The cyanobacteria provide an excellent experimental system for studying the control of gene expression in photoautotrophs. We have been applying the powerful tools of molecular genetics and recombinant DNA technology to the study of the regulation of gene expression in these organisms as well as to the study of protein-protein interactions which are required for the in vivo assembly of the phycobilisome. We have cloned and characterized the genes encoding nine of the twelve structural components of the Synechococcus sp. PCC 7002 phycobilisome; efforts to clone the remaining three genes are currently in progress. Mutations have been constructed in most of these genes. Analyses of these mutant is being performed to define the requirements for assembly of this multiprotein complex as well as to define the pathway of excitation energy migration through the phycobilisome. Two genes, cpcE and cpcF, which apparently encode proteins required for the post-translational chromophorylation of apophycocyanin, and whose products are required for stable accumulation of phycocyanin, have also been cloned, sequenced, and inactivated. Studies to define the role of these proteins in phycocyanin biosynthesis are proposed. a number of genes encoding phycobilisome components have also been isolated and characterized from Cyanophora paradoxa and Pseudanabaena sp. PCC 7409. The expression of some of these genes, cpeBA and cpcB2A2, has been shown to be regulated at the level of mRNA abundance by light wavelength. The proposed experiments are intended to probe two different but related aspects: 1. What specific structural requirements are necessary for phycobilisome assembly and energy transfer? 2. What mechanisms regulate the expression of the various phycobiliprotein genes? The techniques of site-directed mutagenesis will be employed to analyze the function of the L-CM 97 polypeptide. Translational fusions of various phycobiliprotein promoters tot he lacZ gene will be constructed; deletion analyses will be performed in an attempt to define more precisely which sequences are important in controlling the transcription of the phycobiliprotein genes. The constructs generated will be used to examine the effects of light wavelength on phycobiliprotein gene expression. We will initiate attempts to purify the RNA plymerases of Synechococcus sp. PCC 7002 and Pseudanabaena sp. PCC 7409 in an attempt to generate at in vitro system for the analysis of transcriptional regulation in cyanobacteria. The studies proposed should greatly extend out knowledge of mechanisms regulating gene expression in photoautotrophs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031625-11
Application #
3279777
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-03-01
Project End
1994-02-28
Budget Start
1993-03-01
Budget End
1994-02-28
Support Year
11
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
Shen, Gaozhong; Schluchter, Wendy M; Bryant, Donald A (2008) Biogenesis of phycobiliproteins: I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phyococyanobilin lyase specific for beta-phycocyanin and allophycocyanin subunits. J Biol Chem 283:7503-12
Saunee, Nicolle A; Williams, Shervonda R; Bryant, Donald A et al. (2008) Biogenesis of phycobiliproteins: II. CpcS-I and CpcU comprise the heterodimeric bilin lyase that attaches phycocyanobilin to CYS-82 OF beta-phycocyanin and CYS-81 of allophycocyanin subunits in Synechococcus sp. PCC 7002. J Biol Chem 283:7513-22
Inoue-Sakamoto, Kaori; Gruber, Tanja M; Christensen, Suzanne K et al. (2007) Group 3 sigma factors in the marine cyanobacterium Synechococcus sp. strain PCC 7002 are required for growth at low temperature. J Gen Appl Microbiol 53:89-104
Woodger, Fiona J; Bryant, Donald A; Price, G Dean (2007) Transcriptional regulation of the CO2-concentrating mechanism in a euryhaline, coastal marine cyanobacterium, Synechococcus sp. Strain PCC 7002: role of NdhR/CcmR. J Bacteriol 189:3335-47
Shen, Gaozhong; Saunee, Nicolle A; Williams, Shervonda R et al. (2006) Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002. J Biol Chem 281:17768-78
Frigaard, Niels-Ulrik; Sakuragi, Yumiko; Bryant, Donald A (2004) Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation. Methods Mol Biol 274:325-40
Wang, Tao; Shen, Gaozhong; Balasubramanian, Ramakrishnan et al. (2004) The sufR gene (sll0088 in Synechocystis sp. strain PCC 6803) functions as a repressor of the sufBCDS operon in iron-sulfur cluster biogenesis in cyanobacteria. J Bacteriol 186:956-67
Yu, Jianping; Shen, Gaozhong; Wang, Tao et al. (2003) Suppressor mutations in the study of photosystem I biogenesis: sll0088 is a previously unidentified gene involved in reaction center accumulation in Synechocystis sp. strain PCC 6803. J Bacteriol 185:3878-87
Gomez-Lojero, Carlos; Perez-Gomez, Bertha; Shen, Gaozhong et al. (2003) Interaction of ferredoxin:NADP+ oxidoreductase with phycobilisomes and phycobilisome substructures of the cyanobacterium Synechococcus sp. strain PCC 7002. Biochemistry 42:13800-11
Huang, Chenhui; Yuan, Xiaolin; Zhao, Jindong et al. (2003) Kinetic analyses of state transitions of the cyanobacterium Synechococcus sp. PCC 7002 and its mutant strains impaired in electron transport. Biochim Biophys Acta 1607:121-30

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