Gene Regulation in Chromophyte Algae.
We are interested in environmental signals that trigger gene expression leading to chloroplast development in chromophyte algae. Although this process is well characterized in green plants, it is virtually untouched in chromophytes (a general term for eukaryotic algae with red, yellow or brown chloroplasts). We are fortunate to have a nearly ideal model system for this investigation: a unicellular mixotrophic chrysophyte called Poterioochromonas malhamensis, which we can grow heterotrophically in the dark, autotrophically in the light, or anything in between. This gives us the capability to control chloroplast development, allowing us unusual access to the process of gene induction. Thus far, we have concentrated on chloroplast genes (and will continue to do so), but we are presently expanding our approach by beginning a study of nuclear genes involved in this process, as well. (Although chloroplast genomes in all photosynthetic eukaryotes studied thus far encode approximately 100-150 gene products essential to chloroplast function, a far greater number are encoded by the nucleus.) Our findings in the chloroplast indicate that a large number of protein-coding genes are expressed only at very low levels in the dark, and are significantly up-regulated in the light. Light is not the solitary trigger for gene induction, however. Although light is required for high level expression of these genes, carbon starvation is also required. As mixotrophs, these algae are readily capable of taking up glucose as their primary carbon source, and in fact grow at a much higher rate on glucose than they do photosynthetically. Only when carbon becomes limiting do they begin the process of chloroplast development. Current efforts are directed toward characterization of chloroplast promoter regions and the kinetics of chloroplast mRNA accumulation under varying growth conditions, as well as cDNA cloning to attempt to identify nuclear genes expressed preferentially under heterotrophic or autotrophic conditions.
Molecular Biology and Evolution of Bacterial Bioluminescence.
We have for quite some time been interested in the molecular biology and evolution of bacterial bioluminescence. The ability to produce light is widespread in nature, having evolved separately a number of times in organisms as diverse as bacteria, fungi, dinoflagellates, and animals. The bacterial process is restricted to a phylogenetically narrow group in the Gamma Proteobacteria. Only five genes (luxA,B,C,D,and E, which are arranged in an operon) are required to bestow light-generating capability on bacteria. (This was originally demonstrated by Engebrecht et. al. in 1983, by cloning those five genes into E. coli, giving it the capability to glow.) We have done a lot of work on the evolution of the lux operon, carrying out phylogenetic analysis of the luxA gene as well as characterization of the entire operon from Vibrio and Photobacterium species. In addition, we have characterized the lux operon of a taxonomic outlier, Shewanella woodyi, which apparently acquired its lux genes by lateral transfer. More recently, we are moving into the area of lux gene regulation. All lux operons characterized thus far are regulated by a quorum sensing (i.e., cell density-dependent) system. Although the quorum sensing story is still unfolding in several labs, the present understanding is that there are at least two separate quorum sensing systems regulating bacterial bioluminescence. We have become increasingly interested in the evolution of these regulatory systems, the basic question being: "does the evolution of quorum sensing systems in bioluminescent bacteria mirror that of the lux operon itself?" We are presently characterizing promoter regions and regulatory genes in Vibrio and Photobacterium species, in an attempt to discern an evolutionary pattern.
Molecular Characterization of Thermophilic Microbial Communities in Freshwater Hydrothermal Vents.
Yellowstone Lake sits on the mid-continental geothermal hot spot and contains many submerged hydrothermal vent systems. Although a large body of knowledge has accumulated on hot spring microbial communities in Yellowstone National Park, the more difficult access to submerged vents has precluded extensive characterization of microbial communities associated with them. We are working with several other investigators on a multidisciplinary approach to characterizing these submerged vent communities. Microbial habitats throughout the Yellowstone Lake hydrothermal vent system are constrained by vastly different nutrient and chemical inputs. Furthermore, microbial communities in the vent systems are subjected to changing flow rates of hydrothermal fluids. Because different vent fields exhibit dissimilar physical and chemical characteristics, we hypothesize that these differences will be reflected in the composition of microbial communities associated with these vents. We are testing this hypothesis by examining 16S rDNA diversity to elucidate community structure. In addition, we are using primers designed to amplify specific metabolic genes, in order to assess the genetic potential of the communities to carry out various types of metabolism.
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