Role of N-acyl homoserine lactone (AHL)-based quorum sensing (QS) in aerobic sludge granulation

N-acyl homoserine lactone (AHL)-based quorum sensing (QS) has been recognized to play an important role in the formation of biofilm. However, aerobic granular sludge is considered as a special biofilm, and its biological implication and role of AHL-based QS still remain unclear. This study investigated the role of AHL-based QS in aerobic granulation. Results showed that AHLs were necessary to the typical aerobic granulation, and AHL-associated coordination of bacteria in sludge aggregation was sludge density dependent only when it reached a threshold of 1.010 g/mL; AHL-based QS was activated to regulate aerobic granulation. Furthermore, a quorum quenching method was firstly adopted to investigate the role of AHLs in aerobic granules. Results showed inhibition of AHL by acylase that reduced the AHL content in aerobic granules and further weakened its attachment potential, which proved that AHLs play an important role in the formation of aerobic granules. Additionally, the assay of quorum quenching not only proved that AHL-based QS could regulate EPS production but also provided additional evidence for the role of AHLs in aerobic granulation by regulating EPS content and its component proportion.     

酰基高丝氨酸内酯(AHLs)介导群感效应在好氧颗粒污泥中的研究进展

虽然好氧颗粒污泥(Aerobic Granular Sludge,AGS)具有沉降性能好、高效脱氮除磷以及抗冲击负荷等优点,但是该技术仍然存在颗粒化进程缓慢及容易解体等技术瓶颈.因此,如何克服上述瓶颈是实现好氧颗粒污泥技术在实际污水处理推广的关键.近年来,酰基高丝氨酸内酯(Acyl Homoserine Lactone...     

Quorum quenching activity in Anabaena sp. PCC 7120: identification of AiiC, a novel AHL-acylase

Many bacteria use quorum sensing (QS) to coordinate responses to environmental changes. In Gram-negative bacteria, the most extensively studied QS systems rely on the use of N -acylhomoserine lactones (AHLs) signal molecules. Some bacteria produce enzymes that are able to inactivate AHL signals produced by other bacteria and hence interfere with QS-mediated processes via a phenomenon known as quorum quenching. Acylase-type AHL degradation activity has been found in the biomass of the filamentous nitrogen-fixing cyanobacterium Anabaena ( Nostoc ) sp. PCC 7120, being absent from the culture media. The gene all3924 has been identified and cloned whose product exhibits homology to the acylase QuiP of Pseudomonas aeruginosa PAO1, demonstrating that it is at least partially responsible for the AHL-acylase activity. The recombinant enzyme, which was named auto-inducer inhibitor from Cyanobacteria (AiiC), shows broad acyl-chain length specificity. Because the presence of AHLs in the biomass of nitrogen-fixing cultures of Anabaena sp. PCC 7120 has been described recently, AiiC could represent a self-modulatory system to control the response to its own QS signals but could also be involved in the interference of signalling within complex microbial communities in which Cyanobacteria are present.     

Quorum Sensing Signal Production and Microbial Interactions in a Polymicrobial Disease of Corals and the Coral Surface Mucopolysaccharide Layer

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.     

Microbial metabolism of quorum-sensing molecules acyl-homoserine lactones, γ-heptalactone and other lactones

The cell-to-cell communication of microorganisms is known to be via exertion of certain chemical compounds (signal molecules) and is referred to as quorum sensing (QS). QS phenomenon is widespread in microbial communities. Several Gram-positive and Gram-negative bacteria and fungi use lactone-containing compounds (e.g. acyl-homoserine lactones (AHLs), γ-heptalactone, butyrolactone-I) as signalling molecules. The ability of microorganisms to metabolise these compounds and the mechanisms they employ for this purpose are not clearly understood. Many studies, however, have focused on identifying AHL and other lactone-degrading enzymes produced by bacteria and fungi. Various strains that are able to utilise these signalling molecules as carbon and energy sources have also been isolated. In addition, several reports have provided evidence on the involvement of lactones and lactone-degrading enzymes in numerous biological functions. These studies, although focused on processes other than metabolism of lactone signalling molecules, still provide insights into further understanding of the mechanisms employed by various microorganisms to metabolise the QS compounds. In this review, we consider conceivable microbial strategies to metabolise AHL and other lactone-containing signalling molecules such as γ-heptalactones.     

Profiling acylated homoserine lactones in Yersinia ruckeri and influence of exogenous acyl homoserine lactones and known quorum-sensing inhibitors on protease production

AIMS: To profile the quorum-sensing (QS) signals in Yersinia ruckeri and to examine the possible regulatory link between QS signals and a typical QS-regulated virulence phenotype, a protease. METHODS AND RESULTS: Liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) showed that Y. ruckeri produced at least eight different acylated homoserine lactones (AHLs) with N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL) being the dominant molecule. Also, some uncommon AHL, N-(3-oxoheptanoyl)-L-homoserine lactone (3-oxo-C7-HSL) and N-(3-oxononanoyl)-L-homoserine lactone (3-oxo-C9-HSL), were produced. 3-oxo-C8-HSL was detected in organs from fish infected with Y. ruckeri. Protease production was significantly lower at temperatures above 23 degrees C than below although growth was faster at the higher temperatures. Neither addition of sterile filtered high-density Y. ruckeri culture supernatant nor the addition of pure exogenous AHLs induced protease production. Furthermore, three QS inhibitors (QSIs), sulfur-containing AHL analogues, did not inhibit protease production in Y. ruckeri. CONCLUSIONS: Exogenous AHL or sulfur-containing AHL analogues did not influence the protease production indicating that protease production may not be QS regulated in Y. ruckeri. SIGNIFICANCE AND IMPACT OF THE STUDY: The array of different AHLs produced indicates that the QS system of Y. ruckeri is complex and could involve several regulatory systems. In this case, neither AHLs nor QSI would be likely to directly affect a QS-regulated phenotype.     

Transgenic tomato plants alter quorum sensing in plant growth-promoting rhizobacteria

Two Gram-negative, plant growth-promoting rhizobacteria (PGPRs), denominated as M12 and M14, were classified by 16S rDNA sequencing as Burkholderia graminis species. Both strains were shown to produce a variety of N-acyl-homoserine lactone (AHL) quorum sensing (QS) signalling molecules. The involvement of these molecules in plant growth promotion and the induction of protection against salt stress was examined. AHL production was evaluated in vitro by thin-layer chromatography using AHL biosensors, and the identity of the AHLs produced was determined by liquid chromatography-tandem mass spectrometry. The in situ production of AHLs by M12 and M14 in the rhizosphere of Arabidopsis thaliana plants was detected by co-inoculation with green fluorescent protein-based biosensor strains and confocal laser scanning microscopy. To determine whether plant growth promotion and protection against salt stress were mediated by QS, these PGPRs were assayed on wild-type tomato plants, as well as their corresponding transgenics expressing YenI (short-chain AHL producers) and LasI (long-chain AHL producers). In wild-type tomato plants, only M12 promoted plant growth, and this effect disappeared in both transgenic lines. In contrast, M14 did not promote growth in wild-type tomatoes, but did so in the LasI transgenic line. Resistance to salt stress was induced by M14 in wild-type tomato, but this effect disappeared in both transgenic lines. The strain M12, however, did not induce salt resistance in wild-type tomato, but did so in LasI tomato plants. These results reveal that AHL QS signalling molecules mediate the ability of both PGPR strains M12 and M14 to promote plant growth and to induce protection against salt stress.     

Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors

Many Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum-sensing (QS) signal molecules. AHL QS has been the subject of extensive investigation in the last decade and has become a paradigm for bacterial intercellular signaling. Research in AHL QS has been considerably aided by simple methods devised to detect AHLs using bacterial biosensors that phenotypically respond when exposed to exogenous AHLs. This article reviews and discusses the currently available bacterial biosensors which can be used in detecting and studying the different AHLs.     

The quorum sensing molecule N-acyl homoserine lactone produced by Acinetobacter baumannii displays antibacterial and anticancer properties

Secretory N-acyl homoserine lactones (AHLs) mediate quorum sensing (QS) in bacteria. AHLs are shown to be inhibitory for an unrelated group of bacteria and might mimic host signalling elements, thereby subverting the regulatory events in host cells. This study investigated the AHL produced by Acinetobacter baumannii and analysed its effect on other bacterial species and mammalian cells. Chemically characterized AHL had an m/z value of 325 with a molecular formula C₁₈H₃₁NO₄ and showed its inhibitory potential against Staphylococcus aureus. Molecular docking studies identified D-alanine-D-alanine synthetase A, a cell wall synthesizing enzyme of S. aureus having a strong binding affinity towards AHL. Electron microscopy showed the disruption and sloughing off of the S. aureus cell wall when treated with AHL. In vitro experiments revealed that this bacteriostatic AHL showed time-dependent activity and induced apoptosis in cancer cell lines. This compound could be a potential structural backbone for constructing new AHL analogues against S. aureus. The findings emphasize the need to re-evaluate all previously characterized AHLs for any additional new biological functions other than QS.     

N-acyl-l-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge

The role of intercellular signalling in the regulation of genes and phenotypes in a broad range of bacterial species is now firmly established. In contrast, the impact of intercellular signalling on microbial community parameters, such as species diversity and function, is less well understood. In this study the role of N-acyl-l-homoserine lactones (AHLs) in microbial community dynamics in an industrial wastewater treatment system is addressed. Seven proteobacterial strains producing compounds with AHL-like activity were isolated from the treatment plant. Three of these belong to genera with no previously identified AHL producing species. Addition of AHLs at 2 micro M to sludge samples generated changes in both community function (phenol degradation) and composition as determined by length heterogeneity PCR and denaturing gradient gel electrophoresis. Phenol degradation was more stable as a result of the AHL augmentation. A dominant functional member of the Thauera genus was transiently supplanted by a member of the Comomonas genus in response to AHL addition. This suggests that AHLs can play a role in mediating microbial community parameters and has implications for ecosystem function and industrial wastewater treatment.     

Targeting N-acyl-homoserine-lactones to mitigate membrane biofouling based on quorum sensing using a biofouling reducer

Exploring novel biological anti-quorum sensing (QS) agents to control membrane biofouling is of great worth in order to allow sustainable performance of membrane bioreactors (MBRs) for wastewater treatment. In recent studies, QS inhibitors have provided evidence of alternative route to control membrane biofouling. This study investigated the role of Piper betle extract (PBE) as an anti-QS agent to mitigate membrane biofouling. Results demonstrated the occurrence of the N-acyl-homoserine-lactone (AHL) autoinducers (AIs), correlate QS activity and membrane biofouling mitigation. The AIs production in bioreactor was confirmed using an indicator strain Agrobacterium tumefaciens (NTL4) harboring plasmid pZLR4. Moreover, three different AHLs were found in biocake using thin layer chromatographic analysis. An increase in extracellular polymeric substances (EPS) and transmembrane pressure (TMP) was observed with AHL activity of the biocake during continuous MBR operation, which shows that membrane biofouling was in close relationship with QS activity. PBE was verified to mitigate membrane biofouling via inhibiting AIs production. SEM analysis further confirmed the effect of PBE on EPS and biofilm formation. These results exhibited that PBE could be a novel agent to target AIs for mitigation of membrane biofouling. Further work can be carried out to purify the active compound of Piper betle extract to target the QS to mitigate membrane biofouling.     

Multiplicity of Quorum Quenching Enzymes: A Potential Mechanism to Limit Quorum Sensing Bacterial Population

Bacteria express certain of their characteristics especially, pathogenicity factors at high cell densities. The process is termed as quorum sensing (QS). QS operates via signal molecules such as acylhomoserine lactones (AHLs). Other bacteria inhibit QS through the inactivation of AHL signals by producing enzymes like AHL-lactonases and -acylases. Comparative genomic analysis has revealed the multiplicity of genes for AHL lactonases (up to 12 copies per genome) among Bacillus spp. and that of AHL-acylases (up to 5 copies per genome) among Pseudomonas spp. This genetic evolution can be envisaged to enable host to withstand the attacks from bacterial population, which regulates its functioning through QS.     

The presence, type and role of N-acyl homoserine lactone quorum sensing in fluorescent Pseudomonas originally isolated from rice rhizospheres are unpredictable

In Gram-negative bacteria, a typical quorum-sensing (QS) system involves the production and response to N-acyl homoserine lactones (AHLs). It still remains unclear as to how pivotal and conserved AHL QS is in root-colonizing rhizosphere Pseudomonas. We, therefore, performed a systematic study of AHL QS on a set of 50 rice rhizosphere Pseudomonas isolates. We also isolated the AHL QS genes in two representative strains and analyzed the role of AHL QS regulation of various phenotypes. Our results are discussed with the current knowledge of AHL QS of rhizosphere Pseudomonas, implicating a lack of conservation and an unpredictable role played by AHL QS in this group of bacteria.     

Design, synthesis, and biological evaluation of abiotic, non-lactone modulators of LuxR-type quorum sensing

Quorum sensing (QS) is a cell-cell signaling mechanism that allows bacteria to monitor their population size and alter their behavior at high cell densities. Gram-negative bacteria use N-acylated L-homoserine lactones (AHLs) as their primary signals for QS. These signals are susceptible to lactone hydrolysis in biologically relevant media, and the ring-opened products are inactive QS signals. We have previously identified a range of non-native AHLs capable of strongly agonizing and antagonizing QS in Gram-negative bacteria. However, these abiotic AHLs are also prone to hydrolysis and inactivation and thereby have a relatively short time window for use (～12-48 h). Non-native QS modulators with reduced or no hydrolytic instability could have enhanced potencies and would be valuable as tools to study the mechanisms of QS in a range of environments (for example, on eukaryotic hosts). This study reports the design and synthesis of two libraries of new, non-hydrolyzable AHL mimics. The libraries were screened for QS modulatory activity using LasR, LuxR, and TraR bacterial reporter strains, and several new, abiotic agonists and antagonists of these receptors were identified.     

Two dissimilar N-acyl-homoserine lactone acylases of Pseudomonas syringae influence colony and biofilm morphology

Plant aerial surfaces comprise a complex habitat for microorganisms, and many plant-associated bacteria, such as the pathogen Pseudomonas syringae, exhibit density-dependent survival on leaves by utilizing quorum sensing (QS). QS is often mediated by diffusible signals called N-acyl-homoserine lactones (AHLs), and P. syringae utilizes N-3-oxo-hexanoyl-dl-homoserine lactone (3OC6HSL) to control traits influencing epiphytic fitness and virulence. The P. syringae pathovar syringae B728a genome sequence revealed two putative AHL acylases, termed HacA (Psyr_1971) and HacB (Psyr_4858), which are N-terminal nucleophile hydrolases that inactivate AHLs by cleaving their amide bonds. HacA is a secreted AHL acylase that degrades only long-chain (C > or = 8) AHLs, while HacB is not secreted and degrades all tested AHLs. Targeted disruptions of hacA, hacB, and hacA and hacB together do not alter endogenous 3OC6HSL levels under the tested conditions. Surprisingly, targeted disruptions of hacA alone and hacA and hacB together confer complementable phenotypes that are very similar to autoaggregative phenotypes seen in other species. While AHL acylases might enable P. syringae B728a to degrade signals of competing species and block expression of their QS-dependent traits, these enzymes also play fundamental roles in biofilm formation.     

Identification of N-acylhomoserine lactones in mucopurulent respiratory secretions from cystic fibrosis patients

Pseudomonas aeruginosa and species of the Burkholderia cepacia complex are the primary bacterial pathogens contributing to lung disease in patients with cystic fibrosis. Quorum sensing systems using N-acyl homoserine lactone (AHL) signal molecules are involved in the regulation of a number of virulence factors in these species. Extracts of mucopurulent respiratory secretions from 13 cystic fibrosis patients infected with P. aeruginosa and/or strains of the B. cepacia complex were fractionated using reverse-phase fast pressure liquid chromatography and analyzed for the presence of AHLs using a traI-luxCDABE-based reporter that responds to AHLs with acyl chains ranging between 4 and 12 carbons. Using this assay system, a broad range of AHLs were detected and identified despite being present at low concentrations in limited sample volumes. N-(3-oxo-dodecanoyl)-l-homoserine lactone, N-(3-oxo-decanoyl)-l-homoserine lactone and N-octanoyl-l-homoserine lactone (OHL) were the AHLs most frequently identified. OHL and N-decanoyl-l-homoserine lactone were detected in nanomolar concentrations compared to picomolar amounts of the 3-oxo-derivatives of the AHLs identified.