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

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.

Dark Diazotrophy during the Late Summer in Surface Waters of Chile Bay, West Antarctic Peninsula.

Although crucial for the addition of new nitrogen in marine ecosystems, dinitrogen (N2) fixation remains an understudied process, especially under dark conditions and in polar coastal areas, such as the West Antarctic Peninsula (WAP). New measurements of light and dark N2 fixation rates in parallel with carbon (C) fixation rates, as well as analysis of the genetic marker nifH for diazotrophic organisms, were conducted during the late summer in the coastal waters of Chile Bay, South Shetland Islands, WAP. During six late summers (February 2013 to 2019), Chile Bay was characterized by high NO3− concentrations (~20 µM) and an NH4+ content that remained stable near 0.5 µM. The N:P ratio was approximately 14.1, thus close to that of the Redfield ratio (16:1). The presence of Cluster I and Cluster III nifH gene sequences closely related to Alpha-, Delta- and, to a lesser extent, Gammaproteobacteria, suggests that chemosynthetic and heterotrophic bacteria are primarily responsible for N2 fixation in the bay. Photosynthetic carbon assimilation ranged from 51.18 to 1471 nmol C L−1 d−1, while dark chemosynthesis ranged from 9.24 to 805 nmol C L−1 d−1. N2 fixation rates were higher under dark conditions (up to 45.40 nmol N L−1 d−1) than under light conditions (up to 7.70 nmol N L−1 d−1), possibly contributing more than 37% to new nitrogen-based production (≥2.5 g N m−2 y−1). Of all the environmental factors measured, only PO43- exhibited a significant correlation with C and N2 rates, being negatively correlated (p < 0.05) with dark chemosynthesis and N2 fixation under the light condition, revealing the importance of the N:P ratio for these processes in Chile Bay. This significant contribution of N2 fixation expands the ubiquity and biological potential of these marine chemosynthetic diazotrophs. As such, this process should be considered along with the entire N cycle when further reviewing highly productive Antarctic coastal waters and the diazotrophic potential of the global marine ecosystem.

Surface Ammonia-Oxidizer Abundance During the Late Summer in the West Antarctic Coastal System.

Marine ammonia oxidizers that oxidize ammonium to nitrite are abundant in polar waters, especially during the winter in the deeper mixed-layer of West Antarctic Peninsula (WAP) waters. However, the activity and abundance of ammonia-oxidizers during the summer in surface coastal Antarctic waters remain unclear. In this study, the ammonia-oxidation rates, abundance and identity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were evaluated in the marine surface layer (to 30 m depth) in Chile Bay (Greenwich Island, WAP) over three consecutive late-summer periods (2017, 2018, and 2019). Ammonia-oxidation rates of 68.31 nmol N L-1 day-1 (2018) and 37.28 nmol N L-1 day-1 (2019) were detected from illuminated 2 m seawater incubations. However, high ammonia-oxidation rates between 267.75 and 109.38 nmol N L-1 day-1 were obtained under the dark condition at 30 m in 2018 and 2019, respectively. During the late-summer sampling periods both stratifying and mixing events occurring in the water column over short timescales (February-March). Metagenomic analysis of seven nitrogen cycle modules revealed the presence of ammonia-oxidizers, such as the Archaea Nitrosopumilus and the Bacteria Nitrosomonas and Nitrosospira, with AOA often being more abundant than AOB. However, quantification of specific amoA gene transcripts showed number of AOB being two orders of magnitude higher than AOA, with Nitrosomonas representing the most transcriptionally active AOB in the surface waters. Additionally, Candidatus Nitrosopelagicus and Nitrosopumilus, phylogenetically related to surface members of the NP-ε and NP-γ clades respectively, were the predominant AOA. Our findings expand the known distribution of ammonium-oxidizers to the marine surface layer, exposing their potential ecological role in supporting the marine Antarctic system during the productive summer periods.

Proteorhodopsin Phototrophy in Antarctic Coastal Waters.

Microbial proton-pumping rhodopsins are considered the simplest strategy among phototrophs to conserve energy from light. Proteorhodopsins are the most studied rhodopsins thus far because of their ubiquitous presence in the ocean, except in Antarctica, where they remain understudied. We analyzed proteorhodopsin abundance and transcriptional activity in the Western Antarctic coastal seawaters. Combining quantitative PCR (qPCR) and metagenomics, the relative abundance of proteorhodopsin-bearing bacteria accounted on average for 17, 3.5, and 29.7% of the bacterial community in Chile Bay (South Shetland Islands) during 2014, 2016, and 2017 summer-autumn, respectively. The abundance of proteorhodopsin-bearing bacteria changed in relation to environmental conditions such as chlorophyll a and temperature. Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were the main bacteria that transcribed the proteorhodopsin gene during day and night. Although green light-absorbing proteorhodopsin genes were more abundant than blue-absorbing ones, the latter were transcribed more intensely, resulting in >50% of the proteorhodopsin transcripts during the day and night. Flavobacteriia were the most abundant proteorhodopsin-bearing bacteria in the metagenomes; however, Alphaproteobacteria and Gammaproteobacteria were more represented in the metatranscriptomes, with qPCR quantification suggesting the dominance of the active SAR11 clade. Our results show that proteorhodopsin-bearing bacteria are prevalent in Antarctic coastal waters in late austral summer and early autumn, and their ecological relevance needs to be elucidated to better understand how sunlight energy is used in this marine ecosystem. IMPORTANCE Proteorhodopsin-bearing microorganisms in the Southern Ocean have been overlooked since their discovery in 2000. The present study identify taxonomy and quantify the relative abundance of proteorhodopsin-bearing bacteria and proteorhodopsin gene transcription in the West Antarctic Peninsula's coastal waters. This information is crucial to understand better how sunlight enters this marine environment through alternative ways unrelated to chlorophyll-based strategies. The relative abundance of proteorhodopsin-bearing bacteria seems to be related to environmental parameters (e.g., chlorophyll a, temperature) that change yearly at the coastal water of the West Antarctic Peninsula during the austral late summers and early autumns. Proteorhodopsin-bearing bacteria from Antarctic coastal waters are potentially able to exploit both the green and blue spectrum of sunlight and are a prevalent group during the summer in this polar environment.

Fischerella thermalis: a model organism to study thermophilic diazotrophy, photosynthesis and multicellularity in cyanobacteria.

The true-branching cyanobacterium Fischerella thermalis (also known as Mastigocladus laminosus) is widely distributed in hot springs around the world. Morphologically, it has been described as early as 1837. However, its taxonomic placement remains controversial. F. thermalis belongs to the same genus as mesophilic Fischerella species but forms a monophyletic clade of thermophilic Fischerella strains and sequences from hot springs. Their recent divergence from freshwater or soil true-branching species and the ongoing process of specialization inside the thermal gradient make them an interesting evolutionary model to study. F. thermalis is one of the most complex prokaryotes. It forms a cellular network in which the main trichome and branches exchange metabolites and regulators via septal junctions. This species can adapt to a variety of environmental conditions, with its photosynthetic apparatus remaining active in a temperature range from 15 to 58 °C. Together with its nitrogen-fixing ability, this allows it to dominate in hot spring microbial mats and contribute significantly to the de novo carbon and nitrogen input. Here, we review the current knowledge on the taxonomy and distribution of F. thermalis, its morphological complexity, and its physiological adaptations to an extreme environment.

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

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.

Temperature modulates Fischerella thermalis ecotypes in Porcelana Hot Spring.

In the Porcelana Hot Spring (Northern Patagonia), true-branching cyanobacteria are the dominant primary producers in microbial mats, and they are mainly responsible for carbon and nitrogen fixation. However, little is known about their metabolic and genomic adaptations at high temperatures. Therefore, in this study, a total of 81 Fischerella thermalis strains (also known as Mastigocladus laminosus) were isolated from mat samples in a thermal gradient between 61-46°C. The complementary use of proteomic comparisons from these strains, and comparative genomics of F. thermalis pangenomes, suggested that at least two different ecotypes were present within these populations. MALDI-TOF MS analysis separated the strains into three clusters; two with strains obtained from mats within the upper temperature range (61 and 54°C), and a third obtained from mats within the lower temperature range (51 and 46°C). Both groups possessed different but synonymous nifH alleles. The main proteomic differences were associated with the abundance of photosynthesis-related proteins. Three F. thermalis metagenome assembled genomes (MAGs) were described from 66, 58 and 48°C metagenomes. These pangenomes indicated a divergence of orthologous genes and a high abundance of exclusive genes at 66°C. These results improved the current understanding of thermal adaptation of F. thermalis and the evolution of these thermophilic cyanobacterial species.

Microbial activity during a coastal phytoplankton bloom on the Western Antarctic Peninsula in late summer.

Phytoplankton biomass during the austral summer is influenced by freezing and melting cycles as well as oceanographic processes that enable nutrient redistribution in the West Antarctic Peninsula (WAP). Microbial functional capabilities, metagenomic and metatranscriptomic activities as well as inorganic 13C- and 15N-assimilation rates were studied in the surface waters of Chile Bay during two contrasting summer periods in 2014. Concentrations of Chlorophyll a (Chla) varied from 0.3 mg m-3 in February to a maximum of 2.5 mg m-3 in March, together with a decrease in nutrients; however, nutrients were never depleted. The microbial community composition remained similar throughout both sampling periods; however, microbial abundance and activity changed with Chla levels. An increased biomass of Bacillariophyta, Haptophyceae and Cryptophyceae was observed along with night-grazing activity of Dinophyceae and ciliates (Alveolates). During high Chla conditions, HCO3- uptake rates during daytime incubations increased 5-fold (>2516 nmol C L-1 d-1), and increased photosynthetic transcript numbers that were mainly associated with cryptophytes; meanwhile night time NO3- (>706 nmol N L-1 d-1) and NH4+ (41.7 nmol N L-1 d-1) uptake rates were 2- and 3-fold higher, respectively, due to activity from Alpha-/Gammaproteobacteria and Bacteroidetes (Flavobacteriia). Due to a projected acceleration in climate change in the WAP, this information is valuable for predicting the composition and functional changes in Antarctic microbial communities.