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 degrees 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.

FIG. 1.

(Panel I) Schematic map of Yellowstone National Park indicating locations of research sites. (Panel II) Representative light micrograph of the Spray Geyser red layer. The bar represents 20 μm. (Panel III) Site photographs of Hillside II Spring mat (A1) and core (A2), Witch Pond mat (B1) and core (B2), Fairy Spring mat (C1) and core (C2), Shoshone/Western Pool mat (D1) and core (D2), and Spray Geyser mat (E1) and core (E2). The arrows at the right indicate the red layer of each core.

FIG. 2.

Maximum parsimony tree of Yellowstone red-layer GNS-like clones. Representative GNS organisms are indicated in italics, with the GenBank accession number in parentheses. Red-layer clones are signified by site and clone number (abbreviations: Hillside II [Hs2], Witch Pond [Witch], and Shoshone/Western Pool [Shosh]). Three significant clusters are shown in brackets and are labeled YRL-A, YRL-B, and YRL-B/Spray. The bar represents 10 nucleotide changes. In this analysis, there were 980 total characters, and 353 were usable for parsimony analysis. A total of 100 bootstrap replicates were performed, and the bootstrap values are indicated (those that were <50% are not shown).

FIG. 3.

(A) Secondary structure prediction of Yellowstone red-layer GNS-like clones. Alignments were analyzed in conjunction with the secondary structure of T. roseum (B). In this analysis, three areas were found that corresponded to significant phylogenetic clustering (see Fig. 2): YRL-A, YRL-B, and YRL-B/Spray. These three areas are boxed in panel B and labeled 1, 2, and 3. In panel A, these areas are enlarged for T. roseum, and the corresponding predicted areas for the three clusters are shown.