From Exception to Rule: Widespread Production of Methylated Rhamnolipids in the Burkholderia cepacia Complex

Although Burkholderia cenocepacia exhibits biosurfactant-dependent phenotypes and possesses an rhl operon, the chemical identity of its surface-active product has long remained unresolved. The rhl operon of B. cenocepacia harbors an additional gene, rhlM, predicted to encode a methyltransferase, and recently linked to methylated rhamnolipid synthesis in a few Burkholderiaceae. Here, we hypothesized that this biosurfactant remained cryptic due to low expression under standard laboratory conditions. Using a chromosomal Prhl-luxCDABE fusion reporter, we screened diverse nutritional conditions and identified a defined medium containing galactose and urea that strongly induced rhl transcription. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of B. cenocepacia K56-2 extracts identified the biosurfactants as methylated dirhamnolipid congeners, predominantly L-rhamnosyl-L-rhamnosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoate methyl ester (RR-C14-C14Me). Extending our analysis across the Burkholderia cepacia complex (Bcc) revealed that methylated rhamnolipid production is widespread and restricted to rhl-positive species. Comparative genomic and protein structural analyses further demonstrated high conservation of both the rhlM-containing operon architecture and the RhlM protein across the Bcc. Together, our findings identify the long-standing cryptic biosurfactant of B. cenocepacia as methylated rhamnolipids and establish rhamnolipid methylation as a conserved hallmark across the Bcc, shifting the paradigm of rhamnolipid structural diversity from an isolated exception to a widespread rule.