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Mutational analysis and biochemical characterization of the Burkholderia thailandensis DW503 quorum-sensing network.

ABSTRACT: Numerous gram-negative bacteria communicate and regulate gene expression through a cell density-responsive mechanism termed quorum sensing (QS), which involves the synthesis and perception of diffusible N-acyl-homoserine lactones (AHL). In this study we genetically and physiologically characterized the Burkholderia thailandensis DW503 QS network. In silico analysis of the B. thailandensis genome revealed the presence of at least three AHL synthases (AHS) and five transcriptional regulators belonging to the LuxIR family of proteins. Mass spectrometry demonstrated that wild-type B. thailandensis synthesizes N-hexanoyl-homoserine lactone (C6-HSL), N-octanoyl-homoserine lactone (C8-HSL), and N-decanoyl-homoserine lactone (C10-HSL). Mutation of the btaI1 (luxI) AHS gene prevented accumulation of C8-HSL in culture supernatants, enhanced beta-hemolysis of sheep erythrocytes, increased lipase production, and altered colony morphology on swarming and twitching motility plates. Disruption of the btaI3 (luxI) AHS prevented biosynthesis of C6-HSL and increased lipase production and beta-hemolysis, whereas mutagenesis of the btaI2 (luxI) allele eliminated C10-HSL accumulation and reduced lipase production. Complementation of the btaI1 and btaI3 mutants fully restored the synthesis of C8-HSL and C6-HSL to parental levels. In contrast, mutagenesis of the btaR1, btaR3, btaR4, and btaR5 (luxR) transcriptional regulators had no effect on AHL accumulation, enhanced lipase production, and resulted in extensive beta-hemolysis on sheep blood agar plates. Furthermore, interruption of the btaI1, btaR1, and btaR3 genes altered colony morphology on twitching and swarming motility plates and induced pigmentation. Additionally, phenotypic microarray analysis indicated that QS in B. thailandensis both positively and negatively affects the metabolism of numerous substrates, including citric acid, formic acid, glucose 6-phosphate, capric acid, gamma-hydroxybutyric acid, and d-arabinose. These results demonstrate that mutagenesis of the B. thailandensis QS system affects various cellular processes, including lipase production, swarming and twitching motility, beta-hemolysis of sheep erythrocytes, and carbon metabolism and/or transport.

SUBMITTER: Ulrich RL

PROVIDER: S-EPMC421622 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Mutational analysis and biochemical characterization of the Burkholderia thailandensis DW503 quorum-sensing network.

Ulrich Ricky L RL Hines Harry B HB Parthasarathy N N Jeddeloh Jeffrey A JA

Journal of bacteriology 20040701 13

Numerous gram-negative bacteria communicate and regulate gene expression through a cell density-responsive mechanism termed quorum sensing (QS), which involves the synthesis and perception of diffusible N-acyl-homoserine lactones (AHL). In this study we genetically and physiologically characterized the Burkholderia thailandensis DW503 QS network. In silico analysis of the B. thailandensis genome revealed the presence of at least three AHL synthases (AHS) and five transcriptional regulators belon ...[more]

PMID: 15205437

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Project description:UnlabelledMembers of the genus Burkholderia are known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterized Burkholderia thailandensis biofilm development under flow conditions and sought to determine whether QS contributes to this process. B. thailandensis biofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by "dome" structures filled with biofilm matrix material. We showed that this process was dependent on QS. B. thailandensis has three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the three B. thailandensis QS systems, we show that QS-1 is required for proper biofilm development, since a btaR1 mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. The btaR1 mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions.ImportanceThe saprophyte Burkholderia thailandensis is a close relative of the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms, B. thailandensis is an ideal model organism for investigating questions in Burkholderia physiology. In this study, we characterized B. thailandensis biofilm development and sought to determine if quorum sensing (QS) contributes to this process. Our work shows that B. thailandensis produces biofilms with unusual dome structures under flow conditions. Our findings suggest that these dome structures are filled with a QS-regulated, fucose-containing exopolysaccharide that may be involved in the resilience of B. thailandensis biofilms against changes in the nutritional environment.

Project description: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.

Project description:Rhamnolipids are surface-active agents of microbial origin used as alternatives to synthetic surfactants. Burkholderia thailandensis is a non-pathogenic rhamnolipid-producing bacterium that could represent an interesting candidate for use in commercial processes. However, current bioprocesses for rhamnolipid production by this bacterium are not efficient enough, mainly due to low yields. Since regulation of rhamnolipid biosynthesis in B. thailandensis remains poorly understood, identifying new regulatory factors could help increase the production of these valuable metabolites. We performed a random transposon mutagenesis screening to identify genes directing rhamnolipid production in B. thailandensis E264. The most efficient rhamnolipid producer we identified harbored an inactivating transposon insertion in the scmR gene, which was recently described to encode as a secondary metabolite regulator in B. thailandensis. We investigated the impact of scmR loss on rhamnolipid biosynthesis and cell growth. Because biosynthesis of rhamnolipids and polyhydroxyalkanoates (PHAs) could share the same pool of lipid precursors, we also investigate the effect of ScmR on PHA production. We found that production of both rhamnolipids and PHAs are modulated by ScmR during the logarithmic growth phase and demonstrate that ScmR downregulates the production of rhamnolipids by affecting the expression of both rhl biosynthetic operons. Furthermore, our results indicate that PHA biosynthesis is reduced in the scmR- mutant, as ScmR promotes the transcription of the phaC and phaZ genes. By studying the relationship between ScmR and quorum sensing (QS) regulation we reveal that QS acts as an activator of scmR transcription. Finally, we pinpoint the QS-3 system as being involved in the regulation of rhamnolipid and PHA biosynthesis. We conclude that ScmR negatively affects rhamnolipid production, whereas it positively impacts PHAs biosynthesis. This could provide an interesting approach for future strain engineering, leading to improved yields of these valuable metabolites.

Project description: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.

Dataset Information

Mutational analysis and biochemical characterization of the Burkholderia thailandensis DW503 quorum-sensing network.

Publications

Mutational analysis and biochemical characterization of the Burkholderia thailandensis DW503 quorum-sensing network.

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