Sebastian Behrens

The identification of microorganisms by fluorescence in situ hybridisation

Fluorescence in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes facilitates the rapid and specific identification of individual microbial cells in their natural environments. Over the past year there have been a number of methodological developments in this area and new applications of FISH in microbial ecology and biotechnology have been reported.

The interplay of microbially mediated and abiotic reactions in the biogeochemical Fe cycle

Many iron (Fe) redox processes that were previously assumed to be purely abiotic, such as photochemical Fe reactions, are now known to also be microbially mediated. Owing to this overlap, discerning whether biotic or abiotic processes control Fe redox chemistry is a major challenge for geomicrobiologists and biogeochemists alike. Therefore, to understand the network of reactions within the biogeochemical Fe cycle, it is necessary to determine which abiotic or microbially mediated reactions are dominant under various environmental conditions. In this Review, we discuss the major microbially mediated and abiotic reactions in the biogeochemical Fe cycle and provide an integrated overview of biotic and chemically mediated redox transformations.

In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes

ABSTRACT Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp. strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3′ half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae.

Linking N2O emissions from biochar-amended soil to the structure and function of the N-cycling microbial community

Nitrous oxide (N2O) contributes 8% to global greenhouse gas emissions. Agricultural sources represent about 60% of anthropogenic N2O emissions. Most agricultural N2O emissions are due to increased fertilizer application. A considerable fraction of nitrogen fertilizers are converted to N2O by microbiological processes (that is, nitrification and denitrification). Soil amended with biochar (charcoal created by pyrolysis of biomass) has been demonstrated to increase crop yield, improve soil quality and affect greenhouse gas emissions, for example, reduce N2O emissions. Despite several studies on variations in the general microbial community structure due to soil biochar amendment, hitherto the specific role of the nitrogen cycling microbial community in mitigating soil N2O emissions has not been subject of systematic investigation. We performed a microcosm study with a water-saturated soil amended with different amounts (0%, 2% and 10% (w/w)) of high-temperature biochar. By quantifying the abundance and activity of functional marker genes of microbial nitrogen fixation (nifH), nitrification (amoA) and denitrification (nirK, nirS and nosZ) using quantitative PCR we found that biochar addition enhanced microbial nitrous oxide reduction and increased the abundance of microorganisms capable of N2-fixation. Soil biochar amendment increased the relative gene and transcript copy numbers of the nosZ-encoded bacterial N2O reductase, suggesting a mechanistic link to the observed reduction in N2O emissions. Our findings contribute to a better understanding of the impact of biochar on the nitrogen cycling microbial community and the consequences of soil biochar amendment for microbial nitrogen transformation processes and N2O emissions from soil.

Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit?

ABSTRACT Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-Å-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.

Linking environmental processes to the in situ functioning of microorganisms by high‐resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission X‐ray microscopy (STXM)

Environmental microbiology research increasingly focuses on the single microbial cell as the defining entity that drives environmental processes. The interactions of individual microbial cells with each other, the environment and with higher organisms shape microbial communities and control the functioning of whole ecosystems. A single-cell view of microorganisms in their natural environment requires analytical tools that measure both cell function and chemical speciation at the submicrometre scale. Here we review the technical capabilities and limitations of high-resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission (soft) X-ray microscopy (STXM) and give examples of their applications. Whereas NanoSIMS can be combined with isotope-labelling, thereby localizing the distribution of cellular activities (e.g. carbon/nitrogen fixation/turnover), STXM provides information on the location and chemical speciation of metabolites and products of redox reactions. We propose the combined use of both techniques and discuss the technical challenges of their joint application. Both techniques have the potential to enhance our understanding of cellular mechanisms and activities that contribute to microbially mediated processes, such as the biogeochemical cycling of elements, the transformation of contaminants and the precipitation of mineral phases.

Abundance, Distribution, and Activity of Fe(II)-Oxidizing and Fe(III)-Reducing Microorganisms in Hypersaline Sediments of Lake Kasin, Southern Russia

ABSTRACT The extreme osmotic conditions prevailing in hypersaline environments result in decreasing metabolic diversity with increasing salinity. Various microbial metabolisms have been shown to occur even at high salinity, including photosynthesis as well as sulfate and nitrate reduction. However, information about anaerobic microbial iron metabolism in hypersaline environments is scarce. We studied the phylogenetic diversity, distribution, and metabolic activity of iron(II)-oxidizing and iron(III)-reducing Bacteria and Archaea in pH-neutral, iron-rich salt lake sediments (Lake Kasin, southern Russia; salinity, 348.6 g liter−1) using a combination of culture-dependent and -independent techniques. 16S rRNA gene clone libraries for Bacteria and Archaea revealed a microbial community composition typical for hypersaline sediments. Most-probable-number counts confirmed the presence of 4.26 × 102 to 8.32 × 103 iron(II)-oxidizing Bacteria and 4.16 × 102 to 2.13 × 103 iron(III)-reducing microorganisms per gram dry sediment. Microbial iron(III) reduction was detected in the presence of 5 M NaCl, extending the natural habitat boundaries for this important microbial process. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of total Bacteria, total Archaea, and species dominating the iron(III)-reducing enrichment cultures (relatives of Halobaculum gomorrense, Desulfosporosinus lacus, and members of the Bacilli) were highest in an iron oxide-rich sediment layer. Combined with the presented geochemical and mineralogical data, our findings suggest the presence of an active microbial iron cycle at salt concentrations close to the solubility limit of NaCl.

Graphical representation of ribosomal RNA probe accessibility data using ARB software package

BackgroundTaxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments.ResultsThe PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different types of sequence associated information (SAI) can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool.ConclusionUsing this tool, in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of in situ hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page http://www.arb-home.de.

In Situ Accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains

ABSTRACT Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455T. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.

Fate of Cd during Microbial Fe(III) Mineral Reduction by a Novel and Cd-Tolerant Geobacter Species

Fe(III) (oxyhydr)oxides affect the mobility of contaminants in the environment by providing reactive surfaces for sorption. This includes the toxic metal cadmium (Cd), which prevails in agricultural soils and is taken up by crops. Fe(III)-reducing bacteria can mobilize such contaminants by Fe(III) mineral dissolution or immobilize them by sorption to or coprecipitation with secondary Fe minerals. To date, not much is known about the fate of Fe(III) mineral-associated Cd during microbial Fe(III) reduction. Here, we describe the isolation of a new Geobacter sp. strain Cd1 from a Cd-contaminated field site, where the strain accounts for 10(4) cells g(-1) dry soil. Strain Cd1 reduces the poorly crystalline Fe(III) oxyhydroxide ferrihydrite in the presence of at least up to 112 mg Cd L(-1). During initial microbial reduction of Cd-loaded ferrihydrite, sorbed Cd was mobilized. However, during continuous microbial Fe(III) reduction, Cd was immobilized by sorption to and/or coprecipitation within newly formed secondary minerals that contained Ca, Fe, and carbonate, implying the formation of an otavite-siderite-calcite (CdCO3-FeCO3-CaCO3) mixed mineral phase. Our data shows that microbially mediated turnover of Fe minerals affects the mobility of Cd in soils, potentially altering the dynamics of Cd uptake into food or phyto-remediating plants.