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James W. Golden

James W. Golden

Professor
Section of Molecular Biology, UCSD

e-mail: jwgolden@ucsd.edu

Molecular Genetics of Cyanobacterial Development and Nitrogen Fixation

   I am primarily interested in the developmental biology of bacterial microorganisms with an emphasis on the genetic regulation of cellular differentiation and the cell-to-cell signaling mechanisms that control multicellular pattern formation. My research uses methods of genetics and molecular biology to understand basic principles of regulation and signaling pathways that control development in a simple prokaryotic multicellular organism, the filamentous cyanobacterium Anabaena (Nostoc). It is expected that the basic information gained from studying this model system will be applicable to a variety of other areas including those related to health and the environment. Like all cyanobacteria, Anabaena obtains energy by photosynthesis. Anabaena is also capable of nitrogen fixation, a process that is incompatible with photosynthesis because the nitrogenase enzyme is destroyed by the oxygen produced as a byproduct of photosynthesis. Anabaena solves this problem by spatially separating the two processes into different cell types: photosynthetic vegetative cells and nitrogen-fixing heterocysts. Anabaena grows as a very simple multicellular organism organized as filaments of vegetative cells containing about 10 percent heterocysts. Heterocysts differentiate from vegetative cells at semiregular intervals along the filament and supply fixed nitrogen to neighboring vegetative cells to support their growth.

   A new area of interest in my lab is genetic engineering of cyanobacteria for the production of desired products such as biofuels.

figure 1

The following are examples of our current research projects.

   Control of heterocyst pattern by cell-to-cell signals. We identified a gene, patS, which encodes a small peptide that functions as a diffusible inhibitor that partially controls heterocyst pattern formation. This discovery is the basis for several ongoing projects. We are using gfp (green fluorescent protein) reporter fusions to study the timing and cell-type specificity of patS expression and have developed methods for making time-lapse microscopy movies to follow gene expression patterns. We are now examining the regulatory mechanisms that control these expression patterns by identifying the cis-acting DNA elements and trans-acting factors involved in controlling patS transcription. Recent data support a role for HetR as a transcription activator for the patS gene and we have started to examine this possibility with in vitro DNA-protein interaction experiments and transcription assays.

   We have used genetic screens to identify genes that are involved directly or indirectly in patS expression or the PatS signaling pathway. For example, we identified the gene asr1734, which can completely suppress heterocyst development, and we are currently trying to determine the mechanism of its action.

   Using bioinformatics based on the complete genome sequence of our strain of Anabaena (PCC 7120) and that of several other cyanobacteria, we have identified several candidate genes that may be involved in signaling by the PatS peptide. We are using reverse genetics to inactivate these genes to determine if they are required for normal heterocyst development and pattern.

   Identification and analysis of genes required for heterocyst differentiation. Another project in the lab is the use of mutant screens, DNA expression microarrays, and bioinformatics to identify genes involved in the regulation of several aspects of heterocyst differentiation. We have identified novel genes involved in the initiation of heterocyst formation and in heterocyst morphogenesis. We also are determining the role of RNA polymerase sigma factors and proteins involved in cyclic-di-GMP signaling in heterocyst development. Finally, we are identifying genes that regulate the nitrogen-fixation genes in mature heterocysts.

   Metabolic engineering and biofuels. New research projects in the lab are designed to determine the capabilities of cyanobacteria for the production of desired products related to biofuels. It is widely believed that microalgae will ultimately be one of the most efficient methods for the production of liquid biofuels required for transportation. Our research projects are designed to determine what products can be most efficiently produced by cyanobacteria.

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Taton, A., E. Lis, D. M. Adin, G. Dong, S. Cookson, S. A. Kay, S. S. Golden, and J. W. Golden. Gene transfer in Leptolyngbya sp. strain BL0902, a cyanobacterium suitable for biomass and biofuel production. In preparation for Applied and Environmental Microbiology.

Flaherty, B. L., F. Van Nieuwerburgh, S. R. Head, and J. W. Golden. 2011. Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation. BMC Genomics, 12:332.
http://www.biomedcentral.com/1471-2164/12/332

Saha, S. K., and J. W. Golden. 2011. Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120. J. Bacteriol. 
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21421755

Mella-Herrera, R. A., M. R. Neunuebel, K. Kumar, S. K. Saha, and J. W. Golden. 2011. The sigE gene is required for normal expression of heterocyst-specific genes in Anabaena sp. strain PCC 7120. J. Bacteriol. 193:1823-1832. 
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21317330

Mella-Herrera, R. A., M. R. Neunuebel, and J. W. Golden. 2011. Anabaena sp. strain PCC 7120 conR contains a LytR-CpsA-Psr domain, is developmentally regulated, and is essential for diazotrophic growth and heterocyst morphogenesis. Microbiology 157:617-626. 
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21088107

Kumar, K., R. A. Mella, and J. W. Golden. 2010. Cyanobacterial heterocysts. Cold Spring Harb. Perspect. Biol. 2009. 2:a000315.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845205/pdf/cshperspect-PRK-a000315.pdf

Neunuebel, M. R. and J. W. Golden. 2008. The Anabaena sp. strain PCC 7120 gene all2874 encodes a diguanylate cyclase and is required for normal heterocyst development under high-light growth conditions. J. Bacteriol. 190:6829-6836.
http://jb.asm.org/cgi/content/abstract/190/20/6829

Aldea, M. R., K. Kumar, and J. W. Golden, 2008. Heterocyst development and pattern formation, p. 75-90, In S. C. Winans and B. L. Bassler (eds.), Chemical Communication Among Microbes. ASM Press, Washington, D.C.

Aldea, M. R., R. A. Mella, and J. W. Golden, 2007. Sigma factor genes sigC, sigE, and sigG are upregulated in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 189:8392-8396.
http://jb.asm.org/cgi/content/abstract/189/22/8392

Wu, X., D. W. Lee, R. A. Mella, and J. W. Golden. 2007. The Anabaena sp. strain PCC 7120 asr1734 gene encodes a negative regulator of heterocyst development. Mol. Microbiol. 64:782-794.
http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2958.2007.05698.x

Carrasco, C. D., S. D. Holliday, A. Hansel, P. Lindblad, and J. W. Golden. 2005. Heterocyst-specific excision of the Anabaena sp. strain PCC 7120 hupL element requires xisC. J. Bacteriol. 187: 6031-6038.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16109944

Liu, T., J. W. Golden, and D. P. Giedroc. 2005. A Zinc(II)/Lead(II)/Cadmium(II)-Inducible Operon from the Cyanobacterium Anabaena Is Regulated by AztR, an alpha3N ArsR/SmtB Metalloregulator. Biochemistry 44:8673-8683.
http://pubs3.acs.org/acs/journals/doilookup?in_doi=10.1021/bi050450%2b

Khudyakov, I. Y., and J. W. Golden. 2004. Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp. PCC 7120, can be separated by mutation. Proc. Natl. Acad. Sci. USA 101:16040-16045.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15520378


Dr. James W. Golden received a B.S. (1977) in Microbiology from the University of Maryland-College Park and a Ph.D. (1983) in Biology from the University of Missouri-Columbia. After postdoctoral work as an NIH Fellow at The University of Chicago, he joined the Department of Biology at Texas A&M University in 1986. He was promoted to Associate Professor in 1990 and then to Professor in 1996. Dr. Golden moved to the University of California, San Diego in 2008.