Diurnal Changes in Active Carbon and Nitrogen Pathways Along the Temperature Gradient in Porcelana Hot Spring Microbial Mat

Abstract

Composition, carbon and nitrogen uptake, and gene transcription of microbial mat communities in Porcelana neutral hot spring (Northern Chilean Patagonia) were analyzed using metagenomics, metatranscriptomics and isotopically labeled carbon (H¹³CO₃) and nitrogen (¹⁵NH₄Cl and K¹⁵NO₃) assimilation rates. The microbial mat community included 31 phyla, of which only Cyanobacteria and Chloroflexi were dominant. At 58°C both phyla co-occurred, with similar contributions in relative abundances in metagenomes and total transcriptional activity. At 66°C, filamentous anoxygenic phototrophic Chloroflexi were >90% responsible for the total transcriptional activity recovered, while Cyanobacteria contributed most metagenomics and metatranscriptomics reads at 48°C. According to such reads, phototrophy was carried out both through oxygenic photosynthesis by Cyanobacteria (mostly Mastigocladus) and anoxygenic phototrophy due mainly to Chloroflexi. Inorganic carbon assimilation through the Calvin-Benson cycle was almost exclusively due to Mastigocladus, which was the main primary producer at lower temperatures. Two other CO₂ fixation pathways were active at certain times and temperatures as indicated by transcripts: 3-hydroxypropionate (3-HP) bi-cycle due to Chloroflexi and 3-hydroxypropionate-4-hydroxybutyrate (HH) cycle carried out by Thaumarchaeota. The active transcription of the genes involved in these C-fixation pathways correlated with high in situ determined carbon fixation rates. In situ measurements of ammonia assimilation and nitrogen fixation (exclusively attributed to Cyanobacteria and mostly to Mastigocladus sp.) showed these were the most important nitrogen acquisition pathways at 58 and 48°C. At 66°C ammonia oxidation genes were actively transcribed (mostly due to Thaumarchaeota). Reads indicated that denitrification was present as a nitrogen sink at all temperatures and that dissimilatory nitrate reduction to ammonia (DNRA) contributed very little. The combination of metagenomic and metatranscriptomic analysis with in situ assimilation rates, allowed the reconstruction of day and night carbon and nitrogen assimilation pathways together with the contribution of keystone microorganisms in this natural hot spring microbial mat.

Keywords: Cyanobacteria; carbon and nitrogen assimilation; metagenomics; metatranscriptomics; microbial mat; neutral hot spring; photosynthesis.

FIGURE 1

Porcelana hot spring and the pigmented microbial mats growing along the temperature gradient. Sampling sites are indicated by squares: 66°C (orange), 58°C (orange + green) and 48°C (green).

FIGURE 2

Taxonomic assignment at the Phylum level of metagenomic (DNA; white bars) and metatranscriptomic reads (cDNA day: gray bars; cDNA night: black bars) from samples at the three temperatures studied. (A) Percent of Bacteria reads assigned to the most abundant Phyla. (B) Percent of Archaea reads assigned to the most abundant Phyla.

FIGURE 3

Phototrophy in Porcelana mat. (A) Oxygenic (psbA and psaA genes) and (B) Anoxygenic (pufM, fmoA, pscA, and bchC genes) photosynthesis. Cartoons in the middle part show the selected photosynthetic genes. Bar graphs show the percent of the total transcripts of each gene found in each sample, during the day (white bars) and night (black bars) at each temperature. The lower horizontal bars show the percent contribution of the main taxa to transcription for each process at each temperature. The genes considered are shown above the bars.

FIGURE 4

Autotrophy in Porcelana mat. (A) Calvin–Benson–Bassham cycle (rbcL gene); (B) 3-hydroxypropionate bi-cycle (blue arrows: mcl, mcr, and prpE genes), and hydroxypropionate-hydroxybutyrate cycle (yellow arrows: atoB, crt, abfD, sucD genes). Green arrows show the steps shared by the two pathways in Bacteria and Archaea. Bar graphs show the percent of the total transcripts of each gene found in each sample, during the day (white bars) and night (black bars) at each temperature. The lower horizontal bars show the percent contribution of the main taxa to transcription for each process at each temperature.

FIGURE 5

Nitrogen cycle in Porcelana mat. (A) Scheme of the reactions, representative genes, and transcriptional activity for each gene at the specific pathway. Bar graphs show the percent of the total transcripts of each gene found in each sample, during the day (white bars) and night (black bars) at each temperature. (B) Percent contribution of the main Phyla to transcription for each gene is shown by the horizontal bars. The most important genera in each case are noted to the right.

FIGURE 6

Reconstruction of the diurnal changes in active carbon and nitrogen pathways, based in metatranscriptomics analysis, along the temperature gradient in Porcelana microbial mat dominated by Chloroflexi and Cyanobacteria (pie charts). The white (day) and black (night) arrow width represent the total gene transcripts to each pathway and indicates the importance of these at each temperature.