Red Layer Microbial Publications & Presentations Published and Rejected Research Manuscripts pdf Links – Most Recent to Oldest

	Undergraduate Honors Thesis 2006: Attempts to Cultivate Red Layer Bacteria Using Site-Specific Water Chemistry Data Terrance Manning II; Advisor: Dr. Sarah Boomer; Second Reader: Dr. Irja Galvan. Abstract My research focuses on novel filamentous red Chloroflexi bacteria that form mats in Yellowstone hot springs (pH 7.5-9, 37-65°C), Red Layer Microbial Observatory (RLMO) sites. Based on 16S rRNA sequence analysis, red Chloroflexi bacteria are 92-95% similar to Roseiflexus castenholzii, a red filamentous phototroph isolated from comparable hot springs in Japan. Despite these similarities, culturing of Yellowstone red Chloroflexi using medium PE, which was used to culture Roseiflexus, has been unsuccessful. Given that RLMO sites showed distinct water chemistry profiles (using 15 salts and metals), I designed media based on these data, focusing on one RLMO site, Hillside Springs (pH 8, 54°C). I hypothesized that (A) the growth of red Chloroflexi bacteria would be dependent on chemical differences at that are site-specific, (B) a different array of microbial isolates would be retrieved using each medium, and (C) media made using site-specific chemical data would support more diversity than broad-based media such as medium PE. Hillside red Chloroflexi samples were inoculated into: (1) site-specific media plus high yeast extract (0.1 g/L), (2) site-specific media plus low yeast (0.01 g/L), (3) Roseiflexus media (medium PE), (4) and sterilized source water from Hillside Springs. Growth was assessed once a week for four weeks. Microscopic, pigment, DGGE, and 16S rRNA sequence analyses were used to compare diversity enriched by each medium. Although site-specific media did not support the culture of target red Chloroflexi bacteria, these media enriched for different populations of new Proteobacteria, Gram Positives, and green Chloroflexi. Medium PE enriched for new Proteobacteria, Gram Positives, and Nitrospira - but not any red Chloroflexi. Sterilized source water supported the most visible growth of red filaments. Additionally, a co-culture derived from source water enrichments retrieved Chloroflexus species 396-1, a Yellowstone green Chloroflexi. Given that the use of sterilized source water media appeared to be the most promising, the potential environmental impact of source water-based cultivation could be substantial; thus, future cultivation studies should include improved research into the composition of the source water and how to more closely replicate it.
	Red Layer Microbial Observatory Database: A Model for the Integration and Dissemination of Biological and Geochemical Data via the World Wide Web Sarah M. Boomer,Will M. Drury, Bryan E. Dutton, Daniel P. Lodge, Melissa S. Boschee, William M. Kernan Abstract The Red Layer Microbial Observatory (RLMO) aims to survey and compare alkaline mat communities throughout Yellowstone National Park, all of which contain distinct layers of red, filamentous bacteria we have identified as atypical red Chloroflexi using 16S rRNA, pigment spectroscopy, and microscopic methods. The RLMO Database Project is a web-accessible OracleÔ database that integrates physical, chemical, and molecular data from RLMO sites. This application, in its second year of development, was written using PL/SQL, HTML, and Javascript. Each research site in this study is assigned a unique identifier that is linked to the following tables: Geochemical Data (pH, 15 common salts and metals); DNA Sequence Data (16S clone name, GenBank-linked accession number, BLAST-inferred identity); Macroscopic and Microscopic Image Data; and Student Collection Team Information. Using the administrative URL, all data can be entered and edited through restricted web-access. Using the public URL, users can view and query data. Site Query results can be formatted to display any combination of geochemical parameters across one or more years and/or sites. Sequence Query results can be formatted to display inferred identity and GenBank-linked accession numbers across one or more sites. The RLMO Database, in its current form, is designed to accommodate physical, chemical, and molecular information as part of a five-year study to address whether observed variation in geothermal ground water chemistry affects the population distribution of genetically distinct strains of Chloroflexi in red layer communities. This chapter for the First Biannual Workshop on Geothermal Biology and Geochemistry Proceedings is currently in press (June 2005).
	A Survey-Driven Study of Microbial Mats in Joseph’s Coat Thermal Basin, Yellowstone National Park Daniel P. Lodge, Sarah M. Boomer, Bryan E. Dutton Abstract We comprehensively surveyed thermal features in Joseph’s Coat Basin, identifying two red layer communities among 155 aqueous sites. We compared 16S rRNA libraries amplified using two primer sets (R-GNS- and Chloroflexacaea-specific) from these sites, demonstrating novel primer amplification and bias. Analyzing survey and molecular data allowed us to define sub-regions within the basin and suggest potential dispersal patterns. This was rejected by AEM in November 2003. Reviewers felt the data was interesting but too preliminary. Given that we are not able to return Joseph’s Coat, we are posting results on-line.
	Molecular Characterization of Novel Red Green Nonsulfur Bacteria from Five Distinct Hot Spring Communities in Yellowstone National Park Sarah M. Boomer, Daniel P. Lodge,Bryan E. Dutton,Beverly Pierson Abstract We characterized and compared five geographically isolated hot springs with distinct red-layer communities in Yellowstone National Park. Individual red-layer communities were observed to thrive in temperatures ranging from 35 to 60°C and at pH 7 to 9. All communities were dominated by red filamentous bacteria and contained bacteriochlorophyll a (Bchl a), suggesting that they represented novel green nonsulfur (GNS) bacteria. The in vivo absorption spectra of individual sites were different, with two sites showing unusual Bchl a protein absorption bands beyond 900 nm. We prepared and analyzed 16S rRNA libraries from all of these sites by using a combination of general bacterial primers and new GNS-specific primers described here. These studies confirmed the presence of novel GNS-like bacteria in all five communities. All GNS-like clones were most similar to Roseiflexus castenholzii, a red filamentous bacterium from Japan that also contains only Bchl Phylogenies constructed by using GNS-like clones from Yellowstone red-layer communities suggest the presence of a moderately diverse new “red” cluster within the GNS lineage. Within this cluster, at least two well-supported subclusters emerged: YRL-A was most similar to Roseiflexus and YRL-B appeared to be novel, containing no known isolates. While these patterns showed some site specificity, they did not correlate with observed Bchl a spectrum differences or obvious features of the habitat. This was published in the ASM Journal of Applied & Environmental Microbiology in 2002.
	Characterization of novel bacteriochlorophyll-a-containing red filaments from alkaline hot springs in Yellowstone National Park Sarah M. Boomer,Beverly K. Pierson, Ruthann Austinhirst, Richard W. Castenholz Abstract Novel, red, filamentous, gliding bacteria formed deep red layers in several alkaline hot springs in Yellowstone National Park. Filaments contained densely layered intracellular membranes and bacteriochlorophyll a. The in vivo absorption spectrum of the red layer filaments was distinct from other phototrophs, with unusual bacteriochlorophyll a signature peaks in the near-infrared (IR) region (807 nm and 911 nm). These absorption peaks were similar to the wavelengths penetrating to the red layer of the mets as measured with in situ spectroradiometry. The filaments also demonstrated maximal photosynthetic uptake of radiolabeled carbon sources at these wavelengths. The red layer filaments displayed anoxygenic photoheterotrophy, as evidenced by the specific incorporation of acetate, not bicarbonate, and by the absence of oxygen production. Photoheterotrophy was unaffected by sulfide and oxygen, but was diminished by high-intensity visible light. Near-IR radiation supported photoheterotrophy. Morphologically and spectrally similar filaments were observed in several springs in Yellowstone National Park, including Octopus Spring. Taken together, these data suggest that the red layer filaments are most similar to the photoheterotroph, Heliothrix oregonensis. Notable differences include mat position and coloration, absorption spectra, and prominent intracellular membranes. This scanned article was published in Archives of Microbiology in 2000.