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

ABSTRACT 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 P rhl - 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-C 14 -C 14 Me). 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. IMPORTANCE The chemical identity of the long-standing cryptic biosurfactants produced by the Burkholderia cepacia complex (Bcc) has remained unresolved due to low expression under standard laboratory conditions. Using a bioluminescent reporter screening approach, we identified the responsible operon and defined specific nutritional parameters that promote biosurfactant production, showing that Burkholderia cenocepacia synthesizes methylated rhamnolipids. Our findings establish rhamnolipid methylation as a conserved and functionally significant feature among rhl -positive Bcc members, mediated by the conserved RhlM methyltransferase. This work reframes the paradigm of rhamnolipid structural diversity, proposing that methylated congeners may constitute the ancestral glycolipid form. Beyond expanding our understanding of secondary metabolism in opportunistic pathogens, this study highlights RhlM as a promising biocatalytic tool for the engineering of custom non-ionic biosurfactants.