The genome of the bacterium Burkholderia thailandensis encodes three complete LuxI/LuxR-type quorum sensing (QS) systems: BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). The LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3 modulate the expression of target genes in association with various N-acyl-l-homoserine lactones (AHLs) as signaling molecules produced by the LuxI-type synthases BtaI1, BtaI2, and BtaI3. We have systematically dissected the complex QS circuitry of B. thailandensis strain E264. Direct quantification of N-octanoyl-homoserine lactone (C8-HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC10-HSL), and N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-HSL), the primary AHLs produced by this bacterium, was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in QS deletion mutants. This was compared to the transcription of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. Furthermore, the levels of expression of btaR1, btaR2, and btaR3 were monitored by quantitative reverse transcription-PCR (qRT-PCR). We observed that C8-HSL, 3OHC10-HSL, and 3OHC8-HSL are differentially produced over time during bacterial growth and correlate with the btaI1, btaI2, and btaI3 gene expression profiles, revealing a successive activation of the corresponding QS systems. Moreover, the transcription of the btaR1, btaR2, and btaR3 genes is modulated by cognate and noncognate AHLs, showing that their regulation depends on themselves and on other QS systems. We conclude that the three QS systems in B. thailandensis are interdependent, suggesting that they cooperate dynamically and function in a concerted manner in modulating the expression of QS target genes through a successive regulatory network.

Species from the Burkholderia cepacia complex (Bcc) share a canonical LuxI/LuxR quorum sensing (QS) regulation system named CepI/CepR, which mainly relies on the acyl-homoserine lactone (AHL), octanoyl-homoserine lactone (C8-HSL) as signaling molecule. Burkholderia ambifaria is one of the least virulent Bcc species, more often isolated from rhizospheres where it exerts a plant growth-promoting activity. However, clinical strains of B. ambifaria display distinct features, such as phase variation and higher virulence properties. Notably, we previously reported that under laboratory conditions, only clinical strains of the B. ambifaria species produced 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs) via expression of the hmqABCDEFG operon. HMAQs are the methylated counterparts of the 4-hydroxy-2-alkylquinolines (HAQs) produced by the opportunistic human pathogen Pseudomonas aeruginosa, in which they globally contribute to the bacterial virulence and survival. We have found that unlike P. aeruginosa’s HAQs, HMAQs do not induce their own production. However, they indirectly regulate the expression of the hmqABCDEFG operon. In B. ambifaria, a strong link between CepI/CepR-based QS and HMAQs is proposed, as we have previously reported an increased production of C8-HSL in HMAQ-negative mutants. Here, we report the identification of all AHLs produced by the clinical B. ambifaria strain HSJ1, namely C6-HSL, C8-HSL, C10-HSL, 3OHC8-HSL, 3OHC10-HSL, and 3OHC12-HSL. Production of significant levels of hydroxylated AHLs prompted the identification of a second complete LuxI/LuxR-type QS system relying on 3OHC10-HSL and 3OHC12-HSL, that we have named CepI2/CepR2. The connection between these two QS systems and the hmqABCDEFG operon, responsible for HMAQs biosynthesis, was investigated. The CepI/CepR system strongly induced the operon, while the second system appears moderately involved. On the other hand, a HMAQ-negative mutant overproduces AHLs from both QS systems. Even if HMAQs are not classical QS signals, their effect on AHL-based QS system still gives them a part to play in the QS circuitry in B. ambifaria and thus, on regulation of various phenotypes.

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 nonpathogenic soil saprophyte Burkholderia thailandensis is a member of the Burkholderia pseudomallei/B. thailandensis/B. mallei group, which also comprises the closely related human pathogens B. pseudomallei and Burkholderia mallei responsible for the melioidosis and glanders diseases, respectively. ScmR, a recently identified LysR-type transcriptional regulator in B. thailandensis, acts as a global transcriptional regulator throughout the stationary phase and modulates the production of a wide range of secondary metabolites, including N-acyl-l-homoserine lactones and 4-hydroxy-3-methyl-2-alkylquinolines and virulence in the Caenorhabditis elegans nematode worm host model, as well as several quorum sensing (QS)-dependent phenotypes. We have investigated the role of ScmR in B. thailandensis strain E264 during the exponential phase. We used RNA sequencing transcriptomic analyses to identify the ScmR regulon, which was compared to the QS-controlled regulon, showing a considerable overlap between the ScmR-regulated genes and those controlled by QS. We characterized several genes modulated by ScmR using quantitative reverse transcription-PCR or mini-CTX-lux transcriptional reporters, including the oxalate biosynthetic gene obc1 required for pH homeostasis, the orphan LuxR-type transcriptional regulator BtaR5-encoding gene, and the bsa (Burkholderia secretion apparatus) type III secretion system genes essential for both B. pseudomallei and B. mallei pathogenicity, as well as the scmR gene itself. We confirmed that the transcription of scmR is under QS control, presumably ensuring fine-tuned modulation of gene expression. Finally, we demonstrated that ScmR influences virulence using the fruit fly model host Drosophila melanogaster. We conclude that ScmR represents a central component of the B. thailandensis QS regulatory network. IMPORTANCE Coordination of the expression of genes associated with bacterial virulence and environmental adaptation is often dependent on quorum sensing (QS). The QS circuitry of the nonpathogenic bacterium Burkholderia thailandensis, widely used as a model system for the study of the human pathogen Burkholderia pseudomallei, is complex. We found that the LysR-type transcriptional regulator, ScmR, which is highly conserved and involved in the control of virulence/survival factors in the Burkholderia genus, is a global regulator mediating gene expression through the multiple QS systems coexisting in B. thailandensis, as well as QS independently. We conclude that ScmR represents a key QS modulatory network element, ensuring tight regulation of the transcription of QS-controlled genes, particularly those required for acclimatization to the environment.

The genome of the bacterium Burkholderia thailandensis encodes three complete LuxI/LuxR-type quorum sensing (QS) systems: BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). The LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3 modulate the expression of target genes in association with various N-acyl-Lhomoserine lactones (AHLs) as signaling molecules produced by the LuxI-type synthases BtaI1, BtaI2, and BtaI3. We have systematically dissected the complex QS circuitry of B. thailandensis strain E264. Direct quantification of N-octanoyl-homoserine lactone (C 8 -HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC 10 -HSL), and N-3-hydroxyoctanoyl-homoserine lactone (3OHC 8 -HSL), the primary AHLs produced by this bacterium, was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in QS deletion mutants. This was compared to the transcription of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. Furthermore, the levels of expression of btaR1, btaR2, and btaR3 were monitored by quantitative reverse transcription-PCR (qRT-PCR). We observed that C 8 -HSL, 3OHC 10 -HSL, and 3OHC 8 -HSL are differentially produced over time during bacterial growth and correlate with the btaI1, btaI2, and btaI3 gene expression profiles, revealing a successive activation of the corresponding QS systems. Moreover, the transcription of the btaR1, btaR2, and btaR3 genes is modulated by cognate and noncognate AHLs, showing that their regulation depends on themselves and on other QS systems. We conclude that the three QS systems in B. thailandensis are interdependent, suggesting that they cooperate dynamically and function in a concerted manner in modulating the expression of QS target genes through a successive regulatory network.IMPORTANCE Quorum sensing (QS) is a widespread bacterial communication system coordinating the expression of specific genes in a cell density-dependent manner and allowing bacteria to synchronize their activities and to function as multicellular communities. QS plays a crucial role in bacterial pathogenicity by regulating the expression of a wide spectrum of virulence/survival factors and is essential to environmental adaptation. The results presented here demonstrate that the multiple QS systems coexisting in the bacterium Burkholderia thailandensis, which is considered the avirulent version of the human pathogen Burkholderia pseudomallei and thus commonly used as an alternative study model, are hierarchically and homeostatically organized. We found these QS systems to be finely integrated into a complex regulatory network, including transcriptional and posttranscriptional interactions, and further incorporating growth stages and temporal expression. These results provide a unique, comprehensive illustration of a sophisticated QS network and will contribute to a better comprehension of the regulatory mechanisms that can be involved in the expression of QS-controlled genes, in particular those associ...