Frida Lise Daae

Novel techniques for analysing microbial diversity in natural and perturbed environments

Molecular techniques were applied for analysing the entire bacterial community, including both the cultivated and non-cultivated part of the community. DNA was extracted from samples of soils and sediments, and a combination of different molecular methods were used to investigate community structure and diversity in these environments. Reassociation of sheared and thermally denatured DNA in solution was used to measure the total genetical diversity. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of rRNA genes gave information about changes in the numerically dominating bacterial populations. Hybridisation with phylogenetic group specific probes, and sequencing provided information about the affiliation of the bacterial populations. Using DNA reassociation analysis we demonstrated that bacterial communities in pristine soil and sediments may contain more than 10,000 different bacterial types. The diversity of the total soil community was at least 200 times higher than the diversity of bacterial isolates from the same soil. This indicates that the culturing conditions select for a distinct subpopulation of the bacteria present in the environment. Molecular methods were applied to monitor the effects of perturbations due to antropogenic activities and pollution on microbial communities. Our investigations show that agricultural management, fish farming and pollution may lead to profound changes in the community structure and a reduction in the bacterial diversity.

Diversity of life in ocean floor basalt

Abstract Electron microscopy and biomolecular methods have been used to describe and identify microbial communities inhabiting the glassy margins of ocean floor basalts. The investigated samples were collected from a neovolcanic ridge and from older, sediment-covered lava flows in the rift valley of the Knipovich Ridge at a water depth around 3500 m and an ambient seawater temperature of −0.7°C. Successive stages from incipient microbial colonisation, to well-developed biofilms occur on fracture surfaces in the glassy margins. Observed microbial morphologies are various filamentous, coccoidal, oval, rod-shaped and stalked forms. Etch marks in the fresh glass, with form and size resembling the attached microbes, are common. Precipitation of alteration products around microbes has developed hollow subspherical and filamentous structures. These precipitates are often enriched in Fe and Mn. The presence of branching and twisted stalks that resemble those of the iron-oxidising Gallionella , indicate that reduced iron may be utilised in an energy metabolic process. Analysis of 16S-rRNA gene sequences from microbes present in the rock samples, show that the bacterial population inhabiting these samples cluster within the γ- and ϵ-Proteobacteria and the Cytophaga/Flexibacter/Bacteroides subdivision of the Bacteria, while the Archaea all belong to the Crenarchaeota kingdom. This microbial population appears to be characteristic for the rock and their closest relatives have previously been reported from cold marine waters in the Arctic and Antarctic, deep-sea sediments and hydrothermal environments.

Characterization of Microbial Diversity in Hypersaline Environments by Melting Profiles and Reassociation Kinetics in Combination with Terminal Restriction Fragment Length Polymorphism (T-RFLP)

The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA. These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches. Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied. The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments. The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity. At 37% salinity the diversity was reduced again to nearly half that at 22% salinity. Our results revealed that the community “genome” had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome. The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds. They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity. The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT. The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques. In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP. One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber.

Analysis of broad-scale differences in microbial community composition of two pristine forest soils

Broad-scale differences in soil microbial community composition were analyzed in two contrasting soils using DNA reassociation and % G + C profiles for analysis on the community-level, and filter- and whole cell hybridization techniques for a coarse-level characterization of larger phylogenetic groups of bacteria. Reassociation analysis of DNA from bacterial fractions extracted from the organic soil Seim and the mineral soil Hau revealed similar complexity of the communities with 5700 and 4900 different bacterial genomes (g soil [dry wt])-1, respectively. Thermal denaturation studies showed wide % G + C distributions in DNA from bacteria of both soils. Differences in the median % G + C with 55 to 61% for the bacterial community in soil Seim and 61 to 66% for that in soil Hau indicated a higher proportion of bacteria with a high DNA G + C content in soil Hau. In situ hybridization with fluorescent (Cy3-labeled) probes targeting larger phylogenetic groups showed minor differences between both soils, and between direct detection of bacteria in dispersed soil slurries and in bacterial fractions extracted from soils through about 90% of the total bacteria were lost during extraction. In dispersed slurries of both soils, only probes ALF1b, SRB385, and PLA46 hybridized to cells accounting for more than 1% of the DAPI-stained cells, while numbers obtained after hybridization with probes ARCH915, BET42a, GAM42a, HGC69a, and CF319a were below the detection limit set at < 1%. These results were confirmed by in situ hybridization with horseradish peroxidase (HRP)-labeled probes and subsequent Cy3-tyramide signal amplification. In contrast, dot blot hybridization with probe HGC69a indicated significant amounts of Gram-positive bacteria with a high DNA G + C content in both soils. These could subsequently be visualized in non-dispersed soil slurries by in situ hybridization with HRP-labeled probe HGC69a and Cy3-tyramide signal amplification. Filamentous Gram-positive bacteria with a high DNA G + C content, likely actinomycetes, which are present in soil Hau in significant numbers are obviously destroyed by procedures used for soil dispersion.

Microbial metacommunities in the lichen–rock habitat

Lichens are common as colonizers of bare rocks and contribute to weathering, but their associated bacterial communities have been poorly studied. In this study Hydropunctaria maura, Ophioparma ventosa, Pertusaria corallina and Rhizocarpon geographicum were analysed to determine the influence of lichens on lichen-rock-associated microbial metacommunities. For the first time, Archaea were documented to be associated with rock-inhabiting lichens. All the archaeal sequences obtained were affiliated with Crenarchaeota. The Bacteria detected in the lichen-rock samples were affiliated with the major lineages Acidobacteria, Actinobacteria, Alpha-, Beta-, Gammaproteobacteria, Bacteriodetes, Chloroflexi, Deinococcus, Firmicutes, Planctomycetes, Tenericutes and Cyanobacteria. The microbial communities of O. ventosa, P. corallina and R. geographicum were more similar to each other both terms of the number and types of different sequences, than to H. maura. A higher bacterial diversity was observed endolithically than within the epilithic lichen thalli directly above. The abundance of Archaea were also generally higher endolithically than in the epilithic lichen thalli, while the abundance of Bacteria was higher in the lichen thalli compared with within the rock. These results demonstrated that the lichen-rock interfaces are complex habitats, where the macroscopic lichens influence the composition of microbial metacommunities.

Classification of fish-pathogenic vibrios based on comparative 16S rRNA analysis

No systematic classification of fish-pathogenic vibrios has been accomplished previously despite the use of serological, physiological, and genetical classification systems. In this study, a comparative 16S rRNA analysis of 34 strains (representing seven species) of fish-pathogenic vibrios was performed. The 16S rRNA sequences were obtained by using reverse transcriptase. Nearly complete sequences were obtained for nine strains. On the basis of the results of this analysis, the remaining strains were investigated by analyzing selected stretches containing a total of 560 nucleotides. With the exception of a few strains, including ATCC 43313 (serovar O9), our comparative 16S rRNA analysis confirmed that strains preliminarily identified as Vibrio anguillarum were phylogenetically closely related. Strains of V. anguillarum could be divided into groups, with the main group containing serotype O1 and O2 strains isolated from Atlantic salmon, rainbow trout, turbot, cod, and saithe. The other distinctive group was represented by type strain NCMB 6. This strain was nearly indistinguishable from the type strains of Vibrio ordalii and Vibrio damsela on the basis of the 16S rRNA stretches compared. The results of a comparative 16S rRNA analysis justified the status of Vibrio salmonicida as a distinct species. Originally, this species was characterized biochemically as a very homogeneous species. However, two strains, which were isolated from diseased halibut and from the intestines of healthy cod, could not be distinguished from V. salmonicida strains phylogenetically, although they differed from the original species description in several phenotypic traits. Our results indicate that V. salmonicida and Vibrio fischeri form a cluster that is clearly separated from the cluster that includes V. anguillarum.

Nitrogen availability decreases prokaryotic diversity in sandy soils

In this study, the interrelation between nitrogen availability and prokaryotic diversity are studied using a well-characterised system from a long-term field experiment on a loamy sandy soil. The prokaryotic potential functional diversity and community composition were assessed using community-level physiological profiling (CLPP), and their phylogenetic diversity was analysed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in combination with sequencing analysis. Highest prokaryotic potential functional diversity was measured in the control soil receiving no N fertilisation, indicating an efficient as well as versatile utilisation of the substrates in this soil. Both substrate utilisation richness and substrate utilisation evenness, the two constituents of the functional diversity, were decreased with increasing N supply. Furthermore, distinct prokaryotic community compositions were generated in N-enriched soils compared to unfertilised control soils. These differences suggest a dominance of populations adapted to utilising readily available substrates. We demonstrated that the shift in prokaryotic functional diversity was connected to a shift in the phylogenetic structure of the bacterial and archaeal communities. Taken together, our data clearly show that, for the sandy soil system, prokaryotic diversity and N availability were interrelated.

Microbial life associated with low‐temperature alteration of ultramafic rocks in the Leka ophiolite complex

Water-rock interactions in ultramafic lithosphere generate reduced chemical species such as hydrogen that can fuel subsurface microbial communities. Sampling of this environment is expensive and technically demanding. However, highly accessible, uplifted oceanic lithospheres emplaced onto continental margins (ophiolites) are potential model systems for studies of the subsurface biosphere in ultramafic rocks. Here, we describe a microbiological investigation of partially serpentinized dunite from the Leka ophiolite (Norway). We analysed samples of mineral coatings on subsurface fracture surfaces from different depths (10-160 cm) and groundwater from a 50-m-deep borehole that penetrates several major fracture zones in the rock. The samples are suggested to represent subsurface habitats ranging from highly anaerobic to aerobic conditions. Water from a surface pond was analysed for comparison. To explore the microbial diversity and to make assessments about potential metabolisms, the samples were analysed by microscopy, construction of small subunit ribosomal RNA gene clone libraries, culturing and quantitative-PCR. Different microbial communities were observed in the groundwater, the fracture-coating material and the surface water, indicating that distinct microbial ecosystems exist in the rock. Close relatives of hydrogen-oxidizing Hydrogenophaga dominated (30% of the bacterial clones) in the oxic groundwater, indicating that microbial communities in ultramafic rocks at Leka could partially be driven by H2 produced by low-temperature water-rock reactions. Heterotrophic organisms, including close relatives of hydrocarbon degraders possibly feeding on products from Fischer-Tropsch-type reactions, dominated in the fracture-coating material. Putative hydrogen-, ammonia-, manganese- and iron-oxidizers were also detected in fracture coatings and the groundwater. The microbial communities reflect the existence of different subsurface redox conditions generated by differences in fracture size and distribution, and mixing of fluids. The particularly dense microbial communities in the shallow fracture coatings seem to be fuelled by both photosynthesis and oxidation of reduced chemical species produced by water-rock reactions.

Physiological and genomic characterization of Arcobacter anaerophilus IR-1 reveals new metabolic features in Epsilonproteobacteria.

In this study we characterized and sequenced the genome of Arcobacter anaerophilus strain IR-1 isolated from enrichment cultures used in nitrate-amended corrosion experiments. A. anaerophilus IR-1 could grow lithoautotrophically on hydrogen and hydrogen sulfide and lithoheterothrophically on thiosulfate and elemental sulfur. In addition, the strain grew organoheterotrophically on yeast extract, peptone, and various organic acids. We show for the first time that Arcobacter could grow on the complex organic substrate tryptone and oxidize acetate with elemental sulfur as electron acceptor. Electron acceptors utilized by most Epsilonproteobacteria, such as oxygen, nitrate, and sulfur, were also used by A. anaerophilus IR-1. Strain IR-1 was also uniquely able to use iron citrate as electron acceptor. Comparative genomics of the Arcobacter strains A. butzleri RM4018, A. nitrofigilis CI and A. anaerophilus IR-1 revealed that the free-living strains had a wider metabolic range and more genes in common compared to the pathogen strain. The presence of genes for NAD+-reducing hydrogenase (hox) and dissimilatory iron reduction (fre) were unique for A. anaerophilus IR-1 among Epsilonproteobacteria. Finally, the new strain had an incomplete denitrification pathway where the end product was nitrite, which is different from other Arcobacter strains where the end product is ammonia. Altogether, our study shows that traditional characterization in combination with a modern genomics approach can expand our knowledge on free-living Arcobacter, and that this complementary approach could also provide invaluable knowledge about the physiology and metabolic pathways in other Epsilonproteobacteria from various environments.

Functional interactions among filamentous Epsilonproteobacteria and Bacteroidetes in a deep-sea hydrothermal vent biofilm

Little is known about how lithoautotrophic primary production is connected to microbial organotrophic consumption in hydrothermal systems. Using a multifaceted approach, we analysed the structure and metabolic capabilities within a biofilm growing on the surface of a black smoker chimney in the Lokis Castle vent field. Imaging revealed the presence of rod-shaped Bacteroidetes growing as ectobionts on long, sheathed microbial filaments (> 100 μm) affiliated with the Sulfurovum genus within Epsilonproteobacteria. The filaments were composed of a thick (> 200 nm) stable polysaccharide, representing a substantial fraction of organic carbon produced by primary production. An integrated -omics approach enabled us to assess the metabolic potential and in situ metabolism of individual taxonomic and morphological groups identified by imaging. Specifically, we provide evidence that organotrophic Bacteroidetes attach to and glide along the surface of Sulfurovum filaments utilizing organic polymers produced by the lithoautotrophic Sulfurovum. Furthermore, in situ expression of acetyl-CoA synthetase by Sulfurovum suggested the ability to assimilate acetate, indicating recycling of organic matter in the biofilm. This study expands our understanding of the lifestyles of Epsilonproteobacteria in hydrothermal vents, their metabolic properties and co-operative interactions in deep-sea hydrothermal vent food webs.