Species-Specific Quorum Sensing Represses the Chitobiose Utilization Locus in Vibrio cholerae

ABSTRACT The marine facultative pathogen Vibrio cholerae forms complex multicellular communities on the chitinous shells of crustacean zooplankton in its aquatic reservoir. V. cholerae-chitin interactions are critical for the growth, evolution, and waterborne transmission of cholera. This is due, in part, to chitin-induced changes in gene expression in this pathogen. Here, we sought to identify factors that influence chitin-induced expression of one locus, the chitobiose utilization operon (chb), which is required for the uptake and catabolism of the chitin disaccharide. Through a series of genetic screens, we identified that the master regulator of quorum sensing, HapR, is a direct repressor of the chb operon. We also found that the levels of HapR in V. cholerae are regulated by the ClpAP protease. Furthermore, we show that the canonical quorum sensing cascade in V. cholerae regulates chb expression in an HapR-dependent manner. Through this analysis, we found that signaling via the species-specific autoinducer CAI-1, but not the interspecies autoinducer AI-2, influences chb expression. This phenomenon of species-specific regulation may enhance the fitness of this pathogen in its environmental niche. IMPORTANCE In nature, bacteria live in multicellular and multispecies communities. Microbial species can sense the density and composition of their community through chemical cues using a process called quorum sensing (QS). The marine pathogen Vibrio cholerae is found in communities on the chitinous shells of crustaceans in its aquatic reservoir. V. cholerae interactions with chitin are critical for the survival, evolution, and waterborne transmission of this pathogen. Here, we show that V. cholerae uses QS to regulate the expression of one locus required for V. cholerae-chitin interactions.

Download Full-text

Species-specific quorum sensing represses the chitobiose utilization locus in Vibrio cholerae

10.1101/2020.04.16.045526 ◽

2020 ◽

Author(s):

Catherine A. Klancher ◽

Jane D. Newman ◽

Alyssa S. Ball ◽

Julia C. van Kessel ◽

Ankur B. Dalia

Keyword(s):

Quorum Sensing ◽

Vibrio Cholerae ◽

Crustacean Zooplankton ◽

Dependent Manner ◽

Environmental Niche ◽

Clpap Protease ◽

Waterborne Transmission ◽

Specific Regulation ◽

Species Specific ◽

Induced Changes

AbstractThe marine facultative pathogen Vibrio cholerae forms complex multicellular communities on the chitinous shells of crustacean zooplankton in its aquatic reservoir. V. cholerae-chitin interactions are critical for the growth, evolution, and waterborne transmission of cholera. This is due, in part, to chitin-induced changes in gene expression in this pathogen. Here, we sought to identify factors that influence chitin-induced expression of one locus, the chitobiose utilization operon (chb), which is required for the uptake and catabolism of the chitin disaccharide. Through a series of genetic screens, we identified that the master regulator of quorum sensing, HapR, is a direct repressor of the chb operon. We also found that the levels of HapR in V. cholerae are regulated by the ClpAP protease. Furthermore, we show that the canonical quorum sensing cascade in V. cholerae regulates chb expression in a HapR-dependent manner. Through this analysis we found that signaling via the species-specific autoinducer CAI-1, but not the inter-species autoinducer AI-2, influences chb expression. This phenomenon of species-specific regulation may enhance the fitness of this pathogen in its environmental niche.ImportanceIn nature, bacteria live in multicellular and multispecies communities. Microbial species can sense the density and composition of their community through chemical cues using a process called quorum sensing (QS). The marine pathogen Vibrio cholerae is found in communities on the chitinous shells of crustaceans in its aquatic reservoir. V. cholerae interactions with chitin are critical for the survival, evolution, and waterborne transmission of this pathogen. Here, we show that V. cholerae uses QS to regulate the expression of one locus required for V. cholerae-chitin interactions.

Download Full-text

The Transcriptional Regulator VqmA Increases Expression of the Quorum-Sensing Activator HapR in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.188.7.2446-2453.2006 ◽

2006 ◽

Vol 188 (7) ◽

pp. 2446-2453 ◽

Cited By ~ 32

Author(s):

Zhi Liu ◽

Ansel Hsiao ◽

Adam Joelsson ◽

Jun Zhu

Keyword(s):

Quorum Sensing ◽

Vibrio Cholerae ◽

Diarrheal Disease ◽

Constitutive Expression ◽

Physiological Role ◽

Transcriptional Regulator ◽

Protease Production ◽

Dependent Manner ◽

Cell Densities

ABSTRACT Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. A number of environmental stimuli regulate virulence gene expression in V. cholerae, including quorum-sensing signals. At high cell densities, quorum sensing in V. cholerae invokes a series of signal transduction pathways in order to activate the expression of the master regulator HapR, which then represses the virulence regulon and biofilm-related genes and activates protease production. In this study, we identified a transcriptional regulator, VqmA (VCA1078), that activates hapR expression at low cell densities. Under in vitro inducing conditions, constitutive expression of VqmA represses the virulence regulon in a HapR-dependent manner. VqmA increases hapR transcription as measured by the activity of the hapR-lacZ reporter, and it increases HapR production as measured by Western blotting. Using a heterogenous luxCDABE cosmid, we found that VqmA stimulates quorum-sensing regulation at lower cell densities and that this stimulation bypasses the known LuxO-small-RNA regulatory circuits. Furthermore, we showed that VqmA regulates hapR transcription directly by binding to its promoter region and that expression of vqmA is cell density dependent and autoregulated. The physiological role of VqmA is also discussed.

Download Full-text

The Streptomyces filipinensis Gamma-Butyrolactone System Reveals Novel Clues for Understanding the Control of Secondary Metabolism

Applied and Environmental Microbiology ◽

10.1128/aem.00443-20 ◽

2020 ◽

Vol 86 (18) ◽

Author(s):

Eva G. Barreales ◽

Tamara D. Payero ◽

Ester Jambrina ◽

Jesús F. Aparicio

Keyword(s):

Quorum Sensing ◽

Secondary Metabolism ◽

Target Genes ◽

Antibiotic Production ◽

The Other ◽

Antimycin A ◽

Dependent Manner ◽

Biosynthetic Genes ◽

Content Type ◽

Communication Signals

ABSTRACT Streptomyces γ-butyrolactones (GBLs) are quorum sensing communication signals triggering antibiotic production. The GBL system of Streptomyces filipinensis, the producer of the antifungal agent filipin, has been investigated. Inactivation of sfbR (for S. filipinensis γ-butyrolactone receptor), a GBL receptor, resulted in a strong decrease in production of filipin, and deletion of sfbR2, a pseudo-receptor, boosted it, in agreement with lower and higher levels of transcription of filipin biosynthetic genes, respectively. It is noteworthy that none of the mutations affected growth or morphological development. While no ARE (autoregulatory element)-like sequences were found in the promoters of filipin genes, suggesting indirect control of production, five ARE sequences were found in five genes of the GBL cluster, whose transcription has been shown to be controlled by both S. filipinensis SfbR and SfbR2. In vitro binding of recombinant SfbR and SfbR2 to such sequences indicated that such control is direct. Transcription start points were identified by 5′ rapid amplification of cDNA ends, and precise binding regions were investigated by the use of DNase I protection studies. Binding of both regulators took place in the promoter of target genes and at the same sites. Information content analysis of protected sequences in target promoters yielded an 18-nucleotide consensus ARE sequence. Quantitative transcriptional analyses revealed that both regulators are self-regulated and that each represses the transcription of the other as well as that of the remaining target genes. Unlike other GBL receptor homologues, SfbR activates its own transcription whereas SfbR2 has a canonical autorepression profile. Additionally, SfbR2 was found here to bind the antifungal antimycin A as a way to modulate its DNA-binding activity. IMPORTANCE Streptomyces GBLs are important signaling molecules that trigger antibiotic production in a quorum sensing-dependent manner. We have characterized the GBL system from S. filipinensis, finding that two key players of this system, the GBL receptor and the pseudo-receptor, each counteracts the transcription of the other for the modulation of filipin production and that such control over antifungal production involves an indirect effect on the transcription of filipin biosynthetic genes. Additionally, the two regulators bind the same sites, are self-regulated, and repress the transcription of three other genes of the GBL cluster, including that encoding the GBL synthase. In contrast to all the GBL receptors known, SfbR activates its own synthesis. Moreover, the pseudo-receptor was identified as the receptor of antimycin A, thus extending the range of examples supporting the idea of signaling effects of antibiotics in Streptomyces. The intricate regulatory network depicted here should provide important clues for understanding the regulatory mechanism governing secondary metabolism.

Download Full-text

Integration of Cyclic di-GMP and Quorum Sensing in the Control ofvpsTandaphAin Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.05167-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6331-6341 ◽

Cited By ~ 103

Author(s):

Disha Srivastava ◽

Rebecca C. Harris ◽

Christopher M. Waters

Keyword(s):

Quorum Sensing ◽

Binding Site ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Virulence Gene ◽

Transcriptional Activator ◽

Transcriptional Regulators ◽

Content Type ◽

Virulence Gene Expression ◽

Signaling Systems

Vibrio choleraetransitions between aquatic environmental reservoirs and infection in the gastrointestinal tracts of human hosts. The second-messenger molecule cyclic di-GMP (c-di-GMP) and quorum sensing (QS) are important signaling systems that enableV. choleraeto alternate between these distinct environments by controlling biofilm formation and virulence factor expression. Here we identify a conserved regulatory mechanism inV. choleraethat integrates c-di-GMP and QS to control the expression of two transcriptional regulators:aphA, an activator of virulence gene expression and an important regulator of the quorum-sensing pathway, andvpsT, a transcriptional activator that induces biofilm formation. Surprisingly,aphAexpression was induced by c-di-GMP. Activation of bothaphAandvpsTby c-di-GMP requires the transcriptional activator VpsR, which binds to c-di-GMP. The VpsR binding site at each of these promoters overlaps with the binding site of HapR, the master QS regulator at high cell densities. Our results suggest thatV. choleraecombines information conveyed by QS and c-di-GMP to appropriately respond and adapt to divergent environments by modulating the expression of key transcriptional regulators.

Download Full-text

The Vibrio cholerae Quorum-Sensing Protein VqmA Integrates Cell Density, Environmental, and Host-Derived Cues into the Control of Virulence

mBio ◽

10.1128/mbio.01572-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Ameya A. Mashruwala ◽

Bonnie L. Bassler

Keyword(s):

Small Intestine ◽

Quorum Sensing ◽

Vibrio Cholerae ◽

Cell Density ◽

Chemical Communication ◽

Bile Salts ◽

Anaerobic Growth ◽

Signal Molecules ◽

Content Type ◽

Collective Behaviors

ABSTRACT Quorum sensing is a chemical communication process in which bacteria use the production, release, and detection of signal molecules called autoinducers to orchestrate collective behaviors. The human pathogen Vibrio cholerae requires quorum sensing to infect the small intestine. There, V. cholerae encounters the absence of oxygen and the presence of bile salts. We show that these two stimuli differentially affect quorum-sensing function and, in turn, V. cholerae pathogenicity. First, during anaerobic growth, V. cholerae does not produce the CAI-1 autoinducer, while it continues to produce the DPO autoinducer, suggesting that CAI-1 may encode information specific to the aerobic lifestyle of V. cholerae. Second, the quorum-sensing receptor-transcription factor called VqmA, which detects the DPO autoinducer, also detects the lack of oxygen and the presence of bile salts. Detection occurs via oxygen-, bile salt-, and redox-responsive disulfide bonds that alter VqmA DNA binding activity. We propose that VqmA serves as an information processing hub that integrates quorum-sensing information, redox status, the presence or absence of oxygen, and host cues. In response to the information acquired through this mechanism, V. cholerae appropriately modulates its virulence output. IMPORTANCE Quorum sensing (QS) is a process of chemical communication that bacteria use to orchestrate collective behaviors. QS communication relies on chemical signal molecules called autoinducers. QS regulates virulence in Vibrio cholerae, the causative agent of the disease cholera. Transit into the human small intestine, the site of cholera infection, exposes V. cholerae to the host environment. In this study, we show that the combination of two stimuli encountered in the small intestine, the absence of oxygen and the presence of host-produced bile salts, impinge on V. cholerae QS function and, in turn, pathogenicity. We suggest that possessing a QS system that is responsive to multiple environmental, host, and cell density cues enables V. cholerae to fine-tune its virulence capacity in the human intestine.

Download Full-text

VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00234-20 ◽

2020 ◽

Vol 202 (18) ◽

Cited By ~ 2

Author(s):

Meng-Lun Hsieh ◽

Christopher M. Waters ◽

Deborah M. Hinton

Keyword(s):

Rna Polymerase ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Core Promoter ◽

Class Ii ◽

Class I ◽

Dependent Manner ◽

Mobility Shift ◽

Content Type

ABSTRACT Vibrio cholerae biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the Vibrio polysaccharides (vps) operons I and II as well as the cluster of ribomatrix (rbm) genes. Transcriptional control of these genes is a complex process that requires several activators/repressors and the ubiquitous signaling molecule, cyclic di-GMP (c-di-GMP). Previously, we demonstrated that VpsR directly activates RNA polymerase containing σ70 (σ70-RNAP) at the vpsL promoter (PvpsL), which precedes the vps-II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using in vitro transcription, electrophoretic mobility shift assays, and DNase I footprinting, we show here that VpsR also directly activates σ70-RNAP transcription from other promoters within the biofilm formation cluster, including PvpsU, at the beginning of the vps-I operon, PrbmA, at the start of the rbm cluster, and PrbmF, which lies upstream of the divergent rbmF and rbmE genes. In this capacity, we find that VpsR is able to behave both as a class II activator, which functions immediately adjacent/overlapping the core promoter sequence (PvpsL and PvpsU), and as a class I activator, which functions farther upstream (PrbmA and PrbmF). Because these promoters vary in VpsR-DNA binding affinity in the absence and presence of c-di-GMP, we speculate that VpsR’s mechanism of activation is dependent on both the concentration of VpsR and the level of c-di-GMP to increase transcription, resulting in finely tuned regulation. IMPORTANCE Vibrio cholerae, the bacterial pathogen that is responsible for the disease cholera, uses biofilms to aid in survival, dissemination, and persistence. VpsR, which directly senses the second messenger c-di-GMP, is a major regulator of this process. Together with c-di-GMP, VpsR directly activates transcription by RNA polymerase containing σ70 from the vpsL biofilm biogenesis promoter. Using biochemical methods, we demonstrate for the first time that VpsR/c-di-GMP directly activates σ70-RNA polymerase at the first genes of the vps and ribomatrix operons. In this regard, it functions as either a class I or class II activator. Our results broaden the mechanism of c-di-GMP-dependent transcription activation and the specific role of VpsR in biofilm formation.

Download Full-text

Sustained Release of a Novel Anti-Quorum-Sensing Agent against Oral Fungal Biofilms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04212-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2265-2272 ◽

Cited By ~ 17

Author(s):

Mark Feldman ◽

Julia Shenderovich ◽

Abed Al Aziz Al-Quntar ◽

Michael Friedman ◽

Doron Steinberg

Keyword(s):

Quorum Sensing ◽

Sustained Release ◽

Biofilm Formation ◽

Active Form ◽

Time Dependent ◽

Prolonged Release ◽

Dependent Manner ◽

Content Type ◽

Bacteria And Fungi ◽

Membrane Reservoir

ABSTRACTThiazolidinedione-8 (S-8) has recently been identified as a potential anti-quorum-sensing/antibiofilm agent against bacteria and fungi. Based on these results, we investigated the possibility of incorporating S-8 in a sustained-release membrane (SRM) to increase its pharmaceutical potential againstCandida albicansbiofilm. We demonstrated that SRM containing S-8 inhibits fungal biofilm formation in a time-dependent manner for 72 h, due to prolonged release of S-8. Moreover, the SRM effectively delivered the agent in its active form to locations outside the membrane reservoir. In addition, eradication of mature biofilm by the SRM containing S-8 was also significant. Of note, S-8-containing SRM affected the characteristics of matureC. albicansbiofilm, such as thickness, exopolysaccharide (EPS) production, and morphogenesis of fungal cells. The concept of using an antibiofilm agent with no antifungal activity incorporated into a sustained-release delivery system is new in medicine and dentistry. This concept of an SRM containing a quorum-sensing quencher with an antibiofilm effect could pave the way for combating oral fungal infectious diseases.

Download Full-text

Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00127-17 ◽

2017 ◽

Vol 199 (18) ◽

Cited By ~ 5

Author(s):

Shouji Yamamoto ◽

Makoto Ohnishi

Keyword(s):

Gene Expression ◽

Signaling Pathways ◽

Vibrio Cholerae ◽

Nutrient Status ◽

Sensor Kinase ◽

Dependent Manner ◽

Specific Enzyme ◽

Phosphotransferase System ◽

Natural Competence ◽

Content Type

ABSTRACTInVibrio cholerae, the genes required for chitin utilization and natural competence are governed by the chitin-responsive two-component system (TCS) sensor kinase ChiS. In the classical TCS paradigm, a sensor kinase specifically phosphorylates a cognate response regulator to activate gene expression. However, our previous genetic study suggested that ChiS stimulates the non-TCS transcriptional regulator TfoS by using mechanisms distinct from classical phosphorylation reactions (S. Yamamoto, J. Mitobe, T. Ishikawa, S. N. Wai, M. Ohnishi, H. Watanabe, and H. Izumiya, Mol Microbiol 91:326–347, 2014,https://doi.org/10.1111/mmi.12462). TfoS specifically activates the transcription oftfoR, encoding a small regulatory RNA essential for competence gene expression. Whether ChiS and TfoS interact directly remains unknown. To determine if other factors mediate the communication between ChiS and TfoS, we isolated transposon mutants that turned offtfoR::lacZexpression but possessed intactchiSandtfoSgenes. We demonstrated an unexpected association of chitin-induced signaling pathways with the glucose-specific enzyme IIA (EIIAglc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses revealed that dephosphorylated EIIAglcinactivated natural competence andtfoRtranscription. Chitin-induced expression of thechboperon, which is required for chitin transport and catabolism, was also repressed by dephosphorylated EIIAglc. Furthermore, the regulation oftfoRandchbexpression by EIIAglcwas dependent on ChiS and intracellular levels of ChiS were not affected by disruption of the gene encoding EIIAglc. These results define a previously unknown connection between the PTS and chitin signaling pathways inV. choleraeand suggest a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.IMPORTANCEThe EIIAglcprotein of the PTS coordinates a wide variety of physiological functions with carbon availability. In this report, we describe an unexpected association of chitin-activated signaling pathways inV. choleraewith EIIAglc. The signaling pathways are governed by the chitin-responsive TCS sensor kinase ChiS and lead to the induction of chitin utilization and natural competence. We show that dephosphorylated EIIAglcinhibits both signaling pathways in a ChiS-dependent manner. This inhibition is different from classical catabolite repression that is caused by lowered levels of cyclic AMP. This work represents a newly identified connection between the PTS and chitin signaling pathways inV. choleraeand suggests a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.

Download Full-text

Analysis of Autoinducer-2 Quorum Sensing in Yersinia pestis

Infection and Immunity ◽

10.1128/iai.00880-13 ◽

2013 ◽

Vol 81 (11) ◽

pp. 4053-4062 ◽

Cited By ~ 15

Author(s):

Jing Yu ◽

Melissa L. Madsen ◽

Michael D. Carruthers ◽

Gregory J. Phillips ◽

Jeffrey S. Kavanaugh ◽

...

Keyword(s):

Quorum Sensing ◽

Yersinia Pestis ◽

Pathogenic Bacteria ◽

Biochemical Characterization ◽

Global Analysis ◽

Dependent Manner ◽

Logarithmic Growth Phase ◽

Content Type ◽

Stress Genes ◽

Autoinducer 2

ABSTRACTThe autoinducer-2 (AI-2) quorum-sensing system has been linked to diverse phenotypes and regulatory changes in pathogenic bacteria. In the present study, we performed a molecular and biochemical characterization of the AI-2 system inYersinia pestis, the causative agent of plague. In strain CO92, the AI-2 signal is produced in aluxS-dependent manner, reaching maximal levels of 2.5 μM in the late logarithmic growth phase, and both wild-type and pigmentation (pgm) mutant strains made equivalent levels of AI-2. Strain CO92 possesses a chromosomallsrlocus encoding factors involved in the binding and import of AI-2, and confirming this assignment, anlsrdeletion mutant increased extracellular pools of AI-2. To assess the functional role of AI-2 sensing inY. pestis, microarray studies were conducted by comparing Δpgmstrain R88 to a ΔpgmΔluxSmutant or a quorum-sensing-null ΔpgmΔypeIRΔyspIRΔluxSmutant at 37°C. Our data suggest that AI-2 quorum sensing is associated with metabolic activities and oxidative stress genes that may helpY. pestissurvive at the host temperature. This was confirmed by observing that theluxSmutant was more sensitive to killing by hydrogen peroxide, suggesting a potential requirement for AI-2 in evasion of oxidative damage. We also show that a large number of membrane protein genes are controlled by LuxS, suggesting a role for quorum sensing in membrane modeling. Altogether, this study provides the first global analysis of AI-2 signaling inY. pestisand identifies potential roles for the system in controlling genes important to disease.

Download Full-text

Quorum Sensing in a Methane-Oxidizing Bacterium

Journal of Bacteriology ◽

10.1128/jb.00773-16 ◽

2016 ◽

Vol 199 (5) ◽

Cited By ~ 14

Author(s):

Aaron W. Puri ◽

Amy L. Schaefer ◽

Yanfen Fu ◽

David A. C. Beck ◽

E. Peter Greenberg ◽

...

Keyword(s):

Quorum Sensing ◽

Greenhouse Gas ◽

Bacterial Species ◽

Homoserine Lactone ◽

Sole Source ◽

Biosynthetic Gene Cluster ◽

Freshwater Ecosystems ◽

Dependent Manner ◽

Content Type ◽

Methane Oxidizer

ABSTRACT Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Dissecting the molecular details of how these organisms interact in the environment may increase our understanding of how they perform this important ecological role. Many bacterial species use quorum sensing (QS) systems to regulate gene expression in a cell density-dependent manner. We have identified a QS system in the genome of Methylobacter tundripaludum, a dominant methane oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA). We determined that M. tundripaludum produces primarily N-3-hydroxydecanoyl-l-homoserine lactone (3-OH-C10-HSL) and that its production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by QS in this methane oxidizer using transcriptome sequencing (RNA-seq) and discovered that this system regulates the expression of a putative nonribosomal peptide synthetase biosynthetic gene cluster. Finally, we detected an extracellular factor that is produced by M. tundripaludum in a QS-dependent manner. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium. IMPORTANCE Aerobic methanotrophs are critical for sequestering carbon from the potent greenhouse gas methane in the environment, yet the mechanistic details of chemical interactions in methane-oxidizing bacterial communities are not well understood. Understanding these interactions is important in order to maintain, and potentially optimize, the functional potential of the bacteria that perform this vital ecosystem function. In this work, we identify a quorum sensing system in the aerobic methanotroph Methylobacter tundripaludum and use both chemical and genetic methods to characterize this system at the molecular level.

Download Full-text