Quorum Sensing Genes Research Articles

Can rumen bacteria communicate to each other?

BackgroundThe rumen contains a myriad of microbes whose primary role is to degrade and ferment dietary nutrients, which then provide the host with energy and nutrients. Rumen microbes commonly attach to ingested plant materials and form biofilms for effective plant degradation. Quorum sensing (QS) is a well-recognised form of bacterial communication in most biofilm communities, with homoserine lactone (AHL)-based QS commonly being used by Gram-negative bacteria alone and AI-2 Lux-based QS communication being used to communicate across Gram-negative and Gram-positive bacteria. However, bacterial cell to cell communication in the rumen is poorly understood. In this study, rumen bacterial genomes from the Hungate collection and Genbank were prospected for QS-related genes. To check that the discovered QS genes are actually expressed in the rumen, we investigated expression levels in rumen metatranscriptome datasets.ResultsA total of 448 rumen bacterial genomes from the Hungate collection and Genbank, comprised of 311 Gram-positive, 136 Gram-negative and 1 Gram stain variable bacterium, were analysed. Abundance and distribution of AHL and AI-2 signalling genes showed that only one species (Citrobacter sp. NLAE-zl-C269) of a Gram-negative bacteria appeared to possess an AHL synthase gene, while the Lux-based genes (AI-2 QS) were identified in both Gram-positive and Gram-positive bacteria (191 genomes representing 38.2% of total genomes). Of these 192 genomes, 139 are from Gram-positive bactreetteria and 53 from Gram-negative bacteria. We also found that the genera Butyrivibrio, Prevotella, Ruminococcus and Pseudobutyrivibrio, which are well known as the most abundant bacterial genera in the rumen, possessed the most lux-based AI-2 QS genes. Gene expression levels within the metatranscriptome dataset showed that Prevotella, in particular, expressed high levels of LuxS synthase suggesting that this genus plays an important role in QS within the rumen.ConclusionThis is the most comprehensive study of QS in the rumen microbiome to date. This study shows that AI-2-based QS is rife in the rumen. These results allow a greater understanding on plant-microbe interactions in the rumen.

Parallel quorum-sensing system in Vibrio cholerae prevents signal interference inside the host.

Many bacteria use quorum sensing (QS) to regulate virulence factor production in response to changes in population density. QS is mediated through the production, secretion, and detection of signaling molecules called autoinducers (AIs) to modulate population-wide behavioral changes. Four histidine kinases, LuxPQ, CqsS, CqsR and VpsS, have been identified in Vibrio cholerae as QS receptors to activate virulence gene expression at low cell density. Detection of AIs by these receptors leads to virulence gene repression at high cell density. The redundancy among these receptors is puzzling since any one of the four receptors is sufficient to support colonization of V. cholerae in the host small intestine. It is believed that one of the functions of such circuit architecture is to prevent interference on any single QS receptor. However, it is unclear what natural molecules can interfere V. cholerae QS and in what environment interference is detrimental. We show here mutants expressing only CqsR without the other three QS receptors are defective in colonizing the host large intestine. We identified ethanolamine, a common intestinal metabolite that can function as a chemical source of QS interference. Ethanolamine specifically interacts with the ligand-binding CACHE domain of CqsR and induces a premature QS response in V. cholerae mutants expressing only CqsR without the other three QS receptors. The effect of ethanolamine on QS gene expression and host colonization is abolished by mutations that disrupt CqsR signal sensing. V. cholerae defective in producing ethanolamine is still proficient in QS, therefore, ethanolamine functions only as an external cue for CqsR. Our findings suggest the inhibitory effect of ethanolamine on CqsR could be a possible source of QS interference but is masked by the presence of the other parallel QS pathways, allowing V. cholerae to robustly colonize the host.

Resumptive Streptococcus mutans Persisters Induced From Dimethylaminododecyl Methacrylate Elevated the Cariogenic Virulence by Up-Regulating the Quorum-Sensing and VicRK Pathway Genes.

Bacterial persistence has become a worldwide health problem due to its ability to cause the recalcitrance and relapse of infections. The existence of bacterial persistence and their possible mechanisms have been widely reported. However, the following regrowth of persister cells is not clear although the awakening of dormant surviving persisters is the key to reinitialize bacterial infection. In this study, we investigated the growth character and cariogenic virulence during the recovery of Streptococcus mutans drug-tolerant persister cells induced by a novel quaternary ammonium: dimethylaminododecyl methacrylate (DMADDM). A remarkable lag phase was observed in S. mutans persisters when regrew at the first 24 h compared to normal cells. During the entire recovery state, persisters are metabolically active to increase the production of both water-soluble and water-insoluble glucan. The shortage of cell number in persisters resulted in the decrease of lactic acid production, but persisters gradually recovered the normal acid production ability after 72 h. The up-regulated expression of gtf and vicR was in line with comDE circuit and consistent with the virulence change during the regrowth stage. Our findings proved that lethal dosages of DMADDM induced drug-tolerant S. mutans persisters in biofilm, which had a prolonged lag phase and elevated cariogenic virulence during regrowth. The recovery and elevated virulence of persisters were regulated by quorum-sensing and VicRK pathway. This alarmed the elevated cariogenicity of persisters and highlighted the critical requirement for the drug-tolerance evaluation when developing new oral antimicrobial agents. To the best of our knowledge, we characterized the regrowth and cariogenic virulence variation of S. mutans persisters induced by quaternary ammonium for the first time. Our findings suggest that S. mutans persisters with the elevated cariogenic virulence during their regrowth stage highlighted the need of new strategy to overcome bacterial persistence. Meanwhile, the prolonged lag phase and the involvement of quorum-sensing system in the regrowth of S. mutans persisters may provide the potential targets.

Building Quantitative Gene Regulatory Mechanism in Quorum Sensing in <i>Pseudomonas aeruginosa</i> Using Transcriptomic Data

A large amount of transcriptomic data provides opportunities 1) to verify the gene regulatory mechanism, which is usually obtained from a single experiment, at population level; 2) to uncover the gene regulatory mechanism at population level; and 3) to build a quantitatively gene regulatory mechanism. One of the best studied regulatory mechanisms in bacteria is the quorum sensing (QS), which plays an important role in regulation of bacteria population behaviors such as antibiotic production, biofilm formation, bioluminescence, competence, conjugation, motility and sporulation. Pseudomonas aeruginosa is a Gram-negative bacterium causing diseases in plants, animals, humans, and its biofilm and drug-resistance become great concerns in clinics. P. aeruginosa has three QS systems including a specific one for Pseudomonas. In this study, the transcriptomic data of P. aeruginosa were combined from 104 publications and QS gene expressions were analyzed under different experimental conditions. The results demonstrate the quantitatively regulatory mechanisms of QS genes at population level including 1) to rank and group QS-related genes according to their activity; 2) to quantitatively define the role of a single global regulator; 3) to find out the probability that a global regulator impacts QS genes and the probability that a QS gene responds to global regulators; and 4) to search for overlapped genes under four types of experimental conditions. These results provide integrative information on understanding the regulation of QS genes at population level.

Phenotypic and genotypic analysis of biofilm production by Pseudomonas aeruginosa isolates from infection and colonization samples.

INTRODUCTION: Pseudomonas aeruginosa is an opportunistic pathogen associated with healthcare-related infections, affecting mainly patients with underlying diseases and immunosuppression. This microorganism has several virulence mechanisms that favour its pathogenesis, including the production of biofilm. This study aimed to analyze the phenotypic production of biofilms, the occurrence of quorum sensing (QS) genes, and the clonal profile of clinical isolates of P. aeruginosa from colonized/infected patients in a tertiary hospital in Recife-PE. METHODS: We obtained 21 isolates that were classified as infection isolates (II), and 10 colonization isolates (CI). The phenotypic analysis for biofilm production was performed quantitatively. The QS genes were detected by specific PCRs, and the clonal profile was assessed using ERIC-PCR. RESULTS: Of the 31 isolates, 58.1 % (18/31) were biofilm producers, of which 70 % (7/10) were CI and classified as weakly adherent; 52.4 % (11/21) of the II produced biofilms, and were classified as weak (38.1 %, (8/21)), moderate (9.5 %, (2/21)), and strongly adherent (4.8 %, (1/21)). All isolates harbored the QS genes analyzed. In the clonal analysis, 26 distinct genetic profiles were identified, highlighting the presence of a clone in four samples, i.e., one infection isolate, and 3 colonization isolates. CONCLUSIONS: The detection of biofilm formation is important in P. aeruginosa in addition to the identification of colonization and infection isolates, especially from complex environments such as ICUs. Further, we define a strategy for monitoring and analyzing P. aeruginosa strains that can potentially cause infections in hospitalized patients.

Use of Quorum Sensing Gene SdiA as a Molecular Marker for Salmonella Diagnosis

Use of Quorum Sensing Gene SdiA as a Molecular Marker for Salmonella Diagnosis

Natural Anti-Quorum Sensing agents againstPseudomonas aeruginosa

Abuse of antibiotics in therapy has led to development of resistance in the target organisms. Failure of the current antibiotics to control infections makes it essential to discover alternative drugs. The pathogenicity in numerous bacteria is regulated by Quorum sensing (QS) signaling systems. The QS inhibition system may cause the reduction of virulence and defense against the bacterial infections. The QS is the main regulator of virulence and biofilm formation in Pseudomonas aeruginosa. A variety of plants showed their effects on P. aeruginosa virulence. Extract of various plants control the regulatory QS genes and factors with marginal effects on bacterial growth. The quorum-quenching (QQ) mechanisms are unrelated to static or cidal effects. In fact, anti-QS have already shown promise in the battle against P. aeruginosa infections.

Quorum quenching activity of Aerva lanata against Catheter Associated Urinary Tract Infections (CAUTI) Pathogens

Background. CAUTI is the most common hospital infection caused by Gram-negative bacterium owing to their ability to colonize UT mucosa. These uro-pathogens elaborate quorum sensing (QS) molecules that modify the expression and secretion of biofilm matrix compounds in response to rheological and other UT changes during catheterization. Quenching the biological effects of QS molecules will have substantial clinical value. This study identifies and evaluate the antibiofilm/anti-virulence properties of Aerva lanata plant extract against select CAUTI pathogens. Methods. Methanolic extract of Aerva lanata was screened for its anti-urobacterial effects using Chromobacterium violaceum as the test strain. A. lanata extract impact on the QS molecule, acyl homoserine lactone (AHL) regulated bacterial functions essential for biofilm formation was investigated. It includes RT-PCR analysis of QS genes in the bacterium and in silico studies of bacterial compounds. Results. Methanolic extract of A. lanata interfered with AHL regulated physiological functions such as biofilm formation, pellicle inhibition, flagellar motility and exopolysaccharides (EPS) production. In situ-visualization of biofilm with confocal microscopy showed concentration influenced reduction in bacterial biofilm formation in response to plant extract. GC-MS identified 11 compounds and molecular docking analysis predicted their respective host receptors. In silico analysis of plant extract revealed putative compounds with potential to inhibit the bacterial QS AHL system. RT-PCR analysis revealed down regulation of QS related virulence genes viz., las1, lasR, lasB, lasA. Conclusion. Data obtained in this study strongly suggests that Aerva lanata should be further investigated for its therapeutic potentials to treat bacterial CAUTI.

Terpinen-4-ol attenuates quorum sensing regulated virulence factors and biofilm formation in Pseudomonas aeruginosa.

Aim: To investigate the effects of Terpinen-4-ol on quorum sensing (QS)-regulated biofilm formation and virulence factors production in Pseudomonas aeruginosa. Materials & methods: QS inhibition, molecular docking analysis and gene expression studies were performed to check attenuation effect of Terpinen-4-ol on virulence of P. aeruginosa. Production of various virulence factors and biofilm formation were studied at sub-MIC of Terpinen-4-ol alone and in combination with ciprofloxacin. Results: Terpinen-4-ol at sub-MIC exhibited QS inhibition and downregulated all key QS genes. Molecular docking analysis showed high binding affinities of Terpinen-4-ol with QS receptors. Terpinen-4-ol exhibited synergistic interaction with ciprofloxacin and further reduced production of all the virulence factors and biofilms formation. Conclusion: Terpinen-4-ol could be developed into antivirulence drug after its in vivo evaluation for treatment strategies.

Purification of the Vibrio Quorum-Sensing Transcription Factors LuxR, HapR, and SmcR.

Quorum sensing is a cell density-dependent form of cellular communication among bacteria. This signaling process has been heavily studied in vibrios due to their diverse and complex phenotypes and relevance to human and aquaculture disease. Mechanistic studies of Vibrio quorum sensing have required optimization of protein purification techniques to examine the role of key proteins, such as the LuxR/HapR family of transcription factors that control quorum-sensing gene expression. Protein purification is the cornerstone of biochemistry, and it is crucial to consistently produce batches of protein that are pure, active, and concentrated to perform various assays. The methods described here are optimized for purification of the Vibrio master quorum-sensing regulators, LuxR (Vibrio harveyi), HapR (Vibrio cholerae), and SmcR (Vibrio vulnificus). We anticipate that these methods can be applied to other proteins in this family of transcription factors.

SprI/SprR Quorum Sensing System of Serratia proteamaculans 94.

In this study, we investigated the quorum sensing (QS) regulatory system of the psychrotrophic strain Serratia proteamaculans 94 isolated from spoiled refrigerated meat. The strain produced several N-acyl-L-homoserine-lactone (AHL) QS signal molecules, with N-(3-oxo-hexanoyl)-L-homoserine lactone and N-(3-hydroxy-hexanoyl)-L-homoserine lactone as two main types. The sprI and sprR genes encoding an AHL synthase and a receptor regulatory protein, respectively, were cloned and sequenced. Analysis of their nucleotide sequence showed that these genes were transcribed convergently and that their reading frames partly overlapped by 23 bp in the terminal regions. The genes were highly similar to the luxI/luxR-type QS genes of other Gram-negative bacteria. An spr-box (analog of the lux-box) was identified upstream of the sprR gene and found to be overlapped with the sequence of −10 sequence site in the promoter region of this gene. Inactivation of the sprI gene led to the absence of AHL synthesis, chitinolytic activity, and swimming motility; decrease of extracellular proteolytic activity; affected the cellular fatty acid composition; and reduced suppression of the fungal plant pathogen mycelium growth by volatile compounds emitted by strain S. proteamaculans 94. The data obtained demonstrated the important role of the QS system in the regulation of cellular processes in S. proteamaculans 94.

Effect of the luxI/R gene on AHL-signaling molecules and QS regulatory mechanism in Hafnia alvei H4

Hafnia alvei H4 is a bacterium subject to regulation by a N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing system and is closely related to the corruption of instant sea cucumber. Studying the effect of Hafnia alvei H4 quorum sensing regulatory genes on AHLs is necessary for the quality and preservation of instant sea cucumber. In this study, the draft genome of H. alvei H4, which comprises a single chromosome of 4,687,151 bp, was sequenced and analyzed and the types of AHLs were analyzed employing thin-layer chromatography (TLC) and high resolution triple quadrupole liquid chromatography/mass spectrometry (LC/MS). Then the wild-type strain of H. alvei H4 and the luxI/R double mutant (ΔluxIR) were compared by transcriptome sequencing (RNA-seq). The results indicate that the incomplete genome sequence revealed the presence of one quorum-sensing (QS) gene set, designated as lasI/expR. Three major AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL), N-butyryl-l-homoserine lactone (C4-HSL), and N-(3-oxo-octanoyl)-l-homoserine lactone (3-oxo-C8-HSL) were found, with C6-HSL being the most abundant. C6-HSL was not detected in the culture of the luxI mutant (ΔluxI) and higher levels of C4-HSL was found in the culture of the luxR mutant (ΔluxR), which suggested that the luxR gene may have a positive effect on C4-HSL production. It was also found that AHL and QS genes are closely related in the absence of luxIR double deletion. The results of this study can further elucidate at the genetic level that luxI and luxR genes are involved in the regulation of AHL.

The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms.

Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection.IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

Virulence Factors Of Carbapenem-Resistant Pseudomonas aeruginosa In Hospital-Acquired Infections In Mansoura, Egypt.

PurposeThe problem of carbapenem-resistant Pseudomonas aeruginosa in health-care settings is growing worse. This study was conducted to investigate the rate of carbapenemase genes, antibiotic resistance, and virulence factors in carbapenem-resistant P. aeruginosa associated with hospital-acquired infections.Patients and methodsIsolates of P. aeruginosa were collected from patients with hospital-acquired infections at Mansoura University Hospital in Mansoura. Carbapenem susceptibility was done by broth dilution. The presence of carbapenemase genes and quorum-sensing genes was assessed by PCR. Production of protease, pyocyanin, twitching motility, hemolytic activity and biofilm formation was evaluated.ResultsOut of 80 P. aeruginosa isolates, 34 (42.5%) were resistant to carbapenem. Among carbapenem-resistant P. aeruginosa isolates, 21 (61.8%) were carbapenemase producers. The most prevalent gene detected was blaVIM. The frequency of protease, pyocyanin, twitching motility, hemolytic activity and biofilm formation was 76.2%, 58.8%, 83.8%, 93.8% and 77.5%, respectively. Biofilm formation was significantly associated with carbapenem-resistant P. aeruginosa. On the other hand, pyocyanin production was significantly lower in carbapenem-resistant isolates. No correlation existed between carbapenem resistance and any other studied virulence factors or quorum-sensing genes.ConclusionAssociation of carbapenem-resistant P. aeruginosa with other antibiotic resistance or the presence of virulence factors in hospital-acquired infection may represent a warning that enhances the need for a stringent surveillance program.

Quorum Sensing Inhibition: Current Advances of the Natural Antimicrobial Agents

The misuses of antimicrobials in infectious diseases have led to the progress of extensive resistance in the infectious organisms. The failure of accessible antimicrobials to control infections makes it essential to discover alternatives to currently existing drugs. Their connection to infectious diseases and their natural ability to increase antimicrobial resistance in microbes, has led to platforms for research focused on new techniques to control them. Pathogenicity in many bacteria is regulated by quorum sensing (QS). Inhibition of QS system may cause reduction of virulence and protect against bacterial infections. These bacteria rely on chemical communication (or QS) to coordinate activities necessary for their survival in groups by some course of action. Their dependence on QS has made those signaling systems within bacteria an attractive target for the design of new anti-infective agents. Compounds that can interrupt these processes are known as QS inhibitors. The QS is the key regulator of virulence in various bacteria. Various plant extracts and their chemical constituents exhibited their effects on bacterial virulence factors by inhibiting QS genes and QS-controlled factors and effects on bacterial growth. The anti-QS approach is a promising one in the fight against infections pathogens, thereby making the bacteria more susceptible to traditional antimicrobials. The QS inhibitors (QSI) may provide the newest weapon against infections involving drug-resistant bacterial strains. These QSIs come from a variety of sources and have a wide array of structures.

Antimicrobial action of photoactivated C-Phycocyanin against Enterococcus faecalis biofilms: Attenuation of quorum-sensing system.

The limitations of conventional chemo-mechanic therapy in endodontic have given rise to introduce and develop photoactivated disinfection (PAD) as an adjunctive treatment. The objective of this ex vivo study was to evaluate the effects of PAD using C-Phycocyanin, as a natural photosensitizer, on biological activities and expression of quorum-sensing system gene fsrB of Enterococcus faecalis. A total of 52 extracted single-rooted human premolar teeth were divided into five groups (n = 10) and were incubated with E. faecalis for biofilm formation, and they were treated according to following groups; A. C-PC only; B. Diode laser only (at wavelengths of 635 nm); C. PAD (C-PC plus diode laser); and D. Sodium hypochlorite (NaOCl). E. Control group (not treatment). Two remaining teeth were used to confirm E. faecalis biofilm formation in the root canal system by scanning electron microscopic. The effects of each treatment were measured on viability and expression level of fsrB gene of E. faecalis using microbial cell viability and quantitative real-time PCR assays, respectively. Additionally, intracellular reactive oxygen species (ROS) production was measured using 2',7'-dichlorfluorescein-diacetate (DCFH-DA) fluorescent probe. The C-PC alone and C-PC-PAD reduced the culture viability of E. faecalis by 38.1% and 89.45%, respectively (both; P < 0.05). According to the Bonferroni post hoc test, a significant difference in the reduction of E. faecalis count was observed between C-PC-PAD and other groups (P < 0.05), except the NaOCl group (P > 0.05). As well as, photoexcited C-PC in PAD could increase (3.8- fold) the intracellular ROS production in E. faecalis compared to the control group (P < 0.05). C-PC-PAD at sub-significant inhibitory concentration and NaOCl at sub-lethal dose could significantly decrease the expression levels of fsrB by 10.8- and 11.4-fold, respectively. Our data support that C-PC alone and C-PC-PAD can serve as a potent irrigation solution and a disinfection method, respectively for microbial reduction and promising adjunct therapy in endodontic infection treatment against E. faecalis with clinical applications for infection control in endodontics.

Elucidation of quorum sensing components and their role in regulation of symbiotically important traits in Ensifer nodulating pigeon pea

Quorum sensing (QS) in rhizobia regulates diverse processes determining the success and efficiency of association with the legume host. Despite the notable importance of QS as well as the well-known underlying variability in the genomic and metabolic components thereof, its study in rhizobia is largely restricted to few laboratory strains. In this work, QS phenomenon in the rhizobia nodulating pigeon pea– one of the most important legume crops of the global-south, is characterized. Using 16S rRNA and recombinaseA sequencing analysis, the selected QS-positive and host-beneficial isolates were identified to be taxonomically affiliated to the genus Ensifer. Their QS components, including homologues of QS genes, and the repertoire of N-acyl homoserine lactone (AHL) autoinducers were identified. Sequences of the QS homologues showed significant variabilities ranging from 10 to >20% with the known Ensifer sequences. Autoinducer profiling using LC–MS/MS revealed the production of long and short chain AHLs variably by the isolates, including 3-oxo-C12-homoserine lactone (3-O-C12-HSL) and 3-OH-C16-HSL as their first report in Rhizobiaceae. Motility and attachment– two of the most crucial traits for effective establishment on host roots were discovered to be QS dependent in in vitro analysis and the same was confirmed using expression analysis of their regulatory genes using qRT-PCR; both revealing a QS mediated repression of motility and promotion of attachment. This study highlights that Ensifer nodulating pigeon pea, although with significant variance in the anatomy of their QS components, regulate symbiotically crucial cell-processes via QS in a scheme that is conserved in multiple genera.

Simultaneous Visualization of Multiple Gene Expression in Single Cells Using an Engineered Multicolor Reporter Toolbox and Approach of Spectral Crosstalk Correction.

Synthetic biology aims to make biology easier to engineer and focuses on the design and construction of core components that can be modeled, understood, and tuned to meet specific performance criteria, and the assembly of these smaller parts and devices into larger integrated systems to solve specific problems. Here, we designed and engineered a multicolor fluorescent reporter toolbox to simultaneously monitor the activities of multiple genes in single cells. The toolbox contained standardized and well-characterized genetic building blocks for the convenient and reproducible assembly of multiple promoter-reporter fusions (ranging from 1 to 4) into a single plasmid. Given the common problem of spectral crosstalk among multiple fluorescent proteins, we deciphered multiple spectral signatures within cells through a deduced linear unmixing algorithm. Our approach enabled the quantification of gene expression with direct FP concentrations, instead of mix-contributed fluorescence intensities, thus enabling true signal separation with high confidence. This approach performed well in the imaging of mixing cells with single FP labels. Additionally, combining with the multicolor toolbox, we succeeded in simultaneously monitoring the genetic dynamics of four selected quorum-sensing genes in response to the induction of two exogenously added autoinducers and were able to examine gene regulatory connections within the QS signaling network in Pseudomonas aeruginosa. Overall, this synthetic framework (i.e., the genetic toolbox and the well-evaluated approach of spectral correction) will be useful for applied synthetic biology projects, multicolor imaging, and analyzing interactions of multiple genes of natural genetic networks or assembling synthetic ones.

Differential Modulation of Quorum Sensing Signaling through QslA in Pseudomonas aeruginosa Strains PAO1 and PA14.

Two clinical isolates of the opportunist pathogen Pseudomonas aeruginosa named PAO1 and PA14 are commonly studied in research laboratories. Despite the isolates being closely related, PA14 exhibits increased virulence compared to that of PAO1 in various models. To determine which players are responsible for the hypervirulence phenotype of the PA14 strain, we elected a transcriptomic approach through RNA sequencing. We found 2,029 genes that are differentially expressed between the two strains, including several genes that are involved with or regulated by quorum sensing (QS), known to control most of the virulence factors in P. aeruginosa Among them, we chose to focus our study on QslA, an antiactivator of QS whose expression was barely detectable in the PA14 strain according our data. We hypothesized that lack of expression of qslA in PA14 could be responsible for higher QS expression in the PA14 strain, possibly explaining its hypervirulence phenotype. After confirming that QslA protein was highly produced in PAO1 but not in the PA14 strain, we obtained evidence showing that a PAO1 deletion strain of qslA has faster QS gene expression kinetics than PA14. Moreover, known virulence factors activated by QS, such as (i) pyocyanin production, (ii) H2-T6SS (type VI secretion system) gene expression, and (iii) Xcp-T2SS (type II secretion system) machinery production and secretion, were all lower in PAO1 than in PA14, due to higher qslA expression. However, biofilm formation and cytotoxicity toward macrophages, although increased in PA14 compared to PAO1, were independent of QslA control. Together, our findings implicated differential qslA expression as a major determinant of virulence factor expression in P. aeruginosa strains PAO1 and PA14.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen responsible for acute nosocomial infections and chronic pulmonary infections. P. aeruginosa strain PA14 is known to be hypervirulent in different hosts. Despite several studies in the field, the underlining molecular mechanisms sustaining this phenotype remain enigmatic. Here we provide evidence that the PA14 strain has faster quorum sensing (QS) kinetics than the PAO1 strain, due to the lack of QslA expression, an antiactivator of QS. QS is a major regulator of virulence factors in P. aeruginosa; therefore, we propose that the hypervirulent phenotype of the PA14 strain is, at least partially, due to the lack of QslA expression. This mechanism could be of great importance, as it could be conserved among other P. aeruginosa isolates.

In silico Analysis Reveals Distribution of Quorum Sensing Genes and Consistent Presence of LuxR Solos in the Pandoraea Species

The most common quorum sensing (QS) system in Gram-negative bacteria consists of signaling molecules called N-acyl-homoserine lactones (AHLs), which are synthesized by an enzyme AHL synthase (LuxI) and detected by a transcriptional regulator (LuxR) that are usually located in close proximity. However, many recent studies have also evidenced the presence of LuxR solos that are LuxR-related proteins in Proteobacteria that are devoid of a cognate LuxI AHL synthase. Pandoraea species are opportunistic pathogens frequently isolated from sputum specimens of cystic fibrosis (CF) patients. We have previously shown that P. pnomenusa strains possess QS activity. In this study, we examined the presence of QS activity in all type strains of Pandoraea species and acquired their complete genome sequences for holistic bioinformatics analyses of QS-related genes. Only four out of nine type strains (P. pnomenusa, P. sputorum, P. oxalativorans, and P. vervacti) showed QS activity, and C8-HSL was the only AHL detected. A total of 10 canonical luxIs with adjacent luxRs were predicted by bioinformatics from the complete genomes of aforementioned species and publicly available Pandoraea genomes. No orphan luxI was identified in any of the genomes. However, genes for two LuxR solos (LuxR2 and LuxR3 solos) were identified in all Pandoraea genomes (except two draft genomes with one LuxR solo gene), and P. thiooxydans was the only species that harbored no QS-related activity and genes. Except the canonical LuxR genes, LuxIs and LuxR solos of Pandoraea species were distantly related to the other well-characterized QS genes based on phylogenetic clustering. LuxR2 and LuxR3 solos might represent two novel evolutionary branches of LuxR system as they were found exclusively only in the genus. As a few luxR solos were located in close proximity with prophage sequence regions in the genomes, we thus postulated that these luxR solos could be transmitted into genus Pandoraea by transduction process mediated by bacteriophage. The bioinformatics approach developed in this study forms the basis for further characterization of closely related species. Overall, our findings improve the current understanding of QS in Pandoraea species, which is a potential pharmacological target in battling Pandoraea infections in CF patients.