Microbial community structure of sandy intertidal sediments in the North Sea, Sylt-Rømø Basin, Wadden Sea

Molecular biological methods were used to investigate the microbial diversity and community structure in intertidal sandy sediments near the island of Sylt (Wadden Sea) at a site which was characterized for transport and mineralization rates in a parallel study (D. de Beer, F. Wenzhöfer, T. Ferdelman, S.E. Boehme, M. Huettel, J.E.E. van Beusekom, M.E. Böttcher, N. Musat, N. Dubilier, Transport and mineralization rates in North Sea sandy intertidal sediments, Sylt-Romo Basin, Wadden Sea, Limnol. Oceanogr. 50 (2005) 113–127). Comparative 16S rRNA sequence analysis revealed a high bacterial diversity. Most sequences retrieved by PCR with a general bacterial primer set were affiliated with Bacteroidetes, Gammaproteobacteria, Deltaproteobacteria and the Pirellula cluster of Planctomycetales. Fluorescence in situ hybridization (FISH) and slot-blot hybridization with group-specific rRNA-targeted oligonucleotide probes were used to characterize the microbial community structure over depth (0–12 cm) and seasons (March, July, October). We found high abundances of bacteria with total cell numbers up to 3×10⁹ cells ml⁻¹ and a clear seasonal variation, with higher values in July and October versus March. The microbial community was dominated by members of the Planctomycetes, the Cytophaga/Flavobacterium group, Gammaproteobacteria, and bacteria of the Desulfosarcina/Desulfococcus group. The high abundance (1.5×10⁷–1.8×10⁸ cells ml⁻¹ accounting for 3–19% of all cells) of presumably aerobic heterotrophic polymer-degrading planctomycetes is in line with the high permeability, deep oxygen penetration, and the high rates of aerobic mineralization of algal biomass measured in the sandy sediments by de Beer et al. (2005). The high and stable abundance of members of the Desulfosarcina/Desulfococcus group, both over depth and season, suggests that these bacteria may play a more important role than previously assumed based on low sulfate reduction rates in parallel cores (de Beer et al., 2005).     

Modular enzyme design: regulation by mutually exclusive protein folding

A regulatory mechanism is introduced whereupon the catalytic activity of a given enzyme is controlled by ligand binding to a receptor domain of choice. A small enzyme (barnase) and a ligand-binding polypeptide (GCN4) are fused so that a simple topological constraint prevents them from existing simultaneously in their folded states. The two domains consequently engage in a thermodynamic tug-of-war in which the more stable domain forces the less stable domain to unfold. In the absence of ligand, the barnase domain is more stable and is therefore folded and active; the GCN4 domain is substantially unstructured. DNA binding induces folding of GCN4, forcibly unfolding and inactivating the barnase domain. Barnase-GCN4 is thus a "natively unfolded" protein that uses ligand binding to switch between partially folded forms. The key characteristics of each parent protein (catalytic efficiency of barnase, DNA binding affinity and sequence specificity of GCN4) are retained in the chimera. Barnase-GCN4 thus defines a modular approach for assembling enzymes with novel sensor capabilities from a variety of catalytic and ligand binding domains.     

Look@NanoSIMS--a tool for the analysis of nanoSIMS data in environmental microbiology

We describe an open-source freeware programme for high throughput analysis of nanoSIMS (nanometre-scale secondary ion mass spectrometry) data. The programme implements basic data processing and analytical functions, including display and drift-corrected accumulation of scanned planes, interactive and semi-automated definition of regions of interest (ROIs), and export of the ROIs' elemental and isotopic composition in graphical and text-based formats. Additionally, the programme offers new functions that were custom-designed to address the needs of environmental microbiologists. Specifically, it allows manual and automated classification of ROIs based on the information that is derived either from the nanoSIMS dataset itself (e.g. from labelling achieved by halogen in situ hybridization) or is provided externally (e.g. as a fluorescence in situ hybridization image). Moreover, by implementing post-processing routines coupled to built-in statistical tools, the programme allows rapid synthesis and comparative analysis of results from many different datasets. After validation of the programme, we illustrate how these new processing and analytical functions increase flexibility, efficiency and depth of the nanoSIMS data analysis. Through its custom-made and open-source design, the programme provides an efficient, reliable and easily expandable tool that can help a growing community of environmental microbiologists and researchers from other disciplines process and analyse their nanoSIMS data.     

HISH–SIMS analysis of bacterial uptake of algal-derived carbon in the Río de la Plata estuary

One of the main goals of microbial ecologists is to assess the contribution of distinct bacterial groups to biogeochemical processes, e.g. carbon cycling. Until very recently, it was not possible to quantify the uptake of a given compound at single cell level. The advent of nano-scale secondary-ion mass spectrometry (nanoSIMS), and its combination with halogen in situ hybridization (HISH) opened up this possibility. Despite its power, difficulties in cell identification during analysis of environmental samples might render this approach challenging for certain applications. A pilot study, designed to quantify the incorporation of phytoplankton-derived carbon by the main clades of heterotrophic aquatic bacteria (i.e. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes), is used to exemplify and suggest potential solutions to these technical difficulties. The results obtained indicate that the main aquatic bacterial clades quantitatively differ in the incorporation of algae-derived organic matter. From the methodological point of view, they highlight the importance of the concentration of the target cells, which needs to be sufficient to allow for a rapid mapping under the nanoSIMS. Moreover, when working with highly productive waters, organic and inorganic particles pose a serious problem for cell recognition based on HISH–SIMS. In this work several technical suggestions are presented to minimize the above mentioned difficulties, including alternatives to improve the halogen labeling of the cells and proposing the use of a combination of FISH and HISH along with a mapping system. This approach considerably enhances the reliability of cell identification and the speed of the subsequent nanoSIMS analysis in such complex samples.     

Iron corrosion by methanogenic archaea characterized by stable isotope effects and crust mineralogy

Carbon and hydrogen stable isotope effects associated with methane formation by the corrosive archaeon Methanobacterium strain IM1 were determined during growth with hydrogen and iron. Isotope analyses were complemented by structural, elemental and molecular composition analyses of corrosion crusts. During growth with H₂, strain IM1 formed methane with average δ¹³C of −43.5‰ and δ²H of −370‰. Corrosive growth led to methane more depleted in ¹³C, with average δ¹³C ranging from −56‰ to −64‰ during the early and the late growth phase respectively. The corresponding δ²H were less impacted by the growth phase, with average values ranging from −316 to −329‰. The stable isotope fractionation factors, , were 1.026 and 1.042 for hydrogenotrophic and corrosive growth respectively. Corrosion crusts formed by strain IM1 have a domed structure, appeared electrically conductive and were composed of siderite, calcite and iron sulfide, the latter formed by precipitation of sulfide (from culture medium) with ferrous iron generated during corrosion. Strain IM1 cells were found attached to crust surfaces and encrusted deep inside crust domes. Our results may assist to diagnose methanogens-induced corrosion in the field and suggest that intrusion of sulfide in anoxic settings may stimulate corrosion by methanogenic archaea via formation of semiconductive crusts.     

Novel groups of Gammaproteobacteria catalyse sulfur oxidation and carbon fixation in a coastal, intertidal sediment

The oxidation of hydrogen sulfide is essential to sulfur cycling in marine habitats. However, the role of microbial sulfur oxidation in marine sediments and the microorganisms involved are largely unknown, except for the filamentous, mat‐forming bacteria. In this study we explored the diversity, abundance and activity of sulfur‐oxidizing prokaryotes (SOP) in sulfidic intertidal sediments using 16S rRNA and functional gene sequence analyses, fluorescence in situ hybridization (FISH) and microautoradiography. The 16S rRNA gene analysis revealed that distinct clades of uncultured Gammaproteobacteria are important SOP in the tidal sediments. This was supported by the dominance of gammaproteobacterial sequences in clone libraries of genes encoding the reverse dissimilatory sulfite reductase (rDSR) and the adenosine phosphosulfate reductase (APR). Numerous sequences of all three genes grouped with uncultured autotrophic SOP. Accordingly, Gammaproteobacteria accounted for 40–70% of all ¹⁴CO₂‐incorporating cells in surface sediments as shown by microautoradiography. Furthermore, phylogenetic analysis of all three genes consistently suggested a discrete population of SOP that was most closely related to the sulfur‐oxidizing endosymbionts of the tubeworm Oligobrachia spp. FISH showed that members of this population (WS‐Gam209 group) were abundant, reaching up to 1.3 × 10⁸ cells ml^?1 (4.6% of all cells). Approximately 25% of this population incorporated CO₂, consistent with a chemolithoautotrophic metabolism most likely based on sulfur oxidation. Thus, we hypothesize that novel, gammaproteobacterial SOP attached to sediment particles may play a more important role for sulfide removal and primary production in marine sediments than previously assumed.     

Metal ion mediated self-assembly directed formation of protein arrays

Self-assembled inorganic-protein arrays with well-defined and controllable size and structure were obtained through the Fe(II) complexation of protein-conjugated terpyridine units (ligand). The atom-level control of the ligand is obtained through residue-specific conjugation between the complexing unit (terpy) containing an activity-based probe and a corresponding active enzyme (papain). The Fe(II)-based self-assembly performed on this unique building block (ligand) leads to chemical species of unprecedented constitution. The first example presented herein opens the way to a shape and size regime usually reserved to polymers.     

Nitrogen fixation and transfer in open ocean diatom–cyanobacterial symbioses

Many diatoms that inhabit low-nutrient waters of the open ocean live in close association with cyanobacteria. Some of these associations are believed to be mutualistic, where N₂-fixing cyanobacterial symbionts provide N for the diatoms. Rates of N₂ fixation by symbiotic cyanobacteria and the N transfer to their diatom partners were measured using a high-resolution nanometer scale secondary ion mass spectrometry approach in natural populations. Cell-specific rates of N₂ fixation (1.15–71.5 fmol N per cell h⁻¹) were similar amongst the symbioses and rapid transfer (within 30 min) of fixed N was also measured. Similar growth rates for the diatoms and their symbionts were determined and the symbiotic growth rates were higher than those estimated for free-living cells. The N₂ fixation rates estimated for Richelia and Calothrix symbionts were 171–420 times higher when the cells were symbiotic compared with the rates estimated for the cells living freely. When combined, the latter two results suggest that the diatom partners influence the growth and metabolism of their cyanobacterial symbionts. We estimated that Richelia fix 81–744% more N than needed for their own growth and up to 97.3% of the fixed N is transferred to the diatom partners. This study provides new information on the mechanisms controlling N input into the open ocean by symbiotic microorganisms, which are widespread and important for oceanic primary production. Further, this is the first demonstration of N transfer from an N₂ fixer to a unicellular partner. These symbioses are important models for molecular regulation and nutrient exchange in symbiotic systems.