Bacterioplankton & Flow Cytometry

Recently, work has focused on comparisons of FISH and CARD-FISH protocols for single cell identification (Fuchs et al. 2007; Woebken et al. 2007), and the detection of low copy number targets (Moraru, Ph.D. thesis). The optimization of CARD-FISH for various samples has been continued (Tujula et al. 2006). With CARD-FISH, the efficiency of single cell identification is now routinely above 60% and often close to 100%, even in samples from oligotrophic sites. The combination of CARD-FISH and automated microscopy enables large counting studies on the scale of ocean basins with hundreds of individual samples (Schattenhofer et al. unpublished).

For detailed information, please switch to the "FISH" pages.

We work on improving a set of approximately 40 oligonucleotide probes for marine bacterioplankton (e.g. Woebken et al. 2007; Mary et al. 2006). Based on the SILVA-ref data set of the Microbial Genomics group (Prüsse et al. 2007) the group-specific probes for large taxa within the Bacteria and Archaea were re-evaluated against about 200,000 publicly accessible high quality SSU rRNA sequences (Amann & Fuchs, submitted).

We developed an image acquisition and evaluation system for the automated microscopic evaluation of comparatively simple samples, i.e. the counting of bacterial cells in suspension that are filtered and immobilized on membrane filters (Pernthaler et al. 2003). For this we have developed a robust computerized focussing system that combines a proprietary autofocus with custom texture parameters, feedback loops for image contrast maximation, and decisions on image content during acquisition.

The system is capable of excluding inappropriate or badly focussed microscopic fields prior to evaluation without operator interaction with high reliability (>95%), and to evaluate >50 FISH stained samples per day at a total of 2000-3000 counted cells per sample.

Bacterioplankton analysis is routinely done with flow cytometry. Parameters like DNA-content, cell size and protein content are measured and quantified. There are two machines available for flow cytometry. A small benchtop flow cytometer was purchased for shipboard analysis of bacterioplankton.

The second, large research flow cytometer was used for high speed sorting of cell material for 16S rRNA cloning and sequencing.

Flavobacteria

Marine members of the phylum Bacteroidetes are highly diverse. Most belong to the class Flavobacteria (Alonso et al. 2007). Data of our VISION cruise show relative abundances of about 20% in North Atlantic surface waters at the end of the phytoplankton summer blooms (Gomez et al. unpublished). Screening of meta-genomic libraries has been initiated in order to further analyze the genetic potential of this important bacterioplankton group. This will also evaluate the hypotheses generated by Bauer et al. (2006) on the basis of the annotation of the first fully sequenced marine flavobacterium, Gramella forsetii KT0803.

Planctomycetes

In collaboration with Marcel Kuypers (Nutrient group), localization studies of bacteria mediating anaerobic oxidation of ammonia in Namibian upwelling waters were carried out (Woebken et al. 2007a). Anammox bacteria can be found both in aggregates and free-living. A large comparative (meta)genomics study including the collaboration of Dagmar Woebken (PIs: Teeling & Glöckner) suggests taxonomic separation of Anammox from “true” Planctomycetes (Woebken et al. 2007b). This study also corroborates the important role of Planctomycetes in the degradation of algal cell walls. Unpublished results of the new Ph.D. student Ilaria Pizzetti suggest seasonal peaks of probe PLA46-positive cells in aerobic North Sea waters of up to 5%.

The gammaproteobacterial NOR5/OM60 clade

Bacteria of this cosmopolitan group are preferentially found in coastal waters. FISH reveals strong seasonal dynamics. The NOR5/OM60 cells may account for >10 e4 cells per ml (Shi et al., unpublished). The genome of an isolate obtained within the group by Heike Eilers (“Congregibacter litoralis” KT71 = DSMZ 17192) has been sequenced with funds of the Moore Foundation. The annotation of the genome revealed to our surprise a photosynthesis super-operon. KT71 was subsequently shown to be a representative of the gammaproteobacterial aerobic anoxygenic phototrophs predicted by Beja & DeLong based on metagenomics (Fuchs et al. 2007). Three more genomes of related gammaproteobacteria are currently sequenced.