Insights from the Genome Sequence of Quorum-Quenching Staphylococcus sp. Strain AL1, Isolated from Traditional Chinese Soy Sauce Brine Fermentation

We report the draft genome sequence of Staphylococcus sp. strain AL1, which degrades quorum-sensing molecules (namely, N-acyl homoserine lactones). To the best of our knowledge, this is the first documentation that reports the whole genome sequence and quorum-quenching activity of Staphylococcus sp. strain AL1.     

Complete Genome Sequence of Burkholderia sp. Strain GG4, a Betaproteobacterium That Reduces 3-Oxo-N-Acylhomoserine Lactones and Produces Different N-Acylhomoserine Lactones

Burkholderia sp. strain GG4, isolated from the ginger rhizosphere, possesses a unique N-acylhomoserine lactone (AHL)-modifying activity that reduces 3-oxo-AHLs to 3-hydroxy-AHLs. To the best of our knowledge, this is the first sequenced genome from a bacterium of the genus Burkholderia that shows both quorum-sensing and signaling confusion activities.     

Draft Genome Sequence of Pantoea sp. Strain A4, a Rafflesia-Associated Bacterium That Produces N-Acylhomoserine Lactones as Quorum-Sensing Molecules

Pantoea sp. strain A4 is a Gram-negative bacterium isolated from the Rafflesia flower. We present here, for the first time, the genome sequence of Rafflesia-associated Pantoea sp. strain A4, which exhibited quorum-sensing activity.     

AiiA Quorum-Sensing Quenching Controls Proteolytic Activity and Biofilm Formation by Enterobacter cloacae

The aim of this work was to evaluate a quorum-quenching approach to identify functions regulated by quorum sensing in Enterobacter cloacae. We employed an aiiA transconjugant strain of E. cloacae that synthesizes a lactonase enzyme that hydrolyzes N-acyl homoserine lactone signaling molecules to compare bacterial phenotypes in the presence and absence of quorum signals. The aiiA-expressing strain displayed increased proteolytic activity and intensity of a milk-clotting reaction when compared to the wild-type strain. Although both strains growing on polystyrene plates in rich media and a minimal medium of salts formed biofilms, the wild-type strain exhibited a higher number of adhered cells. On the surface of stainless steel coupons that were submerged in culture media, the number of adhered cells of the wild type contained up to one log more cells compared with the aiiA transconjugant. However, after 48?h of incubation, there was no significant difference between the strains. The results demonstrated that the quorum-sensing system negatively regulates proteolytic activity and is likely involved in the early steps of biofilm formation by E. cloacae 067.     

Genome Sequence of Dyella japonica Strain A8, a Quorum-Quenching Bacterium That Degrades N-Acylhomoserine Lactones,Isolated from Malaysian Tropical Soil

Dyella japonica strain A8 is a Malaysian tropical soil bacterial strain which shows N-acylhomoserine lactone-degrading activity. Here, we present its draft genome sequence. A putative quorum-quenching gene was identified based on the genome sequence analysis of strain A8. To the best of our knowledge, this is the first genome announcement of a member from the genus of Dyella, and this is also the first work that reports the quorum-quenching activity of Dyella japonica.     

Utilization of Acyl-Homoserine Lactone Quorum Signals for Growth by a Soil Pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42°C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other γ-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.     

Fighting Acinetobacter baumannii infections with the acylase PvdQ

Acinetobacter baumannii is an opportunistic Gram-negative bacterial pathogen that poses a threat for frail patients worldwide. The high ability to withstand environmental stresses as well as its resistance towards a broad range of antibiotics make A. baumannii an effective hard-to-eradicate pathogen. One of the key mechanisms mediating tolerance against antibiotic treatment is the formation of biofilms, a process that is controlled by a multitude of different regulatory mechanisms. A key factor with major impact on biofilm formation is cell-to-cell communication by quorum-sensing, which in A. baumannii is mediated by acyl homoserine lactone signaling molecules. Here we show that the Ntn-Hydrolase PvdQ from Pseudomonas aeruginosa can reduce biofilm formation by the A. baumannii ATCC 17978 type strain and several clinical isolates on abiotic surfaces. Further, our study shows that a combination treatment of PvdQ-mediated quorum-quenching with the antibiotic gentamicin has a synergistic effect on the clearance of A. baumannii biofilms and possible biofilm dispersal. Moreover, we demonstrate in a Galleria mellonella larval infection model that PvdQ administration significantly prolongs survival of the larvae. Altogether, we conclude that the acylase-mediated irreversible cleavage of quorum-sensing signaling molecules as exemplified with PvdQ can set a profound limit to the progression of A. baumannii infections.     

Genome Sequence of Roseomonas sp. Strain B5, a Quorum-Quenching N-Acylhomoserine Lactone-Degrading Bacterium Isolated from Malaysian Tropical Soil

Roseomonas sp. strain B5 was isolated from Malaysian tropical soil that showed N-acylhomoserine lactone degradation. This is the first genome announcement of a member from the genus of Roseomonas and the first report on the quorum-quenching activity of Roseomonas spp.     

Quorum-quenching microbial infections: mechanisms and implications

The discovery of antibiotics early in the past century marked the beginning of active control and prevention of infectious microbial diseases. However, extensive use of antibiotics has also unavoidably resulted in the emergence of ‘superbugs’ that resist conventional antibiotics. The finding that many pathogens rely on cell-to-cell communication mechanisms, known as quorum sensing, to synchronize microbial activities essential for infection and survival in the host suggests a promising disease control strategy, i.e. quenching microbial quorum sensing or in short, quorum quenching. Work over the past few years has demonstrated that quorum-quenching mechanisms are widely conserved in many prokaryotic and eukaryotic organisms. These naturally occurring quorum-quenching mechanisms appear to play important roles in microbe–microbe and pathogen–host interactions and have been used, or served as lead compounds, in developing and formulating a new generation of antimicrobials. Characterization of the crystal structures of several types of quorum-quenching enzymes has provided valuable information to elucidate the catalytic mechanisms, as well as clues for future protein tailoring and molecular improvement. The discovery of quorum-sensing signal degradation enzymes in mammalian species represents a new milestone in quorum sensing and quorum quenching research. The finding highlights the importance of investigating their roles in host innate defence against infectious diseases and to determine the factors influencing their in vivo concentrations and catalytic activities.     

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k_cat/K_m of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.     

Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 10⁴ transformants per μg of DNA. We validated this protocol by tagging L. rhamnosus GG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression of gfp in L. rhamnosus GG upon induction with nisin. The GFP⁺ strain can be used to monitor the survival and behavior of L. rhamnosus GG in vivo. Moreover, implementation of the NICE system as a gene expression switch in L. rhamnosus GG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate that L. rhamnosus GG is sensitive to human beta-defensin-2 but not to human beta-defensin-1.     

Chemical shift assignments and secondary structure prediction of the phosphorelay protein VanU from Vibrio anguillarum

Vibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the ¹H, ¹³C, and ¹⁵N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum.     

Artificially Constructed Quorum-Sensing Circuits Are Used for Subtle Control of Bacterial Population Density

Vibrio fischeri is a typical quorum-sensing bacterium for which lux box, luxR, and luxI have been identified as the key elements involved in quorum sensing. To decode the quorum-sensing mechanism, an artificially constructed cell–cell communication system has been built. In brief, the system expresses several programmed cell-death BioBricks and quorum-sensing genes driven by the promoters lux pR and P_lacO-1 in Escherichia coli cells. Their transformation and expression was confirmed by gel electrophoresis and sequencing. To evaluate its performance, viable cell numbers at various time periods were investigated. Our results showed that bacteria expressing killer proteins corresponding to ribosome binding site efficiency of 0.07, 0.3, 0.6, or 1.0 successfully sensed each other in a population-dependent manner and communicated with each other to subtly control their population density. This was also validated using a proposed simple mathematical model.     

A Second Quorum-Sensing System Regulates Cell Surface Properties but Not Phenazine Antibiotic Production in Pseudomonas aureofaciens

The root-associated biological control bacterium Pseudomonas aureofaciens 30-84 produces a range of exoproducts, including protease and phenazines. Phenazine antibiotic biosynthesis by phzXYFABCD is regulated in part by the PhzR-PhzI quorum-sensing system. Mutants defective in phzR or phzI produce very low levels of phenazines but wild-type levels of exoprotease. In the present study, a second genomic region of strain 30-84 was identified that, when present in trans, increased β-galactosidase activity in a genomic phzB::lacZ reporter and partially restored phenazine production to a phzR mutant. Sequence analysis identified two adjacent genes, csaR and csaI, that encode members of the LuxR-LuxI family of regulatory proteins. No putative promoter region is present upstream of the csaI start codon and no lux box-like element was found in either the csaR promoter or the 30-bp intergenic region between csaR and csaI. Both the PhzR-PhzI and CsaR-CsaI systems are regulated by the GacS-GacA two-component regulatory system. In contrast to the multicopy effects of csaR and csaI in trans, a genomic csaR mutant (30-84R2) and a csaI mutant (30-84I2) did not exhibit altered phenazine production in vitro or in situ, indicating that the CsaR-CsaI system is not involved in phenazine regulation in strain 30-84. Both mutants also produced wild-type levels of protease. However, disruption of both csaI and phzI or both csaR and phzR eliminated both phenazine and protease production completely. Thus, the two quorum-sensing systems do not interact for phenazine regulation but do interact for protease regulation. Additionally, the CsaI N-acylhomoserine lactone (AHL) signal was not recognized by the phenazine AHL reporter 30-84I/Z but was recognized by the AHL reporters Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136(pCF240). Inactivation of csaR resulted in a smooth mucoid colony phenotype and formation of cell aggregates in broth, suggesting that CsaR is involved in regulating biosynthesis of cell surface components. Strain 30-84I/I2 exhibited mucoid colony and clumping phenotypes similar to those of 30-84R2. Both phenotypes were reversed by complementation with csaR-csaI or by the addition of the CsaI AHL signal. Both quorum-sensing systems play a role in colonization by strain 30-84. Whereas loss of PhzR resulted in a 6.6-fold decrease in colonization by strain 30-84 on wheat roots in natural soil, a phzR csaR double mutant resulted in a 47-fold decrease. These data suggest that gene(s) regulated by the CsaR-CsaI system also plays a role in the rhizosphere competence of P. aureofaciens 30-84.     

Quorum quenching and proactive host defense

Both plants and humans have inducible defense mechanisms. This passive defense strategy leaves the host unprotected for a period of time until resistance is activated. Moreover, many bacterial pathogens have evolved cell-cell communication (quorum-sensing) mechanisms to mount population-density-dependent attacks to overwhelm the host's defense responses. Several chemicals and enzymes have been investigated for years for their potential to target the key components of bacterial quorum-sensing systems. These quorum-quenching reagents, which block bacterial cell-cell communications, can disintegrate a bacterial population-density-dependent attack. It has now been shown that a quorum-quenching mechanism can be engineered in plants and might be used as a strategy in controlling bacterial pathogens and to build up a proactive defense barrier.     

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.     

Implications of Rewiring Bacterial Quorum Sensing

Bacteria employ quorum sensing, a form of cell-cell communication, to sense changes in population density and regulate gene expression accordingly. This work investigated the rewiring of one quorum-sensing module, the lux circuit from the marine bacterium Vibrio fischeri. Steady-state experiments demonstrate that rewiring the network architecture of this module can yield graded, threshold, and bistable gene expression as predicted by a mathematical model. The experiments also show that the native lux operon is most consistent with a threshold, as opposed to a bistable, response. Each of the rewired networks yielded functional population sensors at biologically relevant conditions, suggesting that this operon is particularly robust. These findings (i) permit prediction of the behaviors of quorum-sensing operons in bacterial pathogens and (ii) facilitate forward engineering of synthetic gene circuits.     

Glutathione S-transferase isozymes in Aedes aegypti: Purification,characterization, and isozyme-specific regulation

Glutathione S-transferase (GST) isozymes were purified from the GG strain of Aedes aegypti, a strain having ≥4-fold higher total GST activity compared to the wild-type lab strain. Purification involved S-hexyl-glutathione affinity chromatography in high salt buffer, and GST specific elution with S-(p-bromobenzyl)-glutathione. Final purification was accomplished on DEAE-Sepharose. Two isozymes, GST-1b and GST-2 were purified using this procedure, and an additional isozyme, GST-1a, was partially purified. The GST-2 isozyme has one of the highest specific activities reported for a GST, with a specific activity of 739 μmol/min/mg using 1-chloro-2,4-dinitrobenzene (CDNB), and 16.4 μmol/min/mg using 3,4-dichloronitrobenzene (DCNB) as substrates. GST-2, GST-1a, and GST-1b were analyzed for amino acid composition and subjected to N-terminal sequencing. All three GSTs showed amino acid differences, especially among the nonpolar and polar amino acids. The amino acid composition of GST-1b was found to be more similar to GST 1-1 from Drosophila melanogaster than to GST-2 or GST-1a from Aedes aegypti. Only GST-2 gave N-terminal sequence data, raising the possibility that GST-1a and 1b are N-terminally blocked. The A. aegypti GST-2 showed amino acid sequence identity or similarity in all but one residue between residue numbers 31 through 41 compared to the D. melanogaster and Musca domestica GST 1-1 isozymes. The pattern of GST isozyme expression was analyzed in various tissues and stages of development of the GG and wild type strains using isozyme-specific antisera and substrates. GST-1a was constitutively overexpressed in all tissues examined in the GG strain compared to the wild type strain. The expression of GST-1b was similar in both strains for all tissues and developmental stages examined. GST-2 was constitutively overexpressed in head, thorax and abdomen, but was not detected in ovaries of the GG strain. These results suggest that elevated GST activity in the GG strain is due to constitutive overexpression of GST-2 and GST-1a. GST-1a, GST-1b and GST-2 apparently are the products of 3 independently regulated genes and appear to be expressed in a tissue-specific manner.     

Quorum sensing activity of the plant growth-promoting rhizobacterium <Emphasis Type="Italic">Serratia glossinae</Emphasis> GS2 isolated from the sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) rhizosphere

Plant growth-promoting rhizobacteria (PGPR) affect plant growth through various mechanisms, such as indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and biofilm formation. The aim of the study reported here was to isolate and characterize rhizobacteria that produce quorum-sensing signal molecules and other PGPR-related molecules. A biofilm-forming bacterium, GS2, was isolated from the rhizosphere of a sesame plant and subsequently found to produce two quorum-sensing signal molecules that were identified as N-hexanoyl-L-homoserine lactone (m/z 200) and N-octanoyl-L-homoserine lactone (m/z 228) by liquid chromatography–tandem mass spectrometry analysis. The strain was also found to produce IAA (17.2 μg mL^?1), gibberellins (113.7 μg mL^?1), and ACC deaminase (9.7 μM α-ketobutyrate mg^?1 protein h^?1). The strain was identified as Serratia glossinae based on a comparison of 16S rRNA gene sequences. Inoculation of the strain promoted growth of a gibberellin-deficient rice dwarf mutant (Waito-C). Different growth attributes, including shoot and root elongation, chlorophyll content, and plant weight could be attributed to the PGPR characteristics of strain GS2. These results suggest that S. glossinae strain GS2 can serve as a microbial agent that improves plant growth.