AHL Signal Molecules Research Articles

YtnP: One novel quorum quenching enzyme from Bacillus amyloliquefaciens W11 inhibits biofilms and spoilage of white radish by Serratia marcescens

The spoilage of fruits and vegetables caused by microorganisms poses significant challenges to food safety and food industry. Serratia marcescens, a microorganism commonly found in the environment and food production facilities, exhibits strong spoilage activity on fruits and vegetables. In this study, one novel lactonase (YtnP) derived from Bacillus amyloliquefaciens W11 was successfully expressed and utilized to combat the spoilage of white radish caused by S. marcescens. The recombinant YtnP demonstrated broad-spectrum activity against various AHL signaling molecules. Notably, YtnP exhibited remarkable thermal stability, retaining over 60% enzyme activity after exposure to 75 °C for 1 h. Through mutagenesis of key amino acid residues, mutants H111A and H113A exhibited negligible enzyme activity against C6-HSL. H115A and H191A demonstrated reduced catalytic activity compared to the wild type. Conversely, mutant S154K exhibited enhanced catalytic activity against C6-HSL. Furthermore, infection models involving white radish and Galleria mellonella larvae confirmed that YtnP significantly reduced the toxicity of S. marcescens towards the host. Consequently, these findings highlight the potential of YtnP from B. amyloliquefaciens W11 as a food preservative against S. marcescens.

An Integrated Quorum Quenching Biocatalytic Nanoplatform for Synergistic Chemo-Photothermal Eradication of P. aeruginosa Biofilm Infections.

Decontamination of biofilm-associated infections presents a significant challenge due to the physical and chemical barrier created by the formation of extracellular matrices. This barrier restricts the access of antibiotics to the bacterial communities within the biofilm and provides protection to the persister cells, potentially leading to antibiotic resistance. In this study, we have developed an integrated quorum quenching biocatalytic nanoplatform for the synergistic chemo-photothermal eradication of P. aeruginosa biofilm infections. Ciprofloxacin (Cip), a model antibiotic, was absorbed onto PDA NPs through π-π stacking. Additionally, acylase (AC) was immobilized on PDA NPs through Schiff base reaction and Michael addition, resulting in the formation of the biocatalytic nanoplatform (PDA-Cip-AC NPs). This biocatalytic nanoplatform was able to enzymatically degrade AHL signaling molecules, thus achieving efficient quorum quenching activity to prevent biofilm formation. Furthermore, the NIR light-triggered on-demand Ciprofloxacin release further enhanced the eradication of P. aeruginosa biofilm infections with a synergy of local hyperthermia. We envision that this integrated quorum quenching nanoplatform provides a reliable tool for combating P. aeruginosa biofilm infections. STATEMENT OF SIGNIFICANCE: : An integrated quorum quenching biocatalytic nanoplatform has been developed for the eradication of P. aeruginosa biofilm infections. Quorum-sensing signals play a crucial role in modulating bacterial cell-to-cell communication, biofilm formation, and secretion of virulence factors. This biocatalytic nanoplatform efficiently degrades AHL signaling molecules, thereby blocking cell-to-cell communication and preventing biofilm formation. Additionally, local hyperthermia and on-demand Ciprofloxacin release were achieved through NIR irradiation, working synergistically to eradicate P. aeruginosa biofilm infections.

Unraveling the Diverse Profile of N-Acyl Homoserine Lactone Signals and Their Role in the Regulation of Biofilm Formation in Porphyra haitanensis-Associated Pseudoalteromonas galatheae.

N-acyl homoserine lactones (AHLs) are small, diffusible chemical signal molecules that serve as social interaction tools for bacteria, enabling them to synchronize their collective actions in a density-dependent manner through quorum sensing (QS). The QS activity from epiphytic bacteria of the red macroalgae Porphyra haitanensis, along with its involvement in biofilm formation and regulation, remains unexplored in prior scientific inquiries. Therefore, this study explores the AHL signal molecules produced by epiphytic bacteria. The bacterium isolated from the surface of P. haitanensis was identified as Pseudoalteromonas galatheae by 16s rRNA gene sequencing and screened for AHLs using two AHL reporter strains, Agrobacterium tumefaciens A136 and Chromobacterium violaceum CV026. The crystal violet assay was used for the biofilm-forming phenotype. The inferences revealed that P. galatheae produces four different types of AHL molecules, i.e., C4-HSL, C8-HSL, C18-HSL, and 3-oxo-C16-HSL, and it was observed that its biofilm formation phenotype is regulated by QS molecules. This is the first study providing insights into the QS activity, diverse AHL profile, and regulatory mechanisms that govern the biofilm formation phenotype of P. galatheae. These findings offer valuable insights for future investigations exploring the role of AHL producing epiphytes and biofilms in the life cycle of P. haitanensis.

Accurate Identification of Diverse N-acyl Homoserine Lactones in Marine Vibrio fluvialis by UHPLC-MS/MS.

Vibrio fluvialis is a marine opportunistic pathogen that frequently causes diseases in aquatic animals and humans. V. fluvialis can produce quorum sensing signaling molecules to coordinate cell density-dependent behavioral changes, including N-acyl homoserine lactone (AHL), which acts as a vital mediator of virulence-associated gene expression. Currently, several AHL molecules in V. fluvialis have been detected via biological and physicochemical methods, although different detection approaches have generated diverse AHL profiles. Here, we describe the AHL-producing bacterium, V. fluvialis BJ-1, which was isolated from marine sediments from the East China Sea. V. fluvialis BJ-1 could stimulate AHL-mediated β-galactosidase synthesis of the biosensor Agrobacterium tumefaciens NTL4 (pZLR4) but couldnot induce violacein production in the AHL reporter strain, Chromobacterium violaceum CV026. This bacterial isolate exhibited strong AHL-producing activity atlowcelldensity; however, the AHL activity declined when populationdensity remained at high levels. Analysis of the AHLs by Ultra-High-Performance Liquid Chromatography tandem Mass Spectrometry demonstrated that V. fluvialis BJ-1 produced five different AHL signaling molecules, including two linear chain AHL products (C8- and C10-HSL), and three β-carbon-oxidative AHL products (3-O-C8-, 3-O-C10- and 3-O-C12-HSL). Significantly, the present study is the first to accurately define the AHL profile of marine V. fluvialis. In future, the coupling of UHPLC to ESI-MS/MS is expected to be utilized for the accurate determination of AHL profiles in marine Vibrio.

A novel quorum quencher, Rhodococcus pyridinivorans XN-36, is a powerful agent for the biocontrol of soft rot disease in various host plants

Quorum sensing (QS) is a microbial cell–cell communication mechanism that regulates the virulence factor production in numerous pathogens, which plays a key role in microbial infections. QS-mediated microbial infections can be blocked via quorum quenching (QQ), which disrupts QS either by degradation of QS signals or interference of signal generation or perception. The pathogenicity of various diseases, including Pectobacterium carotovorum, is regulated by N-acyl homoserine lactone (AHL), a widely conserved QS signaling molecule. P. carotovorum can induce soft rot in a variety of crops and cause great economic losses to agricultural production. In this study, a newly isolated bacterium of Rhodococcus pyridinivorans XN-36 was identified as a novel QQ strain that can effectively degrade AHL and reduce the disease severity of P. carotovorum in host plants. Strain XN-36 was capable of rapidly degrading AHL without a lag phase, and over 85% of AHL (0.5 mmol·L−1) was degraded within 96 h. The metabolic intermediates of AHL biodegradation were detected and identified by gas chromatography-mass spectrometry (GC–MS) as lactamide and propenamide. Based on the chemical structure of AHL and intermediate metabolites, a novel metabolic pathway of AHL in strain XN-36 was proposed, which suggests that AHL could be degraded first by cleavage of its amide bond, followed by hydrolysis of the lactonic ring and subsequent metabolism. In addition, strain XN-36 participated in efficient degradation of a wide range of AHL signaling molecules including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), N-hexanoyl-L-homoserine lactone (C6HSL), N-(3-oxooctanoyl)-L-homoserine lactone (3OC8HSL), and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12HSL). Furthermore, the application of strain XN-36 as a biocontrol agent could substantially alleviate the soft rot disease caused by P. carotovorum to suppress tissue maceration in a variety of host plants, including Chinese cabbage (Brassica pekinensis(Lour.) Rupr.), carrot (Daucus carota L.) and potato (Solanum tuberosum L.). Similarly, the application of crude enzymes of strain XN-36 significantly attenuated the disease incidence and severity in host plants. Enzyme activity test indicated that strain XN-36 has acylase activity. These findings expand the application of QQ strains as biocontrol agents, while opening up new prospects for the control of QS-mediated bacterial diseases.

Roles of autoinducer-2 mediated quorum sensing in wastewater treatment

Abstract Quorum sensing (QS) is considered to be a promising regulation method for biological wastewater treatment (WWT) due to its regulation in extracellular substances (EPS) production, biofilm formation, granulation, colonization and bacterial activity and stability. Recently, autoinducer-2 (AI-2), a kind of interspecies communication QS signal molecule, is being increasingly reported for its roles in regulating bacterial gene expression and aggregation. Consequently, AI-2 mediated QS system is considered as a promising regulatory approach in WWT processes. This article systematically reviews the effects of AI-2-mediated QS system on bacterial behavior and its high potential for real-world applications in different WWT systems. Given the extensive presence of AI-2, AI-2 mediated QS could cooperate with other signal molecules in WWT processes, which suggests that the interactions among multiple signal molecules might be underestimated in the previous studies. The differences between AI-2 and AHL signaling molecules are also compared. Furthermore, the attempts at AI-2 regulated QS in pollution control of different WWT systems are summarized, while some challenges and defects still require targeted research in the future.

Detection of Quorum Sensing N-Acyl-Homoserine Lactone Molecules Produced by Different Resistant Klebsiella pneumoniae Isolates Recovered from Poultry and Different Environmental Niches.

This study aimed to detect and identify the N-acyl-homoserine lactones molecules (AHLs) produced by different resistant Klebsiella pneumoniae isolates recovered from poultry and environmental samples using a modified validated high-performance liquid chromatography method. A total of 56 K. pneumoniae isolates were recovered, investigated for their antibiotic susceptibility, and screened for AHLs production using the Agrobacterium tumefaciens NTL4 biosensor system and a validated high-performance liquid chromatography method. The results revealed the detection of different short- and long-chain AHLs molecules among 39 K. pneumoniae isolates recovered from poultry and environmental samples. All environmental isolates produced nine peaks with retention times for C4-HSL, C6-HSL, C12-HSL, C8-HSL, C14-HSL, C8-oxo-HSL, C10-HSL, C6-oxo-HSL, and C7-HSL. The most quantifiable AHL signal molecules in poultry isolates were C4-HSL, C6-HSL, and C12-HSL. No statistical correlation between the AHL-producing ability of K. pneumoniae isolates and antibiotic resistance was reported. To the best of our knowledge, this study provides the first detailed report on the detection and identification of AHLs in K. pneumoniae isolates recovered from poultry and environmental samples. Furthermore, it provides a new insight available tool other than LC-MS/MS for detection and identification of AHL molecules.

Functional characterization of quorum sensing LuxR-type transcriptional regulator, EasR in Enterobacter asburiae strain L1.

Over the past decades, Enterobacter spp. have been identified as challenging and important pathogens. The emergence of multidrug-resistant Enterobacteria especially those that produce Klebsiella pneumoniae carbapenemase has been a very worrying health crisis. Although efforts have been made to unravel the complex mechanisms that contribute to the pathogenicity of different Enterobacter spp., there is very little information associated with AHL-type QS mechanism in Enterobacter spp. Signaling via N-acyl homoserine lactone (AHL) is the most common quorum sensing (QS) mechanism utilized by Proteobacteria. A typical AHL-based QS system involves two key players: a luxI gene homolog to synthesize AHLs and a luxR gene homolog, an AHL-dependent transcriptional regulator. These signaling molecules enable inter-species and intra-species interaction in response to external stimuli according to population density. In our recent study, we reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. Whole genome sequencing and in silico analysis revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easI/R, in strain L1. In a QS system, a LuxR transcriptional protein detects and responds to the concentration of a specific AHL controlling gene expression. In E. asburiae strain L1, EasR protein binds to its cognate AHLs, N-butanoyl homoserine lactone (C4-HSL) and N–hexanoyl homoserine lactone (C6-HSL), modulating the expression of targeted genes. In this current work, we have cloned the 693 bp luxR homolog of strain L1 for further characterization. The functionality and specificity of EasR protein in response to different AHL signaling molecules to activate gene transcription were tested and validated with β-galactosidase assays. Higher β-galactosidase activities were detected for cells harboring EasR, indicating EasR is a functional transcriptional regulator. This is the first report documenting the cloning and characterization of transcriptional regulator, luxR homolog of E. asburiae.

Flexibility and Adaptability of Quorum Sensing in Nature.

Quorum sensing (QS), a type of chemical communication, allows bacteria to sense and coordinate activities in natural biofilm communities using N-acyl homoserine lactones (AHLs) as one type of signaling molecule. For AHL-based communication to occur, bacteria must produce and recognize the same signals, which activate similar genes in different species. Our current understanding of AHL-QS suggests that signaling between species would arise randomly, which is not probable. We propose that AHL-QS signaling is a mutable and adaptable process, within limits. AHLs are highly-conserved signals, however, their corresponding receptor proteins (LuxR) are highly variable. We suggest that both flexibility and adaptation occur among receptor proteins, allowing for complex signaling networks to develop in biofilms over time.

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

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

Heterologous Expression of the Marine-Derived Quorum Quenching Enzyme MomL Can Expand the Antibacterial Spectrum of Bacillus brevis.

Quorum sensing (QS) is closely associated with the production of multiple virulence factors in bacterial pathogens. N-acyl homoserine lactones (AHLs) are important QS signal molecules that modulate the virulence of gram-negative pathogenic bacteria. Enzymatic degradation of AHLs to interrupt QS, termed quorum quenching (QQ), has been considered a novel strategy for reduction of pathogenicity and prevention of bacterial disease. However, the low expression levels of QQ proteins in the original host bacteria has affected the applications of these proteins. Previously, we identified a novel marine QQ enzyme, named MomL, with high activity and promising biocontrol function. In this study, we linked the target fragment momL to pNCMO2, which provided a basis for the first heterologous expression of MomL in the antifungal and anti-gram-positive-bacteria biocontrol strain Bacillus brevis, and obtaining the recombinant strain named BbMomL. The QQ activity of BbMomL was confirmed using a series of bioassays. BbMomL could not only degrade the exogenous signal molecule C6-HSL, but also the AHL signal molecules produced by the gram-negative pathogens Pectobacterium carotovorum subsp. carotovorum (Pcc) and Pseudomonas aeruginosa PAO1. In addition, BbMomL significantly reduced the secretion of pathogenic factors and the pathogenicity of Pcc and P. aeruginosa PAO1. We tested the biocontrol function of BbMomL for prevention of plant diseases in vitro. The result indicates that BbMomL has a broad antibacterial spectrum. Compared with wild-type B. brevis, BbMomL not only inhibited fungi and gram-positive bacterial pathogens but also considerably inhibited gram-negative bacterial pathogens. Moreover, the Bacillus brevis expression system has good application prospects and is an ideal host for expression and secretion of foreign proteins.

High intracellular c-di-GMP levels antagonize quorum sensing and virulence gene expression in Burkholderia cenocepacia H111.

The opportunistic human pathogen Burkholderia cenocepacia H111 uses two chemically distinct signal molecules for controlling gene expression in a cell density-dependent manner: N-acyl-homoserine lactones (AHLs) and cis-2-dodecenoic acid (BDSF). Binding of BDSF to its cognate receptor RpfR lowers the intracellular c-di-GMP level, which in turn leads to differential expression of target genes. In this study we analysed the transcriptional profile of B. cenocepacia H111 upon artificially altering the cellular c-di-GMP level. One hundred and eleven genes were shown to be differentially expressed, 96 of which were downregulated at a high c-di-GMP concentration. Our analysis revealed that the BDSF, AHL and c-di-GMP regulons overlap for the regulation of 24 genes and that a high c-di-GMP level suppresses expression of AHL-regulated genes. Phenotypic analyses confirmed changes in the expression of virulence factors, the production of AHL signal molecules and the biosynthesis of different biofilm matrix components upon altered c-di-GMP levels. We also demonstrate that the intracellular c-di-GMP level determines the virulence of B. cenocepacia to Caenorhabditis elegans and Galleria mellonella.

Isolation of Marine Paracoccus sp. Ss63 from the Sponge Sarcotragus sp. and Characterization of its Quorum‐Sensing Chemical‐Signaling Molecules by LC‐MS/MS Analysis

AbstractGram‐negative bacteria often use N‐acyl homoserine lactones (AHLs) as quorum‐sensing (QS) signal molecules. The present study aimed to identify and characterize the production of AHLs by Paracoccus sp. Ss63, which was isolated from the Mediterranean sponge Sarcotragus sp. High performance liquid chromatography – high resolution tandem mass spectrometry (LC‐HRMS/MS) provided evidence for the production, by Paracoccus sp. Ss63, of twelve saturated long‐chain AHLs (3‐OH‐C10‐HSL, 3‐OH‐C12‐HSL, C10‐HSL, 3‐OH‐C14‐HSL, C12‐HSL, 3‐O‐C16‐HSL, 3‐OH‐C16‐HSL, C14‐HSL, 3‐OH‐C18‐HSL, C16‐HSL, 3‐O‐C18‐HSL, and C18‐HSL), along with four putative unsaturated long‐chain AHLs (C10 : 1‐HSL, 3‐OH‐C18 : 1‐HSL, C16 : 1‐HSL, and C18 : 1‐HSL). The distribution of Paracoccus sp. Ss63 in different sponge species, as well as in seawater and marine sediment samples, suggests a mixed lifestyle for this bacterium, i.e., free‐living and host‐associated. The high pH of seawater is likely to inactivate the AHL signal molecules, and limit the possibility of AHLs to accumulate to the concentration required for QS‐based gene regulation. We hypothesize that Paracoccus sp. Ss63 utilizes quorum sensing to sense and respond to the different environments it experiences, with an active QS only when found inside enclosed niches (host‐associated and at lower pH).

Interference with the germination and growth ofUlvazoospores by quorum‐sensing molecules fromUlva‐associated epiphytic bacteria

Ulva zoospores preferentially settle on N-acylhomoserine lactone (AHL) producing marine bacterial biofilms. To investigate whether AHL signal molecules also affect the success and rate of zoospore germination in addition to zoospore attraction, the epiphytic bacteria associated with mature Ulva linza were characterized and bacterial isolates representative of this community tested for the ability to produce AHLs. Two of these AHL-producing isolates, Sulfitobacter spp. 376 and Shewanella spp. 79, were transformed with plasmids expressing the Bacillus spp. AHL lactonase gene aiiA to generate AHL-deficient variants. The germination and growth of U. linza zoospores was studied in the presence of these AHL-deficient strains and their AHL-producing counterparts. This revealed that the AHLs produced by Sulfitobacter spp. and Shewanella spp. or the bacterial products they regulate have a negative impact on both zoospore germination and the early growth of the Ulva germling. Further experiments with Escherichia coli biofilms expressing recombinant AHL synthases and synthetic AHLs provide data to demonstrate that zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. These results reveal an additional role for AHLs per se in the interactive relationships between marine bacteria and Ulva zoospores.

Thematic Issue: Protists

Ulva zoospores preferentially settle on N-acylhomoserine lactone (AHL) producing marine bacterial biofilms. To investigate whether AHL signal molecules also affect the success and rate of zoospore germination in addition to zoospore attraction, the epiphytic bacteria associated with mature Ulva linza were characterized and bacterial isolates representative of this community tested for the ability to produce AHLs. Two of these AHL-producing isolates, Sulfitobacter spp. 376 and Shewanella spp. 79, were transformed with plasmids expressing the Bacillus spp. AHL lactonase gene aiiA to generate AHL-deficient variants. The germination and growth of U. linza zoospores was studied in the presence of these AHL-deficient strains and their AHL-producing counterparts. This revealed that the AHLs produced by Sulfitobacter spp. and Shewanella spp. or the bacterial products they regulate have a negative impact on both zoospore germination and the early growth of the Ulva germling. Further experiments with Escherichia coli biofilms expressing recombinant AHL synthases and synthetic AHLs provide data to demonstrate that zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. These results reveal an additional role for AHLs per se in the interactive relationships between marine bacteria and Ulva zoospores.

Cis-2-dodecenoic acid quorum sensing system modulates N-acyl homoserine lactone production through RpfR and cyclic di-GMP turnover in Burkholderia cenocepacia

BackgroundBurkholderia cenocepacia employs both N-Acyl homoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) quorum sensing (QS) systems in regulation of bacterial virulence. It was shown recently that disruption of BDSF synthase RpfFBc caused a reduction of AHL signal production in B. cenocepacia. However, how BDSF system influences AHL system is still not clear.ResultsWe show here that BDSF system controls AHL system through a novel signaling mechanism. Null mutation of either the BDSF synthase, RpfFBc, or the BDSF receptor, RpfR, caused a substantial down-regulation of AHL signal production in B. cenocepacia strain H111. Genetic and biochemical analyses showed that BDSF system controls AHL signal production through the transcriptional regulation of the AHL synthase gene cepI by modulating the intracellular level of second messenger cyclic di-GMP (c-di-GMP). Furthermore, we show that BDSF and AHL systems have a cumulative role in the regulation of various biological functions, including swarming motility, biofilm formation and virulence factor production, and exogenous addition of either BDSF or AHL signal molecules could only partially rescue the changed phenotypes of the double deletion mutant defective in BDSF and AHL signal production.ConclusionsThese results, together with our previous findings, thus depict a molecular mechanism with which BDSF regulates AHL signal production and bacterial virulence through modulating the phosphodiesterase activity of its receptor RpfR to influence the intracellular level of c-di-GMP.

Investigation of N-acyl homoserine lactone (AHL) molecule production in Gram-negative bacteria isolated from cooling tower water and biofilm samples

In this study, 99 Gram-negative rod bacteria were isolated from cooling tower water, and biofilm samples were examined for cell-to-cell signaling systems, N-acyl homoserine lactone (AHL) signal molecule types, and biofilm formation capacity. Four of 39 (10 %) strains isolated from water samples and 14 of 60 (23 %) strains isolated from biofilm samples were found to be producing a variety of AHL signal molecules. It was determined that the AHL signal molecule production ability and the biofilm formation capacity of sessile bacteria is higher than planktonic bacteria, and there was a statistically significant difference between the AHL signal molecule production of these two groups (p < 0.05). In addition, it was found that bacteria belonging to the same species isolated from cooling tower water and biofilm samples produced different types of AHL signal molecules and that there were different types of AHL signal molecules in an AHL extract of bacteria. In the present study, it was observed that different isolates of the same strains did not produce the same AHLs or did not produce AHL molecules, and bacteria known as AHL producers did not produce AHL. These findings suggest that detection of signal molecules in bacteria isolated from cooling towers may contribute to prevention of biofilm formation, elimination of communication among bacteria in water systems, and blockage of quorum-sensing controlled virulence of these bacteria.

AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans

Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.

A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies.

Isolation and identification of quorum quenching bacteria from environmental samples

A large number of Gram-negative pathogens produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell–cell communication system allows them to coordinate gene expression and regulate virulence. Therefore, strategies to inhibit QS are promising for the control of infectious diseases. The aim of the present study was to develop a high-throughput method for the isolation and identification of AHL-degrading bacteria from environmental samples. Samples were cultured in a microtitre plate in a minimal medium containing 1mM N-(3-oxo-dodecanoyl)-l-homoserine lactone and 2mM N-(3-oxo-hexanoyl)-l-homoserine lactone as the sole sources of carbon and nitrogen. Isolates growing on this minimal medium were subcultured and identified by partial 16S rRNA gene sequencing. Subsequently, the AHL-degrading capacity of each isolate was evaluated in the Pseudomonas aeruginosa QSIS2 biosensor assay, as such or after treatment with heat or proteinase K. The 16 samples tested yielded a total of 59 isolates which are, either alone or as part of a consortium, able to use AHL signal molecules as sole sources of carbon and nitrogen. Follow-up experiments have shown that in each sample there is at least one isolate with quorum quenching (QQ) activity, and that for all samples combined, 41 isolates have QQ activity. Furthermore, heat treatment did not fully inhibit QQ activity in all isolates. In some isolates, QQ activity was lost after proteinase K treatment, while others remained able to quench QS. Therefore, it is likely that some isolates produce and secrete (a) heat-stable, low molecular weight inhibitory compound(s).