Production of the antibacterial compound TDA in Phaeobacter inhibens DSM17395

In the marine environment, micro-organisms rapidly colonise surfaces that have a high nutrient concentration, as compared to the surroundings. As a result, the competition for attachment space on surfaces is high and bacteria have developed different strategies to face such competition such as the production of bioactive compounds

Several studies have shown co-occurrence of bacterial biofilms and production of antibacterial compounds, and biofilm cultivation of bacteria originally isolated from gravel, algae and invertebrates induces antimicrobial activity. Biofilm formation and antibiotic production is linked in several species belonging to the marine Roseobacter clade. Phaeobacter gallaeciensis, P. inhibens and Ruegeria mobilis produce the broad-spectrum antibiotic tropodithietic acid (TDA), and its production in marine broth appears facilitated by stagnant conditions allowing the formation of a thick biofilm at the air–liquid interface. In R. mobilis, agitation completely abolishes TDA production, while most Phaeobacter strains are able to produce TDA under shaken conditions, which coincides with formation of small suspended biofilm floccules.

It has been observed in a previous study that the expression of one of the TDA biosynthesis genes (tdaC) and TDA production coincided with high c-di-GMP levels and with biofilm formation. However, it remained unclear whether TDA production truly depended on biofilm formation, or whether the two are merely phenotypically linked.

In this study Garcia et al. set to determine whether the ability to form biofilms on community level is a true prerequisite for TDA production, or whether they just co-occur under the respective culture conditions. Under a random transposon mutagenesis approach in P. inhibens DSM17395, they also identified some of the genes likely involved in attachment and biofilm formation in this strain.

A mutant library was created by Tn5 transposon insertion as previously described by Geng et al. (2008). Both nonpigmented and pigmented mutants were randomly selected, as pigmentation indicates TDA production. The mutated loci were identified by rescue cloning. As the goal of this study was to investigate whether biofilm formation was a prerequisite for TDA production, the authors deliberately selected brown (TDA+) mutants with reduced adhesion or biofilm formation and white (TDA  ̶ )mutants with increased adhesion or biofilm formation for further studies. Twenty-two TDA-positive (brown) mutants with decreased biofilm formation or adhesion, and eight TDA-negative (white) mutants with increased biofilm formation or adhesion were selected.

The results of this study demonstrated that the production of the antagonistic compound TDA does not require population attachment or biofilm formation in P. inhibens DSM17395, and biofilm formation is not a physiological prerequisite for TDA production. Most mutant strains deficient in biofilm formation did not produce significantly less TDA, as measured by inhibition or pigment formation, nor did disruption of the ability to synthesize TDA result in reduced biofilm formation. None of the selected biofilm over producing white mutants showed any antibiotic activity, while all brown mutants with reduced or disabled biofilm formation produced the antibacterial compound.

As mutations in single genes can have pleiotropic effects, it can be difficult to explain how a mutation in a certain gene produces the associated phenotype. This becomes evident in the example of the white mutants with increased adhesion and biofilm formation generated in this study. Although the lack of colony pigmentation on marine agar indicates defects in TDA biosynthesis, it is in all but one mutant, which has a mutated tdaB gene, not obvious that the mutations have affected TDA production. Sequencing analysis also gives indications that the genes involved in the production of EPS/LPS, particularly extracellular polysaccharides, motility and chemotaxis, as well as sensing and modification of the redox level play important roles in biofilm formation and/or adhesion P. inhibens DSM17395.

Understanding how TDA production is regulated and how it is influenced by environmental factors is important as TDA-producing Roseobacter clade bacteria are commonly found in aquaculture systems, where they are believed to be important for fish health, as they can antagonise fish and shellfish pathogens. Phaeobacter or other TDA-producing bacteria are also considered very suitable as probiotics for marine larviculture. Furthermore, Porsby et al. (2011) reported that TDA does not appear to induce resistance or tolerance in the target organism. Along with aquaculture, this could have huge benefits to the whole health industry and provide new avenues of research into new potential antibiotics in a world of ever more resistant and tolerant bacteria.

Prol García, M. J., D'Alvise, P. W., Rygaard, A. M., & Gram, L. (2014). Biofilm formation is not a prerequisite for production of the antibacterial compound tropodithietic acid in Phaeobacter inhibens DSM17395. Journal of applied microbiology, 117(6), 1592-1600.

http://onlinelibrary.wiley.com/doi/10.1111/jam.12659/abstract?deniedAccessCustomisedMessage=&userIsAuthenticated=false