Sigridur Hjorleifsdottir

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.

Rhodothermus marinus, gen. nov., sp. nov., a Thermophilic, Halophilic Bacterium from Submarine Hot Springs in Iceland

SUMMARY: Thermophilic, reddish-coloured heterotrophic bacteria different from Thermus were isolated from submarine alkaline hot springs in Iceland. The bacteria were obligately aerobic, moderately halophilic, Gram-negative rods, about 0.5 μm in diameter and 2-2.5 μm long. Neither spores, flagella nor lipid granules were observed, but a slime capsule was formed on carbohydrate-rich medium. Optimum growth was at 65°C, pH 7.0, and at about 2% (w/v) NaCl. The bacteria were oxidase negative, catalase positive and contained a carotenoid pigment with the main absorbance peak at 476 nm and shoulders at 456 and 502 nm. The GC content of the DNA was about 64 mol%. Electron micrographs clearly showed an outer membrane, about 9 nm thick, and the cytoplasmic membrane together with the peptidoglycan layer was about 14 nm in thickness. The isolates were nutritionally different from Thermus. They utilized several common sugars but glutamate and aspartate were the only amino acids that most strains used. These bacteria are considered to represent a new genus which we name Rhodothermus, with the type species Rhodothermus marinus.

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.

Thermus scotoductus, sp.nov., a pigment-producing thermophilic bacterium from hot tap water in Iceland and including Thermus sp. X-1

Summary Thermophilic, aerobic heterotrophic bacteria, producing a water soluble dark brown melanin-like pigment, were isolated from a hot water pipeline in a small town in southern Iceland. The bacteria stained Gram-negative, are short rods, 1.5 urn long and 0.5 um in diameter. The cells vare usually single or in pairs. A very dense peptidoglycan layer was seen as well as cytoplasmic membrane and an outer membrane. The bacteria are nonmotile and do not form spores. They could grow at 42-73 °C with optimum growth temperature of 65 °C and optimum pH at 7.5. The bacteria were oxidase and catalase positive, sensitive to the antibiotics penicillin, erythromycin, tetracyclin, chloramphenicol and streptomycin but resistant to polymyxin B and rifampicin. The GC was about 64.5%. The isolates were compared to other known aerobic, heterotrophic bacteria and turned out to resemble to colourless Thermus strain X-1, which was also found to produce the brown pigment. The DNA : DNA similarity between the new isolates and Thermus X-1 was about 83%, whereas the similarity with Thermus aquaticus was about 46%. The new isolates together with strain X-1 are therefore considered to represent a new species, Thermus scotoductus .

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.

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.

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.

Novel set of multiplex assays (SalPrint15) for efficient analysis of 15 microsatellite loci of contemporary samples of the Atlantic salmon (Salmo salar)

We report the development of new PCR assays and loading panels for the Atlantic salmon using 15 microsatellite loci. A total of 8, 3 and 4 loci were coamplified in three separate PCRs using labelled primers and loaded on the ABI DNA analyzer in two separate panels. Amplified alleles were clearly typed, and easily interpretable results were obtained. The method was successfully applied in different laboratories, even when different types of DNA polymerase were employed. The method is useful for analysing paternity, population genetics and conservation as well as for selective breeding programmes.

A Novel Type of Monoheme Cytochrome c : Biochemical and Structural Characterization at 1.23 Å Resolution of Rhodothermus marinus Cytochrome c

Monoheme cytochromes of the C-type are involved in a large number of electron transfer processes, which play an essential role in multiple pathways, such as respiratory chains, either aerobic or anaerobic, and the photosynthetic electron transport chains. This study reports the biochemical characterization and the crystallographic structure, at 1.23 A resolution, of a monoheme cytochrome c from the thermohalophilic bacterium Rhodothermus marinus. In addition to an alpha-helical core folded around the heme, common for this type of cytochrome, the X-ray structure reveals one unusual alpha-helix and a unique N-terminal extension, which wraps around the back of the molecule. Based on a thorough structural and amino acid sequence comparison, we propose R. marinus cytochrome c as the first characterized member of a new class of C-type cytochromes.