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 a. 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.The green nonsulfur (GNS) lineage has long been recognized as an evolutionarily and environmentally significant group of bacteria (12, 16). Representative GNS isolates include Chloroflexus, Chloronema, and Oscillochloris spp., which are all filamentous phototrophs that use bacteriochlorophyll (Bchl) c and Bchl a (17). However, the GNS lineage contains several members that defy the traditional "green" GNS type strain phenotype. These include chemotrophic genera, such as Herpetosiphon and Thermomicrobium (10, 16), as well as phototrophic Heliothrix and Roseiflexus, both of which use only Bchl a and appear orange and red, respectively (8,20). Recently, several dozen uncultivated GNS-like sequences, all inferred chemotrophs, were redesignated as members of TM7, a proposed candidate division separate from the GNS (11, 13). Similar studies of Herpetosiphon isolates now suggest comparable novel clustering within this atypical GNS group (24).In contrast with studies and information about chemotrophic GNS bacteria and GNS-like sequences, there is a relative paucity of comparable molecular diversity studies about phototrophic GNS organisms and sequences in natural environments. Improving our knowledge of diversity among traditional GNS should better elucidate the relationship between chemotrophs, "green" phototrophs, and "red-orange" phototrophs, a question with broad implications for better understanding the evolution of photosynthesis. In particular, the limited number of Bchl a-containing GNS representives in the database makes it difficult to assess whether these phototrophs represent ...

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We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods. Molecular biology courses typically include laboratory components that enable students to learn methods in DNA analysis. Many curricula utilize defined materials that lead students through the process of verifying previous results. One such text-based curriculum involves cloning and transferring lux genes from Vibrio fischeri to Escherichia coli (12). Given the immense microbial diversity in most environments and the simplicity of molecular procedures afforded by straight-forward kits and equipment, we implemented a research-driven curriculum for our molecular biology course that is based on analyzing 16S rRNA genes (13). We contend that this research-based approach can be modified to any sample source to elucidate microbial diversity that relates to medical, applied, or environmental issues. DNAbased methods are even more appropriate because traditional culture-based approaches have been estimated to detect as little as 1% of bacteria from many environments (1, 9). Applications using rRNA information include inferring the identity of novel sequences, predicting metabolic lifestyles for organisms that are not amenable to pure culture, and improving media development (7, 9).The goal of our particular research project is to improve our understanding of unusual and as yet uncultured red filamentous Green Nonsulfur (GNS) phototrophs from hot springs in Yellowstone National Park. Based on the approach of Woese, students analyzed and compared novel bacterial 16S rRNA genes from red bacterial communities against available DNA databases to make predictions about microbial identity, diversity, and metabolism (13). They described novel sequences, expanding our collective understanding of microbial diversity. Student involvement in this project has led to research publications, presentations, and funding opportunities that have supported the acquisition of major course equipment. Reciprocally, these outcomes have fostered an increase in biology majors earning research-oriented molecular...

We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.