Projects

Main themes of scientific work
The department aims at an understanding of microbial processes on the level of growth, metabolism and gene regulation. Approaches are usually experimental (in vitro) and involve cultivation, but some work is also done with natural populations in sediments and mats. The connection with biogeochemistry and genome projects is of great value. Most research so far was centered around two themes: (a) Microorganisms with the capacity for anaerobic degradation of natural compounds with low reactivity, in particular hydrocarbons (including methane), which involves an intriguing biochemistrly; and (b) microorganisms that transform redox-active inorganic compounds such as sulfur and iron species (including metallic iron).

(Judith Amann, Dörte Gade, Daniela Lange, Ralf Rabus*)
With the increasing significance of genomics (see Dept. Molecular Ecology) for the study of aquatic bacteria, the previously established laboratory for proteomics (20) covers protein synthesis and patterns. The new 2D difference gel electrophoresis with cyanine dyes for quantification of differences in protein patterns has been established (2).
For one study bacterium, Rhodopirellula, a master gel with more than 500 identified proteins was established; from this, central catabolic routes and the regulation of substrate-specific proteins were elaborated, and about 100 proteins regulated in response to growth phases were identified. With two other study bacteria in the common genome project, Desulfotalea psychrophila (genome recently completed) and Desulfobacterium autotrophicum (genome to be completed), a survey of catabolic proteins and the induction of proteins with selected substrates or under high (deep-sea like) pressure began recently. External collaboration has been established with the MPI for Molecular Genetics (Richard Reinhardt), Epidauros (Peter Klenk), and the University of Göttingen (Gerhard Gottschalk).
A former proteomic "spin-off" task dealing with a hydrocarbon-degrading denitrifier has developed into an own project (see next section).

(Judith Amann, Simon Kühner, Daniela Lange, Ralf Rabus*, Lars Wöhlbrand)
The isolation of anaerobic aquatic bacteria with hydrocarbons and polar aromatics during the past years has revealed a great diversity of novel Betaproteobacteria, the "biodegradative" Azoarcus branch. The Microbiology group has participated in or achieved the elucidation of pathways in the anaerobic degradation of hydrocarbons.
With the increasing availability of genomic information, the connection between degradative capacities and their genetic regulation is presently gaining much interest. In a representative model organisms, strain EbN1, separate genomic islands for the anaerobic degradation of ethylbenzene and toluene have been identified (12, 21). Operon and mRNA analyses as well as 2D-electrophoresis not only indicate a separate regulation of ethylbenzene and toluene degradation via hydrocarbon-specific two-component systems, but also a more refined, sequential regulation of an "upper" and a "lower" pathway in ethylbenzene degradation (Fig. 1). There is an intensive external collaboration with the MPI for Molecular Genetics (Richard Reinhardt).
A future goal will be the establishment of a genetic system for gene mutation. Another versatile species of the branch, strain pCyN1, degrades p-isopropyltoluene (and several other terpenes) as well as toluene, possibly via rather different pathways.

(Olav Grundmann, Ingrid Kunze, Ralf Rabus, Friedrich Widdel*)
n-Alkanes [H3C-(CH2)n-CH3], an important class of petroleum and biogenic hydrocarbons with low reactivity, can be degraded anaerobically by denitrifying and sulfate-reducing bacteria as well as by methanogenic communities. A pathway for n-hexane degradation via activation in a radicalic mechanism with fumarate as co-substrate has been suggested upon metabolite analyses in the denitrifying Azoarcus-like strain HxN1 (29, 30).

Friederike Heinrich, Thomas Holler, Martin Krüger, Katja Nauhaus, Friedrich Widdel*,
Heike Wolters)
The study of the anaerobic oxidation of methane (AOM) in a collaborative project of the MPI (previously also BMBF-funded as MUMM) has been continued. After the experimental demonstration of the net reaction (CH4 + SO42 ? HCO3 + HS + H2O) and upon obtaining evidence that H2, acetate or methanol are not free intermediates in AOM (16, 28), further cultivation attempts were made. Whereas microbial mats from the Black Sea are problematic for long-term maintenance in vitro, consortia of Archaea (ANME-2) and Desulfosarcinales from the Cascadia Margin (NE Pacific) could be slowly enriched (tenfold) at 10 bar methane over a period of two years. Still, the mat material from the Black Sea was well-suited for the extraction of a nickel compound (M = 951 Da) resembling F430 (M = 905 Da), and of the protein that harbors this compound. The protein analysis in collaboration with the MPI Marburg (Rolf Thauer) and the in situ gene analysis by the Department of Molecular Ecology (see that report) revealed a relationship to methyl-coenzyme-M reductase (MCR), the key enzyme in methanogenesis; however, the nickel protein from the mat represents a distinct phylogenetic line among MCRs (11).

(Ramona Appel, Galina Gorbik-Weiß, Jens Harder*, Christina Probian, Annika Wülfing)
Interest in degradative pathways of various alicyclic or highly branched natural compounds has led to the identification of structural elements that deserve particular interest from a biochemical point of view. Enrichment and isolation with appropriate model compounds representing such structural elements resulted in the isolation of novel bacteria. Present research efforts are devoted to: (a) the characterization of genes for the anaerobic utilization of natural alkene-monoterpenes; (b) the enzymology of the degradation of quarternary carbon atoms (2,2-dimethylpropionate) in a collaboration with the Univ. of Zürich (J.A. Robinson); (c) the structure of a unique alpha-ketolase of the 1,2-cyclohexanediol-pathway in collaboration with the Univ. of Konstanz (A. Steinbach / P. Kroneck).

(Florin Musat, Friedrich Widdel*)
Oil spills in the sea usually lead to gradually solidifying oil-sediment mixtures on shorelines that may be populated by cyanobacteria. Under these, layers of aerobic chemotrophs including hydrocarbon degraders and a deeper anoxic zone of intense sulfate reduction are situated. Such "oil mats" colonized by cyanobacteria was the topic of the EU project, MATBIOPOL. In this, our group established microcosms with experimental, reproducible surface pollution of sediment and studied the development of cyanobacteria, chemotrophs and sulfate-reducing bacteria (still in progress) (15).

Christine Flies, Karen Grünberg, Udo Heyen, Katja Junge, Oliver Menke, Ekaterina Schmidt, Dirk Schüler*, Daniel Schultheiss, Sabrina Schübbe, Cornelia Stumpf, Cathrin Wawer)
A unique form of biological iron transformation is the finely controlled synthesis of nano-sized ferromagnetic magnetite (Fe3O4, formally FeO·Fe2O3) crystals in the magnetosomes of magnetotactic bacteria (MTB).
A challenging research theme is the biochemistry and genetic regulation of magnetite synthesis (biomineralization). Analysis of proteins from the magnetosome membrane in Magnetospirillum gryphiswaldense revealed 20 magnetosome-specific polypeptides (MMPs) with presumptive functions in magnetosome formation (3, 22). MMPs are encoded by several mam-operons within a large genomic "magnetosome island"; this is genetically unstable, as revealed by mutant analyses (22). Site-directed mutagenesis (26, 27) indicated an essential function of mam-genes magnetosome assembly, e.g. in magnetosome-directed iron transport. Furthermore, the subcellular topology and regulation of MMPs in magnetic and non-magnetic strains is under study. This can now be achieved by transcriptional and translational profiling as well as by the use of gfp-gene fusions.
The perfect crystalline and magnetic properties of magnetosomes make them a promising biomaterial for applications. Using an oxystat for growth (7), we produce magnetosomes from M. gryphiswaldense such that their biomedical and nanobiotechnological potential can be studied in detail by external partners (6, 8).
Since most MTB have not been cultivated, we also study their phylogenetic affiliation and behavior directly in aquatic habitats. Microelectrode and geochemical analyses revealed that the vertical distribution of MTB in sediments correlated with O2- and Fe(II)-gradients.

(Hang T. Dinh, Friedrich Widdel*)
A form of iron that is not part of natural biogeochemical cycles but belongs the most important technical materials is the element itself (Fe). Unfortunately, iron as a base metal undergoes rusting at the air. In anoxic sterile water, iron is rather stable. However, sulfate-reducing bacteria (SRB) corrode iron anaerobically and cause significant technical damage, e.g. in pipelines. According to text books, the scavenge by Desulfovibrio species of hydrogen formed on iron (Fe + 2H2O ? Fe2+ + H2 + 2HO ) is regarded as a central mechanism in iron corrosion by SRB. However, the recent isolation of distinctly corrosive SRB (in particular a Desulfobacterium type) differing from classical Desulfovibrio species as well as kinetic measurements suggest that H2 utilization is not decisive for anaerobic corrosion; rather the novel, surface-attached SRB seem to withdraw electrons from iron more directly, presumably via an overall mechanism according to Fe (? Fe2+) ? 2e ? electron transport system ? 2e ? sulfate reduction enzymes (1). In this area, we collaborate with the Institute for Material Testing (Jan Kuever), and the MPI for Iron Research (Achim Hassel).