The goals of this project are to understand how certain types of bacteria are capable of supplying themselves with nitrogen fertilizer, which is required for the growth of all organisms. The project will determine at the cellular and molecular level, how these bacteria, called cyanobacteria, produce the cells and enzymes that "fix" nitrogen from the air to produce compounds that can enter cell metabolism. Cyanobacteria are green in color and obtain energy from sunlight with photosynthesis similarly to plants. Like algae, cyanobacteria are important primary producers, which are food for other organisms, in many environments, especially the oceans, and they remove carbon dioxide from the atmosphere, which reduces effects of global warming. Nitrogen-fixing cyanobacteria provide biologically available nitrogen in natural environments and are used as nitrogen fertilizer for rice cultivation in some parts of the world. Recently, there has been enormous interest in using cyanobacteria and algae to capture energy from sunlight to produce biomass, biofuels such as biodiesel and ethanol, and hydrogen gas. This project will use the well-established model organism Anabaena to determine how specialized cells called heterocysts produce the enzymes required for nitrogen fixation. The project will use methods of genetics, molecular biology, and biochemistry to study these processes. This project is expected to reveal new mechanisms of genetic regulation and gene expression in this large and important group of microorganisms. Understanding the mechanisms that control nitrogen-fixation genes will enhance our knowledge of basic microbial biology, genetics, and metabolism, and will provide knowledge that can be applied to industrial carbon capture and biofuel production. During the course of this project, undergraduate, graduate, and postdoctoral students will receive training in state-of-the-art research methods. The research will also produce materials and methods that will be useful to other researchers in both academia and industry, and the long-term goals of this project will benefit society by providing basic knowledge related to natural ecosystems, agriculture, global warming, and biofuels.
This project has improved our understanding of the genetic and molecular mechanisms that regulate gene expression related to bacterial cellular differentiation (how cells change their cell type) and nitrogen fixation, using heterocyst development in the cyanobacterium Anabaena as a model system. During this project, we have developed new and improved genetic and molecular methods to study gene expression, and we have used these and other established methods to better understand the mechanisms that control changes in gene expression in response to deprivation of the essential nutrient nitrogen, with a focus on the nitrogen fixation (nif) genes and other genes required for heterocyst development. Cyanobacteria are important and ubiquitous microalgae that are primary producers in diverse environments including fresh water and the oceans. Cyanobacteria fix carbon dioxide by oxygenic photosynthesis (similar to plants) for their growth. Some species are also capable of obtaining nitrogen from air by nitrogen fixation, which occurs in specialized cells called heterocysts. As primary producers, nitrogen-fixing cyanobacteria provide organic compounds and nitrogen to their natural environments and they are also used as low-cost nitrogen fertilizer, which is increasingly important as energy cost rise. In addition to their ecological importance, there is great interest in using cyanobacteria to produce renewable biomass, biochemicals, biofuels, and hydrogen. New knowledge on the basic cellular mechanisms that control gene expression and metabolism in these organisms will enhance our understanding of their growth and how they respond to nutritional and other challenges, and will provide information that can be applied to emerging biotechnology fields related to carbon sequestration and renewable production of commercial products. The nitrogen-fixing filamentous cyanobacterium Anabaena is a well-established model organism used to study cyanobacterial genetics, physiology, and developmental biology. Anabaena fixes nitrogen in heterocysts, which are required to protect the nitrogenase enzymes from oxygen, which is produced by photosynthesis. A developmental pattern of heterocysts spaced along filaments of vegetative cells, like beads on a string, forms a simple multicellular organism. This project has contributed directly to the scientific education and training of five undergraduate students, five graduate students, and two postdoctoral scientists. In addition, project personnel have presented the results of this project to students in university classes, and in science outreach programs for high-school teachers, interested community members, and elementary-school students. Four major scientific findings resulted from this research project. One, a successful gfp reporter-gene construct for the nitrogen-fixation gene cluster was designed and tested. This new genetic tool was then used to study the regulated expression of the expression of the nitrogen fixation genes and led to the unexpected result that expression required DNA sequences (the nifB promoter) present far away from the major nitrogen fixation genes. This result can be exploited in the future to identify regulatory DNA sequences and protein factors that regulate the nitrogen fixation genes during heterocyst differentiation. Two, the sigE gene, which encodes a DNA expression regulator called a sigma factor, is required for expression of the nitrogen fixation genes as well as other heterocyst-specific genes. These data are the first that link a specific sigma factor with expression of heterocyst-specific genes. Three, we have examined the expression of all Anabaena genes by a technique called directional RNA-seq to study response of cells to nitrogen deprivation. These systems-level data have provided information for the entire genome on mRNA levels (products of gene expression), the mRNA start and end points, operon structure (groups of genes), and noncoding and antisense RNAs (new types of genetic elements). And four, HetR ChIP-seq, a method to examine how proteins interact with genes to control their activity, has allowed us to produce improved information on how genes are regulated during heterocyst development and nitrogen fixation.
