Methylobacterium extorquens AM1 is a methylotrophic bacteria ubiquitous in the environment and in particular on the undersides of leaves. This organism is able to metabolize both single carbon compounds like methanol and multi-carbon compounds such as succinate and pyruvate. M. extorquens has been studied for decades because of its potential for not only creating value added compounds from methanol, but important regulatory systems including the regulatory pathway of methanol metabolism, which has yet to be fully elaborated. Our laboratory focuses on single carbon compound metabolism and the regulation of the pathways required for growth. The first step in methylotrophic growth is the oxidation of methanol via methanol dehydrogenase encoded by the mxaFI genes. Promoter fusion data suggests a complex regulatory system involving two two-component systems MxbDM and MxcQE, two homologues of the main methanol dehydrogenase XoxF12, and an unknown transcriptional repressor. In order to elucidate this process, the direct binding of a transcriptional regulator to the mxaF promoter region must be demonstrated. Unfortunately all putative regulators have historically failed to show activity in vitro. In order to isolate direct regulators, whole cell extract will be washed over an abundance of the mxa promoter region that is bound to magnetic beads. Then using a magnet, the promoter region can be removed along with proteins that have bound from the cell extract, any non-specific binding proteins can be washed off using a low salt buffer, and specific binding proteins removed using a high salt buffer. The elutions containing the proteins of interest can be run out on an SDS PAGE gel, and the unique bands of interest excised and sent for mass spectrometry analysis for identification. Our work will define transcriptional regulators of the mxa operand, determining which specific proteins are binding the promoter region of that operand.

This project focuses on using microbiota to determine environmental changes in Puget Sound, specifically benthic foraminifera. Benthic foraminifera are single celled organisms that produce a shell and live at the bottom of the ocean. Because foraminifera have characteristic ecologic requirements, they can be used to document environmental changes over time. In addition, foraminifera’s short reproductive cycle (weeks to months) and species specific reactions to environmental changes enable high resolution studies of ecosystem health and reaction to mitigation efforts. By examining the density, diversity and assemblage composition of foraminifera in samples of sediment several determinations can be made about environment in the local region at the time the sediment was deposited. This study focuses on stations in Bellingham Bay, WA using 11 sediment samples taken between 1997 and 2011 by the Washington state Department of Ecology. From 2002 to 2006 a clear drop in foraminiferal density is observed in Bellingham Bay. This indicates a serious change in environment for the area during this period; changes that may include an increase or decrease in pollution, an increase in pH, or changing climatic conditions. In the Bellingham Bay area a very low species diversity is also observed. This lack of diversity can also indicate various climatic or other environmental conditions. For example, the most dominant species in these samples, Eggerella advena, is known to colonize areas of sewage pollution in other bays. By comparing the Bellingham Bay stations to others in Puget Sound, it will be possible to document environmental shifts over the last 15 years.

Puget Sound is home to countless aquatic species, all of whom are dependent on healthy water conditions. At the bottom of the trophic web are foraminifera—single celled organisms that directly reflect the environment they live in, because each species has a very narrow range of acceptable habitats. Foraminifera can be used to assess local conditions on a very fine scale, and to assess the large-scale health of the body of water. This type of study has been done on numerous bodies of water throughout the world and is well proven, but has never been applied to Puget Sound. As a small part of this overarching project, I focus on identifying and analyzing foraminifera from Port Madison, a part of Puget Sound relatively sheltered from anthropogenic impact. Port Madison thus acts as a control with which to compare samples from other locations in Puget Sound. Preliminary results indicate that Port Madison has a very high diversity of foraminifera with at least 12 different species immediately apparent, but a relatively low abundance of about 28 foraminifera per gram of sediment. In comparison with other locations throughout Puget Sound, this shows that anthropogenic conditions decrease the diversity of foraminifera, and areas like Port Madison where little anthropogenic activity is present yield higher diversity. Data from sheltered areas help build a knowledge base for a healthy Puget Sound, which in turn gives us an idea of what to work toward in future attempts to promote a healthier Puget Sound.

The harmful bloom-forming alga, Heterosigma akashiwo, has a dual-stage life cycle, transitioning between a vegetative, motile stage in the water column and a resting, non-motile stage in the sediments. Previous observations have revealed a large shift in neutral lipid content as cells transition between vegetative and resting life stages. The relative neutral lipid content per cell decreases as cells enter the resting stage, followed by a rapid synthesis of neutral lipids once cells emerge from the resting stage. Neutral lipid bodies are thought to be a source of cell energy reserves. The purpose of my research is to study how fatty acid profiles shift in H. akashiwo during these life cycle transitions. Understanding changes in fatty acid profiles is important in understanding how cells respond to changes in environmental conditions and how lipids are used in metabolic processes. In this study, two strains of H. akashiwo (CCMP 452 and UWC 13.03) are signaled to enter the resting stage by putting cell cultures into the dark at 10 °C for 18 days. Resting cells are activated by exposing the cultures to growth supporting conditions of light and increased temperature (12 °C). Cell viability and relative neutral lipid content is determined by staining with the SYTOX-Green DNA stain and the BODIPY505/515 neutral lipid stain, respectively, using flow cytometry. Fatty acid profiles are determined using an established gas chromatography- mass spectrometry method developed in the Cattolico laboratory. I will report on the outcomes of the fatty acid profiling for H. akashiwo over the alga’s dual-stage life cycle. I anticipate distinct change in the fatty acid profiles between these life stages.