Two rsaM homologues encode central regulatory elements modulating quorum sensing expression in Burkholderia thailandensis
The bacterium Burkholderia thailandensis possesses three conserved N-acyl-L-homoserine lactone (AHL) quorum sensing (QS) systems designated BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). These QS-systems are associated with the biosynthesis of N-octanoyl-homoserine lactone (C8-HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC10-HSL), as well as N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-HSL), which are produced by the LuxI-type synthase BtaI1, BtaI2, and BtaI3, and modulated by the LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3. Both btaR1/btaI1 and btaR2/btaI2 gene clusters contain an additional gene that is conserved in the Burkholderia genus, homologous to a gene coding for the negative AHL biosynthesis modulatory protein RsaM originally identified in the phytopathogen Pseudomonas fuscovaginae, and hence designated rsaM1 and rsaM2. We have characterized the function of these two rsaM homologues and demonstrated their involvement in the regulation of AHLs biosynthesis in B. thailandensis strain E264. We measured the production of C8-HSL, 3OHC10-HSL, and 3OHC8-HSL by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in the rsaM1- and rsaM2- mutants, and monitored the transcription of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. The expression of btaR1, btaR2, and btaR3 was also measured by quantitative everse-transcription PCR (qRT-PCR). We demonstrate that the QS-1 system is repressed by RsaM1, whereas RsaM2 principally represses the QS-2 system. We also found that both rsaM1 and rsaM2 are QS-controlled, as well as negatively auto-regulated. We conclude that RsaM1 and RsaM2 are an integral part of the QS modulatory circuitry of B. thailandensis, and play a major role in the hierarchical and homeostatic organization of the QS-1, QS-2, and QS-3 systems.\n\nImportanceQuorum sensing (QS) is a global regulatory mechanism of genes expression depending on bacterial density. QS is commonly involved in the coordination of genes expression associated with the establishment of host-pathogen interactions and acclimatization to the environment. We present the functional characterization of the two rsaM homologues designated rsaM1 and rsaM2 in the regulation of the multiple QS systems coexisting in the non-pathogenic bacterium Burkholderia thailandensis, widely used as a model system for the study of the pathogen Burkholderia pseudomallei. We found that inactivation of these rsaM homologues, which are clustered with the other QS genes, profoundly affects the QS regulatory circuity of B. thailandensis. It is proposed that these genes code for QS repressors and we conclude that they constitute essential regulatory components of the QS modulatory network of B. thailandensis, and provide additional layers of regulation to modulate the expression of QS-controlled genes, including those encoding virulence/survival factors and linked to environmental adaptation in B. pseudomallei.