Molecular Characterization of Novel Red Green Nonsulfur Bacteria from Five Distinct Hot Spring Communities in Yellowstone National Park

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 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 ...

Cultivation and Genomic, Nutritional, and Lipid Biomarker Characterization of Roseiflexus Strains Closely Related to Predominant In Situ Populations Inhabiting Yellowstone Hot Spring Microbial Mats

“…Nübel et al (35) suggested the possibility of temperature-adapted strains of Roseiflexus spp. based on differences in phylogeny and temperature distribution of SSU rRNA sequences cloned from hot spring mats (6,16). Differences in light absorption between strains RS1 and RS2 may also reflect differential adaptation that might help explain the cooccurrence of many Roseiflexus-like SSU rRNA sequences at a single mat location.…”

Chlorobium tepidum Mutant Lacking Bacteriochlorophyll c Made by Inactivation of the bchK Gene, Encoding Bacteriochlorophyll c Synthase

“…Such bacteria could thrive in sulfide-rich environments similar to those preferred by green sulfur bacteria except that they would possibly require a much higher light intensity to sustain reasonable growth rates. Interestingly, such bacteria that are phylogenetically related to the green filamentous bacteria have already been identified and isolated (4,17,29). Heliothrix oregonensis (29) and Roseiflexus castenholzii (17) are photoheterotrophs isolated from hot springs in Oregon and Japan, respectively.…”

Microscopic Examination of Distribution and Phenotypic Properties of Phylogenetically Diverse Chloroflexaceae -Related Bacteria in Hot Spring Microbial Mats

“…1), and assumed that these were filamentous phototrophs since they were recovered from Bchl-rich mat layers (7). Interestingly, they observed some site-specific phylogenetic clustering and did not detect any type C-like organisms in their samples (7). It will be interesting to unravel the environmental conditions that govern the differential compositions of these microbial mats, which are still little understood.…”

“…Recently, related 16S rRNA gene sequences from low-temperature Yellowstone hot springs and small geyser splash zones containing thick mats exhibiting distinct red layers (clusters YRL-A and YRL-B in Fig. 1) have been reported (7).…”