Expression of Tropodithietic Acid Biosynthesis Is Controlled by a Novel Autoinducer

Abstract

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.

Global occurrence and heterogeneity of the Roseobacter-clade species Ruegeria mobilis

“…It has been demonstrated that TDA induces its own production in Ruegeria (Silicibacter) sp. TM1040 (Geng and Belas, 2010), and we speculate that this would also be true for the R. mobilis strains of the present study.…”

“…It has been hypothesized that QS regulates the production of TDA (Geng and Belas, 2010;Berger et al, 2011). One of the classical groups of QS molecules, the AHLs, was not detected in any of the tested R. mobilis strains.…”

“…No TDA, or only traces, was detected in MB cultures from non-inhibitory strains or under conditions that abolished activity, such as aeration of the R. mobilis strains. As QS can be involved in regulating TDA production (Bruhn et al, 2005;Geng and Belas, 2010;Zan et al, 2014), we specifically searched for OH-C 10 -HSL, the AHL inducing TDA biosynthesis in P. inhibens (Berger et al, 2011;Wilson et al, 2016), and its known open lactone form in sterile-filtered supernatants, however, no AHL compounds were produced by R. mobilis under the tested conditions, but were easily detected in the three Phaeobacter reference strains using the same method (Supplementary Table S1). Furthermore, when concentrated acidified ethyl acetate extracts of selected R. mobilis strains of the four sub-clusters were analyzed by high performance liquid chromatography-time of flight MS, OH-C 10 -HSL or other AHLs were not detected (data not shown).…”

“…In P. gallaeciensis, TDA production is influenced by acylated homoserine lactones (Berger et al, 2011) and in Ruegeria (Silicibacter) sp. TM1040, TDA upregulates its own production (Geng and Belas, 2010). The antibacterial activity of these Roseobacter-clade strains could have a role in their natural niche, for example, during growth on particles, such as algae and marine snow, and it allows them to invade already formed bacterial biofilms (Rao et al, 2005); however, it is unknown how wide these specific bioactive strains are distributed on a global scale and in which environmental niches they appear.…”

“…TDA is also bactericidal against human pathogens including Salmonella enterica and Staphylococcus aureus (Porsby et al, 2011) and it has been recently been demonstrated that TDA acts by disrupting the proton motive force (Wilson et al, 2016). TDA is produced by R. mobilis, P. inhibens and Phaeobacter gallaeciensis, as well as by other alphaproteobacteria belonging to Pseudovibrio (Porsby et al, 2008;Geng and Belas, 2010;Penesyan et al, 2011;Harrington et al, 2014). Strains of Ruegeria and Phaeobacter produce TDA when grown under stagnant conditions in marine broth (MB) and TDA production coincides with bacterial rosette formation, a biofilm at the air-liquid interface, and production of a brown pigment that is an iron-TDA complex (Bruhn et al, 2007;Geng et al, 2008;Porsby et al, 2008;D'Alvise et al, 2015).…”