Bacterial Cell-cell Communication Research Articles

Synthetic Homoserine Lactone Sensors for Gram-Positive Bacillus subtilis Using LuxR-Type Regulators.

A universal biochemical signal for bacterial cell-cell communication could facilitate programming dynamic responses in diverse bacterial consortia. However, the classical quorum sensing paradigm is that Gram-negative and Gram-positive bacteria generally communicate via homoserine lactones (HSLs) or oligopeptide molecular signals, respectively, to elicit population responses. Here, we create synthetic HSL sensors for Gram-positive Bacillus subtilis 168 using allosteric LuxR-type regulators (RpaR, LuxR, RhlR, and CinR) and synthetic promoters. Promoters were combinatorially designed from different sequence elements (-35, -16, -10, and transcriptional start regions). We quantified the effects of these combinatorial promoters on sensor activity and determined how regulator expression affects its activation, achieving up to 293-fold activation. Using the statistical design of experiments, we identified significant effects of promoter regions and pairwise interactions on sensor activity, which helped to understand the sequence-function relationships for synthetic promoter design. We present the first known set of functional HSL sensors (≥20-fold dynamic range) in B. subtilis for four different HSL chemical signals: p-coumaroyl-HSL, 3-oxohexanoyl-HSL, n-butyryl-HSL, and n-(3-hydroxytetradecanoyl)-HSL. This set of synthetic HSL sensors for a Gram-positive bacterium can pave the way for designable interspecies communication within microbial consortia.

Promoter selectivity of the RhlR quorum-sensing transcription factor receptor in Pseudomonas aeruginosa is coordinated by distinct and overlapping dependencies on C4-homoserine lactone and PqsE.

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-β-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa.

Quorum sensing system effectively enhances DegU-mediated degradation of pyrethroids by Bacillus subtilis

The contamination of the natural environment is a growing concern that threatens all life forms, including microorganisms. Bacteria protect themselves by initiating quorum sensing (QS), a bacterial cell-cell communication, to generate adaptive responses to these pollutants. Bacillus subtilis has a typical QS ComQXPA system that regulates the phosphorylation of the transcription factor DegU (DegU-P), and thus can mediate the expression of various downstream genes under different stress conditions. Herein, we found that cesB, a gene of Bacillus subtilis 168, plays a key role in pyrethroid degradation, and cesB-mediated degradation could be enhanced by coordinating with the ComX communication system. Using β-cypermethrin (β-CP) as a paradigm, we demonstrated that DegU-P increased upon exposure to β-CP, thus facilitating β-CP degradation by binding to the upstream regulatory regions of cesB, leading to the activation of the expression of cesB. Further, we showed that the expression of different levels of phosphorylated DegU in a degU deletion strain resulted in varying degrees of β-CP degradation efficiency, with phosphorylated DegUH12L achieving 78.39% degradation efficiency on the first day, surpassing the 56.27% degradation efficiency in the wild type strain. Consequently, based on the conserved regulatory mechanism of ComQXPA system, we propose that DegU-P-dependent regulation serves as a conserved defense mechanism owing to its ability to fine-tune the expression of genes involved in the degradation of pollutants upon exposure to different pesticides.

TapA acts as specific chaperone in TasA filament formation by strand complementation

Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell-cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of β-sandwich subunits. The secondary structure around the intercalated N-terminal strand β0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface.

Agrobacterium tumefaciens: a Transformative Agent for Fundamental Insights into Host-Microbe Interactions, Genome Biology, Chemical Signaling, and Cell Biology.

Agrobacterium tumefaciens incites the formation of readily visible macroscopic structures known as crown galls on plant tissues that it infects. Records from biologists as early as the 17th century noted these unusual plant growths and began examining the basis for their formation. These studies eventually led to isolation of the infectious agent, A. tumefaciens, and decades of study revealed the remarkable mechanisms by which A. tumefaciens causes crown gall through stable horizontal genetic transfer to plants. This fundamental discovery generated a barrage of applications in the genetic manipulation of plants that is still under way. As a consequence of the intense study of A. tumefaciens and its role in plant disease, this pathogen was developed as a model for the study of critical processes that are shared by many bacteria, including host perception during pathogenesis, DNA transfer and toxin secretion, bacterial cell-cell communication, plasmid biology, and more recently, asymmetric cell biology and composite genome coordination and evolution. As such, studies of A. tumefaciens have had an outsized impact on diverse areas within microbiology and plant biology that extend far beyond its remarkable agricultural applications. In this review, we attempt to highlight the colorful history of A. tumefaciens as a study system, as well as current areas that are actively demonstrating its value and utility as a model microorganism.

The Multifaceted MEP Pathway: Towards New Therapeutic Perspectives.

Isoprenoids, a diverse class of natural products, are present in all living organisms. Their two universal building blocks are synthesized via two independent pathways: the mevalonate pathway and the 2-C-methyl-ᴅ-erythritol 4-phosphate (MEP) pathway. The presence of the latter in pathogenic bacteria and its absence in humans make all its enzymes suitable targets for the development of novel antibacterial drugs. (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), the last intermediate of this pathway, is a natural ligand for the human Vγ9Vδ2 T cells and the most potent natural phosphoantigen known to date. Moreover, 5-hydroxypentane-2,3-dione, a metabolite produced by Escherichia coli 1-deoxy-ᴅ-xylulose 5-phosphate synthase (DXS), the first enzyme of the MEP pathway, structurally resembles (S)-4,5-dihydroxy-2,3-pentanedione, a signal molecule implied in bacterial cell communication. In this review, we shed light on the diversity of potential uses of the MEP pathway in antibacterial therapies, starting with an overview of the antibacterials developed for each of its enzymes. Then, we provide insight into HMBPP, its synthetic analogs, and their prodrugs. Finally, we discuss the potential contribution of the MEP pathway to quorum sensing mechanisms. The MEP pathway, providing simultaneously antibacterial drug targets and potent immunostimulants, coupled with its potential role in bacterial cell-cell communication, opens new therapeutic perspectives.

Structure of the RhlR-PqsE complex from Pseudomonas aeruginosa reveals mechanistic insights into quorum-sensing gene regulation

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P.aeruginosa virulence factor production is mediated by quorum sensing, a mechanism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers. In P.aeruginosa, the transcription factor receptor RhlR is activated by a RhlI-synthesized autoinducer. We recently showed that RhlR-dependent transcription is enhanced by a physical interaction with the enzyme PqsE via increased affinity of RhlR for promoter DNA. However, the molecular basis for complex formation and how complex formation enhanced RhlR transcriptional activity remained unclear. Here, we report the structure of ligand-bound RhlR in complex with PqsE. Additionally, we determined the structure of the complex bound with DNA, revealing the mechanism by which RhlR-mediated transcription is enhanced by PqsE, thereby establishing the molecular basis for RhlR-dependent virulence factor production in P.aeruginosa.

Comparative genomic and functional analysis of Arthrobacter sp. UMCV2 reveals the presence of luxR-related genes inducible by the biocompound N, N-dimethylhexadecilamine.

Quorum sensing (QS) is a bacterial cell-cell communication system with genetically regulated mechanisms dependent on cell density. Canonical QS systems in gram-negative bacteria possess an autoinducer synthase (LuxI family) and a transcriptional regulator (LuxR family) that respond to an autoinducer molecule. In Gram-positive bacteria, the LuxR transcriptional regulators "solo" (not associated with a LuxI homolog) may play key roles in intracellular communication. Arthrobacter sp. UMCV2 is an actinobacterium that promotes plant growth by emitting the volatile organic compound N, N-dimethylhexadecylamine (DMHDA). This compound induces iron deficiency, defense responses in plants, and swarming motility in Arthrobacter sp. UMCV2. In this study, the draft genome of this bacterium was assembled and compared with the genomes of type strains of the Arthrobacter genus, finding that it does not belong to any previously described species. Genome explorations also revealed the presence of 16 luxR-related genes, but no luxI homologs were discovered. Eleven of these sequences possess the LuxR characteristic DNA-binding domain with a helix-turn-helix motif and were designated as auto-inducer-related regulators (AirR). Four sequences possessed LuxR analogous domains and were designated as auto-inducer analogous regulators (AiaR). When swarming motility was induced with DMHDA, eight airR genes and two aiaR genes were upregulated. These results indicate that the expression of multiple luxR-related genes is induced in actinobacteria, such as Arthrobacter sp. UMCV2, by the action of the bacterial biocompound DMHDA when QS behavior is produced.

Communication Breakdown: Into the Molecular Mechanism of Biofilm Inhibition by CeO2 Nanocrystal Enzyme Mimics and How It Can Be Exploited.

Bacterial biofilm formation is a huge problem in industry and medicine. Therefore, the discovery of anti-biofilm agents may hold great promise. Biofilm formation is usually a consequence of bacterial cell-cell communication, a process called quorum sensing (QS). CeO2 nanocrystals (NCs) have been established as haloperoxidase (HPO) mimics and ecologically beneficial biofilm inhibitors. They were suggested to interfere with QS, a mechanism termed quorum quenching (QQ), but their molecular mechanism remained elusive. We show that CeO2 NCs are effective QQ agents, inactivating QS signals by bromination. Catalytic bromination of 3-oxo-C12-AHL a QS signaling compound used by Pseudomonas aeruginosa, was detected in the presence of CeO2 NCs, bromide ions, and hydrogen peroxide. Brominated acyl-homoserine lactones (AHLs) no longer act as QS signals but were not detected in the bacterial cultures. Externally added brominated AHLs also disappeared in P. aeruginosa cultures within minutes of their addition, indicating that they are rapidly degraded by the bacteria. Moreover, we detected the catalytic bromination of 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO), a multifunctional non-AHL QS signal from P. aeruginosa with antibacterial and algicidal properties controlling the expression of many virulence genes. Brominated HQNO was not degraded by the bacteria in vivo. The repression of the Pseudomonas quinolone signal (PQS) production and biofilm formation in P. aeruginosa through the catalytic formation of Br-HQNO on surfaces with coatings containing CeO2 enzyme mimics validates the non-toxic strategy for the development of anti-infectives.

Dynamics of the protein search for targets on DNA in quorum-sensing cells

Quorum sensing is a bacterial cell-cell communication process that regulates gene expression. The search and binding of the autoinducer molecule (AHL)-bound LuxR-type proteins to specific sites on DNA in quorum-sensing cells in Gram-negative bacteria is a complex process and has been theoretically investigated based on a discrete-state stochastic approach. It is shown that several factors such as the rate of formation of the AHL-bound LuxR protein within the cells and its dissociation to freely diffusing AHL, the diffusion of the latter in and out of the cells, positive feedback loops, and the cell population density play important roles in the protein target search and can control the gene regulation processes. Physical-chemical arguments to explain these observations are presented. Our calculations of the dynamic properties are also supplemented by Monte Carlo computer simulations. Our theoretical model provides physical insights into the complex mechanisms of protein target search in quorum-sensing cells.

The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa.

ABSTRACTPseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals and individuals with underlying pulmonary disorders. P. aeruginosa virulence is controlled by quorum sensing (QS), a bacterial cell-cell communication mechanism that underpins transitions between individual and group behaviors. In P. aeruginosa, the PqsE enzyme and the QS receptor RhlR directly interact to control the expression of genes involved in virulence. Here, we show that three surface-exposed arginine residues on PqsE comprise the site required for interaction with RhlR. We show that a noninteracting PqsE variant [PqsE(NI)] possesses catalytic activity, but is incapable of promoting virulence phenotypes, indicating that interaction with RhlR, and not catalysis, drives these PqsE-dependent behaviors. Biochemical characterization of the PqsE-RhlR interaction coupled with RNA-seq analyses demonstrates that the PqsE-RhlR complex increases the affinity of RhlR for DNA, enabling enhanced expression of genes encoding key virulence factors. These findings provide the mechanism for PqsE-dependent regulation of RhlR and identify a unique regulatory feature of P. aeruginosa QS and its connection to virulence.IMPORTANCE Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI). QS is required for virulence in the human pathogen Pseudomonas aeruginosa, which can cause fatal infections in patients with underlying pulmonary disorders. In this study, we determine the molecular basis for the physical interaction between two virulence-driving QS components, PqsE and RhlR. We find that the ability of PqsE to bind RhlR correlates with virulence factor production. Since current antimicrobial therapies exacerbate the growing antibiotic resistance problem because they target bacterial growth, we suggest that the PqsE-RhlR interface discovered here represents a new candidate for targeting with small molecule inhibition. Therapeutics that disrupt the PqsE-RhlR interaction should suppress virulence. Targeting bacterial behaviors such as QS, rather than bacterial growth, represents an attractive alternative for exploration because such therapies could potentially minimize the development of resistance.

Transparent polycarbonate coated with CeO2 nanozymes repel Pseudomonas aeruginosa PA14 biofilms.

Highly transparent CeO2/polycarbonate surfaces were fabricated that prevent adhesion, proliferation, and the spread of bacteria. CeO2 nanoparticles with diameters of 10-15 nm and lengths of 100-200 nm for this application were prepared by oxidizing aqueous dispersions of Ce(OH)3 with H2O2 in the presence of nitrilotriacetic acid (NTA) as the capping agent. The surface-functionalized water-dispersible CeO2 nanorods showed high catalytic activity in the halogenation reactions, which makes them highly efficient functional mimics of haloperoxidases. These enzymes are used in nature to prevent the formation of biofilms through the halogenation of signaling compounds that interfere with bacterial cell-cell communication ("quorum sensing"). Bacteria-repellent CeO2/polycarbonate plates were prepared by dip-coating plasma-treated polycarbonate plates in aqueous CeO2 particle dispersions. The quasi-enzymatic activity of the CeO2 coating was demonstrated using phenol red enzyme assays. The monolayer coating of CeO2 nanorods (1.6 μg cm-2) and the bacteria repellent properties were demonstrated by atomic force microscopy, biofilm assays, and fluorescence measurements. The engineered polymer surfaces have the ability to repel biofilms as green antimicrobials on plastics, where H2O2 is present in humid environments such as automotive parts, greenhouses, or plastic containers for rainwater.

Analysis and application of bacterial cell-cell communication system

Analysis and application of bacterial cell-cell communication system

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria.

Biofilms of heterotrophic bacteria cover organic matter aggregates and constitute hotspots of mineralization, primarily acting through extracellular hydrolytic enzyme production. Nevertheless, regulation of both biofilm and hydrolytic enzyme synthesis remains poorly investigated, especially in estuarine ecosystems. In this study, various bioassays, mass spectrometry and genomics approaches were combined to test the possible involvement of quorum sensing (QS) in these mechanisms. QS is a bacterial cell-cell communication system that relies notably on the emission of N-acylhomoserine lactones (AHLs). In our estuarine bacterial collection, we found that 28 strains (9%), mainly Vibrio, Pseudomonas and Acinetobacter isolates, produced at least 14 different types of AHLs encoded by various luxI genes. We then inhibited the AHL QS circuits of those 28 strains using a broad-spectrum lactonase preparation and tested whether biofilm production as well as β-glucosidase and leucine-aminopeptidase activities were impacted. Interestingly, we recorded contrasted responses, as biofilm production, dissolved and cell-bound β-glucosidase and leucine-aminopeptidase activities significantly increased in 4%-68% of strains but decreased in 0%-21% of strains. These findings highlight the key role of AHL-based QS in estuarine bacterial physiology and ultimately on biogeochemical cycles. They also point out the complexity of QS regulations within natural microbial assemblages.

Genomic Evolution of the Marine Bacterium Phaeobacter inhibens during Biofilm Growth.

Phaeobacter inhibens 2.10 is an effective biofilm former on marine surfaces and has the ability to outcompete other microorganisms, possibly due to the production of the plasmid-encoded secondary metabolite tropodithietic acid (TDA). P. inhibens 2.10 biofilms produce phenotypic variants with reduced competitiveness compared to the wild type. In the present study, we used longitudinal, genome-wide deep sequencing to uncover the genetic foundation that contributes to the emergent phenotypic diversity in P. inhibens 2.10 biofilm dispersants. Our results show that phenotypic variation is not due to the loss of the plasmid that carries the genes for TDA synthesis but instead show that P. inhibens 2.10 biofilm populations become rapidly enriched in single nucleotide variations in genes involved in the synthesis of TDA. While variants in genes previously linked to other phenotypes, such as lipopolysaccharide production (i.e., rfbA) and cellular persistence (i.e., metG), also appear to be selected for during biofilm dispersal, the number and consistency of variations found for genes involved in TDA production suggest that this metabolite imposes a burden on P. inhibens 2.10 cells. Our results indicate a strong selection pressure for the loss of TDA in monospecies biofilm populations and provide insight into how competition (or a lack thereof) in biofilms might shape genome evolution in bacteria. IMPORTANCE Biofilm formation and dispersal are important survival strategies for environmental bacteria. During biofilm dispersal, cells often display stable and heritable variants from the parental biofilm. Phaeobacter inhibens is an effective colonizer of marine surfaces, in which a subpopulation of its biofilm dispersal cells displays a noncompetitive phenotype. This study aimed to elucidate the genetic basis of these phenotypic changes. Despite the progress made to date in characterizing the dispersal variants in P. inhibens, little is understood about the underlying genetic changes that result in the development of the specific variants. Here, P. inhibens phenotypic variation was linked to single nucleotide polymorphisms (SNPs), in particular in genes affecting the competitive ability of P. inhibens, including genes related to the production of the antibiotic tropodithietic acid (TDA) and bacterial cell-cell communication (e.g., quorum sensing). This work is significant as it reveals how the biofilm lifestyle might shape genome evolution in a cosmopolitan bacterium.

A novel anti-infective molecule nesfactin identified from sponge associated bacteria Nesterenkonia sp. MSA31 against multidrug resistant Pseudomonas aeruginosa

Overuse of antibiotics coupled with biofilm-forming ability has led to the emergence of multi-drug P. aeruginosa strains worldwide. Quorum sensing is a bacterial cell-cell communication system that regulates the expression of genes, including virulence factors, through production of acyl-homoserine lactones (AHLs) in Pseudomonas aeruginosa. The phenotypic expression of virulence factors in P. aeruginosa is mediated by quorum sensing systems (las and rhl). In this study an anti-infective molecule produced by a marine actinomycetes Nesterenkonia sp. MSA31 was elucidated as lipopeptide by NMR and LC-MS/MS analysis. The new lipopeptide molecule was named Nesfactin. This molecule effectively inhibited virulence phenotypes including production of hemolysin, protease, lipase, phospholipase, esterase, elastase, rhamnolipid, alginate, and pyocyanin, as well as motility and biofilm formation in P. aeruginosa. The high-performance thin layer chromatography (HPTLC) analysis revealed that the lipopeptide (50 μg/mL) inhibited production of the AHLs produced by the las and rhl quorum sensing systems (3-oxo-C12-HSL and C4-HSL, respectively). Docking analysis showed the binding affinity of the ligand towards the quorum sensing receptor molecules. The confocal laser scanning microscopy images showed the anti-biofilm effect of lipopeptide against P. aeruginosa. Nesfactin based hydrogel showed a significant antibiofilm effect on the catheter. This study suggests that the lipopeptide may be an effective anti-virulence treatment for Pseudomonas aeruginosa infections.

Quorum Sensing Inhibition Attenuates the Virulence of the Plant Pathogen Ralstonia solanacearum Species Complex.

Strains of Ralstonia solanacearum species complex (RSSC) cause "bacterial wilt" on a wide range of plant species and thus lead to marked economic losses in agriculture. Quorum sensing (QS), a bacterial cell-cell communication mechanism, controls the virulence of RSSC strains by regulating the production of extracellular polysaccharide (EPS) and secondary metabolites, biofilm formation, and cellular motility. R. solanacearum strain OE1-1 employs (R)-methyl 3-hydroxymyristate (3-OH MAME) as a QS signal, which is synthesized by the PhcB methyltransferase and sensed by the PhcS/PhcRQ two-component system. We describe the design, synthesis, and biological evaluation of inhibitors of the phc QS system. Initial screening of a small set of QS signal analogues revealed that methyl 3-hydroxy-8-phenyloctanoate, named, PQI-1 (phc quorum sensing inhibitor-1), inhibited biofilm formation by strain OE1-1. To improve its inhibitory activity, the derivatives of PQI-1 were synthesized, and their QS inhibition activities were evaluated. PQIs-2-5 evolved from PQI-1 more strongly inhibited not only biofilm formation but also the production of ralfuranone and EPS. Furthermore, RNA-Seq analysis revealed that the PQIs effectively inhibited QS-dependent gene expression and repression in strain OE1-1. On the other hand, the PQIs did not affect the canonical QS systems of the representative reporter bacteria. These antagonists, especially PQI-5, reduced wilting symptoms of the tomato plants infected with strain OE1-1. Taken together, we suggest that targeting the phc QS system has potential for the development of chemicals that protect agricultural crops from bacterial wilt disease.

The Roles of Microbial Cell-Cell Chemical Communication Systems in the Modulation of Antimicrobial Resistance.

Rapid emergence of antimicrobial resistance (AMR) has become a critical challenge worldwide. It is of great importance to understand how AMR is modulated genetically in order to explore new antimicrobial strategies. Recent studies have unveiled that microbial communication systems, which are known to play key roles in regulation of bacterial virulence, are also associated with the formation and regulation of AMR. These microbial cell-to-cell chemical communication systems, including quorum sensing (QS) and pathogen–host communication mechanisms, rely on detection and response of various chemical signal molecules, which are generated either by the microbe itself or host cells, to activate the expression of virulence and AMR genes. This article summarizes the generic signaling mechanisms of representative QS and pathogen–host communications systems, reviews the current knowledge regarding the roles of these chemical communication systems in regulation of AMR, and describes the strategies developed over the years for blocking bacterial chemical communication systems in disease control. The research progress in this field suggests that the bacterial cell-cell communication systems are a promising target not only for disease control but also for curbing the problem of microbial drug resistance.

Quorum quenching acylase impacts the viability and morphological change of Agrobacterium tumefaciens cells

Acylase is known as a quorum quenching enzyme that degrades N-acyl-homoserine lactones (AHLs), a key signaling molecule in a quorum sensing (QS) mechanism. Acylase I cleaves the acyl-chain in the chemical structures of AHLs, thereby exerting an anti-biofilm effect by the inhibition of bacterial cell-cell communication and resultant secretion of extracellular polymeric substances (EPS). However, the physical and physiological impacts of acylase on bacterial cells remain to be systematically elucidated. This study, therefore, investigated the effect of active and inactive acylase addition on the growth, viability, and cell morphologies of Agrobacterium tumefaciens. For comparison, active and inactive lysozymes were taken as positive controls. The results showed that active acylase inhibited A.tumefaciens cell growth at concentrations ranging from 0.1 to 1000μgmL-1, and so did active lysozyme. Fluorescent detection by Live/Dead staining underpinned that cell viability of A.tumefaciens decreased at concentrations higher than 0.1μgmL-1 for both acylase and lysozyme, although lysozyme inflicted higher degree of cellular damage. Moreover, atomic force microscopy unraveled a noticeable distortion of A.tumefaciens cells by both acylase and lysozyme. Together, the results showed that acylase not only blocked AHLs-based QS mechanisms but also compromised cell viability and altered surface morphology of A.tumefaciens cells, as observed by the addition of hydrolase.

Standard operating procedure (SOP) for disk diffusion-based quorum sensing inhibition assays

Introduction: The emergence of multidrug-resistant bacterial strains is a severe global health issue, which is worsened by the inability of new antibiotics. Virulence inhibition is one of the novel strategies that have been proposed to combat bacterial pathogens more effectively, without the risk of exerting selection pressure on these microorganisms. Inhibition of bacterial cell-cell communication (quorum sensing; QS) is a promising approach however, rapid and cost-effective screening for compounds with QS-inhibitory activity is not yet well-established. Aims: The aim of the present study is to determine the ideal experimental conditions for the disk-diffusion based QS-inhibitory assay with the most frequently used QS-signal molecule-producing and reporter strains. Methods: In our study, the effects of growth characteristics, incubation time, temperature and the used culture media were studied on the used bacterial strains and results of the disk-diffusion based QS-inhibitory assay. Results: Based on our results, the ideal experimental setting includes a modified Luria-Bertani medium (LB*; complemented with nutrients and microelements), incubation at room temperature (25 °C) for 48 hours before the reading of results, where the density of the starting inocula has less influence of the results of the assay. Conclusion: Establishing standard operating procedures (SOPs) is a way to help carry out various operations, aiming to increase precision and efficiency. Adherence to the experimental settings defined based on our results may aid in improving the reproducibility, comparability and reliability of results obtained by this method.