Geographic and Ecological Diversity of Green Sulfur Bacteria in Hot Spring Mat Communities

Abstract

Three strains of thermophilic green sulfur bacteria (GSB) are known; all are from microbial mats in hot springs in Rotorua, New Zealand (NZ) and belong to the species Chlorobaculum tepidum. Here, we describe diverse populations of GSB inhabiting Travel Lodge Spring (TLS) (NZ) and hot springs ranging from 36.1 °C to 51.1 °C in the Republic of the Philippines (PHL) and Yellowstone National Park (YNP), Wyoming, USA. Using targeted amplification and restriction fragment length polymorphism analysis, GSB 16S rRNA sequences were detected in mats in TLS, one PHL site, and three regions of YNP. GSB enrichments from YNP and PHL mats contained small, green, nonmotile rods possessing chlorosomes, chlorobactene, and bacteriochlorophyll c. Partial 16S rRNA gene sequences from YNP, NZ, and PHL mats and enrichments from YNP and PHL samples formed distinct phylogenetic clades, suggesting geographic isolation, and were associated with samples differing in temperature and pH, suggesting adaptations to these parameters. Sequences from enrichments and corresponding mats formed clades that were sometimes distinct, increasing the diversity detected. Sequence differences, monophyly, distribution patterns, and evolutionary simulation modeling support our discovery of at least four new putative moderately thermophilic Chlorobaculum species that grew rapidly at 40 °C to 44 °C.

Keywords: Chlorobaculum tepidum; bacteriochlorophyll c; biogeographic distribution; chlorosomes; ecological species; photosynthesis; thermophile.

Figure 1

Locations sampled are (A) New Zealand (green star) and the Philippines (red star), and (B) Yellowstone National Park, USA (blue stars).

Figure 2

Sites where mats containing green sulfur bacteria were found were in New Zealand and the Philippines. (A) Dick Castenholz at Travel Lodge Stream, Rotorua, NZ, in 1986. (B) Core of the Travel Lodge Spring mat. (C) Lipayo hot spring, the Philippines. (D) Thin yellowish-green veil of GSB near the shore at site Lipayo 5.

Figure 3

Sites where mats containing green sulfur bacteria were found in Yellowstone National Park. (A) “Big Spring.” The arrows show the collection points where GSB mats were found in this pool. (B) Comparison of the GSB mat and a cyanobacterial mat at site BS1. (C) The effluent stream originating from Obsidian Pool (OP) in the Mud Volcano region. Sites where mats containing GSB were found are indicated by the arrows. SP, side pool, where sites MV1 and MV2 were located in 2000 and sites MV4 and MV5 were located in 2001 (see panel F). (D) Site MV3 bubbling spring and GSB mat. Note the 25 mm coin for scale (unlabeled black arrow). (E) Pool at the source of site MV6 with numerous bubbling springs with mats containing GSB. Scale bar (25 mm) (F) Shallow side pool (SP) and sediment covered with a GSB mat in 2001. Source of samples MV4 and MV5; the same pool was the source of samples MV1 and MV2 in 2000, when they were discrete mats. Note the 25 mm coin for scale (unlabeled black arrow).

Figure 4

Whole-cell absorbance spectra from GSB enrichments. (A) From Lipayo 5, grown at 35 °C. (B) From sites MV1 and BS3.2M, grown at 44 °C. The absorbance peaks at 753 nm and 756 nm are due to BChl c aggregates in chlorosomes. Absorbance peaks at 456–458 are due to chlorobactene. Spectra for all enrichments were taken and showed the characteristic peaks for BCl c and chlorobactene.

Figure 5

Thin-section electron micrographs show chlorosomes in cells from GSB enrichments from YNP hot springs. (A,B) cells from site BS1 enrichment. (C,D) cells from site BS3.2M enrichment. (E,F) cells from site MV4 enrichment. Arrows show examples of well-resolved chlorosomes, which are the more lightly-stained, irregular, sac-like objects closely appressed to the inner surface of the cytoplasmic membranes. The magnification for all micrographs was 80,000×. The size bars represent 250 nm.

Figure 6

A phylogenetic tree of 16S rRNA gene sequences shows relationships among hot spring variants and cultivated Chlorobaculum strains. The tree was rooted by sequences of several Chlorobium strains. Sequence labels include site, year collected (00, 2000; 02, 2002), whether from mat (MAT) or enrichment (ENR), temperature grown at (enrichments only), sequence designation, number of copies of the sequence (if more than 1), sequence length (nt), and accession number. Sequences and circles indicating association with mat (closed) or enrichment (open) are color-highlighted based on the samples from which they were derived. Colored vertical bars and associated text show relationships of clades to mat and enrichment samples from different geographic regions and environmental conditions. Black vertical bars represent putative ecotypes/geotypes demarcated by Ecotype Simulation 2 analysis. Numbers at nodes are bootstrap values from 100 replicates. The scale bar indicates the number of substitutions per nt position.

Figure 7

The expanded phylogeny of the YNP-2 clade is shown in Figure 6. Sequence labels include site, year collected (00, 2000; 02, 2002), whether from mat (MAT, closed circles) or enrichment (ENR, open circles), temperature grown at (enrichments only), sequence designation, number of copies of the sequence (if more than 1), sequence length (nt), and accession number. Sequences and circles indicating association with mat (closed) or enrichment (open) are color-highlighted based on the samples from which they were derived. Blue stars highlight sequences that came from the site YNP-1-BS3.1/3.2 mat (filled) and enrichment (unfilled) samples. Colored vertical bars and associated text show relationships of clades to mat and enrichment samples from springs with different environmental conditions. Black vertical bars represent putative ecotypes/geotypes demarcated by Ecotype Simulation 2 analysis. Numbers at nodes are bootstrap values from 100 replicates. The scale bar indicates the number of substitutions per nt position.