Alkaline Siliceous Hot Springs Research Articles

Exploring the world hot springs: A bibliometric analysis of global trends in metagenomics research

The aim of the present study is to utilize bibliometric analytical tools to examine the trends in research publications within the hot spring metagenomics field. To achieve this, an in-depth exploration of the Scopus and Web of Science (WoS) databases was undertaken to recognize scholarly articles within this research domain that were published from 2005 to 2022. As a result, a dataset consisting of 377 articles was obtained. The VOSviewer, RStudio, MS Excel, and MS Access were utilized to investigate the annual publication trend, the most productive journals, organizations/authors/countries, authorship and citation patterns, the most cited documents/articles, and the co-occurrence of keywords. According to the findings, the initial article related to hot spring metagenomics was published in 2005. At the end of 2010, the number of published articles was merely 27. However, since then, there has been a significant rise in the yearly number of publications, and post-2010, more than 20 articles have been published every year. The countries with the highest publication output in the field of hot spring metagenomics from 2005 to 2022 were the United States, India, China, and Japan. Montana State university, University of Nevada, Pennsylvania State University, and Bigelow laboratory for ocean sciences all from US have been most active organizations working in this field. The authors who have the highest number of citations, with Ward M. having 1202 and Bryant A. having 1173, also have the greatest total link strength (TLS) of 643 and 653, respectively. Around 11.40 % of the articles published across various sources and journals were contributed by Frontiers in Microbiology, which had the highest number of publications. Bryant's research article, published in 2007 in the Science journal, about phototrophic microbial mats in alkaline siliceous hot springs, has the highest citation score of 292 as of 2022. This research provides a comprehensive overview of the research landscape in hot spring metagenomics and to identify research trends, patterns, and knowledge gaps in this field. This information could be used by researchers, policymakers, and other stakeholders to make informed decisions about future research directions and priorities in hot spring metagenomics.

Acclimation of the photosynthetic apparatus to low light in a thermophilic Synechococcus sp. strain.

Depending upon their growth responses to high and low irradiance, respectively, thermophilic Synechococcus sp. isolates from microbial mats associated with the effluent channels of Mushroom Spring, an alkaline siliceous hot spring in Yellowstone National Park, can be described as either high-light (HL) or low-light (LL) ecotypes. Strains isolated from the bottom of the photic zone grow more rapidly at low irradiance compared to strains isolated from the uppermost layer of the mat, which conversely grow better at high irradiance. The LL-ecotypes develop far-red absorbance and fluorescence emission features after growth in LL. These isolates have a unique gene cluster that encodes a putative cyanobacteriochrome denoted LcyA, a putative sensor histidine kinase; an allophycocyanin (FRL-AP; ApcD4-ApcB3) that absorbs far-red light; and a putative chlorophyll a-binding protein, denoted IsiX, which is homologous to IsiA. The emergence of FRL absorbance in LL-adapted cells of Synechococcus sp. strain A1463 was analyzed in cultures responding to differences in light intensity. The far-red absorbance phenotype arises from expression of a novel antenna complex containing the FRL-AP, ApcD4-ApcB3, which is produced when cells were grown at very low irradiance. Additionally, the two GAF domains of LcyA were shown to bind phycocyanobilin and a [4Fe-4S] cluster, respectively. These ligands potentially enable this photoreceptor to respond to a variety of environmental factors including irradiance, redox potential, and/or oxygen concentration. The products of the gene clusters specific to LL-ecotypes likely facilitate growth in low-light environments through a process called Low-Light Photoacclimation.

Elioraea tepida, sp. nov., a Moderately Thermophilic Aerobic Anoxygenic Phototrophic Bacterium Isolated from the Mat Community of an Alkaline Siliceous Hot Spring in Yellowstone National Park, WY, USA.

Strain MS-P2T was isolated from microbial mats associated with Mushroom Spring, an alkaline siliceous hot spring in Yellowstone National Park, WY, USA. The isolate grows chemoheterotrophically by oxygen-dependent respiration, and light stimulates photoheterotrophic growth under strictly oxic conditions. Strain MS-P2T synthesizes bacteriochlorophyll a and the carotenoid spirilloxanthin. However, photoautotrophic growth did not occur under oxic or anoxic conditions, suggesting that this strain should be classified as an aerobic anoxygenic phototrophic bacterium. Strain MS-P2T cells are motile, curved rods about 0.5 to 1.0 μm wide and 1.0 to 1.5 μm long. The optimum growth temperature is 45–50 °C, and the optimum pH for growth is circum-neutral (pH 7.0–7.5). Sequence analysis of the 16S rRNA gene revealed that strain MS-P2T is closely related to Elioraea species, members of the class Alphaproteobacteria, with a sequence identity of 96.58 to 98%. The genome of strain MS-P2T is a single circular DNA molecule of 3,367,643 bp with a mol% guanine-plus-cytosine content of 70.6%. Based on phylogenetic, physiological, biochemical, and genomic characteristics, we propose this bacteriochlorophyll a-containing isolate is a new species belonging to the genus Elioraea, with the suggested name Elioraea tepida. The type-strain is strain MS-P2T (= JCM33060T = ATCC TSD-174T).

Relationship between Microorganisms Inhabiting Alkaline Siliceous Hot Spring Mat Communities and Overflowing Water.

The compositions of Octopus Spring and Mushroom Spring (Yellowstone National Park, Wyoming, USA) microbial mats have been thoroughly studied, but the compositions of the effluent waters that flow above the mats have not. In this study, cells in the mats and overflowing waters of both springs were investigated at multiple sites where Synechococcus spp. are the dominant cyanobacteria (ca. 72°C to ca. 50°C), and on several dates. In addition to microscopic analyses of stained and autofluorescent cells, 16S rRNA gene sequencing was used to characterize the major taxa present and a protein-encoding gene (psaA) was sequenced and analyzed by ecotype simulation to predict species of Synechococcus The mats of both springs were similar in terms of the downstream distribution of predominant taxa detected previously. However, waters above these mats were predominated by taxa that reside in upstream mats or communities above the upper-temperature limit of the mat. A disturbance/recolonization study was performed at a site normally predominated by Synechococcus species adapted to low temperatures. After removing indigenous Synechococcus cells, Synechococcus species adapted to higher temperatures, which were predominant in the water overflowing this site, colonized the newly forming mat. Differences in recolonization under reduced and UV-screened irradiance suggested that, in addition to physical transport, environmental conditions likely select for species that are better adapted to these different conditions and can influence mat recovery. A transport model was developed and used to predict that, in Mushroom Spring, erosion predominates in the narrower and deeper upstream effluents and deposition predominates over erosion in wider and shallower downstream effluents.IMPORTANCE In flowing aquatic systems, cell erosion and deposition are important to the dispersal of cells from one location to another. Very little is known about microbial dispersal and the physical processes that underlie it. This study demonstrates its importance to colonization of downstream surfaces and especially to the recolonization and functioning of disturbed sites. Ecological systems in flowing environments are often, roughly speaking, pseudosteady, in that nutrients enter the system and by-products leave at relatively steady rates. Over time, material inputs and outputs must balance. Measurements of input fluxes (e.g., growth rates and proxies, such as photosynthesis rates) are frequent. However, erosion and deposition of cells are seldom measured and ecological significance is sometimes neglected. The importance of these parameters is immediately evident in any attempt to construct a model of long-time community behavior, as spatial ecological structure is significantly impacted and can be dominated by migration of organisms, even in small numbers.

‘Candidatus Thermochlorobacter aerophilum:’ an aerobic chlorophotoheterotrophic member of the phylum Chlorobi defined by metagenomics and metatranscriptomics

An uncultured member of the phylum Chlorobi, provisionally named 'Candidatus Thermochlorobacter aerophilum', occurs in the microbial mats of alkaline siliceous hot springs at the Yellowstone National Park. 'Ca. T. aerophilum' was investigated through metagenomic and metatranscriptomic approaches. 'Ca. T. aerophilum' is a member of a novel, family-level lineage of Chlorobi, a chlorophototroph that synthesizes type-1 reaction centers and chlorosomes similar to cultivated relatives among the green sulfur bacteria, but is otherwise very different physiologically. 'Ca. T. aerophilum' is proposed to be an aerobic photoheterotroph that cannot oxidize sulfur compounds, cannot fix N(2), and does not fix CO(2) autotrophically. Metagenomic analyses suggest that 'Ca. T. aerophilum' depends on other mat organisms for fixed carbon and nitrogen, several amino acids, and other important nutrients. The failure to detect bchU suggests that 'Ca. T. aerophilum' synthesizes bacteriochlorophyll (BChl) d, and thus it occupies a different ecological niche than other chlorosome-containing chlorophototrophs in the mat. Transcription profiling throughout a diel cycle revealed distinctive gene expression patterns. Although 'Ca. T. aerophilum' probably photoassimilates organic carbon sources and synthesizes most of its cell materials during the day, it mainly transcribes genes for BChl synthesis during late afternoon and early morning, and it synthesizes and assembles its photosynthetic apparatus during the night.

Complete genome of Candidatus Chloracidobacterium thermophilum, a chlorophyll‐based photoheterotroph belonging to the phylum Acidobacteria

Candidatus Chloracidobacterium thermophilum, which naturally inhabits microbial mats of alkaline siliceous hot springs in Yellowstone National Park, is the only known chlorophototroph in the phylum Acidobacteria. The Ca. C. thermophilum genome was composed of two chromosomes (2,683,362 bp and 1,012,010 bp), and both encoded essential genes. The genome included genes to produce chlorosomes, the Fenna-Matthews-Olson protein, bacteriochlorophylls a and c as principal pigments, and type-1, homodimeric reaction centres. Ca. C. thermophilum is an aerobic photoheterotroph that lacks the ability to synthesize several essential nutrients. Key genes of all known carbon fixation pathways were absent, as were genes for assimilatory nitrate and sulfate reduction and vitamin B(12) synthesis. Genes for the synthesis of branched-chain amino acids (valine, isoleucine and leucine) were also absent, but genes for catabolism of these compounds were present. This observation suggested that Ca. C. thermophilum may synthesize branched-chain amino acids from an intermediate(s) of the catabolic pathway by reversing these reactions. The genome encoded an aerobic respiratory electron transport chain that included NADH dehydrogenase, alternative complex III and cytochrome oxidase. The chromosomes of the laboratory isolate were compared with assembled, metagenomic scaffolds from the major Ca. C. thermophilum population in hot-spring mats. The larger chromosomes of the two populations were highly syntenous but significantly divergent (~13%) in sequence. In contrast, the smaller chromosomes have undergone numerous rearrangements, contained many transposases, and might be less constrained by purifying selection than the large chromosomes. Some transposases were homologous to those of mat community members from other phyla.

Metatranscriptomic analyses of chlorophototrophs of a hot-spring microbial mat

The phototrophic microbial mat community of Mushroom Spring, an alkaline siliceous hot spring in Yellowstone National Park, was studied by metatranscriptomic methods. RNA was extracted from mat specimens collected at four timepoints during light-to-dark and dark-to-light transitions in one diel cycle, and these RNA samples were analyzed by both pyrosequencing and SOLiD technologies. Pyrosequencing was used to assess the community composition, which showed that ~84% of the rRNA was derived from members of four kingdoms Cyanobacteria, Chloroflexi, Chlorobi and Acidobacteria. Transcription of photosynthesis-related genes conclusively demonstrated the phototrophic nature of two newly discovered populations; these organisms, which were discovered through metagenomics, are currently uncultured and previously undescribed members of Chloroflexi and Chlorobi. Data sets produced by SOLiD sequencing of complementary DNA provided >100-fold greater sequence coverage. The much greater sequencing depth allowed transcripts to be detected from ~15,000 genes and could be used to demonstrate statistically significant differential transcription of thousands of genes. Temporal differences for in situ transcription patterns of photosynthesis-related genes suggested that the six types of chlorophototrophs in the mats may use different strategies for maximizing their solar-energy capture, usage and growth. On the basis of both temporal pattern and transcript abundance, intra-guild gene expression differences were also detected for two populations of the oxygenic photosynthesis guild. This study showed that, when community-relevant genomes and metagenomes are available, SOLiD sequencing technology can be used for metatranscriptomic analyses, and the results suggested that this method can potentially reveal new insights into the ecophysiology of this model microbial community.

Influence of molecular resolution on sequence-based discovery of ecological diversity among Synechococcus populations in an alkaline siliceous hot spring microbial mat.

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel.

Cultivation and genomic, nutritional, and lipid biomarker characterization of Roseiflexus strains closely related to predominant in situ populations inhabiting Yellowstone hot spring microbial mats.

Roseiflexus sp. strains were cultivated from a microbial mat of an alkaline siliceous hot spring in Yellowstone National Park. These strains are closely related to predominant filamentous anoxygenic phototrophs found in the mat, as judged by the similarity of small-subunit rRNA, lipid distributions, and genomic and metagenomic sequences. Like a Japanese isolate, R. castenholzii, the Yellowstone isolates contain bacteriochlorophyll a, but not bacteriochlorophyll c or chlorosomes, and grow photoheterotrophically or chemoheterotrophically under dark aerobic conditions. The genome of one isolate, Roseiflexus sp. strain RS1, contains genes necessary to support these metabolisms. This genome also contains genes encoding the 3-hydroxypropionate pathway for CO(2) fixation and a hydrogenase, which might enable photoautotrophic metabolism, even though neither isolate could be grown photoautotrophically with H(2) or H(2)S as a possible electron donor. The isolates exhibit temperature, pH, and sulfide preferences typical of their habitat. Lipids produced by these isolates matched much better with mat lipids than do lipids produced by R. castenholzii or Chloroflexus isolates.

Candidatus Chloracidobacterium thermophilum: An Aerobic Phototrophic Acidobacterium

Only five bacterial phyla with members capable of chlorophyll (Chl)-based phototrophy are presently known. Metagenomic data from the phototrophic microbial mats of alkaline siliceous hot springs in Yellowstone National Park revealed the existence of a distinctive bacteriochlorophyll (BChl)-synthesizing, phototrophic bacterium. A highly enriched culture of this bacterium grew photoheterotrophically, synthesized BChls a and c under oxic conditions, and had chlorosomes and type 1 reaction centers. "Candidatus Chloracidobacterium thermophilum" is a BChl-producing member of the poorly characterized phylum Acidobacteria.

Comparative genomics provides evidence for the 3‐hydroxypropionate autotrophic pathway in filamentous anoxygenic phototrophic bacteria and in hot spring microbial mats

Stable carbon isotope signatures of diagnostic lipid biomarkers have suggested that Roseiflexus spp., the dominant filamentous anoxygenic phototrophic bacteria inhabiting microbial mats of alkaline siliceous hot springs, may be capable of fixing bicarbonate via the 3-hydroxypropionate pathway, which has been characterized in their distant relative, Chloroflexus aurantiacus. The genomes of three filamentous anoxygenic phototrophic Chloroflexi isolates (Roseiflexus sp. RS-1, Roseiflexus castenholzii and Chloroflexus aggregans), but not that of a non-photosynthetic Chloroflexi isolate (Herpetosiphon aurantiacus), were found to contain open reading frames that show a high degree of sequence similarity to genes encoding enzymes in the C. aurantiacus pathway. Metagenomic DNA sequences from the microbial mats of alkaline siliceous hot springs also contain homologues of these genes that are highly similar to genes in both Roseiflexus spp. and Chloroflexus spp. Thus, Roseiflexus spp. appear to have the genetic capacity for carbon dioxide reduction via the 3-hydroxypropionate pathway. This may contribute to heavier carbon isotopic signatures of the cell components of native Roseiflexus populations in mats compared with the signatures of cyanobacterial cell components, as a similar isotopic signature would be expected if Roseiflexus spp. were participating in photoheterotrophic uptake of cyanobacterial photosynthate produced by the reductive pentose phosphate cycle.

Impact of carbon metabolism on 13C signatures of cyanobacteria and green non‐sulfur‐like bacteria inhabiting a microbial mat from an alkaline siliceous hot spring in Yellowstone National Park (USA)

Alkaline siliceous hot spring microbial mats in Yellowstone National Park are composed of two dominant phototropic groups, cyanobacteria and green non-sulfur-like bacteria (GNSLB). While cyanobacteria are thought to cross-feed low-molecular-weight organic compounds to support photoheterotrophic metabolism in GNSLB, it is unclear how this could lead to the heavier stable carbon isotopic signatures in GNSLB lipids compared with cyanobacterial lipids found in previous studies. The two groups of phototrophs were separated using percoll density gradient centrifugation and subsequent lipid and stable carbon isotopic analysis revealed that we obtained fractions with a approximately 60-fold enrichment in cyanobacterial and an approximately twofold enrichment in GNSLB biomass, respectively, compared with the mat itself. This technique was used to study the diel cycling and 13C content of the glucose pools in and the uptake of 13C-bicarbonate by the cyanobacteria and GNSLB, as well as the transfer of incorporated 13C from cyanobacteria to GNSLB. The results show that cyanobacteria have the highest bicarbonate uptake rates and accumulate glucose during the afternoon in full light conditions. In contrast, GNSLB have relatively higher bicarbonate uptake rates compared with cyanobacteria in the morning at low light levels. During the night GNSLB take up carbon that is likely derived through fermentation of cyanobacterial glucose enriched in 13C. The assimilation of 13C-enriched cyanobacterial carbon may thus lead to enriched 13C-contents of GNSLB cell components.

Cyanobacterial ecotypes in the microbial mat community of Mushroom Spring (Yellowstone National Park, Wyoming) as species-like units linking microbial community composition, structure and function

We have investigated microbial mats of alkaline siliceous hot springs in Yellowstone National Park as natural model communities to learn how microbial populations group into species-like fundamental units. Here, we bring together empirical patterns of the distribution of molecular variation in predominant mat cyanobacterial populations, theory-based modelling of how to demarcate phylogenetic clusters that correspond to ecological species and the dynamic patterns of the physical and chemical microenvironments these populations inhabit and towards which they have evolved adaptations. We show that putative ecotypes predicted by the theory-based model correspond well with distribution patterns, suggesting populations with distinct ecologies, as expected of ecological species. Further, we show that increased molecular resolution enhances our ability to detect ecotypes in this way, though yet higher molecular resolution is probably needed to detect all ecotypes in this microbial community.

Diel variations in carbon metabolism by green nonsulfur-like bacteria in alkaline siliceous hot spring microbial mats from Yellowstone National Park.

Green nonsulfur-like bacteria (GNSLB) in hot spring microbial mats are thought to be mainly photoheterotrophic, using cyanobacterial metabolites as carbon sources. However, the stable carbon isotopic composition of typical Chloroflexus and Roseiflexus lipids suggests photoautotrophic metabolism of GNSLB. One possible explanation for this apparent discrepancy might be that GNSLB fix inorganic carbon only during certain times of the day. In order to study temporal variability in carbon metabolism by GNSLB, labeling experiments with [13C]bicarbonate, [14C]bicarbonate, and [13C]acetate were performed during different times of the day. [14C]bicarbonate labeling indicated that during the morning, incorporation of label was light dependent and that both cyanobacteria and GNSLB were involved in bicarbonate uptake. 13C-labeling experiments indicated that during the morning, GNSLB incorporated labeled bicarbonate at least to the same degree as cyanobacteria. The incorporation of [13C]bicarbonate into specific lipids could be stimulated by the addition of sulfide or hydrogen, which both were present in the morning photic zone. The results suggest that GNSLB have the potential for photoautotrophic metabolism during low-light periods. In high-light periods, inorganic carbon was incorporated primarily into Cyanobacteria-specific lipids. The results of a pulse-labeling experiment were consistent with overnight transfer of label to GNSLB, which could be interrupted by the addition of unlabeled acetate and glycolate. In addition, we observed direct incorporation of [13C]acetate into GNSLB lipids in the morning. This suggests that GNSLB also have a potential for photoheterotrophy in situ.

Highly ordered vertical structure of Synechococcus populations within the one-millimeter-thick photic zone of a hot spring cyanobacterial mat.

A variety of contemporary techniques were used to investigate the vertical distribution of thermophilic unicellular cyanobacteria, Synechococcus spp., and their activity within the upper 1-mm-thick photic zone of the mat community found in an alkaline siliceous hot spring in Yellowstone National Park in Wyoming. Detailed measurements were made over a diel cycle at a 61 degrees C site. Net oxygenic photosynthesis measured with oxygen microelectrodes was highest within the uppermost 100- to 200-microm-thick layer until midmorning, but as the day progressed, the peak of net activity shifted to deeper layers, stabilizing at a depth of 300 microm from midday throughout the afternoon. Examination of vertical thin sections by bright-field and autofluorescence microscopy revealed the existence of different populations of Synechococcus which form discrete bands at different vertical positions. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments from horizontal cryosections obtained at 100-microm-thick vertical intervals also suggested vertical stratification of cyanobacterial, green sulfur bacterium-like, and green nonsulfur bacterium-like populations. There was no evidence of diel migration. However, image analysis of vertical thin sections revealed the presence of a narrow band of rod-shaped Synechococcus cells in which the cells assumed an upright position. These upright cells, located 400 to 800 microm below the surface, were observed only in mat samples obtained around noon. In mat samples obtained at other time points, the cells were randomly oriented throughout the mat. These combined observations reveal the existence of a highly ordered structure within the very thin photic zone of this hot spring microbial mat, consisting of morphologically similar Synechococcus populations that are likely to be differentially adapted, some co-occurring with green sulfur bacterium-like populations, and all overlying green nonsulfur bacterium-like populations.