James W. Golden
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. 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.