Hot Spring Microbial Mats Research Articles

Mineralogical and microbial characterization of alkali hot spring microbial mats and deposits in Pong Dueat Pa Pae hot spring, Northern Thailand.

Hot spring environments encompass broad physicochemical ranges, in which temperature and pH account for crucial factors shaping hot spring microbial community and diversity. However, the presence of photosynthetic microbial mats adjacent to boiling hot spring vents, where fluid temperatures extend beyond photosynthetic capability, questions the microbial profiles and the actual temperatures of such adjacent mats. Therefore, this study aims to characterize thermophilic microbial communities at Pong Dueat Pa Pae hot spring using next-generation sequencing, including investigating hot spring mineralogy. Results suggest that Pong Dueat Pa Pae hot spring precipitates comprise mainly silica which also acts as the main preservative medium for microbial permineralization. Molecular results revealed the presence of cyanobacterial and Chloroflexi species in the thick, orange and green subaerial mats surrounding the vents, suggesting the mats would be at least 30°C cooler than source vents despite constantly receiving geyser splashes. Bacterial abundance was considerably higher than archaeal (97.9% versus 2.1%). Cyanobacterial (mainly Synechococcus and Leptolygbya) and Chloroflexi species (mainly Roseiflexus) accounted for almost half (40.04%) of the bacterial community, while DHVEG-6 and Thaumarchaeota comprised dominant members (> 90%) of the archaeal fraction. This study updates and provides insights into thermophilic microbial community composition and mineralogy of hot springs in Thailand.

Chelatococcus albus sp. nov., a bacterium isolated from hot spring microbial mat.

A Gram-stain-negative, non-endospore-forming, motile, short rod-shaped strain, designated SYSU G07232T, was isolated from a hot spring microbial mat, sampled from Rehai National Park, Tengchong, Yunnan Province, south-western China. Strain SYSU G07232T grew at 25-50 °C (optimum, 37 °C), at pH 5.5-9.0 (optimum, pH 6.0) and tolerated NaCl concentrations up to 1.0 % (w/v). Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SYSU G07232T showed closest genetic affinity with Chelatococcus daeguensis K106T. The genomic features and taxonomic status of this strain were determined through whole-genome sequencing and a polyphasic approach. The predominant quinone of this strain was Q-10. Major cellular fatty acids comprised C19 : 0 cyclo ω8c and summed feature 8. The whole-genome length of strain SYSU G07232T was 4.02 Mbp, and the DNA G+C content was 69.26 mol%. The average nucleotide identity (ANIm ≤84.85 % and ANIb ≤76.08 %) and digital DNA-DNA hybridization (≤ 21.9 %) values between strain SYSU G07232T and the reference species were lower than the threshold values recommended for distinguishing novel prokaryotic species. Thus, based on the provided phenotypic, phylogenetic, and genetic data, it is proposed that strain SYSU G07232T (=KCTC 8141T=GDMCC 1.4178T) be designated as representing a novel species within the genus Chelatococcus, named Chelatococcus albus sp. nov.

Caldovatus aquaticus sp. nov., a moderately thermophilic bacterium isolated from hot spring microbial mat.

Two closely related, aerobic, Gram-stain-negative, motile, oval-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05006T and SYSU G05005, were isolated from a bioreactor enrichment and the original sample was collected from Rehai National Park, Tengchong, Yunnan Province, PR China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these two strains were closely related to Caldovatus sediminis YIM 72346T (96.75 and 96.89 % sequence similarity, respectively). The whole genome size of strain SYSU G05006T was 3.87 Mbp with a DNA G+C content of 75.33 mol%. The average nucleotide identity (ANI based on the MUMmer algorithm≤90.31 % and ANI based on blast≤89.36 %) and digital DNA-DNA hybridization (≤35.10 %) values between strain SYSU G05006T and other members of the family Acetobacteraceae were all lower than the threshold values recommended for distinguishing novel prokaryotic species. Optimal growth of the strain was observed at 55 °C and pH 6.0. Ubiquinone-10 was the predominant respiratory lipoquinone. The major cellular fatty acids included iso-C14 : 0, C16 : 1 ω5c, summed feature 5 and summed feature 7. The major polar lipids consisted of diphosphatidylglycerol, one unidentified aminolipid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified phospholipids and two unidentified lipids. Based on results of phylogenetic analyses, comparative genomics and phenotypic characteristics, we describe a new species of the genus Caldovatus represented by strain SYSU G05006T (=KCTC 82831T=MCCC 1K06125T), for which we propose the name Caldovatus aquaticus sp. nov.

Global phylogenomic novelty of the Cas1 gene from hot spring microbial communities.

The Cas1 protein is essential for the functioning of CRISPR-Cas adaptive systems. However, despite the high prevalence of CRISPR-Cas systems in thermophilic microorganisms, few studies have investigated the occurrence and diversity of Cas1 across hot spring microbial communities. Phylogenomic analysis of 2,150 Cas1 sequences recovered from 48 metagenomes representing hot springs (42-80°C, pH 6-9) from three continents, revealed similar ecological diversity of Cas1 and 16S rRNA associated with geographic location. Furthermore, phylogenetic analysis of the Cas1 sequences exposed a broad taxonomic distribution in thermophilic bacteria, with new clades of Cas1 homologs branching at the root of the tree or at the root of known clades harboring reference Cas1 types. Additionally, a new family of casposases was identified from hot springs, which further completes the evolutionary landscape of the Cas1 superfamily. This ecological study contributes new Cas1 sequences from known and novel locations worldwide, mainly focusing on under-sampled hot spring microbial mat taxa. Results herein show that circumneutral hot springs are environments harboring high diversity and novelty related to adaptive immunity systems.

Genome mining, phylogenetic, and functional analysis of arsenic (As) resistance operons in Bacillus strains, isolated from As-rich hot spring microbial mats

The geothermal zone of Araró, México, is located within the trans-Mexican volcanic belt, an area with numerous arsenic (As)-rich hot springs. In this study, the draft genome sequence of two endemic Bacillus strains (ZAP17 and ZAP62) from Araró microbial mat hot springs was determined, which were able to grow on arsenate As(V) (up to 64 mM) and arsenite As(III) (up to 32 mM). Phylogenetic analysis based on 16 S rRNA and gyrB sequences, as well as genome sequence analysis based on average nucleotide identity (>96 %) and digital DNA–DNA hybridization (>70 %), indicated that these strains belong to the Bacillus paralicheniformis ZAP17 and Bacillus altitudinis ZAP62. Furthermore, through genome mining, it was identified two arsenic resistance operons, arsRBC, and arsRBCDA in both strains as potential determinants of As resistance. Predicted ArsA (arsenial pump-driving ATPase), ArsB (Arsenical efflux pump protein), ArsC (Arsenate reductase), ArsD (Arsenical efflux pump protein) and ArsR (Metalloregulator/ars operon repressor) proteins, clearly grouped with their respective clades corresponding to other characterized bacterial species, mainly Firmicutes. To further evaluate the functionality of the ars operons in ZAP17 and ZAP62 strains, our results showed that arsRBC and arsRBCDA genes were expressed in the presence of As(III). Finally, the presence of ars operons in the genome of Bacillus species residing in As-rich environments, such as the Araró hot springs, might be a potential mechanism to survive under such harsh conditions.

Hydrogenophilus thiooxidans sp. nov., a moderately thermophilic chemotrophic bacterium unable to grow on hydrogen gas, isolated from hot spring microbial mats.

A novel thermophilic chemotrophic bacterium, strain SS56T, was isolated from Nakabusa Hot Spring, Japan. The isolate was a rod-shaped (1.5-2.1×0.6-0.8 µm), Gram-stain-negative bacterium. The cells of this strain grew chemoheterotrophically under aerobic and anaerobic conditions. Autotrophic growth was observed with thiosulphate and elemental sulphur under aerobic conditions but not with H2 as the electron donor. Heterotrophic growth in the presence of O2 occurred on yeast extract, tryptone, polypeptone and organic acids. Strain SS56T used nitrite as an alternative electron acceptor under anaerobic chemoheterotrophic conditions. The isolate grew between 35 and 65 °C, with the optimum at 55 °C. The pH range for growth was pH 6.0-9.0; optimal growth occurred at pH 7.0-8.0. The 16S rRNA gene sequence of strain SS56T was 98.9% identical to that of Hydrogenophilus thermoluteolus TH-1T. The draft genome sequence of 2401804 bp for strain SS56T gave values of 53.7% for digital DNA-DNA hybridization, 92.9% for average nucleotide identity and 93.6% for average amino acid identity compared with the genome sequence of 2223143 bp for H. thermoluteolus TH-1T. Based on the information described above, strain SS56T (=DSM 111892T=JCM 34254T) is proposed as the type strain of a novel species, Hydrogenophilus thiooxidans sp. nov.

Thermus brevis sp. nov., a moderately thermophilic bacterium isolated from a hot spring microbial mat.

Three closely related, facultative anaerobic, Gram-stain-negative, twitching motile, short rod-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05001T, SYSU G05003 and SYSU G05004, were isolated from a hot spring microbial mat, collected from Rehai National Park, Tengchong, Yunnan Province, south-western China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these three strains were closely related to Thermus scotoductus SE-1T (97.97, 98.18, 97.90 % sequence similarity). Whole genome sequencing and polyphasic taxonomic approach were used to determine the genomic profile and taxonomic status of the novel strain SYSU G05001T. Cell growth occurred at 37-80 °C (optimum, 55 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum, 1%). Thiosulfate enhanced cell growth. MK-8 was the predominant menaquinone. The major cellular fatty acids included iso-C15 : 0, iso-C17 : 0 and anteiso-C15 : 0. The major polar lipids were consisted of aminophospholipid, glycolipid and phospholipids. The whole genome of strain SYSU G05001T consisted of 2.55 Mbp and the DNA G+C content was 64.94 mol%. The average nucleotide identity (≤94.95 %) and digital DNA-DNA hybridization (≤62.3 %) values between strain SYSU G05001T and other members of the genus Thermus were all lower than the threshold values recommended for distinguishing novel prokaryotic species. On the basis of the presented polyphasic evidence and genotypic data, it is proposed that strain SYSU G05001T (=KCTC 82627T=MCCC 1K06118T) represents a novel species of the genus Thermus, for which the name Thermus brevis sp. nov. is proposed.

Molecular Components of Nitrogen Fixation Gene Cluster and Associated Enzymatic Activities of Non-Heterocystous Thermophilic Cyanobacterium Thermoleptolyngbya sp.

Thermoleptolyngbya is a genus of non-heterocystous cyanobacteria that are typical inhabitants of hot spring microbial mats. These filamentous cyanobacteria are capable of nitrogen fixation. In this study, we examined the genome sequences of five publicly available Thermoleptolyngbya strains to explore their nitrogen fixation gene cluster. Analysis of the nitrogen-fixation clusters in these extremophilic strains revealed that the cluster is located in a single locus in Thermoleptolyngbyace. The average nucleotide and amino acid identities of the nitrogen-fixation cluster combined with phylogenetic reconstructions support that nitrogen fixation genes in Thermoleptolyngbyaceae are closely related to one another but also heterogeneous within the genus. The strains from Asia, and China more specifically, generate a separate clade within the genus. Among these strains Thermoleptolyngbya sp. PKUAC-SCTB121 has been selected for experimental validation of clade’s nitrogen fixation capacity. The acetylene reduction experiments of that strain shown that the strain can reduce acetylene to ethylene, indicating a fully functional nitrogenase. The activity of nitrogenase has been tested using different gas compositions across 72 h and exhibited a two-phase trend, high nitrogenase activity at the beginning of the assay that slowed down in the second phase of the analysis.

Taxonomic Novelty and Distinctive Genomic Features of Hot Spring Cyanobacteria.

Several cyanobacterial species are dominant primary producers in hot spring microbial mats. To date, hot spring cyanobacterial taxonomy, as well as the evolution of their genomic adaptations to high temperatures, are poorly understood, with genomic information currently available for only a few dominant genera, including Fischerella and Synechococcus. To address this knowledge gap, the present study expands the genomic landscape of hot spring cyanobacteria and traces the phylum-wide genomic consequences of evolution in high temperature environments. From 21 globally distributed hot spring metagenomes, with temperatures between 32 and 75°C, 57 medium- and high-quality cyanobacterial metagenome-assembled genomes were recovered, representing taxonomic novelty for 1 order, 3 families, 15 genera and 36 species. Comparative genomics of 93 hot spring genomes (including the 57 metagenome-assembled genomes) and 66 non-thermal genomes, showed that the former have smaller genomes and a higher GC content, as well as shorter proteins that are more hydrophilic and basic, when compared to the non-thermal genomes. Additionally, the core accessory orthogroups from the hot spring genomes of some genera had a greater abundance of functional categories, such as inorganic ion metabolism, translation and post-translational modifications. Moreover, hot spring genomes showed increased abundances of inorganic ion transport and amino acid metabolism, as well as less replication and transcription functions in the protein coding sequences. Furthermore, they showed a higher dependence on the CRISPR-Cas defense system against exogenous nucleic acids, and a reduction in secondary metabolism biosynthetic gene clusters. This suggests differences in the cyanobacterial response to environment-specific microbial communities. This phylum-wide study provides new insights into cyanobacterial genomic adaptations to a specific niche where they are dominant, which could be essential to trace bacterial evolution pathways in a warmer world, such as the current global warming scenario.

Insights into the dynamics between viruses and their hosts in a hot spring microbial mat

Our current knowledge of host–virus interactions in biofilms is limited to computational predictions based on laboratory experiments with a small number of cultured bacteria. However, natural biofilms are diverse and chiefly composed of uncultured bacteria and archaea with no viral infection patterns and lifestyle predictions described to date. Herein, we predict the first DNA sequence-based host–virus interactions in a natural biofilm. Using single-cell genomics and metagenomics applied to a hot spring mat of the Cone Pool in Mono County, California, we provide insights into virus–host range, lifestyle and distribution across different mat layers. Thirty-four out of 130 single cells contained at least one viral contig (26%), which, together with the metagenome-assembled genomes, resulted in detection of 59 viruses linked to 34 host species. Analysis of single-cell amplification kinetics revealed a lack of active viral replication on the single-cell level. These findings were further supported by mapping metagenomic reads from different mat layers to the obtained host–virus pairs, which indicated a low copy number of viral genomes compared to their hosts. Lastly, the metagenomic data revealed high layer specificity of viruses, suggesting limited diffusion to other mat layers. Taken together, these observations indicate that in low mobility environments with high microbial abundance, lysogeny is the predominant viral lifestyle, in line with the previously proposed “Piggyback-the-Winner” theory.

Disproportionation of inorganic sulfur compounds by a novel autotrophic bacterium belonging to Nitrospirota

The phylum Nitrospirota (previously known as Nitrospirae or Nitrospira) currently encompasses a limited number of bacterial species with validly published names, including sulfate-reducing bacteria (SRB) of the genus Thermodesulfovibrio. Some metagenome-assembled genomes (MAGs) of bacteria occur in this phylum, and genes involved in dissimilatory sulfur metabolism have been identified in them. Currently, however, there is no established way to discriminate SRB and sulfur-disproportionating bacteria (SDB), which obtain energy from the disproportionation of inorganic sulfur compounds. In this study, a thiosulfate-disproportionating enrichment culture was established from a hot spring microbial mat. The culture was dominated by a single species belonging to the phylum Nitrospirota, and growth of the novel bacterium was supported by disproportionation of thiosulfate and elemental sulfur. Its growth was not observed under sulfate-reducing conditions. Therefore, a comparative genomic analysis of SDB and SRB was performed using its draft genome sequence, in order to identify any genetic element that could be used as a marker for SDB. As a result, a characteristic gene cluster was identified as a putative genetic element that characterized the genomes of SDB. The gene cluster was found in some MAGs of the phylum Nitrospirota, and their corresponding bacteria may also be capable of the disproportionation of inorganic sulfur compounds.

Network-directed efficient isolation of previously uncultivated Chloroflexi and related bacteria in hot spring microbial mats

The perplexity of the complex multispecies community interactions is one of the many reasons why majority of the microorganisms are still uncultivated. We analyzed the entire co-occurrence networks between the OTUs of Tibet and Yunnan hot spring samples, and found that less abundant OTUs such as genus Tepidimonas (relative abundant <1%) had high-degree centricity (key nodes), while dominant OTUs particularly genus Chloroflexus (relative abundant, 13.9%) formed the peripheral vertexes. A preliminary growth-promotion assay determined that Tepidimonas sp. strain SYSU G00190W enhanced the growth of Chloroflexus sp. SYSU G00190R. Exploiting this result, an ameliorated isolation medium containing 10% spent-culture supernatant of Tepidimonas sp. strain SYSU G00190W was prepared for targeted isolation of Chloroflexi in the Tibet and Yunnan hot spring samples. 16S rRNA gene fingerprinting characterized majority of the colonies isolated from these media as previously uncultivated Chloroflexi, of which 36 are potential novel species (16S rRNA sequence identity <98.5%). Metabolomes studies indicated that the spent-culture supernatant comprises several low-molecular-weight organic substrates that can be utilized as potential nutrients for the growth of these bacteria. These findings suggested that limited knowledge on the interaction of microbes provide threshold to traditional isolation method.

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.

Blastochloris tepida, sp. nov., a thermophilic species of the bacteriochlorophyll b-containing genus Blastochloris.

A new taxon is created for the thermophilic purple nonsulfur bacterium previously designated as Rhodopseudomonas strain GI. Strain GI was isolated from a New Mexico (USA) hot spring microbial mat and grows optimally above 40°C and to a maximum of 47°C. Strain GI is a bacteriochlorophyll b-containing species of purple nonsulfur bacteria and displays a budding morphology, typical of species of the genus Blastochloris. Although resembling the species Blc. viridis in many respects, the absorption spectrum, carotenoid content, and lipid fatty acid profile of strain GI is distinct from that of Blc. viridis strain DSM133T and other recognized Blastochloris species. Strain GI forms its own subclade within the Blastochloris clade of purple nonsulfur bacteria based on comparative 16S rRNA gene sequences, and its genome is significantly larger than that of strain DSM133T; average nucleotide identity between the genomes of Blc. viridis and strain GI was below 85%. Moreover, concatenated sequence analyses of PufLM and DnaK clearly showed strain GI to be distinct from both Blc. viridis and Blc. sulfoviridis. Because of its unique assortment of properties, it is proposed to classify strain GI as a new species of the genus Blastochloris, as Blc. tepida, sp.n., with strain GIT designated as the type strain (= ATCC TSD-138 = DSM 106918).

Arsenite Oxidation by a Newly Isolated Betaproteobacterium Possessing arx Genes and Diversity of the arx Gene Cluster in Bacterial Genomes.

Microbes play essential roles in arsenic transformation in the environment. Microbial arsenite oxidation is catalyzed by either of two distantly related arsenite oxidases, referred to as AIO and ARX. The arx genes encoding ARX and its regulatory proteins were originally defined in the genomes of gammaproteobacteria isolated from an alkaline soda lake. The arx gene cluster has been identified in a limited number of bacteria, predominantly in gammaproteobacteria isolated from lakes characterized by high pH and high salinity. In the present study, a novel arsenite-oxidizing betaproteobacterium, strain M52, was isolated from a hot spring microbial mat. The strain oxidized arsenite under both microaerophilic and nitrate-reducing conditions at nearly neutral pH. Genome analysis revealed that the strain possesses the arx gene cluster in its genome and lacks genes encoding AIO. Inspection of the bacterial genomes available in the GenBank database revealed that the presence of this gene cluster is restricted to genomes of Proteobacteria, mainly in the classes Gammaproteobacteria and Betaproteobacteria. In these genomes, the structure of the gene cluster was generally well-conserved, but genes for regulatory proteins were lacking in genomes of strains belonging to a specific lineage. Phylogenetic analysis suggested that ARX encoded in the genomes can be divided into three groups, and strain M52 belongs to a group specific for organisms living in low-salt environments. The ArxA protein encoded in the genome of strain M52 was characterized by the presence of a long insertion, which was specifically observed in the same group of ARX. In clone library analyses with a newly designed primer pair, a diverse ArxA sequence with a long insertion was detected in samples of lake water and hot spring microbial mat, characterized by low salinity and a nearly neutral pH. Among the isolated bacterial strains whose arsenite oxidation has been demonstrated, strain M52 is the first betaproteobacterium that possesses the arx genes, the first strain encoding ARX of the group specific for low-salt environments, and the first organism possessing the gene encoding ArxA with a long insertion.

"Candidatus Thermonerobacter thiotrophicus," A Non-phototrophic Member of the Bacteroidetes/Chlorobi With Dissimilatory Sulfur Metabolism in Hot Spring Mat Communities.

In this study we present evidence for a novel, thermophilic bacterium with dissimilatory sulfur metabolism, tentatively named “Candidatus Thermonerobacter thiotrophicus,” which is affiliated with the Bacteroides/Ignavibacteria/Chlorobi and which we predict to be a sulfate reducer. Dissimilatory sulfate reduction (DSR) is an important and ancient metabolic process for energy conservation with global importance for geochemical sulfur and carbon cycling. Characterized sulfate-reducing microorganisms (SRM) are found in a limited number of bacterial and archaeal phyla. However, based on highly diverse environmental dsrAB sequences, a variety of uncultivated and unidentified SRM must exist. The recent development of high-throughput sequencing methods allows the phylogenetic identification of some of these uncultured SRM. In this study, we identified a novel putative SRM inhabiting hot spring microbial mats that is a member of the OPB56 clade (“Ca. Kapabacteria”) within the Bacteroidetes/Chlorobi superphylum. Partial genomes for this new organism were retrieved from metagenomes from three different hot springs in Yellowstone National Park, United States, and Japan. Supporting the prediction of a sulfate-reducing metabolism for this organism during period of anoxia, diel metatranscriptomic analyses indicate highest relative transcript levels in situ for all DSR-related genes at night. The presence of terminal oxidases, which are transcribed during the day, further suggests that these organisms might also perform aerobic respiration. The relative phylogenetic proximity to the sulfur-oxidizing, chlorophototrophic Chlorobi further raises new questions about the evolution of dissimilatory sulfur metabolism.

Sulfide-dependent Photoautotrophy in the Filamentous Anoxygenic Phototrophic Bacterium, Chloroflexus aggregans.

Chloroflexus aggregans is a thermophilic filamentous anoxygenic phototrophic bacterium frequently found in microbial mats in natural hot springs. C. aggregans often thrives with cyanobacteria that engage in photosynthesis to provide it with an organic substrate; however, it sometimes appears as the dominant phototroph in microbial mats without cyanobacteria. This suggests that C. aggregans has the ability to grow photoautotrophically. However, photoautotrophic growth has not been observed in any cultured strains of C. aggregans. We herein attempted to isolate a photoautotrophic strain from C. aggregansdominated microbial mats in Nakabusa hot spring in Japan. Using an inorganic medium, we succeeded in isolating a new strain that we designated “ACA-12”. A phylogenetic analysis based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences revealed that strain ACA-12 was closely related to known C. aggregans strains. Strain ACA-12 showed sulfide consumption along with autotrophic growth under anaerobic light conditions. The deposited elemental sulfur particles observed by microscopy indicated that sulfide oxidation occurred, similar to that in photoautotrophic strains in the related species, C. aurantiacus. Moreover, we found that other strains of C. aggregans, including the type strain, also exhibited a slight photoautotrophic growing ability, whereas strain ACA-12 showed the fastest growth rate. This is the first demonstration of photoautotrophic growth with sulfide in C. aggregans. The present results strongly indicate that C. aggregans is associated with inorganic carbon incorporation using sulfide as an electron donor in hot spring microbial mats.

Vertical Distribution and Diversity of Phototrophic Bacteria within a Hot Spring Microbial Mat (Nakabusa Hot Springs, Japan).

Phototrophic microbial mats are assemblages of vertically layered microbial populations dominated by photosynthetic microorganisms. In order to elucidate the vertical distribution and diversity of phototrophic microorganisms in a hot spring-associated microbial mat in Nakabusa (Japan), we analyzed the 16S rRNA gene amplicon sequences of the microbial mat separated into five depth horizons, and correlated them with microsensor measurements of O2 and spectral scalar irradiance. A stable core community and high diversity of phototrophic organisms dominated by the filamentous anoxygenic phototrophs, Roseiflexus castenholzii and Chloroflexus aggregans were identified together with the spectral signatures of bacteriochlorophylls (BChls) a and c absorption in all mat layers. In the upper mat layers, a high abundance of cyanobacteria (Thermosynechococcus sp.) correlated with strong spectral signatures of chlorophyll a and phycobiliprotein absorption near the surface in a zone of high O2 concentrations during the day. Deeper mat layers were dominated by uncultured chemotrophic Chlorobi such as the novel putatively sulfate-reducing “Ca. Thermonerobacter sp.”, which showed increasing abundance with depth correlating with low O2 in these layers enabling anaerobic metabolism. Oxygen tolerance and requirements for the novel phototroph “Ca. Chloroanaerofilum sp.” and the uncultured chemotrophic Armatimonadetes member type OS-L detected in Nakabusa hot springs, Japan appeared to differ from previously suggested lifestyles for close relatives identified in hot springs in Yellowstone National Park, USA. The present study identified various microenvironmental gradients and niche differentiation enabling the co-existence of diverse chlorophototrophs in metabolically diverse communities in hot springs.

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.

Discovery of a diverse set of esterases from hot spring microbial mat and sea sediment metagenomes

Esterases are an important group of biocatalysts for synthetic organic chemistry. Functional metagenomics allows discovery of novel biocatalysts by providing access to the gene pool of the microbial community of a habitat. Two metagenomic libraries representing the gene pool of sea sediment and hot spring microbial mat were constructed. Functional screening of these libraries resulted in the isolation of total 8 clones with tributyrin hydrolytic activity. Sequence analysis revealed 10 putative lipolytic proteins with 42–99% homology to the protein sequences in the databases, nine of which represented six known esterase families. Four of the encoded proteins represented Family V and amongst others, one each represented the Family VIII, pectin acetylesterase, enterobactin esterase, G-D-S-L family and OsmC domain containing esterase. One unusual lipolytic protein possessed poly-(3-hydroxybutyrate) depolymerase domain fused to lipase/esterase domain. Two phylogenetically related esterases (MLC3 and SLC5) belonging to family V were expressed and purified to homogeneity. The enzymes exhibited environment-adapted temperature optimum and thermostability. MLC3 was able to stereoselectively hydrolyze R-methyl mandelate to produce R-mandelic acid, an important chiral building block, which suggests MLC3 has potential commercial application.