Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

Feeding and grazing impact by small marine heterotrophic dinoflagellates on heterotrophic bacteria.

We investigated the feeding of the small heterotrophic dinoflagellates (HTDs) Oxyrrhis marina, Gyrodinium cf. guttula, Gyrodinium sp., Pfiesteria piscicida, and Protoperidinium bipes on marine heterotrophic bacteria. To investigate whether they are able to feed on bacteria, we observed the protoplasm of target heterotrophic dinoflagellate cells under an epifluorescence microscope and transmission electron microscope. In addition, we measured ingestion rates of the dominant heterotrophic dinoflagellate, Gyrodinium spp., on natural populations of marine bacteria (mostly heterotrophic bacteria) in Masan Bay, Korea in 2006-2007. Furthermore, we measured the ingestion rates of O. marina, G. cf. guttula, and P. piscicida on bacteria as a function of bacterial concentration under laboratory conditions. All HTDs tested were able to feed on a single bacterium. Oxyrrhis marina and Gyrodinium spp. intercepted and then ingested a single bacterial cell in feeding currents that were generated by the flagella of the predators. During the field experiments, the ingestion rates and grazing coefficients of Gyrodinium spp. on natural populations of bacteria were 14-61 bacteria/dinoflagellate/h and 0.003-0.972 day(-1), respectively. With increasing prey concentration, the ingestion rates of O. marina, G. cf. guttula, and P. piscicida on bacteria increased rapidly at prey concentrations of ca 0.7-2.2 x 10(6) cells/ml, but increased only slowly or became saturated at higher prey concentrations. The maximum ingestion rate of O. marina on bacteria was much higher than those of G. cf. guttula and P. piscicida. Bacteria alone supported the growth of O. marina. The results of the present study suggest that some HTDs may sometimes have a considerable grazing impact on populations of marine bacteria, and that bacteria may be important prey.

Production of antibacterial compounds and biofilm formation by Roseobacter species are influenced by culture conditions.

Bacterial communities associated with marine algae are often dominated by members of the Roseobacter clade, and in the present study, we describe Roseobacter phenotypes that may provide this group of bacteria with selective advantages when colonizing this niche. Nine of 14 members of the Roseobacter clade, of which half were isolated from cultures of the dinoflagellate Pfiesteria piscicida, produced antibacterial compounds. Many non-Roseobacter marine bacteria were inhibited by sterile filtered supernatants of Silicibacter sp. TM1040 and Phaeobacter (formerly Roseobacter) strain 27-4, which had the highest production of antibacterial compound. In contrast, Roseobacter strains were susceptible only when exposed to concentrated compound. The production of antibacterial compound was influenced by the growth conditions, as production was most pronounced when bacteria were grown in liquid medium under static conditions. Under these conditions, Silicibacter sp. TM1040 cells attached to one another, forming rosettes, as has previously been reported for Phaeobacter 27-4. A spontaneous Phaeobacter 27-4 mutant unable to form rosettes was also defective in biofilm formation and the production of antibacterial compound, indicating a possible link between these phenotypes. Rosette formation was observed in 8 of 14 Roseobacter clade strains examined and was very pronounced under static growth in 5 of these strains. Attachment to surfaces and biofilm formation at the air-liquid interface by these five strains was greatly facilitated by growth conditions that favored rosette formation, and rosette-forming strains were 13 to 30 times more efficient in attaching to glass compared to strains under conditions where rosette formation was not pronounced. We hypothesize that the ability to produce antibacterial compounds that principally inhibit non-Roseobacter species, combined with an enhancement in biofilm formation, may give members of the Roseobacter clade a selective advantage and help to explain the dominance of members of this clade in association with marine algal microbiota.

Motility is involved in Silicibacter sp. TM1040 interaction with dinoflagellates.

Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot-) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla-), while CtrA- cells possess flagella, but have reduced motility due to increased cell length. All three Mot- mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla- mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040-P. piscicida interaction.

Predator/prey interaction between Pfiesteria piscicida and Rhodomonas mediated by a marine alpha proteobacterium.

The dinoflagellate Pfiesteria piscicida coexists with bacteria in aquatic environments and as such, may interact with them at the physiological level. This study was designed to investigate the influence of bacteria, present in a clonal culture of Pfiesteria piscicida, on the predator/prey relationship of this dinoflagellate with the alga Rhodomonas. A series of replenishment experiments with bacteria isolated from P. piscicida clonal culture and the bacteria-free P. piscicida derived from the same culture were carried out. In the presence of bacteria, the number of P. piscicida increased significantly when incubated with alga Rhodomonas. This enhanced growth was almost entirely due to the increased consumption rate of Rhodomonas by P. piscicida since in bacteria-free (axenic) cultures Rhodomonas were consumed at significantly reduced rates relative to cultures with bacteria. Subsequent replenishment experiments with individual bacterial isolates showed that a single isolate was responsible for the increased predation rate of P. piscicida. The presence or absence of this specific bacterium determined the outcome of the interaction between P. piscicida and Rhodomonas. Partial sequence analysis of the 16S rDNA of this isolate indicated that it was a novel marine alpha proteobacterium with sequence similarities to a Roseobacter sp. and a bacterium recently isolated from a toxic dinoflagellate Alexandrium sp.

Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp.

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 microM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 microM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.