GENETIC ANALYSIS OF TROPODITHIETIC ACID BIOSYNTHESIS IN MARINE BACTERIA

Abstract

Members of the Roseobacter clade of alphaproteobacteria are among the most abundant and ecologically relevant marine bacteria. The antibiotic tropodithietic acid (TDA) produced by roseobacters is hypothesized to be a critical component of the roseobacter-phytoplankton symbiosis. TDA production is influenced by environmental conditions. Specifically, in the lab, TDA activity is highest when bacteria are cultured in standing liquid nutrient broth, whereas cells produce negligible TDA in nutrient broth with shaking. Random mutagenesis was used to construct loss-of-function mutants defective in TDA activity (Tda<super>-</super>). Twelve genes were identified as required for Tda activity. Six tda genes, tdaA-F, are physically linked and are carried on pSTM3, a ca. 130-kb plasmid, while the remaining 6 genes are located on the sequenced genome. Genetic and molecular biological evidence demonstrates that tdaA and tdaB form a bicistronic message, tdaCDE are part of a separate operon, and tdaF is likely a part of a third operon.

The expression of tdaAB is constitutive, whereas tdaCDE and tdaF mRNA are regulated, showing significantly increased levels when cells are grown in standing liquid broth compared to shaking liquid culturing. Expression of tdaCDE is lost in Tda<super>-</super> strains, but could be restored- tdaA and tdaH failed to respond - by placing wild-type Tda<super>+</super> strains in close proximity or by adding exogenous TDA to the mutant. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

Next, I focused attention on the only known regulatory protein, TdaA, involved in tda expression. Disruption of tdaA results in loss of tdaCDE expression, and expression of tdaA in an Escherichia coli background is sufficient to transcribe tdaCDE. Transcriptional activation of the tdaC promoter by TdaA is supported by data from electrophoretic mobility shift assays (EMSA) showing that purified TdaA protein binds specifically to a fragment of DNA containing the tdaC promoter. These results support a hypothesis that TdaA is a positive transcriptional regulator of tdaCDE gene expression whose function requires binding to the tdaC promoter region. One of the mechanisms used by TM1040 to regulate TDA biosynthesis is therefore through TdaA regulator.