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The Tibetan Plateau in Northwest China hosts a number of hot springs that represent a biodiversity hotspot for thermophiles, yet their diversity and relationship to environmental conditions are poorly explored in these habitats. In this study we investigated microbial diversity and community composition in 13 Tibetan hot springs with a wide range of temperatures (22.1-75°C) and other geochemical conditions by using the 16S rRNA gene pyrosequencing approach. Bacteria (10(8)-10(11) copy/g; 42 bacterial phyla) in Tibetan hot springs were more abundant and far more diverse than Archaea (10(7)-10(10) copy/g; 5 archaeal phyla). The dominant bacterial phyla systematically varied with temperature. Moderate temperatures (75-66°C) favored Aquificae, GAL35, and novel Bacteria, whereas low temperatures (60-22.1°C) selected for Deinococcus-Thermus, Cyanobacteria, and Chloroflexi. The relative abundance of Aquificae was correlated positively with temperature, but the abundances of Deinococcus-Thermus, Cyanobacteria, and Chloroflexi were negatively correlated with temperature. Cyanobacteria and Chloroflexi were abundant in Tibetan hot springs and their abundances were positively correlated at low temperatures (55-43°C) but negatively correlated at moderate temperatures (75-55°C). These correlation patterns suggest a complex physiological relationship between these two phyla. Most archaeal sequences were related to Crenarchaeota with only a few related to Euryarchaeota and Thaumarchaeota. Despite the fact that microbial composition in Tibetan hot springs was strongly shaped by temperature, microbial diversity (richness, evenness and Shannon diversity) was not significantly correlated with temperature change. The results of this study expand our current understanding of microbial ecology in Tibetan hot springs and provide a basis for a global comparison.

The phylogeny of nitrogenase has only been analyzed using the structural proteins NifHDK. As nifHDKENB has been established as the minimum number of genes necessary for in silico prediction of diazotrophy, we present an updated phylogeny of diazotrophs using both structural (NifHDK) and cofactor assembly proteins (NifENB). Annotated Nif sequences were obtained from InterPro from 963 culture-derived genomes. Nif sequences were aligned individually and concatenated to form one NifHDKENB sequence. Phylogenies obtained using PhyML, FastTree, RapidNJ, and ASTRAL from individuals and concatenated protein sequences were compared and analyzed. All six genes were found across the Actinobacteria, Aquificae, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, Fusobacteria, Nitrospira, Proteobacteria, PVC group, and Spirochaetes, as well as the Euryarchaeota. The phylogenies of individual Nif proteins were very similar to the overall NifHDKENB phylogeny, indicating the assembly proteins have evolved together. Our higher resolution database upheld the three cluster phylogeny, but revealed undocumented horizontal gene transfers across phyla. Only 48% of the 325 genera containing all six nif genes are currently supported by biochemical evidence of diazotrophy. In addition, this work provides reference for any inter-phyla comparison of Nif sequences and a quality database of Nif proteins that can be used for identifying new Nif sequences.

Trans-Himalayan hot spring waters rich in boron, chlorine, sodium and sulfur (but poor in calcium and silicon) are known based on PCR-amplified 16S rRNA gene sequence data to harbor high diversities of infiltrating bacterial mesophiles. Yet, little is known about the community structure and functions, primary productivity, mutual interactions, and thermal adaptations of the microorganisms present in the steaming waters discharged by these geochemically peculiar spring systems. We revealed these aspects of a bacteria-dominated microbiome (microbial cell density ~8.5 × 10 4 mL -1 ; live:dead cell ratio 1.7) thriving in the boiling (85°C) fluid vented by a sulfur-borax spring called Lotus Pond, situated at 4436 m above the mean sea-level, in the Puga valley of eastern Ladakh, on the Changthang plateau. Assembly, annotation, and population-binning of >15-GB metagenomic sequence illuminated the numeral predominance of Aquificae. While members of this phylum accounted for 80% of all 16S rRNA-encoding reads within the metagenomic dataset, 14% of such reads were attributed to Proteobacteria. Post assembly, only 25% of all protein-coding genes identified were attributable to Aquificae, whereas 41% was ascribed to Proteobacteria. Annotation of metagenomic reads encoding 16S rRNAs, and/or PCR-amplified 16S rRNA genes, identified 163 bacterial genera, out of which 66 had been detected in past investigations of Lotus Pond’s vent-water via 16S amplicon sequencing. Among these 66, Fervidobacterium , Halomonas , Hydrogenobacter , Paracoccus , Sulfurihydrogenibium , Tepidimonas , Thermus and Thiofaba (or their close phylogenomic relatives) were presently detected as metagenome-assembled genomes (MAGs). Remarkably, the Hydrogenobacter related MAG alone accounted for ~56% of the entire metagenome, even though only 15 out of the 66 genera consistently present in Lotus Pond’s vent-water have strains growing in the laboratory at >45°C, reflecting the continued existence of the mesophiles in the ecosystem. Furthermore, the metagenome was replete with genes crucial for thermal adaptation in the context of Lotus Pond’s geochemistry and topography. In terms of sequence similarity, a majority of those genes were attributable to phylogenetic relatives of mesophilic bacteria, while functionally they rendered functions such as encoding heat shock proteins, molecular chaperones, and chaperonin complexes; proteins controlling/modulating/inhibiting DNA gyrase; universal stress proteins; methionine sulfoxide reductases; fatty acid desaturases; different toxin-antitoxin systems; enzymes protecting against oxidative damage; proteins conferring flagellar structure/function, chemotaxis, cell adhesion/aggregation, biofilm formation, and quorum sensing. The Lotus Pond Aquificae not only dominated the microbiome numerically but also acted potentially as the main primary producers of the ecosystem, with chemolithotrophic sulfur oxidation (Sox) being the fundamental bioenergetic mechanism, and reductive tricarboxylic acid (rTCA) cycle the predominant carbon fixation pathway. The Lotus Pond metagenome contained several genes directly or indirectly related to virulence functions, biosynthesis of secondary metabolites including antibiotics, antibiotic resistance, and multi-drug efflux pumping. A large proportion of these genes being attributable to Aquificae, and Proteobacteria (very few were ascribed to Archaea), it could be worth exploring in the future whether antibiosis helped the Aquificae overcome niche overlap with other thermophiles (especially those belonging to Archaea), besides exacerbating the bioenergetic costs of thermal endurance for the mesophilic intruders of the ecosystem.

We report here detailed phylogenetic and comparative analyses on 11 sequenced genomes from the phylum Aquificae to identify molecular markers that are specific for the species from this phylum or its different families (viz. Aquificaceae, Hydrogenothermaceae and Desulfurobacteriaceae ). In phylogenetic trees based on 16S rRNA gene or concatenated sequences for 32 conserved proteins, species from the three Aquificae families formed distinct clades. These trees also supported a strong relationship between the Aquificaceae and Hydrogenothermaceae families. In parallel, comparative analyses on protein sequences from Aquificae genomes have identified 46 conserved signature indels (CSIs) in broadly distributed proteins that are either exclusively or mainly found in members of the phylum Aquificae or its different families and subclades. Four of these CSIs, which are found in all sequenced Aquificae species, provide potential molecular markers for this phylum. Twelve, six and thirteen other CSIs that respectively are specific for the sequenced Aquificaceae , Hydrogenothermaceae and Desulfurobacteriaceae species provide molecular markers and novel tools for the identification of members of these families and for genetic and biochemical studies on them. Lastly, these studies have identified 11 CSIs in divergent proteins that are uniquely shared by members of the Aquificaceae and Hydrogenothermaceae families providing strong evidence that these two groups of bacteria shared a common ancestor exclusive of all other Aquificae (bacteria). The species from these two families are also very similar in their metabolic and physiological properties and they consist of aerobic or microaerophilic bacteria, which generally obtain energy by oxidation of hydrogen or reduced sulfur compounds by molecular oxygen. Based upon their strong association in phylogenetic trees, unique shared presence of large numbers of CSIs in different proteins, and similarities in their metabolic and physiological properties, it is proposed that the order Aquificales should be emended to include only the members of the families Aquificaceae and Hydrogenothermaceae . The members of the family Desulfurobacteriaceae , which are obligate anaerobes that strictly use hydrogen as electron donor, are now transferred to a new order Desulfurobacteriales ord. nov. The emended descriptions of the phylum Aquificae and its three families incorporating information for different molecular signatures are also provided.

Anaerobic thermophiles inhabit relic environments that resemble the early Earth. However, the lineage of these modern organisms co-evolved with our planet. Hence, these organisms carry both ancestral and acquired genes and serve as models to reconstruct early metabolism. Based on comparative genomic and proteomic analyses, we identified two distinct groups of genes in Thermovibrio ammonificans: the first codes for enzymes that do not require oxygen and use substrates of geothermal origin; the second appears to be a more recent acquisition, and may reflect adaptations to cope with the rise of oxygen on Earth. We propose that the ancestor of the Aquificae was originally a hydrogen oxidizing, sulfur reducing bacterium that used a hybrid pathway for CO2 fixation. With the gradual rise of oxygen in the atmosphere, more efficient terminal electron acceptors became available and this lineage acquired genes that increased its metabolic flexibility while retaining ancestral metabolic traits. Life may have arisen on our planet as far back as four billion years ago. Unlike today, the Earth’s atmosphere at the time had no oxygen and an abundance of volcanic emissions including hydrogen, carbon dioxide and sulfur gases. These dramatic differences have led scientists to wonder: how did the ancient microorganisms that inhabited our early planet make a living? And how has microbial life co-evolved with the Earth? One way to answer these questions is to study bacteria that live today in environments that resemble the early Earth. Deep-sea hydrothermal vents are regions of the deep ocean where active volcanic processes recreate primordial conditions. These habitats support microorganisms that are highly adapted to live off hydrogen, carbon dioxide and sulfur gases, and studying these modern-day microorganisms could give insights into the earliest life on Earth. Thermovibrio ammonificans is a bacterium that was obtained from an underwater volcanic system in the East Pacific. Giovannelli et al. have now asked if T. ammonificans might have inherited some of its genetic traits from a long-gone ancestor that also thrived off volcanic gases. The genetic makeup of this microorganism was examined for genes that would help it thrive at a deep-sea hydrothermal vent. Next, Giovannelli et al. compared these genes to related copies in other species of bacteria to reconstruct how the metabolism of T. ammonificans might have changed over time. This approach identified a group of likely ancient genesthat allow a microorganism to use chemicals like hydrogen, carbon dioxide and sulfur to fuel its growth and metabolism. These findings support the hypothesis that an ancestor of T. ammonificans could live off volcanic gases and that the core set of genes involved in those activities had been passed on, through the generations, to this modern-day microorganism. Giovannelli et al. also identified a second group of genes in T. ammonificans that indicate that this bacterium also co-evolved with Earth’s changing conditions, in particular the rise in the concentration of oxygen. The findings of Giovannelli et al. provide insight into how the metabolism of microbes has co-evolved with the Earth’s changing conditions, and will allow others to formulate new hypotheses that can be tested in laboratory experiments.

Arsenic biogeochemistry has been studied extensively in acid sulfate-chloride hot springs, but not in acid sulfate hot springs with low chloride. In this study, Zhenzhuquan in Tengchong geothermal area, a representative acid sulfate hot spring with low chloride, was chosen to study arsenic geochemistry and microbial community structure using Illumina MiSeq sequencing. Over 0.3 million 16S rRNA sequence reads were obtained from 6-paired parallel water and sediment samples along its outflow channel. Arsenic oxidation occurred in the Zhenxhuquan pool, with distinctly high ratios of arsenate to total dissolved arsenic (0.73-0.86). Coupled with iron and sulfur oxidation along the outflow channel, arsenic accumulated in downstream sediments with concentrations up to 16.44 g/kg and appeared to significantly constrain their microbial community diversity. These oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (arsenic oxidation), Sulfolobus (sulfur and iron oxidation), Metallosphaera and Acidicaldus (iron oxidation). Temperature, total organic carbon and dissolved oxygen significantly shaped the microbial community structure of upstream and downstream samples. In the upstream outflow channel region, most microbial populations were microaerophilic/anaerobic thermophiles and hyperthermophiles, such as Sulfolobus, Nocardia, Fervidicoccus, Delftia, and Ralstonia. In the downstream region, aerobic heterotrophic mesophiles and thermophiles were identified, including Ktedonobacteria, Acidicaldus, Chthonomonas and Sphingobacteria. A total of 72.41-95.91% unassigned-genus sequences were derived from the downstream high arsenic sediments 16S rRNA clone libraries. This study could enable us to achieve an integrated understanding on arsenic biogeochemistry in acid hot springs.

Arsenic biogeochemistry has been studied extensively in acid sulfate-chloride hot springs, but not in acid sulfate hot springs with low chloride. In this study, Zhenzhuquan in Tengchong geothermal area, a representative acid sulfate hot spring with low chloride, was chosen to study arsenic geochemistry and microbial community structure using Illumina MiSeq sequencing. Over 0.3 million 16S rRNA sequence reads were obtained from 6-paired parallel water and sediment samples along its outflow channel. Arsenic oxidation occurred in the Zhenxhuquan pool, with distinctly high ratios of arsenate to total dissolved arsenic (0.73-0.86). Coupled with iron and sulfur oxidation along the outflow channel, arsenic accumulated in downstream sediments with concentrations up to 16.44 g/kg and appeared to significantly constrain their microbial community diversity. These oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (arsenic oxidation), Sulfolobus (sulfur and iron oxidation), Metallosphaera and Acidicaldus (iron oxidation). Temperature, total organic carbon and dissolved oxygen significantly shaped the microbial community structure of upstream and downstream samples. In the upstream outflow channel region, most microbial populations were microaerophilic/anaerobic thermophiles and hyperthermophiles, such as Sulfolobus, Nocardia, Fervidicoccus, Delftia, and Ralstonia. In the downstream region, aerobic heterotrophic mesophiles and thermophiles were identified, including Ktedonobacteria, Acidicaldus, Chthonomonas and Sphingobacteria. A total of 72.41-95.91% unassigned-genus sequences were derived from the downstream high arsenic sediments 16S rRNA clone libraries. This study could enable us to achieve an integrated understanding on arsenic biogeochemistry in acid hot springs.

Soil microbial communities are essential in the cycling of nutrients that aect crop production. Our goal was to characterize the microbial community structure following 34 years of nitrogen (N) fertilization treatments in continuous maize production in highly fertile soils. Using 16S rRNA gene-based analysis of the V4 region via Illumina HiSeq2500 technology with downstream bioinformatics processing and analysis with QIIME 2.0, we aimed to characterize the prokaryotic communities under three increasing N fertilization rates. Factor analyses indicated that a high N level decreased the diversity of soil bacterial and archaeal communities and altered the relative abundance (RA) of the dominant (>1% RA) and minor (<1% RA) phyla. Among the 12 major phyla, we determined increases in Gemmatimonadetes, Proteobacteria, and Euryarchaeota, accompanied by reductions in Cyanobacteria, Chloroflexi, Firmicutes, and Planctomycetes with increasing N. Within the 29 minor phyla, N fertilization led to increases in Aquificae, WPS2, Parvarchaeota, AD3, FCPU426, Armatimonadetes, TM7, Chlamydiae, and OD1, along with reductions of Nitrospirae, WS3, Tenericutes, Lentisphaerae, OP3, Synergistetes, Thermotogae, and prokaryotes that could not be reliably assigned to a phylum (classified as Other).

The microbial community of a Colombian high mountain hot spring, El Coquito, was analyzed using three different culture-independent assessments of 16S ribosomal RNA genes: clone libraries, pyrosequencing of the V5-V6 hypervariable region, and microarray. This acidic spring had a diverse community composed mainly oí Bacteria that shared characteristics with those from other hot springs and extreme acidic environments. The microbial community was dominated by Proteobacteria, Firmicutes, and Planctomycetes and contained chemotrophic bacteria potentially involved in cycling of ferrous and sulfur-containing minerals and phototrophic organisms, most of which were eukaryotic micro-algae. Despite the presence of a large proportion of novel, unclassified sequences, the taxonomic profiles obtained with each strategy showed similarities at higher taxonomic levels. However, some groups, such as Spirochaetes and Aquificae, were identified using only one methodology, and more taxa were detected with the gene array, which also shared more groups with the pyrosequencing data. Overall, the combined use of different approaches provided a broader view of the microbial community in this acidic hot spring.

In Tengchong County, springs with wide physicochemical diversity provide a multitude of niches for extremophilic microorganisms. In this study, eight middle-low temperature spring sites along two continuous small streams with low water flow and slow speed in the fourth geothermal experience area of Rehai scenic spot were chosen, and geochemical characteristics and HTS of the 16S rRNA V4 region were used to analyze the prokaryotic community structure and diversity in the water and sediment of these sites. The effect of environmental factors on the microbial communities was explored via redundancy analysis (RDA). All sediment samples had higher alpha diversity values than the corresponding water samples. Twenty-five phyla were annotated; Euryarchaeota, Crenarchaeota, Aquificae, Thermotogae and Proteobacteria were the dominant phyla, accounting for 95.31% of all prokaryotes, with relative abundances above 5%. Aquificae dominated in water samples, while Euryarchaeota dominated in sediment samples. RDA indicated that temperature was the main factor influencing the microbial communities in the two streams. The study expands the current understanding of the microbiology of Tengchong hot springs and provides a basis for further mining of hot spring microbial and functional gene resources.