Spring 2007): 
Jesse Allen (3 cr, Spring 2007):
Christopher Loftin (4 cr, Summer 2006) 
Andrea Skinner: (2 cr) Spring 2006: 
Katrina Price:  (2 cr, Summer 2004)

Student paid workers who performed lab tasks and experiments:

Pamela Carter: Summer 2008
Leon Harrison, Jr.: Fall 2006-Spring 2007, Fall 2007
Joni Swenson: Fall 2004-Fall 2005
Suehelm Mendoza: Aug 2003-Fall 2004.

Undergraduates (AMRI sponsored research students):

Leon Harrison, Jr.  Summer 2007.

High school students (AMRI sponsored research students):

Leon Harrison, Jr.  Summer 2005 and Summer 2006.
Tiffany Wei:  Summer 2007. 
Jennifer Roque.  Summer 2008. 

Publications:

Miller, C. A., Leonard, H. S., Pinsky, I. G., Turner, B. M., Williams, S. R., Harrison Jr., L., Fletcher, A. F., Shen, G., Bryant, D. A., and Schluchter, W. M. (2008)  Biogenesis of Phycobiliproteins. III: CpcM is the Asparagine Methyltransferase for Phycobiliprotein b-subunits in Cyanobacteria. J. Biol. Chem. 283; 19293-19300.

Shen, G., Leonard, H. R., Schluchter, W. M.  and D. A. Bryant. 2008. Characterization of cpcM mutants that are unable to methylate Asparagine-71/72 of phycobiliprotein beta subunits in Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. J. Bacteriol. . 190; 4808-4817.

Shen, G., Schluchter, W. M.  and D. A. Bryant. 2008. Biogenesis of phycobiliproteins. I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phycocaynobilin lyase specific for beta -phycocyanin and allophycocyanin subunits, J. Biol. Chem. 283; 7503-7512. Mar 21.

Saunée, N. A., Williams, S. R., Bryant, D. A. and W. M. Schluchter. 2008.  Biogenesis of phycobiliproteins. II. CpcS-I and CpcU comprise the heterodimeric bilin lyase that attaches phycocyanobilin to Cys-82 of b-phycocyanin and Cys-81 of allophycocyanin subunits in Synechococcus sp. PCC 7002.  J. Biol. Chem. 283; 7513-7522. Mar 21

Shen, G, Saunée, N. A., Williams, S. R., Gallo, E. F., Schluchter, W. M. and D. A. Bryant.  2006.  Identification and Characterization of a New Class of Bilin Lyase Responsible for Attachment of Phycocyanobilin to Cys-153 on the b-subunit of Phycocyanin in Synechococcus sp. PCC 7002.  J. Biol. Chem.  281; 17768-17778.

Gomez-Lojero, C., Perez-Gomez, B.,  Shen, G., Schluchter, W. M. and D. A. Bryant.  2003. Interaction of Ferredoxin-NADP+ Oxidoreductase with Phycobilisome Substructures of the Cyanobacterium Synechococcus sp PCC 7002. Biochemistry 42. 13800-13811

Yang, F., Shen, G., Schluchter, W. M., Zybailov, B. L., Ganago, A. O., Vassiliev, I. R., Bryant, D. A., Golbeck, J. H. 1998. Deletion of the PsaF Polypeptide Modifies the Environment of the Redox-Active Phylloquinone (A1). Evidence for Unidirectionality of Electron Transfer in Photosystem I, The J. of Phys. Chem. B;; 102(42); 8288-8299.

Schluchter, W. M., and A. N. Glazer  1997.  Characterization of Cyanobacterial  Biliverdin Reductase. Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis. J. Biol. Chem. 272: 13562-13569 

Löffelhardt, W., Stirewalt, V. L., Michalowski, C. B., Annarella, M., Farley, J. F., Schluchter, W. M., Chung, S. Newmann-Spallart, C., Steiner, J. M., Jakowitsch, J., Bohnert, H. B., and Bryant, D. A.  1996.  The complete sequence of the cyanelle genome of Cyanophora paradoxa:  the genetic complexity of a primitive plastid.  Endocytobiology VI.

Schluchter, W. M., Shen, G. H., Zhao, J. D. and D. A. Bryant.  1996.  Characterization of psaI and psaL mutants of Synechococcus sp. PCC 7002:  A new model for state transitions in cyanobacteria.  Photochemistry and Photobiology 64:  53-66.

Schluchter, W. M., Zhao, J. and Bryant, D. A.  1993.  Isolation and characterization of the ndhF gene of Synechococcus sp. PCC 7002 and initial characterization of an interposon mutant.  J. Bacteriol. 175:  3343-3352.

Schluchter, W. M. and Bryant, D. A.  1992. Molecular characterization of ferredoxin-NADP+ reductase in cyanobacteria: cloning and sequence of the petH gene of Synechococcus sp. PCC 7002 and studies on the gene product.  Biochemistry 31: 3092-3102.

Bryant, D. A., Schluchter, W. M. and Stirewalt, V. L.  1991.  Ferredoxin and ribosomal protein S10 are encoded on the cyanelle genome of Cyanophora paradoxa.  Gene 98:  169-175.