Tectonic and geological setting influence hot spring microbiology.

Hydrothermal systems form at divergent and convergent boundaries of lithospheric plates and within plates due to weakened crust and mantle plumes, playing host to diverse microbial ecosystems. Little is known of how differences in tectonic setting influence the geochemical and microbial compositions of these hydrothermal ecosystems. Here, coordinated geochemical and microbial community analyses were conducted on 87 high-temperature (>65°C) water and sediment samples from hot springs in Yellowstone National Park, Wyoming, USA (n = 41; mantle plume setting), Iceland (n = 41, divergent boundary), and Japan (n = 5; convergent boundary). Region-specific variation in geochemistry and sediment-associated 16S rRNA gene amplicon sequence variant (ASV) composition was observed, with 16S rRNA gene assemblages being nearly completely distinguished by region and pH being the most explanatory parameter within regions. Several low abundance ASVs exhibited cosmopolitan distributions across regions, while most high-abundance ASVs were only identified in specific regions. The presence of some cosmopolitan ASVs across regions argues against dispersal limitation primarily shaping the distribution of taxa among regions. Rather, the results point to local tectonic and geologic characteristics shaping the geochemistry of continental hydrothermal systems that then select for distinct microbial assemblages. These results provide new insights into the co-evolution of hydrothermal systems and their microbial communities.

Microbial Response to Increased Temperatures Within a Lava-Induced Hydrothermal System in Iceland: An Analogue for the Habitability of Volcanic Terrains on Mars.

Fossil hydrothermal systems on Mars are important exploration targets because they may have once been habitable and could still preserve evidence of microbial life. We investigated microbial communities within an active lava-induced hydrothermal system associated with the 2014-2015 eruption of Holuhraun in Iceland as a Mars analogue. In 2016, the microbial composition in the lava-heated water differed substantially from that of the glacial river and spring water sources that fed into the system. Several taxonomic and metabolic groups were confined to the water emerging from the lava and some showed the highest sequence similarities to subsurface ecosystems, including to the predicted thermophilic and deeply branching Candidatus Acetothermum autotrophicum. Measurements show that the communities were affected by temperature and other environmental factors. In particular, comparing glacial river water incubated in situ (5.7°C, control) with glacial water incubated within a lava-heated stream (17.5°C, warm) showed that microbial abundance, richness, and diversity increased in the warm treatment compared with the control, with the predicted major metabolism shifting from lithotrophy toward organotrophy and possibly phototrophy. In addition, thermophilic bacteria isolated from the lava-heated water and a nearby acidic hydrothermal system included the known endospore-formers Geobacillus stearothermophilus and Paenibacillus cisolokensis as well as a potentially novel taxon within the order Hyphomicrobiales. Similar lava-water interactions on Mars could therefore have generated habitable environments for microbial communities.

Rhodocaloribacter litoris gen. nov., sp. nov., isolated from an intertidal hot spring.

Red-pigmented strains of non-sporeforming, aerobic, chemoorganotrophic bacteria were isolated from intertidal hot springs in Laugarvík, NW-Iceland. Cells stained Gram-negative and formed pleomorphic rods that often had swollen ends and occurred singly or in filaments. Growth was observed at 40-65 °C (optimum at 60 °C), pH 6-9 (optimum at 6.5-8) and 0.5-5% (optimum at 1-2%) (w/v) NaCl. Strain ISCAR-4553T contained MK-7 as the main respiratory quinone and saturated iso and anteiso branched chains of 17 and 15 carbons as the main cellular fatty acids (83.4%). The G+C content of the DNA is 67.3 mol%. The highest 16S rRNA gene sequence similarity was with the genus Roseithermus (92.0%) and followed by Rhodothermus, Rubrivirga and Rubricoccus (88-90%). Genome and phenotype comparisons supported the affiliation of the novel isolates and the genus Roseithermus to the family Rhodothermaceae of the phylum Rhodothermaeota. The described isolates are proposed to be classified as representatives of a novel species belonging to a novel genus, with the name Rhodocaloribacter litoris gen. nov., sp. nov. The type strain is ISCAR-4553T (=DSM 110790T = ATCC TSD-179T).

Engineering the carotenoid biosynthetic pathway in Rhodothermus marinus for lycopene production.

Rhodothermus marinus has the potential to be well suited for biorefineries, as an aerobic thermophile that produces thermostable enzymes and is able to utilize polysaccharides from different 2nd and 3rd generation biomass. The bacterium produces valuable chemicals such as carotenoids. However, the native carotenoids are not established for industrial production and R. marinus needs to be genetically modified to produce higher value carotenoids. Here we genetically modified the carotenoid biosynthetic gene cluster resulting in three different mutants, most importantly the lycopene producing mutant TK-3 (ΔtrpBΔpurAΔcruFcrtB::trpBcrtB T.thermophilus ). The genetic modifications and subsequent structural analysis of carotenoids helped clarify the carotenoid biosynthetic pathway in R. marinus. The nucleotide sequences encoding the enzymes phytoene synthase (CrtB) and the previously unidentified 1',2'-hydratase (CruF) were found fused together and encoded by a single gene in R. marinus. Deleting only the cruF part of the gene did not result in an active CrtB enzyme. However, by deleting the entire gene and inserting the crtB gene from Thermus thermophilus, a mutant strain was obtained, producing lycopene as the sole carotenoid. The lycopene produced by TK-3 was quantified as 0.49 â€‹g/kg CDW (cell dry weight).

Comparative Analysis of Microbial Diversity Across Temperature Gradients in Hot Springs From Yellowstone and Iceland.

Geothermal hot springs are a natural setting to study microbial adaptation to a wide range of temperatures reaching up to boiling. Temperature gradients lead to distinct microbial communities that inhabit their optimum niches. We sampled three alkaline, high temperature (80-100°C) hot springs in Yellowstone and Iceland that had cooling outflows and whose microbial communities had not been studied previously. The microbial composition in sediments and mats was determined by DNA sequencing of rRNA gene amplicons. Over three dozen phyla of Archaea and Bacteria were identified, representing over 1700 distinct organisms. We observed a significant non-linear reduction in the number of microbial taxa as the temperature increased from warm (38°C) to boiling. At high taxonomic levels, the community structure was similar between the Yellowstone and Iceland hot springs. We identified potential endemism at the genus level, especially in thermophilic phototrophs, which may have been potentially driven by distinct environmental conditions and dispersal limitations.

Adaptive Laboratory Evolution for Enhanced Carotenoid Production in Microalgae.

In order to produce natural pigments with competitive prices, algal strains employed in industrial production need to be improved for increasing the productivity of valuable metabolites, thereby reducing the overall production cost. Adaptive laboratory evolution (ALE) is a traditional method for strain improvement, which has been effectively utilized in bacteria and fungi. With the growing interest in algal biotechnology, attempts have recently been put forward to improve microalgal strains with ALE approach. This chapter describes a stepwise adaptive evolution strategy that enhances carotenoid yield from microalgae.

Characterization of carotenoids in Rhodothermus marinus.

Rhodothermus marinus, a marine aerobic thermophile, was first isolated from an intertidal hot spring in Iceland. In recent years, the R. marinus strain PRI 493 has been genetically modified, which opens up possibilities for targeted metabolic engineering of the species, such as of the carotenoid biosynthetic pathway. In this study, the carotenoids of the R. marinus type-strain DSM 4252T , strain DSM 4253, and strain PRI 493 were characterized. Bioreactor cultivations were used for pressurized liquid extraction and analyzed by ultra-high performance supercritical fluid chromatography with diode array and quadropole time-of-flight mass spectrometry detection (UHPSFC-DAD-QTOF/MS). Salinixanthin, a carotenoid originally found in Salinibacter ruber and previously detected in strain DSM 4253, was identified in all three R. marinus strains, both in the hydroxylated and nonhydroxylated form. Furthermore, an additional and structurally distinct carotenoid was detected in the three strains. MS/MS fragmentation implied that the mass difference between salinixanthin and the novel carotenoid structure corresponded to the absence of a 4-keto group on the ß-ionone ring. The study confirmed the lack of carotenoids for the strain SB-71 (ΔtrpBΔpurAcrtBI'::trpB) in which genes encoding two enzymes of the proposed pathway are partially deleted. Moreover, antioxidant capacity was detected in extracts of all the examined R. marinus strains and found to be 2-4 times lower for the knock-out strain SB-71. A gene cluster with 11 genes in two operons in the R. marinusDSM 4252T genome was identified and analyzed, in which several genes were matched with carotenoid biosynthetic pathway genes in other organisms.

Evaluation of the production of exopolysaccharides by two strains of the thermophilic bacterium Rhodothermus marinus.

The thermophile Rhodothermus marinus produces extracellular polysaccharides (EPSs) that forms a distinct cellular capsule. Here, the first data on EPS production in strains DSM4252T and MAT493 are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that the EPS were produced both in the exponential and stationary growth phase and that production in the exponential phase was boosted by maltose supplementation, while stationary phase production was boosted by lactose. The latter was higher, resulting in 8.8 (DSM4252T) and 13.7mg EPS/g cell dry weight (MAT493) in cultures in marine broth supplemented with 10g/L lactose. The EPSs were heteropolymeric with an average molecular weight of 8×104Da and different monosaccharides, including arabinose and xylose. FT-IR spectroscopy revealed presence of hydroxyl, carboxyl, N-acetyl, amine, and sulfate ester groups, showing that R. marinus produces unusual sulfated EPS with high arabinose and xylose content.

Litorilinea aerophila gen. nov., sp. nov., an aerobic member of the class Caldilineae, phylum Chloroflexi, isolated from an intertidal hot spring.

A thermophilic, aerobic, Gram-stain-negative, filamentous bacterium, strain PRI-4131(T), was isolated from an intertidal hot spring in Isafjardardjup, NW Iceland. The strain grew chemo-organotrophically on various carbohydrates. The temperature range for growth was 40-65 °C (optimum 55 °C), the pH range was pH 6.5-9.0 (optimum pH 7.0) and the NaCl range was 0-3 % (w/v) (optimum 0.5 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain PRI-4131(T) represented a distinct lineage within the class Caldilineae of the phylum http://dx.doi.org/10.1601/nm.550Chloroflexi. The highest levels of sequence similarity, about 91 %, were with Caldilinea aerophila STL-6-O1(T) and Caldilinea tarbellica D1-25-10-4(T). Fermentative growth was not observed for strain PRI-4131(T), which, in addition to other characteristics, distinguished it from the two Caldilinea species. Owing to both phylogenetic and phenotypic differences from the described members of the class Caldilineae, we propose to accommodate strain PRI-4131(T) in a novel species in a new genus, Litorilinea aerophila gen. nov., sp. nov. The type strain of Litorilinea aerophila is PRI-4131(T) ( = DSM 25763(T)  = ATCC BAA-2444(T)).

Generation of targeted deletions in the genome of Rhodothermus marinus.

The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermus marinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R. marinus trpB gene. The current work identified an additional selective marker, purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp(+) selection was used during the deletion of purA from the R. marinus chromosome. The alternative Ade(+) selection was in turn used while deleting the endogenous trpB gene. Since both deletions are unmarked, the purA and trpB markers may be reused. Through the double deletant SB-62 (ΔtrpB ΔpurA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in R. marinus strain SB-62 (ΔtrpB ΔpurA) was demonstrated by targeting putative carotenoid biosynthesis genes, crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5' and 3' homologous sequences, respectively. The resulting Trp(+) transformants were colorless rather than orange-red. The correct replacement of an internal crtBI fragment with the trpB marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the R. marinus chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.

Detection and mapping of mtDNA SNPs in Atlantic salmon using high throughput DNA sequencing.

BACKGROUND: Approximately half of the mitochondrial genome inherent within 546 individual Atlantic salmon (Salmo salar) derived from across the species' North Atlantic range, was selectively amplified with a novel combination of standard PCR and pyro-sequencing in a single run using 454 Titanium FLX technology (Roche, 454 Life Sciences). A unique combination of barcoded primers and a partitioned sequencing plate was employed to designate each sequence read to its original sample. The sequence reads were aligned according to the S. salar mitochondrial reference sequence (NC_001960.1), with the objective of identifying single nucleotide polymorphisms (SNPs). They were validated if they met with the following three stringent criteria: (i) sequence reads were produced from both DNA strands; (ii) SNPs were confirmed in a minimum of 90% of replicate sequence reads; and (iii) SNPs occurred in more than one individual. RESULTS: Pyrosequencing generated a total of 179,826,884 bp of data, and 10,765 of the total 10,920 S. salar sequences (98.6%) were assigned back to their original samples. The approach taken resulted in a total of 216 SNPs and 2 indels, which were validated and mapped onto the S. salar mitochondrial genome, including 107 SNPs and one indel not previously reported. An average of 27.3 sequence reads with a standard deviation of 11.7 supported each SNP per individual. CONCLUSION: The study generated a mitochondrial SNP panel from a large sample group across a broad geographical area, reducing the potential for ascertainment bias, which has hampered previous studies. The SNPs identified here validate those identified in previous studies, and also contribute additional potentially informative loci for the future study of phylogeography and evolution in the Atlantic salmon. The overall success experienced with this novel application of HT sequencing of targeted regions suggests that the same approach could be successfully applied for SNP mining in other species.

Rhodothermus profundi sp. nov., a thermophilic bacterium isolated from a deep-sea hydrothermal vent in the Pacific Ocean.

Nine thermophilic strains of aerobic, non-sporulating, heterotrophic bacteria were isolated after enrichment of chimney material sampled from a deep-sea hydrothermal field at a depth of 2634m on the East-Pacific Rise (1 °N). The bacteria stained Gram-negative. They were rod-shaped and measured approximately 0.5µm in width and 1.5-3.5µm in length. They grew at 55-80°C, pH 6-8 and 1-6 % NaCl. Optimal growth was observed at 70-75°C, pH7.0 and 1-3 % NaCl. The organisms were identified as members of the genus Rhodothermus, having a 16S rRNA gene similarity of 98.1 % with Rhodothermus marinus DSM 4252(T). The novel isolates differed morphologically, physiologically and chemotaxonomically from R. marinus, e.g. in lack of pigmentation, response to hydrostatic pressure, maximum growth temperature and DNA G+C content. DNA-DNA hybridization revealed a reassociation value of 37.2 % between strain PRI 2902(T) and R. marinus DSM 4252(T), which strongly suggested that they represent different species. Furthermore, AFLP fingerprinting separated the novel strains from R. marinus reference strains. It is therefore concluded that the strains described here should be classified as representatives of a novel species for which the name Rhodothermus profundi sp. nov. is proposed; the type strain is PRI 2902(T) (=DSM 22212(T) =JCM 15944(T)).

Analysis of the unique geothermal microbial ecosystem of the Blue Lagoon.

Cultivation and culture-independent techniques were used to describe the geothermal ecosystem of the Blue Lagoon in Iceland. The lagoon contains both seawater and freshwater of geothermal origin and is extremely high in silica content. Water samples were collected repeatedly in summer and autumn in 2003 and 2005 and in winter 2006 were analyzed for species composition. The study revealed the typical traits of an extreme ecosystem characterized by dominating species and other species represented in low numbers. A total of 35 taxa were identified. The calculated biodiversity index of the samples was 2.1-2.5. The majority (83%) of analyzed taxa were closely related to bacteria of marine and geothermal origin reflecting a marine character of the ecosystem and the origin of the Blue Lagoon hydrothermal fluid. A high ratio (63%) of analyzed taxa represented putative novel bacterial species. The majority (71%) of analyzed clones were Alphaproteobacteria, of which 80% belonged to the Roseobacter lineage within the family of Rhodobacteraceae. Of seven cultivated species, the two most abundant ones belonged to this lineage. Silicibacter lacuscaerulensis was confirmed as a dominating species in the Blue Lagoon. One group of isolates represented a recently identified species within the genus of Nitratireductor within Rhizobiales. This study implies an annually stable and seasonally dynamic ecosystem in the Blue Lagoon.

Thermus islandicus sp. nov., a mixotrophic sulfur-oxidizing bacterium isolated from the Torfajokull geothermal area.

Strains PRI 2268 and PRI 3838(T) were isolated from two separate hot springs in the Torfajokull geothermal area of South Iceland. The cells were non-motile rods, approximately 0.3 microm in width and 1.5-2.5 microm in length. Electron microscopy revealed a Gram-negative cell-wall structure. The strains grew at 45-79 degrees C (optimum, 65 degrees C) and pH 5.5-10.5 (optimum, pH 6.0-7.0). 16S rRNA gene sequence analysis indicated that they formed a separate branch within the genus Thermus with 'Thermus kawarayensis' KW11 as their closest cultured relative (96.5 % similarity). The gene sequence similarities of both new isolates to Thermus aquaticus YT-1(T) and Thermus igniterrae RF-4(T) were 96.1 % and 95.5 %, respectively. DNA-DNA relatedness between strain PRI 3838(T) and 'T. kawarayensis' was 46.1 %. The DNA G+C content of strain PRI 3838(T) was 69.0 mol%. The predominant menaquinones, pigmentation, fatty acid profiles and phospholipid profiles of the novel strains were similar to those of other members of the genus Thermus. However, the new strains could be differentiated from the type strains of all other species of the genus Thermus by their lack of catalase activity and their utilization of only a few carbon sources. Furthermore, the novel strains exhibited mixotrophic growth with sulfur oxidation. On the basis of 16S rRNA gene sequence comparisons, DNA-DNA hybridization and physiological and biochemical characteristics, the new isolates represent a novel species. Since the species appears to be ubiquitous in Icelandic hot springs, the name Thermus islandicus sp. nov. is proposed. The type strain is PRI 3838(T) (=DSM 21543(T)=ATCC BAA-1677(T)).

Cloning and expression of heterologous genes in Rhodothermus marinus.

The construction of a shuttle cloning system for Rhodothermus marinus and Escherichia coli is described. It is based on the shuttle vector pRM3000, which contains a multiple cloning site as well as the shuttle marker trpB and TrpB(-) recipients of both species. The vector is stable and in 25 +/- 2 and 91 +/- 3 copies in R. marinus SB-1 and E. coli SDH-1, respectively. Three different R. marinus regulatory sequences of the dnaJ, dnaK and recA genes were integrated into pRM3000 to make the expression vectors pRM5100, pRM5200 and pRM5300, respectively. Genes encoding alpha- and beta-galactosidase (agaT and bgaT) from Thermus brockianus were cloned in R. marinus. Expression of both recombinant genes in R. marinus was demonstrated. The agaT gene was used as a reporter to study transcription from the expression vectors. Induced expression by ten- and twentyfold was observed from the dnaK and dnaJ regulatory sequences, respectively, after a temperature shift from 63 to 77 degrees C. This is the first report of cloning and expression of heterologous genes in R. marinus and the first study of promoter activity in the species.

Rhodothermus marinus: physiology and molecular biology.

Rhodothermus marinus has been the subject of many studies in recent years. It is a thermohalophilic bacterium and is the only validly described species in the genus Rhodothermus. It is not closely related to other well-known thermophiles and is the only thermophile within the family Crenotrichaceae. R. marinus has been isolated from several similar but distantly located geothermal habitats, many of which are subject to large fluctuations in environmental conditions. This presumably affects the physiology of R. marinus. Many of its enzymes show optimum activity at temperatures considerably higher than 65 degrees C, the optimum for growth, and some are active over a broad temperature range. Studies have found distinguishing components in the R. marinus electron transport chain as well as in its pool of intracellular solutes, which accumulate during osmotic stress. The species hosts both bacteriophages and plasmids and a functional intein has been isolated from its chromosome. Despite these interesting features and its unknown genetics, interest in R. marinus has been mostly stimulated by its thermostable enzymes, particularly polysaccharide hydrolysing enzymes and enzymes of DNA synthesis which may be useful in industry and in the laboratory. R. marinus has not been amenable to genetic analysis until recently when a system for gene transfer was established. Here, we review the current literature on R. marinus.

Identification and nucleotide sequence analysis of a cryptic plasmid, pRM21, from Rhodothermus marinus.

Here we report the identification and nucleotide sequence analysis of pRM21, a plasmid isolated from the thermophilic eubacterium Rhodothermus marinus. pRM21 consists of 2935 bp, has a G+C content of 58.2% and one major open reading frame whose deduced product shows significant similarities to RepA proteins from several plasmids, the highest being to the RepA of pSa from Escherichia coli. A region with the characteristics of iteron-containing replicons, three 19 bp repeats, DnaA boxes, an A+T rich region and GATC sequences, was identified. Of 40 additional R. marinus strains screened for plasmids, six (15%) were found to harbour plasmids with the same size and restriction pattern as pRM21.