Biology subjects

Deziel, E.

Publications and source records attributed to Deziel, E..

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bioRxiv2021

Development and Validation of a High-throughput Short Sequence Typing Scheme for Serratia marcescens Pure Culture and Environmental DNA

Molecular typing methods are used to characterize the relatedness between bacterial isolates involved in infections. These approaches rely mostly on discrete loci or whole genome sequences (WGS) analyses of pure cultures. On the other hand, their application to environmental DNA profiling to evaluate epidemiological relatedness amongst patients and environments has received less attention. We developed a specific, high-throughput short sequence typing (HiSST) method for the opportunistic human pathogen Serratia marcescens. Genes displaying the highest polymorphism were retrieved from the core genome of 60 S. marcescens strains. Bioinformatics analyses showed that use of only three loci (within bssA, gabR and dhaM) distinguished strains with the same level of efficiency than average nucleotide identity scores of whole genomes. This HiSST scheme was applied to an epidemiological survey of S. marcescens in a neonatal intensive care unit (NICU). In a first case study, a strain responsible for an outbreak in the NICU was found in a sink drain of this unit, by using HiSST scheme and confirmed by WGS. The HiSST scheme was also applied to environmental DNA extracted from sink-environment samples. Diversity of S. marcescens was modest, with 11, 6 and 4 different sequence types (ST) of gabR, bssA and dhaM loci amongst 19 sink drains, respectively. Epidemiological relationships amongst sinks were inferred on the basis of pairwise comparisons of ST profiles. Further research aimed at relating ST distribution patterns to environmental features encompassing sink location, utilization and microbial diversity is needed to improve the surveillance and management of opportunistic pathogens.

molecular biology↗

bioRxiv2021

Pseudomonas aeruginosa isolates defective in function of the LasR quorum sensing regulator are frequent in diverse environmental niches

The saprophyte Pseudomonas aeruginosa is a versatile opportunistic pathogen causing infections in immunocompromised individuals. To facilitate its adaptation to a large variety of niches, this bacterium exploits population density-dependant gene regulation systems called quorum sensing. In P. aeruginosa, three distinct but interrelated quorum sensing systems (las, rhl and pqs) regulate the production of many survival and virulence functions. In prototypical strains, the las system, through its transcriptional regulator LasR, is important for the full activation of the rhl and pqs systems. Still, LasR-deficient isolates have been reported, mostly sampled from the lungs of people with cystic fibrosis, where they are considered selected by the chronic infection environment. In this study, we show that a defect in LasR activity appears to be an actually widespread mechanism of adaptation in this bacterium. Indeed, we found abundant LasR-defective isolates sampled from hydrocarbon-contaminated soils, hospital sink drains, and meat/fish market environments, using an approach based on phenotypic profiling, supported by gene sequencing. Interestingly, several LasR-defective isolates maintain an active rhl system or are deficient in pqs system signaling. The high prevalence of a LasR-defective phenotype among environmental P. aeruginosa isolates questions the role of quorum sensing in niche adaptation.

microbiology↗

bioRxiv2021

The ability of Pseudomonas aeruginosa to adopt a Small Colony Variant (SCV) phenotype is conserved, and not restricted to clinical isolates

A subpopulation of Small Colony Variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) lungs biofilms. SCVs have almost exclusively been reported from infected hosts, essentially CF individuals or, by extension, from laboratory cultivation of strains originated from infected hosts. We previously reported the identification of P. aeruginosa SCVs emerging from a non-clinical strain and displaying features shared with clinical SCVs. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to, under laboratory culture conditions, spontaneously adopt a SCV-like smaller alternative morphotype distinguishable from the ancestral parent strain. Unexpectedly, we found that all the P. aeruginosa strains tested have the ability to adopt a SCV morphotype, regardless of their origin. Based on the phenotypes already described for SCVs, the SCV-like morphotypes obtained were clustered in two groups displaying various phenotypic profiles, including one characteristic of already described SCVs. We conclude that the ability to switch to a SCV phenotype is a conserved feature in Pseudomonas aeruginosa. IMPORTANCEP. aeruginosa is an opportunistic pathogen that thrives in many environments. It is significant public health concern, notably because it is the most prevalent pathogen found in the lungs of people with cystic fibrosis (CF). In infected hosts, its persistence is believed to be related to the emergence of an alternative small colony variant (SCV) phenotype. By reporting the distribution of P. aeruginosa SCVs in various non-clinical environments, this work contributes to understanding a conserved adaptation mechanism used by P. aeruginosa to rapidly adapt in all environments. Counteraction of this strategy could prevent P. aeruginosa persistent infection in the future.

microbiology↗

bioRxiv2020

Rhamnolipids and their 3-(3-hydroxyalkanoyloxy)alkanoic acid precursors activate Arabidopsis innate immunity through two independent mechanisms

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from L- rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa mostly comprising RLs and HAAs stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29) that also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception in the plant Arabidopsis thaliana. HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, albeit abolishing sensing by LORE, do not impair their ability to trigger plant defense. In addition, our results show that RL-triggered immune response is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms. SignificanceActivation of plant innate immunity relies on the perception of microorganisms through self and nonself elicitors. Rhamnolipids and their precursor HAAs are exoproducts produced by beneficial and pathogenic bacteria. They are involved in bacterial surface dissemination and biofilm development. As these compounds are released in the extracellular milieu, they have the potential to be perceived by the plant immune system. Our work shows that both compounds independently activate plant immunity. We demonstrate that HAAs are perceived by the receptor protein kinase LORE. By contrast, rhamnolipids are not senses by LORE but activate a non-canonical immune response affected by the sphingolipid composition of the plant plasma membrane. Thus, plants are able to sense bacterial molecules as well as their direct precursors to trigger a distinct immune response.

plant biology↗

bioRxiv2020

Presence of the Hmq system and production of 4-hydroxy-3-methyl-2-alkylquinolines is heterogeneously distributed between Burkholderia cepacia complex species and more prevalent among environmental than clinical isolates

Some Burkholderia cepacia complex (Bcc) strains have been reported to produce 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), analogous to the 4-hydroxy-2-alkylquinolines of Pseudomonas aeruginosa. Using in silico analyses, we previously showed that the hmqABCDEFG operon, which encodes enzymes involved in the biosynthesis of HMAQs, is carried by about one-third of Bcc strains, with considerable inter- and intra-species variability. In the present study, we investigated by PCR, using consensus primers, the distribution of hmqABCDEFG in a collection of 313 Bcc strains (222 of clinical and 91 of environmental origins) - belonging to 18 Bcc species. We confirmed that the distribution is species-specific, although not all strains within a species carry the hmqABCDEFG operon. Among the 30% of strains bearing the hmqABCDEFG operon, we measured the total HMAQs production and showed that 90% of environmental isolates and 68% of clinically isolated Bcc produce detectable levels of HMAQs when cultured in TSB medium. For the strains having the hmqABCDEFG operon but not producing HMAQs, we studied the transcription and showed that none expressed the hmqA gene under the specified culture conditions. Interestingly, the hmqABCDEFG operon is more prevalent among plant root environment species (e.g. B. ambifaria, B. cepacia) and absent in species commonly found in chronically colonized individuals with cystic fibrosis (e.g. B. cenocepacia, B. multivorans), suggesting that the Hmq system could play a role in niche adaptation by influencing rhizosphere microbial community and could have been lost through evolution. Understanding the Hmq system and its regulation will provide clues concerning the production of HMAQs and their functions in Bcc.

microbiology↗

bioRxiv2020

An experimentally evolved variant of RsmA confirms its central role in the control of Pseudomonas aeruginosa social motility

Bacteria can colonize a variety of different environments by modulating their gene regulation using two-component systems. The versatile opportunistic pathogen Pseudomonas aeruginosa has been studied for its capacity to adapt to a broad range of environmental conditions. The Gac/Rsm pathway is composed of the sensor kinase GacS, that detects environmental cues, and the response regulator GacA, that modulates the expression of a specific genes. This system, through the sRNA repressors RsmY and RsmZ, negatively controls the activity of the protein RsmA, which is centrally involved in the transition from chronic to acute infections by post-transcriptionally regulating several virulence functions. RsmA positively regulates swarming motility, a social surface behaviour. Through a poorly defined mechanism, RsmA is also indirectly regulated by HptB, and a {Delta}hptB mutant exhibits a severe swarming defect. Since a {Delta}hptB mutant retains all the known functions required for that type of motility, we used an experimental evolution approach to identify elements responsible for its swarming defect. After a few passages under swarming conditions, the defect of the {Delta}hptB mutant was rescued by the emergence of spontaneous single nucleotide substitutions in the gacA and rsmA genes. Since GacA indirectly represses RsmA activity, it was coherent that an inactivating mutation in gacA would compensate the {Delta}hptB swarming defect. However, the effect of the mutation in rsmA was unexpected since RsmA promotes swarming; indeed, using expression reporters, we found that the mutation that does not abolish its activity. Instead, using electrophoretic mobility shift assays and molecular simulations, we show that this variant of RsmA is actually less amenable to titration by its cognate repressor RsmY, supporting the other phenotypes observed for this mutant. These results confirm the central role of RsmA as a regulator of swarming motility in P. aeruginosa and identify residues crucial for RsmA function in social motility. Author summaryBacteria need to readily adapt to their environment. Two-component systems (TCS) allow such adaption by triggering bacterial regulation changes through the detection of environmental cues. The opportunistic pathogen Pseudomonas aeruginosa possesses more than 60 TCS in its genome. The Gac/Rsm is a TCS extensively studied for its implication in virulence regulation. This system regulates the transition between chronic and acute bacterial infection behaviours. To acquire a better understanding of this regulation, we performed a directed experimental evolution on a swarming-deficient mutant in a poorly understood regulatory component of the Gac/Rsm pathway. We observed single nucleotide substitutions that allowed restoration of a swarming phenotype similar to the wild-type behaviour. More specifically, mutations were found in the gacA and rsmA genes. Interestingly, the observed mutation in rsmA does not result in loss of function of the protein but rather alters its susceptibility to repression by its cognate interfering sRNA. Since modification in the RNA sequence of RsmA results in the rescue of swarming motility, we confirm the central role of this posttranscriptional repressor in this social lifestyle.

microbiology↗

bioRxiv2020

Burkholderia thailandensis methylated hydroxy-alkylquinolines: biosynthesis and antimicrobial activity in co-culture experiments

The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In liquid co-culture experiments, B. thailandensis secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576. Results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NO) in B. thailandensis and that HMAQ-NOs are sufficient to eliminate B. subtilis in co-cultures. Moreover, synthetic HMAQ-NO is [~]50-fold more active than HMAQ. Both the methyl group and the length of the carbon side chain account for high activity of HMAQ-NO against B. subtilis. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensis. IMPORTANCEThe soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones, mostly methylated 4-hydroxy-alkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa. Several of the methylated 4-hydroxy-alkenylquinolines have antimicrobial activity against other species. We show that N-oxidated methyl-alkenylquinolines are particularly antimicrobial and sufficient to kill Bacillus subtilis in co-cultures. We confirmed their biosynthesis requires the previously unstudied protein HmqL. These results provide new information about the biology of 2-alkyl-4(1H)-quinolones, particularly the methylated 4-hydroxy-alkenylquinolines, which are unique to B. thailandensis. This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

microbiology↗

bioRxiv2019

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-thailandensis-mallei (Bptm) group, which also comprises the closely related human pathogens Burkholderia pseudomallei and Burkholderia mallei responsible for the diseases melioidosis and glanders, respectively. ScmR, a recently identified LysR-type transcriptional regulator (LTTR) 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 (AHLs) and 4-hydroxy-3-methyl-2-alkylquinoline (HMAQ), virulence in the model host Caenorhabditis elegans, 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 (RNA-Seq) 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 (qRT-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, the bsa (Burkholderia secretion apparatus) type III secretion system (T3SS) 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 demonstrate 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. ImportanceCoordination 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, which is widely used as a model system for the study of the human pathogen Burkholderia pseudomallei, is complex. We found that the recently identified LysR-type transcriptional regulator (LTTR), 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 independently of QS. 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.

microbiology↗

bioRxiv2019

Phenylacetyl-CoA, not phenylacetic acid, mediates quorum sensing-regulated virulence in Burkholderia cenocepacia

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl-CoA (PAA-CoA) by a ligase, PaaK, and then epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation to the TCA cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under control of the LuxIR-like quorum sensing system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography/mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than the wild type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in internal accumulation, compared to wild type. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in supressing B. cenocepacia CepIR-activated virulence.\n\nImportanceThe opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR, which upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that, in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

microbiology↗

bioRxiv2019

Polyhydroxyalkanoate granule accumulation makes optical density measurement an unreliable method for bacterial growth assessment in Burkholderia thailandensis

Optical density (OD) measurement is the standard method used in microbiology for estimating bacterial concentrations in cultures. However, most studies do not compare these measurements with viable cell counts and assume that they reflect the real cell concentration. Burkholderia thailandensis was recently identified as a polyhydroxyalkanoate (PHA) producer. PHA biosynthesis seems to be coded by an ortholog of the Cupriavidus necator phaC gene. When growing cultures of wildtype strain E264 and an isogenic phaC- mutant, we noted a difference in their OD600 values, although viable cell counts indicated similar growth. Investigating the cellular morphologies of both strains, we found that under our conditions the wildtype strain was full of PHA granules, deforming the cells, while the mutant contained no granules. These factors apparently affected the light scattering, making the OD600 values no longer representative of cell density. We show a direct correlation between OD600 values and the accumulation of PHA. We conclude that OD measurement is unreliable for growth evaluation of B. thailandensis because of PHA production. This study also suggests that B. thailandensis could represent an excellent candidate for PHA bioproduction. Correlation between OD measurements and viable cell counts should be verified on any study realized in B. thailandensis.

microbiology↗

bioRxiv2018

The symbiotic complex of Dendroctonus simplex: implications in the beetle attack and its life cycle

The eastern larch beetle (Dendroctonus simplex Le Conte) is recognized as a serious destructive forest pest in the upper part of North America. Under epidemic conditions, this beetle can attack healthy trees, causing severe damages to larch stands. Dendroctonus species are considered as holobionts, as they engage in multipartite interactions with microorganisms, such as bacteria, filamentous fungi, and yeasts, which are implicated in physiological processes of the insect, such as nutrition. They also play a key role in the beetles attack, as they are responsible for the detoxification of the subcortical environment and weaken the trees defense mechanisms. The eastern larch beetle is associated with bacteria and fungi, but their implication in the success of the beetle remains unknown. Here, we investigated the bacterial and fungal microbiota of this beetle pest throughout its ontogeny (pioneer adults, larvae and pupae) by high-throughput sequencing. A successional microbial assemblage was identified throughout the beetle developmental stages, reflecting the beetles requirements. These results indicate that a symbiotic association between the eastern larch beetle and some of these microorganisms takes place and that this D. simplex symbiotic complex is helping the insect to colonize its host tree and survive the conditions encountered.

ecology↗

bioRxiv2017

Fungal communities associated with the eastern larch beetle: diversity and variation within developmental stages

Bacterial and fungal communities associated with bark beetles, especially some Dendroctonus species, mediate challenging aspects of the subcortical habitat for their hosts. Filamentous fungi and yeasts are important in various metabolic processes of certain bark beetles, with involvement in nutrition, protection and detoxification of plant defense compounds. The eastern larch beetle, Dendroctonus simplex, is recognized as a serious forest pest in the upper part of North America. Although D. simplex is well studied, the fungal communities and their ecological role remain to be investigated. Here, using 18S rRNA gene pyrosequencing, we provide a comprehensive overview of the yeasts and filamentous fungi associated with the eastern larch beetle and compare fungal communities between different developmental stages and microenvironments. Fungal mycobiome associated with the galleries was also investigated. Our study has unveiled an unexpected fungal diversity associated with the developmental stages. Significant differences in species richness between the developmental stages were determined. Yeasts were found to be predominant in the adult and larval stages, whereas filamentous fungi were most prevalent in the pupae. Our results indicate a possible implication of yeasts in the eastern larch beetle nutrition.

microbiology↗

bioRxiv2017

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.

microbiology↗

bioRxiv2017

The complex quorum sensing circuitry of Burkholderia thailandensis is both hierarchically and homeostatically organized

The genome of the bacterium Burkholderia thailandensis encodes for 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 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 in the wild-type strain and in QS deletion mutants. This was compared to the expression of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. Furthermore, transcription of btaR1, btaR2, and btaR3 was 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 genes expression profiles, revealing a sequential activation of the corresponding QS systems. Moreover, transcription of the btaR1, btaR2, and btaR3 genes is modulated by AHLs, showing that their regulation depend on themselves, and on other 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 sequential regulatory network.\n\nImportanceQuorum 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, considered as 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 finely integrated into a complex regulatory network, including transcriptional and post-transcriptional 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.

microbiology↗