Gudmundur O. Hreggvidsson

Influence of Sulfide and Temperature on Species Composition and Community Structure of Hot Spring Microbial Mats

ABSTRACT In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80°C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90°C, and a Chloroflexusmat is formed at 65 to 70°C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and oneChloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in theChloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter−1) below 70°C, whereasAquificales were dominant in the high-sulfide spring (12 mg liter−1) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.

Identifying fishes through DNA barcodes and microarrays.

Background International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

Phylogenetic diversity analysis of subterranean hot springs in Iceland.

ABSTRACT Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130°C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-μm-pore-size filter. Cells were observed in wells RG-39 (91.4°C) and MG-18 (71.8°C) and in hot tap water (76°C), but no cells were detected in wells SN-4, SN-5 (95 to 117°C), and RV-5 (130°C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea andBacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85°C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to theThermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.

DNA Microarrays for Identifying Fishes

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

Investigation of the Microbial Ecology of Intertidal Hot Springs by Using Diversity Analysis of 16S rRNA and Chitinase Genes

ABSTRACT The microbial diversity of intertidal hot springs on the seashore of northwest Iceland was examined by combining directed in situ enrichments, artificial support colonization, and mat sampling. Analysis of 16S rRNA genes revealed the presence of clones related to both marine and terrestrial, thermophilic, mesophilic, and psychrophilic microorganisms scattered among 11 bacterial divisions. No archaea were found. The species composition of the enrichments was affected by the length of the hot periods experienced at low tide and was very different from those found in the biomass. A total of 36 chitinase genes were detected by molecular screening of the samples with degenerate primers for glycoside hydrolase family 18. The chitinase gene diversity was at least twofold higher in the enrichment samples than in the controls, indicating that a much higher diversity of hydrolytic genes can be accessed with this approach.

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

Abstract A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C.

The alternative complex III from Rhodothermus marinus – A prototype of a new family of quinol:electron acceptor oxidoreductases

The biochemical and genetic search for a bc 1 complex in Rhodothermus marinus was always fruitless; however, a functional equivalent, i.e. having quinol:cytochrome c oxidoreductase activity was characterized. Now, with the sequencing of R. marinus genome, it was possible to assign the N‐terminal sequences of several proteins of this complex to its coding genes. The alternative complex III from R. marinus has the same genomic organization of the so‐called MFIcc complexes, proposed to be oxidoreductases of the respiratory and photosynthetic electron transfer chains. In this report, we establish undoubtedly the existence of an alternative complex III, a functional substitute of the bc 1 complex, by its identification at both the biochemical and genomic level.

The structure of Rhodothermus marinus Cel12A, a highly thermostable family 12 endoglucanase, at 1.8 Å resolution

Cellulose is one of the most abundant polysaccharides in nature and microorganisms have developed a comprehensive system for enzymatic breakdown of this ubiquitous carbon source, a subject of much interest in the biotechnology industry. Rhodothermus marinus produces a hyperthermostable cellulase, with a temperature optimum of more than 90 degrees C, the structure of which is presented here to 1.8 A resolution. The enzyme has been classified into glycoside hydrolase family 12; this is the first structure of a thermophilic member of this family to have been solved. The beta-jelly roll fold observed has identical topology to those of the two mesophilic members of the family whose structures have been elucidated previously. A Hepes buffer molecule bound in the active site may have triggered a conformational change to an active configuration as the two catalytic residues Glu124 and Glu207, together with dependent residues, are observed in a conformation similar to that seen in the structure of Streptomyces lividans CelB2 complexed with an inhibitor. The structural similarity between this cellulase and the mesophilic enzymes serves to highlight features that may be responsible for its thermostability, chiefly an increase in ion pair number and the considerable stabilisation of a mobile region seen in S. lividans CelB2. Additional aromatic residues in the active site region may also contribute to the difference in thermophilicity.

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°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.