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Bacterial Carbon Storage to Value Added Products

Author(s)
Brigham, Christopher J.; Kurosawa, Kazuhiko; Rha, Chokyun; Sinskey, Anthony J
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Abstract
PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).
Date issued
2011-12
URI
http://hdl.handle.net/1721.1/75741
Department
Harvard University--MIT Division of Health Sciences and Technology; Massachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Materials Science and Engineering; Massachusetts Institute of Technology. Engineering Systems Division
Journal
Journal of Microbial and Biochemical Technology
Publisher
OMICS Publishing Group
Citation
Brigham, Christopher J. et al. “Bacterial Carbon Storage to Value Added Products.” Applied Microbiology and Biotechnology 83 (2011). © 2011 OMICS Group
Version: Final published version
ISSN
1948-5948

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