ScmR, a Global Regulator of Gene Expression, Quorum Sensing, pH Homeostasis, and Virulence in Burkholderia thailandensis.

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 theB. 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.

Le Guillouzer S ，Groleau MC ，Mauffrey F ，Déziel E ... -  《-》

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

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.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 associated with the establishment of host-pathogen interactions and acclimatization to the environment.

Le Guillouzer S ，Groleau MC ，Déziel E 《-》

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

The bacterium Burkholderia thailandensis possesses three 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), and N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-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 btaR1-btaI1 and btaR2-btaI2 gene clusters each carry an additional gene encoding a homologue of the QS repressor RsaM originally identified in the phytopathogen Pseudomonas fuscovaginae and thus here named rsaM1 and rsaM2, respectively. We have characterized the functions of these two conserved rsaM homologues and demonstrated their involvement in the regulation of AHL biosynthesis in B. thailandensis strain E264. We quantified 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 we monitored btaI1, btaI2, and btaI3 expression using chromosomal mini-CTX-lux transcriptional reporters. The transcription of btaR1, btaR2, and btaR3 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 rsaM1 and rsaM2 are QS controlled and negatively autoregulated. We conclude that RsaM1 and RsaM2 are an integral part of the QS 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.IMPORTANCE 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 rsaM homologues in the regulation of the multiple QS systems coexisting in the nonpathogenic bacterium Burkholderia thailandensis, which is widely used as a model system for the study of the human pathogen Burkholderia pseudomallei We found that inactivation of these rsaM homologues, which are clustered with the other QS genes, profoundly affects the QS circuitry of B. thailandensis 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.

Le Guillouzer S ，Groleau MC ，Déziel E 《-》

Discovery of scmR as a global regulator of secondary metabolism and virulence in Burkholderia thailandensis E264.

Mao D ，Bushin LB ，Moon K ，Wu Y ，Seyedsayamdost MR ... -  《-》

Cross-species comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei quorum-sensing regulons.

Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei.

Majerczyk CD ，Brittnacher MJ ，Jacobs MA ，Armour CD ，Radey MC ，Bunt R ，Hayden HS ，Bydalek R ，Greenberg EP ... -  《-》