Distribution and Activity of Sulfur-Metabolizing Bacteria along the Temperature Gradient in Phototrophic Mats of the Chilean Hot Spring Porcelana

Abstract

In terrestrial hot springs, some members of the microbial mat community utilize sulfur chemical species for reduction and oxidization metabolism. In this study, the diversity and activity of sulfur-metabolizing bacteria were evaluated along a temperature gradient (48-69 °C) in non-acidic phototrophic mats of the Porcelana hot spring (Northern Patagonia, Chile) using complementary meta-omic methodologies and specific amplification of the aprA (APS reductase) and soxB (thiosulfohydrolase) genes. Overall, the key players in sulfur metabolism varied mostly in abundance along the temperature gradient, which is relevant for evaluating the possible implications of microorganisms associated with sulfur cycling under the current global climate change scenario. Our results strongly suggest that sulfate reduction occurs throughout the whole temperature gradient, being supported by different taxa depending on temperature. Assimilative sulfate reduction is the most relevant pathway in terms of taxonomic abundance and activity, whereas the sulfur-oxidizing system (Sox) is likely to be more diverse at low rather than at high temperatures. Members of the phylum Chloroflexota showed higher sulfur cycle-related transcriptional activity at 66 °C, with a potential contribution to sulfate reduction and oxidation to thiosulfate. In contrast, at the lowest temperature (48 °C), Burkholderiales and Acetobacterales (both Pseudomonadota, also known as Proteobacteria) showed a higher contribution to dissimilative sulfate reduction/oxidation as well as to thiosulfate metabolism. Cyanobacteriota and Planctomycetota were especially active in assimilatory sulfate reduction. Analysis of the aprA and soxB genes pointed to members of the order Burkholderiales (Gammaproteobacteria) as the most dominant and active along the temperature gradient for these genes. Changes in the diversity and activity of different sulfur-metabolizing bacteria in photoautotrophic microbial mats along a temperature gradient revealed their important role in hot spring environments, especially the main primary producers (Chloroflexota/Cyanobacteriota) and diazotrophs (Cyanobacteriota), showing that carbon, nitrogen, and sulfur cycles are highly linked in these extreme systems.

Keywords: aprA; metagenomics; phototrophic hot spring mat; soxB; sulfur oxidation/reduction.

Figure 1

Abundance of sulfur metabolism genes in Porcelana hot spring metagenomes. Genes from the sulfur cycle are grouped based on the pathway ((A,C) for ASR; (B,D) for DSR; and (E) for Sox) and at which step of the sulfur cycle reaction they are involved ((A,B) for sulfide to sulfite; (C,D) for sulfite to sulfate; (E) for sulfate to thiosulfate). Orders with their respective phyla are shown on the Y-axis and samples are shown on the X-axis. The reads counts are normalized by quantiles and Log2-transformed for metagenomes from the three sampled temperatures (48, 58, and 66 °C). Analyzed genes are listed in Table S3. * C denote “Candidatus”.

Figure 2

Activity of sulfur metabolism genes in Porcelana hot spring metatranscriptomes. The genes from the sulfur cycle are grouped based on the pathway ((A,C) for ASR; (B,D) for DSR; and (E) for Sox) and at which step of the sulfur cycle reaction they are involved ((A,B) for sulfide to sulfite; (C,D) for sulfite to sulfate; (E) for sulfate to thiosulfate). Orders with their respective phyla are shown on the Y-axis and samples are shown on the X-axis. The read counts are normalized by quantiles and Log2-transformed for metatranscriptomes from the three sampled temperatures (48, 58, and 66 °C). Analyzed genes are listed in Table S3. * C denote “Candidatus”.

Figure 3

Phylogenetic reconstruction of the aprA marker gene. Maximum-likelihood tree reconstruction for the alignment of 307 sequences that include 275 CDS from the GTDB R95 protein database, 26 CDS from the Porcelana metagenomes (blue circles), and 6 clone library aprA OTUs (blue triangles). The tree reconstruction was performed with IQtree software using the LG + R9 substitution model and a non-parametric UF-bootstrap support of 1000 replicates. The leaf labels are colored according to GTDB phyla, and specifically by class for sequences from the phylum Pseudomonadota (Proteobacteria). The tree was rooted in the node between a clade of 69 outgroup sequences (fumarate reductase/succinate dehydrogenase flavoprotein subunit) and a separate clade of 16 Gammaproteobacteria sequences and 30 Cyanobacteriota sequences. Purple dots in the tree represent > 95% bootstrap support for that node.

Figure 4

Phylogenetic reconstruction of the soxB marker gene. Maximum-likelihood tree reconstruction for the alignment of 246 sequences that include 189 CDS from the GTDB R95 protein database, 50 CDS from the Porcelana metagenomes (blue circles), and two soxB OTUs from the clone libraries (blue triangles). The tree reconstruction was performed with IQtree software using the LG + F + R7 substitution model and a non-parametric UF-bootstrap support of 1000 replicates. The leaf labels are colored according to GTDB phyla, and specifically by class for sequences from the phylum Pseudomonadota (Proteobacteria). The tree was rooted in the node between a clade of 9 outgroup sequences (trifunctional nucleotide phosphoesterase protein YfkN) and the other 246 sequences. Purple dots represent > 95% bootstrap support for that node.

Figure 5

Graphical summary of the microbial community associated with the sulfur cycle at the Porcelana hot spring. In the left panel, a dot plot was built using the gene counts of each sulfur pathway (Table S4) for 8 different phyla and sulfur cycle steps. The size of the colored circle denotes abundance, and the color denotes the respective phylum. Temperature is shown on the Y-axis and sulfur cycle steps on the X-axis. The phylum names are written on the Y-axis, represented as a function of temperature and the sulfur cycle step where they appeared most relevant. The colored area denoting the most relevant Porcelana sulfur-associated bacteria (Pseudomonadota, Chloroflexota, and Cyanobacteriota) was hand-drawn behind the dots. In the central panel, concentrations of three sulfur species are shown based on field-measured concentrations (Table S2). In the right panel, the abundance level of photosynthesis-associated genes the of phyla Cyanobacteria, Chloroflexota, and Pseudomonadota. Temperature is shown on the Y-axis and concentration on the X-axis. * Based on data obtained from Alcamán-Arias et al. [36].