Tropodithietic Acid Production in Phaeobacter gallaeciensis Is Regulated by N-Acyl Homoserine Lactone-Mediated Quorum Sensing

Abstract

The production of N-acyl homoserine lactones (AHLs) is widely distributed within the marine Roseobacter clade, and it was proposed that AHL-mediated quorum sensing (QS) is one of the most common cell-to-cell communication mechanisms in roseobacters. The traits regulated by AHL-mediated QS are yet not known for members of the Roseobacter clade, but production of the antibiotic tropodithietic acid (TDA) was supposed to be controlled by AHL-mediated QS in Phaeobacter spp. We describe here for the first time the functional role of luxR and luxI homologous genes of an organism of the Roseobacter clade, i.e., pgaR and pgaI in Phaeobacter gallaeciensis. Our results demonstrate that the AHL synthase gene pgaI is responsible for production of N-3-hydroxydecanoylhomoserine lactone (3OHC 10 -HSL). Insertion mutants of pgaI and pgaR are both deficient in TDA biosynthesis and the formation of a yellow-brown pigment when grown in liquid marine broth medium. This indicates that in P. gallaeciensis the production of both secondary metabolites is controlled by AHLmediated QS. Quantitative real-time PCR showed that the transcription level of tdaA, which encodes an essential transcriptional regulator for TDA biosynthesis, decreased 28-and 51-fold in pgaI and pgaR genetic backgrounds, respectively. These results suggest that both the response regulator PgaR and the 3OHC 10 -HSL produced by PgaI induce expression of tdaA, which in turn positively regulates expression of the tda genes. Moreover, we confirmed that TDA can also act as autoinducer in P. gallaeciensis, as previously described for Silicibacter sp. strain TM1040, but only in the presence of the response regulator PgaR.

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

“…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).…”

“…Several roseobacters produce compounds potentially involved in quorum sensing (QS) (Wagner-Döbler and Biebl, 2006;Martens et al, 2007;Zan et al, 2014). 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).…”

Phaeobacter gallaeciensis genomes from globally opposite locations reveal high similarity of adaptation to surface life

“…Thus, the mutualistic relation between this bacterium and its host can switch to parasitism. In addition, other secondary metabolites, like acylated homoserine lactones (Bruhn et al, 2005;Berger et al, 2011), are produced by Phaeobacter species and it was shown that P. gallaeciensis BS107 harbors a hybrid polyketide synthase/non-ribosomal peptide synthetase (NRPS) cluster (Martens et al, 2007). However, none of the above-mentioned traits is unique to Phaeobacter strains, hence, limiting the knowledge of features delineating P. gallaeciensis from other roseobacters.…”

“…In order to elucidate the pathways leading to TDA synthesis in strain DSM 17395, we conducted transposon insertion mutagenesis and screened for mutants with reduced pigmentation, which was shown to correlate with TDA production (Brinkhoff et al, 2004;Geng et al, 2008;Berger et al, 2011). Within the framework of this study, we identified 26 genes involved in TDA biosynthesis and pigment production, of which 18 are essential ( Table 2).…”

“…Transposon Tn5 mutagenesis of P. gallaeciensis DSM 17395 Random insertion libraries were generated using EZ-Tn5 mutagenesis as described by Berger et al (2011). TDA-negative mutants were screened for reduced pigmentation, which is known to coincide with loss of antibiotic production.…”