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Virulence assay on potato tubers. Potato tuber slices inoculated with (a) Pectobacterium carotovorum without plasmid, (b) P. carotovorum (pME6010), (c) P. carotovorum expressing the hqiA gene and (d) cell-free LB medium.
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The screening of a metagenomic library of 250,000 clones generated from a hypersaline soil (Spain) allowed us to identify a single positive clone which confers the ability to degrade N-Acyl homoserine lactones (AHLs). The sequencing of the fosmid revealed a 42,318 bp environmental insert characterized by 46 ORFs. The subcloning of these ORFs demons...
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Context 1
... ability of hqiA on P. carotovorum was then tested using the potato tuber assay. This bioassay highlighted that P. carotovorum car- rying pME6010::hqiA was not able to cause soft rot symptoms (0% maceration on the surface of the potato slice), whilst the same bacterial strain carrying or not the empty plasmid caused a large maceration zone (39%) (Fig. ...
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Necrotizing enterocolitis (NEC) is a devastating intestinal disease that occurs primarily in premature infants. We performed genome-resolved metagenomic analysis of 1,163 fecal samples from premature infants to identify microbial features predictive of NEC. Features considered include genes, bacterial strain types, eukaryotes, bacteriophages, plasm...
Citations
... This reaction is reversible naturally under acid pH, although it requires a relatively stringent condition, that the open ring product needs 24 h to resume the cyclic structure under a pH less than 2 (Yates et al., 2002). In contrast, the reactions catalyzed by acylase and oxidoreductase are irreversible (Torres et al., 2017). The Escherichia coli or Pectobacterium transformants expressing QQ enzymes either significantly inhibited soft rot development or had much lower virulence in ex vivo plant tissues, such as potato, cucumber and carrot slices, and cabbage leaves (Garge and Nerurkar, 2016). ...
Quorum sensing (QS) is a conserved cell–cell communication mechanism widely distributed in bacteria, and is oftentimes tightly correlated with pathogen virulence. Quorum quenching enzymes, which interfere with QS through degrading the QS signaling molecules, could attenuate virulence instead of killing the pathogens, and thus are less likely to induce drug resistance. Many Gram-negative bacteria produce N-acyl homoserine lactones (AHLs) for interspecies communication. In this study, we isolated and identified a bacterial strain, Mesoflavibacter zeaxanthinifaciens XY-85, from an Onchidium sp. collected from the intertidal zone of Dapeng Reserve in Shenzhen, China, and found it had strong AHL degradative activity. Whole genome sequencing and blast analysis revealed that XY-85 harbors an AHL lactonase (designated MzmL), which is predicted to have an N-terminal signal peptide and share the “HXHXDH” motif with known AHL lactonases belonging to the Metallo-β-lactamase superfamily. Phylogenetic studies showed MzmL was closest to marine lactonase cluster members, MomL and Aii20J, instead of the AiiA type lactonases. Ultra performance liquid chromatography-mass spectrometry analysis confirmed that MzmL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MzmL could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, and retained full bioactivity under a wide range of temperatures (28–100°C) and pHs (4–11). Furthermore, MzmL significantly reduced Pectobacterium carotovorum subsp. carotovorum virulence factor production in vitro, such as biofilm formation and plant cell wall degrading enzyme production, and inhibited soft rot development on potato slices. These results demonstrated that MzmL may be a novel type of AHL lactonase with good environmental stability, and has great potential to be developed into a novel biological control agent for bacterial disease management.
... Co-culture assays of strain AA1EC1 and the phytopathogens were carried out according to previous studies (Torres et al., 2017). Briefly, 24 h cultures of each pathogen (10 9 CFU mL −1 ) were co-cultured with strain AA1EC1 (10 9 CFU mL −1 ) at a ratio of 1:100 in LB medium and incubated for 24 h at 28°C and at 120 rpm in a rotary shaker. ...
... The ability of strain AA1EC1 to interfere with soft rot caused by D. solani IPO 2222 T and P. atrosepticum CECT 314 T was tested on potato slices while the infection produced by P. carotovorum subsp. carotovorum CECT 225 T was tested on potato and carrot slices (Garge & Nerurkar, 2016;Torres et al., 2017). Briefly, surface-sterilized, and sliced potato tuber (Solanum tuberosum) and carrots (Daucus carota) were inoculated with AA1EC1-phytopathogen co-cultures and incubated at 28°C for 48 h. ...
The use of fertilizers and pesticides to control plant diseases is widespread in intensive farming causing adverse effects together with the development of antimicrobial resistance pathogens. As the virulence of many Gram‐negative phytopathogens is controlled by N‐acyl‐homoserine lactones (AHLs), the enzymatic disruption of this type of quorum‐sensing (QS) signal molecules, mechanism known as quorum quenching (QQ), has been proposed as a promising alternative antivirulence therapy. In this study, a novel strain of Bacillus toyonensis isolated from the halophyte plant Arthrocaulon sp. exhibited numerous traits associated with plant growth promotion (PGP) and degraded a broad range of AHLs. Three lactonases and an acylase enzymes were identified in the bacterial genome and verified in vitro. The AHL‐degrading activity of strain AA1EC1 significantly attenuated the virulence of relevant phytopathogens causing reduction of soft rot symptoms on potato and carrots. In vivo assays showed that strain AA1EC1 significantly increased plant length, stem width, root and aerial dry weights and total weight of tomato and protected plants against Pseudomonas syringae pv. tomato. To our knowledge, this is the first report to demonstrate PGP and QQ activities in the species B. toyonensis that make this strain as a promising phytostimulant and biocontrol agent.
... In an rsaL mutant, 3-oxo-C12-HSL concentrations continue to increase substantially with growth rather than reaching a plateau. Alternatively, AHL homoeostasis could be achieved via the regulated expression or activity of AHL-degrading enzymes such as lactonase, acylases or oxido-reductases whose expression may be under the control of NagC [43], although to our knowledge none have yet been identified in Yersinia spp. It is also noteworthy that in contrast to most LuxR/I-based QS systems, for Y. pseudotuberculosis and Yersinia enterocolitica, the addition of exogenous AHLs to AHL synthase mutants does not restore QS phenotypes despite the AHLs being confirmed (through reporter assays and by using tritiated AHLs) as entering the cells [22,31]. ...
Biofilm formation by Yersinia pseudotuberculosis is regulated by quorum sensing (QS) and dependent on the haemin storage locus hms , required for the extracellular polysaccharide poly- N -acetylglucosamine (poly-GlcNAc) production. In Escherichia coli NagC regulates both GlcNAc biosynthesis and metabolism with GlcNAc acting as a signal that co-ordinates these and other activities. However, the contribution of NagC and GlcNAc to biofilm development in Y. pseudotuberculosis is not known. Here we show that a Y. pseudotuberculosis nagC mutant is impaired for biofilm production on abiotic (glass) and biotic ( Caenorhabitis elegans ) surfaces. Genetic complementation restored poly-GlcNAc production and biofilm formation on C. elegans . Using lux -based promoter fusions, hmsHFRS expression was found to be nagC dependent. Given that NagC and QS both regulate aggregation and biofilm formation, we investigated their regulatory relationship using lux -based promoter fusions. These revealed that (i) nagC is negatively autoregulated, but expression can be partially restored in the nagC mutant by exogenous GlcNAc, (ii) NagC negatively regulates the ytbI and ypsI QS genes and (iii) nagC expression is reduced in the ytbI , ypsI and ypsR mutants but not the ytbR mutant. These data establish the existence of a reciprocal regulatory relationship between NagC and QS, which in the case of the luxRI pair ytbRI , is also GlcNAc-dependent. NagC and GlcNAc are therefore components of a regulatory system involving QS that modulates biofilm formation and aggregation.
... Pcc uses a cell-to-cell communication system called quorum sensing (QS) to regulate many virulence genes and pathogenesis from the initial infection process (Bellieny-Rabelo et al., 2020;Mole et al., 2007). It has been known that the production of PCWDEs in Pcc is tightly modulated by QS system LuxI/LuxR (ExpI/ExpR in the species) through a signal molecule 3-oxo-C 6 -HSL and the secretion is mainly through the type II secretion system (Abbott and Boraston, 2008;Bell et al., 2004;Bellieny-Rabelo et al., 2020;Lee et al., 2013;Mole et al., 2007;Torres et al., 2017). In addition to PCWDEs, other virulence determinants and factors contributing to bacterial invasion, colonization, proliferation, and host resistance evasion in plants have been characterized. ...
... To evaluate the role of MetC in the virulence activity of Pcc21, we performed the pathogenicity test using Chinese cabbage (Lee et al., 2013) and potato tuber (Torres et al., 2017) as described above. To evaluate the bacterial multiplication on Chinese cabbages, the lesion was cut into 1 × 1 cm 2 size and resuspended in 10 ml of M9 basal medium at designated time points. ...
Plant pathogenic Pectobacterium species cause severe soft rot/blackleg diseases in many economically important crops worldwide. Pectobacterium utilizes plant cell wall degrading enzymes (PCWDEs) as the main virulence determinants for its pathogenicity. In this study, we screened a random mutant, M29 is a transposon insertion mutation in the metC gene encoding cystathionine β-lyase that catalyzes cystathionine to homocysteine at the penultimate step in methionine biosynthesis. M29 became a methionine auxotroph and resulted in growth defects in methionine-limited conditions. Impaired growth was restored with exogenous methionine or homocysteine rather than cystathionine. The mutant exhibited reduced soft rot symptoms in Chinese cabbages and potato tubers, maintaining activities of PCWDEs and swimming motility. The mutant was unable to proliferate in both Chinese cabbages and potato tubers. The reduced virulence was partially restored by a complemented strain or 100 µM of methionine, whereas it was fully restored by the extremely high concentration (1 mM). Our transcriptomic analysis showed that genes involved in methionine biosynthesis or transporter were downregulated in the mutant. Our results demonstrate that MetC is important for methionine biosynthesis and transporter and influences its virulence through Pcc21 multiplication in plant hosts.
... Unfortunately, the results of studies in which compounds are extracted and identified from bacterial cultures are limited by the lack of information on uncultured microbial species, which in some environments, constitute more than 99% of the organisms. It is anticipated that the number of new QS and QQ systems identified by metagenomic methods will exceed the number identified from single cultures of microorganisms [142][143][144][145]. The usefulness of metagenomic studies is confirmed by many examples in the literature; for example, interesting metagenomic studies on the microbial community, and the distribution of QS and QQ genes in particles of organic material collected from the Yellow Sea. ...
... One example is the thermostable esterase Est816 from the Tuban basin [148]. On the other hand, the construction of a metagenomic library consisting of several clones from hypersaline soils allowed the identification of the AHL-degrading enzyme with a new family related to the cysteine hydrolase (CHase) group [145]. The analysis of a metagenomic library from pasture soils allowed the identification of a new metallohydrolase with NAHL lactonase activity [149]. ...
Quorum sensing (QS) is a system of communication of bacterial cells by means of chemical signals called autoinducers, which modulate the behavior of entire populations of Gram-negative and Gram-positive bacteria. Three classes of signaling molecules have been recognized, Al-1, Al-2, Al-3, whose functions are slightly different. However, the phenomenon of quorum sensing is not only concerned with the interactions between bacteria, but the whole spectrum of interspecies interactions. A growing number of research results confirm the important role of QS molecules in the growth stimulation and defense responses in plants. Although many of the details concerning the signaling metabolites of the rhizosphere microflora and plant host are still unknown, Al-1 compounds should be considered as important components of bacterial–plant interactions, leading to the stimulation of plant growth and the biological control of phytopathogens. The use of class 1 autoinducers in plants to induce beneficial activity may be a practical solution to improve plant productivity under field conditions. In addition, researchers are also interested in tools that offer the possibility of regulating the activity of autoinducers by means of degrading enzymes or specific inhibitors (QSI). Current knowledge of QS and QSI provides an excellent foundation for the application of research to biopreparations in agriculture, containing a consortia of AHL-producing bacteria and QS inhibitors and limiting the growth of phytopathogenic organisms.
... Unfortunately, the results of studies in which compounds are extracted and identified from bacterial cultures are limited by the lack of information on uncultured microbial species, which in some environments, constitute more than 99% of the organisms. It is anticipated that the number of new QS and QQ systems identified by metagenomic methods will exceed the number identified from single cultures of microorganisms [142][143][144][145]. The usefulness of metagenomic studies is confirmed by many examples in the literature; for example, interesting metagenomic studies on the microbial community, and the distribution of QS and QQ genes in particles of organic material collected from the Yellow Sea. ...
... One example is the thermostable esterase Est816 from the Tuban basin [148]. On the other hand, the construction of a metagenomic library consisting of several clones from hypersaline soils allowed the identification of the AHL-degrading enzyme with a new family related to the cysteine hydrolase (CHase) group [145]. The analysis of a metagenomic library from pasture soils allowed the identification of a new metallohydrolase with NAHL lactonase activity [149]. ...
Quorum sensing (QS) is a system of communication of bacterial cells by means of chemical signals called autoinducers, which modulate the behavior of entire populations of Gram-negative and Gram-positive bacteria. Three classes of signaling molecules have been recognized, Al-1, Al-2, Al-3, whose functions are slightly different. However, the phenomenon of quorum sensing is not only concerned with the interactions between bacteria, but the whole spectrum of interspecies interactions. A growing number of research results confirm the important role of QS molecules in the growth stimulation and defense responses in plants. Although many of the details concerning the signaling metabolites of the rhizosphere microflora and plant host are still unknown, Al-1 compounds should be considered as important components of bacterial–plant interactions, leading to the stimulation of plant growth and the biological control of phytopathogens. The use of class 1 autoinducers in plants to induce beneficial activity may be a practical solution to improve plant productivity under field conditions. In addition, researchers are also interested in tools that offer the possibility of regulating the activity of autoinducers by means of degrading enzymes or specific inhibitors (QSI). Current knowledge of QS and QSI provides an excellent foundation for the application of research to biopreparations in agriculture, containing a consortia of AHL-producing bacteria and QS inhibitors and limiting the growth of phytopathogenic organisms.
... According to the different mechanisms of action on AHLs, QQ enzymes are conventionally divided into lactonases, acylases, and oxidoreductases [12,13]. Among them, AHL lactonases (AHLases) have received increasing attention due to their widespread presence [14][15][16][17][18]. A large group of AHLases with a broad substrate spectrum Graphical Abstract and excellent catalytic performance have been isolated and characterized. ...
Background
The disease caused by plant pathogenic bacteria in the production, transportation, and storage of many crops has brought huge losses to agricultural production. N -acylhomoserine lactonases (AHLases) can quench quorum-sensing (QS) by hydrolyzing acylhomoserine lactones (AHLs), which makes them the promising candidates for controlling infections of QS-dependent pathogenic bacteria. Although many AHLases have been isolated and considered as a potentially effective preventive and therapeutic agents for bacterial diseases, the intrinsically poor ambient stability has seriously restricted its application.
Results
Herein, we showed that a spheroid enzyme-based hybrid nanoflower (EHNF), AhlX@Ni 3 (PO 4 ) 2 , can be easily synthesized, and it exhibited 10 times AHL (3OC8-HSL) degradation activity than that with free AhlX (a thermostable AHL lactonase). In addition, it showed intriguing stability even at the working concentration, and retained ~ 100% activity after incubation at room temperature (25 °C) for 40 days and approximately 80% activity after incubation at 60 °C for 48 h. Furthermore, it exhibited better organic solvent tolerance and long-term stability in a complicated ecological environment than that of AhlX. To reduce the cost and streamline production processes, CSA@Ni 3 (PO 4 ) 2 , which was assembled from the crude supernatants of AhlX and Ni 3 (PO 4 ) 2 , was synthesized. Both AhlX@Ni 3 (PO 4 ) 2 and CSA@Ni 3 (PO 4 ) 2 efficiently attenuated pathogenic bacterial infection.
Conclusions
In this study, we have developed N -acylhomoserine lactonase-based hybrid nanoflowers as a novel and efficient biocontrol reagent with significant control effect, outstanding environmental adaptability and tolerance. It was expected to overcome the bottlenecks of poor stability and limited environmental tolerance that have existed for over two decades and pioneered the practical application of EHNFs in the field of biological control.
Graphical Abstract
... In particular, C. violaceum CV026 was used to screen a metagenomic library of 250,000 clones from a hypersaline soil located in Spain with a pool strategy of 50 clones per well, which resulted in the identification of a single lactonase, called HqiA which had no homology with any known lactonase or acylase. HqiA shared homology with enzymes from the cysteine hydrolase (CSHase) group as isochorismatase-like and N-carbamoylsarcosine amidaselike enzymes but more studies are required to unravel its function and mode of action (92). ...
Quorum sensing (QS) is a communication system used by most bacteria to adapt their behavior according to population density and synchronize different actions such as virulence and biofilm formation. QS inhibition, also called quorum quenching (QQ), is a promising strategy to control bacterial infections in humans, animals or plants. Among these strategies, the use of enzymes able to interfere with the molecular signals of bacteria is particularly interesting. The thermostable enzyme SsoPox is able to degrade certain communication molecules of Gram-negative bacteria: acyl-homoserine lactones (AHL). An enzymatic engineering strategy was conducted to increase the efficiency of this lactonase to degrade a certain type of lactones: short-chain AHL, used by a large number of pathogenic bacteria. Nine screening methods were first optimized to allow the identification of potentially highly active variants. Eight mutagenesis libraries targeting residues of loop 8, known to modulate the lactonase activity of the enzyme, were then screened leading to the isolation of variants improved up to 297-fold on the targeted AHL compared to the wild type enzyme. Resolution of the three-dimensional structure of the best performing variants allowed the identification of the molecular determinants responsible for this improved activity. The best variant was then studied in vitro to assess its ability to block the QS of Serratia sp. 39006, a model bacterium using short-chain AHL. A multiple approach combining phenotypic, proteomic and metabolomic analyses allowed to demonstrate the ability of the enzyme to disrupt many behaviors of the strain such as its ability to form biofilm, to produce antibiotics or to form the gas vesicles involved in its flotation. Finally, the activity spectrum of SsoPox and several variants was studied in depth and allowed the identification of new substrates such as peptides or β-lactams. Strong modulations of promiscuous activities caused by enzyme mutations were then revealed, highlighting their potential role in the acquisition of new enzymatic functions.
... The marine environment is considered an excellent source of novel enzymes or compounds that interfere with QS systems [6,[29][30][31][32][33]. Indeed, AHL-degrading activity has been described in many marine bacteria belonging to different genera, such as Alteromomas, Bacillus, Paracoccus, Pseudoalteromonas, Psychrobacter, Roseovarius, Ruegeria, Stenotrophomonas, Tenacibaculum, and Thalassomonas [6,30,[34][35][36], as well as to marine metagenomic collections [37][38][39]. ...
Interference with quorum-sensing (QS) intercellular communication systems by the enzymatic disruption of N-acylhomoserine lactones (AHLs) in Gram-negative bacteria has become a promising strategy to fight bacterial infections. In this study, seven strains previously isolated from marine invertebrates and selected for their ability to degrade C6 and C10-HSL, were identified as Acinetobacter junii, Ruegeria atlantica, Microbulbifer echini, Reinheimera aquimaris, and Pseudomonas sihuiensis. AHL-degrading activity against a wide range of synthetic AHLs were identified by using an agar well diffusion assay and Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 and VIR07 as biosensors. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis indicated that this activity was not due to an AHL lactonase. All the strains degraded Vibrio coralliilyticus AHLs in coculture experiments, while some strains reduced or abolished the production of virulence factors. In vivo assays showed that strains M3-111 and M3-127 reduced this pathogen’s virulence and increased the survival rate of Artemia salina up to 3-fold, indicating its potential use for biotechnological purposes. To our knowledge, this is the first study to describe AHL-degrading activities in some of these marine species. These findings highlight that the microbiota associated with marine invertebrates constitute an important underexplored source of biological valuable compounds.
... However, the lack of amplification of the aiiA gene in some strains can be derived from the polymorphisms of the aiiA gene or the presence of novel QQ enzymes not related to AiiA. Indeed, a novel enzyme QQ HqiA has been identified from a metagenomic library of a hypersaline environment (Torres et al., 2017). In addition, a novel lactonase enzyme YtnP was isolated from a strain of B. licheniformis, not related to AiiA (Peng et al., 2021). ...
Aims:
The current study aimed to screen Bacillus strains with wide-spectrum quorum quenching (QQ) activity against N-acyl-l-homoserine lactones (AHLs), helpful in controlling virulence traits in Gram-negatives, including biofilm formation and also with anti-biofilm activity against Gram-positives.
Methods and results:
94 halotolerant strains of Bacillus isolated from soil and salt-lake sediment samples in Algeria were examined for the presence of QQ activity against AHLs, the presence of the aiiA gene, encoding an AHL lactonase enzyme typical of Bacillus spp., antimicrobial, and anti-biofilm activities against Pseudomonas aeruginosa and Streptococcus mutans. Of all strains of Bacillus spp. isolated, 48.9% showed antibacterial activity. In addition, 40% of these isolates showed a positive QQ activity against long-chain AHLs, of which seven strains presented the aiiA gene. Among the species with broad-spectrum QQ activity, the cell extract of Bacillus thuringiensis DZ16 showed antibiofilm activity against P. aeruginosa PAO1, reducing 60% using the Amsterdam active attachment (AAA) biofilm cultivation model. In addition, the cell extract of Bacillus subtilis DZ17, also presenting a broad-spectrum QQ activity, significantly reduced S. mutans ATCC 25175 biofilm formations by 63% and 53% in the xCELLigence and the AAA model, respectively, without affecting growth. Strain DZ17 is of particular interest due to its explicit halophilic nature since it can thrive at salinities in the range of 6%-30%.
Conclusions:
B. thuringiensis DZ16 and B. subtilis DZ17 strains have interesting antibacterial, QQ, and anti-biofilm activities. The high range of salinities accepted by these strains increases their biotechnological potential. This may open up their use as probiotics, the treatment and prevention of conventional and emerging infectious diseases.
Significance and impact of study:
The use of safe, economical, and effective probiotics is limited to control the infections related to multi-resistant bacteria. In our study, we provide two promising agents with QQ, anti-biofilm and antibacterial activities.
