Two rsaM Homologues Encode Central Regulatory Elements Modulating Quorum Sensing in Burkholderia thailandensis

Abstract

The bacterium possesses three-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 -octanoyl-homoserine lactone (C-HSL), -3-hydroxy-decanoyl-homoserine lactone (3OHC-HSL), and -3-hydroxy-octanoyl-homoserine lactone (3OHC-HSL), which are produced by the LuxI-type synthases BtaI1, BtaI2, and BtaI3 and modulated by the LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3. The and gene clusters each carry an additional gene encoding a homologue of the QS repressor RsaM originally identified in the phytopathogen and thus here named and , respectively. We have characterized the functions of these two conserved homologues and demonstrated their involvement in the regulation of AHL biosynthesis in strain E264. We quantified the production of C-HSL, 3OHC-HSL, and 3OHC-HSL by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in the and mutants, and we monitored ,, and expression using chromosomal mini-CTX- transcriptional reporters. The transcription of ,, and was also measured by quantitative reverse transcription-PCR (qRT-PCR). We observed that RsaM1 mainly represses the QS-1 system, whereas RsaM2 principally represses the QS-2 system. We also found that both and are QS controlled and negatively autoregulated. We conclude that RsaM1 and RsaM2 are an integral part of the QS circuitry of and play a major role in the hierarchical and homeostatic organization of the QS-1, QS-2, and QS-3 systems. Quorum sensing (QS) is commonly involved in the coordination of gene transcription associated with the establishment of host-pathogen interactions and acclimatization to the environment. We present the functional characterization of two homologues in the regulation of the multiple QS systems coexisting in the nonpathogenic bacterium, which is widely used as a model system for the study of the human pathogen We found that inactivation of these homologues, which are clustered with the other QS genes, profoundly affects the QS circuitry of We conclude that they constitute essential regulatory components of the QS modulatory network and provide additional layers of regulation to modulate the transcription of QS-controlled genes, particularly those linked to environmental adaptation.

The Complex Quorum Sensing Circuitry of Burkholderia thailandensis Is Both Hierarchically and Homeostatically Organized

“…S5 ). Accordingly, we observed that C 8 -HSL, 3OHC 10 -HSL, and 3OHC 8 -HSL concentrations were all increased in an rsaM2 mutant compared to the wild-type strain ( 24 ), indicating that RsaM2 likely intervenes in the regulation of all QS systems of B. thailandensis E264.…”

ScmR, a global regulator of gene expression, quorum sensing, pH homeostasis, and virulence inBurkholderia thailandensis

“…Chromosomal integration of the mini-CTX- scmR-lux transcriptional reporter at the attB locus in B. thailandensis E264 strains was performed through conjugation with the auxotrophic E. coli χ7213, as described previously (12). Successful chromosomal insertion of scmR - lux was confirmed by PCR using appropriate primers.…”

“…These QS systems are referred to as the BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3) QS systems in B. thailandensis (10, 11). The QS-1 system is composed of the BtaR1 transcriptional regulator and the BtaI1 synthase, which synthesizes N- octanoyl-L-homoserine lactone (C 8 -HSL) (12, 13). The BtaR2 transcriptional regulator and the BtaI2 synthase that catalyze the biosynthesis of both N- 3-hydroxy-decanoyl-L-homoserine lactone (3OHC 10 -HSL) and N- 3-hydroxy-octanoyl-L-homoserine lactone (3OHC 8 -HSL) constitute the QS-2 system (12, 14).…”

“…The QS-1 system is composed of the BtaR1 transcriptional regulator and the BtaI1 synthase, which synthesizes N- octanoyl-L-homoserine lactone (C 8 -HSL) (12, 13). The BtaR2 transcriptional regulator and the BtaI2 synthase that catalyze the biosynthesis of both N- 3-hydroxy-decanoyl-L-homoserine lactone (3OHC 10 -HSL) and N- 3-hydroxy-octanoyl-L-homoserine lactone (3OHC 8 -HSL) constitute the QS-2 system (12, 14). The QS-3 system is composed of the BtaR3 transcriptional regulator and the BtaI3 synthase is also responsible for 3OHC 8 -HSL production (12, 13).…”

“…The BtaR2 transcriptional regulator and the BtaI2 synthase that catalyze the biosynthesis of both N- 3-hydroxy-decanoyl-L-homoserine lactone (3OHC 10 -HSL) and N- 3-hydroxy-octanoyl-L-homoserine lactone (3OHC 8 -HSL) constitute the QS-2 system (12, 14). The QS-3 system is composed of the BtaR3 transcriptional regulator and the BtaI3 synthase is also responsible for 3OHC 8 -HSL production (12, 13). Furthermore, B. thailandensis , B. pseudomallei , and B. mallei carry orphan luxR homologues, namely, btaR4 ( malR ) and btaR5 in B. thailandensis (15, 16).…”