Christian Schütt

Bacteria of the genus Roseobacter associated with the toxic dinoflagellate Prorocentrum lima

The dinoflagellate Prorocentrum lima is known to produce diarrhetic shellfish poisons. However, it is yet unclear if the dinoflagellates themselves or the bacteria associated with them produce the toxins. Here we analyze the toxicity as well as the spectrum of bacteria in two cultures of P. lima, namely P. lima-SY and P. lima-ST, which initially derived from the same P. lima strain PL2V. Toxicity tests, applying the Artemia bioassay revealed in both cultures high levels of toxins. The bacteria, associated with the two cultures, were identified by PCR/nucleotide sequence analysis of the 16S rRNA gene. From cultures of P. lima-SY the dominant sequence was found to share a 93.7% similarity with the sequence of Roseobacter algocolus [R. algicola]; the relative abundance was determined to be 83%. In addition three further sequences of bacteria, grouped to the α-Protobacteria have been identified: Paracoccus denitrificans [90.8%], R. algocolus [94.4%] and Rhizobium huakuii [92.6%]. The identification of bacteria in P. lima-ST revealed that most share highest similarity with Bartonella taylorii but with a relatively low score of 87%. In addition to this sequence, two sequences with high similarity to the genus Roseobacter were obtained. The other sequences identified have not been detected in P. lima-SY. Studies with pure bacterial strains, previously isolated from a culture of P. lima-ST and subsequently cultured on agar plates, revealed that none of them was identical to those identified in the dinoflagellate culture itself. An explanation for the change of the spectrum of bacteria in the different cultures can only be expected when axenic cultures from P. lima are available.

40-year long-term study of microbial parameters near Helgoland (German Bight, North Sea): historical view and future perspectives

Since 1873, the waters at Helgoland Roads (sampling station “Kabeltonne”) have been sampled daily to determine temperature and salinity. In 1962, microbiological parameters were determined for the first time to establish microbiological long-term studies on marine bacteria, starting with the colony-forming units (CFU). In the following years, several other microbiological parameters were integrated for different periods of time (e.g. activity parameters like ATP and ectoenzymatic activity, marine yeasts, oil-degrading bacteria, flagellates and molecular methods like PCR followed by denaturing gradient gel electrophoresis). To date, the total count of bacteria, flagellates and viruses have been acquired using fluorescent DNA dyes and epifluorescence microscopy. Here we present both a historical overview of the methods used and examples of results obtained over the past 40 years. Furthermore, we try to evaluate challenging new methods for marine microbial ecology, appropriate for long-term studies of marine bacteria.

Neuroactive compounds produced by bacteria from the marine sponge Halichondria panicea: activation of the neuronal NMDA receptor

Previous studies revealed that the marine sponge Halichondria panicea habors symbiotic- and commensalic bacteria (Althoff et al., 1998. Marine Biol. 130, 529-536). In the present study the hypothesis was tested whether some of those bacteria synthesize neuroactive compounds. For the first time the effect of bacterial bioactive compounds on the neuronal ionotropic glutamate receptors [iGluR], subtype N-methyl-d-aspartate (NMDA) receptor, was checked. In cortical neurons from rats as cell system the supernatant of two bacterial cultures isolated from H. panicea proved to agonize the NMDA receptor. The response of the NMDA receptor to the bioactive compounds was determined by measuring the intracellular Ca(2+) level. The supernatants of cultures 697 and 698 were found to upregulate the intracellular Ca(2+) level. To validate the specificity of the effects, inhibition studies with Memantine and d-AP5 were performed. The two bacteria were identified by polymerase chain reaction-amplification of the 16S rDNA genes and subsequent sequencing; they displayed highest identity to Antarcticum vesiculatum and to Psychroserpens burtonensis, respectively. Based on these data first experimental evidence is presented indicating that bacteria associated with sponges display neuroactivity by agonizing the NMDA receptor.

A secondary metabolite, 4,5-dibromopyrrole-2-carboxylic acid, from marine sponges of the genus Agelas alters cellular calcium signals

A secondary metabolite from sponges of the genus Agelas, 4,5-dibromopyrrole-2-carboxylic acid, which is well known as feeding deterrent, was investigated for effects on the cellular calcium homeostasis in PC12 cells. 4,5-Dibromopyrrole-2-carboxylic acid did not change intracellular calcium levels if applied alone without cell depolarization. During depolarization of the cellular membrane using high potassium solution, a dose dependent reduction of intracellular calcium elevation was revealed utilizing Fura II as calcium indicator. Significant reduction was seen at concentrations higher than 30μM in a series of experiments, but in single experiments a concentration of 300nM was still reversible effective. In the same concentration range, the onset of depolarization induced calcium elevations was significantly delayed by 4,5-dibromopyrrole-2-carboxylic acid. Dose dependent reduction and delay of depolarization evoked calcium elevations are probably due to a reduction of calcium entry via voltage operated calcium channels. One cellular mode of action of the feeding deterrent potential of 4,5-dibromopyrrole-2-carboxylic acid to fishes may be an interaction with the cellular calcium homeostasis of exposed cells.