As N-acyl Homoserine Lactones Research Articles

Impaired microbial N-acyl homoserine lactone signalling increases plant resistance to aphids across variable abiotic and biotic environments.

Beneficial bacteria interact with plants using signalling molecules, such as N-acyl homoserine-lactones (AHLs). Although there is evidence that these molecules affect plant responses to pathogens, few studies have examined their effect on plant-insect and microbiome interactions, especially under variable soil conditions. We investigated the effect of the AHL-producing rhizobacterium Acidovorax radicis and its AHL-negative mutant (does not produce AHLs) on modulating barley (Hordeum vulgare) plant interactions with cereal aphids (Sitobion avenae) and earthworms (Dendrobaena veneta) across variable nutrient soils. Acidovorax radicis inoculation increased plant growth and suppressed aphids, with stronger effects by the AHL-negative mutant. However, effects varied between barley cultivars and the presence of earthworms altered interaction outcomes. Bacteria-induced plant defences differed between cultivars, and aphid exposure, with pathogenesis-related and WRKY pathways partly explaining the ecological effects in the more resistant cultivars. Additionally, we observed few but specific indirect effects via the wider root microbiome where the AHL-mutant strain influenced rare OTU abundances. We conclude that bacterial AHL-signalling disruption affects plant-microbial interactions by inducing different plant pathways, leading to increased insect resistance, also mediated by the surrounding biotic and abiotic environment. Understanding the mechanisms by which beneficial bacteria can reduce insect pests is a key research area for developing effective insect pest management strategies in sustainable agriculture.

N-3-Oxo-Octanoyl Homoserine Lactone Primes Plant Resistance Against Necrotrophic Pathogen Pectobacterium carotovorum by Coordinating Jasmonic Acid and Auxin-Signaling Pathways.

Many Gram-negative bacteria use small signal molecules, such as N-acyl-homoserine lactones (AHLs), to communicate with each other and coordinate their collective behaviors. Recently, increasing evidence has demonstrated that long-chained quorum-sensing signals play roles in priming defense responses in plants. Our previous work indicated that a short-chained signal, N-3-oxo-octanoyl homoserine lactone (3OC8-HSL), enhanced Arabidopsis resistance to the hemi-biotrophic bacteria Pseudomonas syringae pv. tomato DC3000 through priming the salicylic acid (SA) pathway. Here, we found that 3OC8-HSL could also prime resistance to the necrotrophic bacterium Pectobacterium carotovorum ssp. carotovorum (Pcc) through the jasmonic acid (JA) pathway, and is dependent on auxin responses, in both Chinese cabbage and Arabidopsis. The subsequent Pcc invasion triggered JA accumulation and increased the down-stream genes’ expressions of JA synthesis genes (LOX, AOS, and AOC) and JA response genes (PDF1.2 and VSP2). The primed state was not observed in the Arabidopsis coi1-1 and jar1-1 mutants, which indicated that the primed resistance to Pcc was dependent on the JA pathway. The 3OC8-HSL was not transmitted from roots to leaves and it induced indoleacetic acid (IAA) accumulation and the DR5 and SAUR auxin-responsive genes’ expressions in seedlings. When Arabidopsis and Chinese cabbage roots were pretreated with exogenous IAA (10 μM), the plants had activated the JA pathway and enhanced resistance to Pcc, which implied that the JA pathway was involved in AHL priming by coordinating with the auxin pathway. Our findings provide a new strategy for the prevention and control of soft rot in Chinese cabbage and provide theoretical support for the use of the quorum-sensing AHL signal molecule as a new elicitor.

Importance of N-Acyl-Homoserine Lactone-Based Quorum Sensing and Quorum Quenching in Pathogen Control and Plant Growth Promotion.

The biological control of plant pathogens is linked to the composition and activity of the plant microbiome. Plant-associated microbiomes co-evolved with land plants, leading to plant holobionts with plant-beneficial microbes but also with plant pathogens. A diverse range of plant-beneficial microbes assists plants to reach their optimal development and growth under both abiotic and biotic stress conditions. Communication within the plant holobiont plays an important role, and besides plant hormonal interactions, quorum-sensing signalling of plant-associated microbes plays a central role. Quorum-sensing (QS) autoinducers, such as N-acyl-homoserine lactones (AHL) of Gram-negative bacteria, cause a pronounced interkingdom signalling effect on plants, provoking priming processes of pathogen defence and insect pest control. However, plant pathogenic bacteria also use QS signalling to optimise their virulence; these QS activities can be controlled by quorum quenching (QQ) and quorum-sensing inhibition (QSI) approaches by accompanying microbes and also by plants. Plant growth-promoting bacteria (PGPB) have also been shown to demonstrate QQ activity. In addition, some PGPB only harbour genes for AHL receptors, so-called luxR-solo genes, which can contribute to plant growth promotion and biological control. The presence of autoinducer solo receptors may reflect ongoing microevolution processes in microbe–plant interactions. Different aspects of QS systems in bacteria–plant interactions of plant-beneficial and pathogenic bacteria will be discussed, and practical applications of bacteria with AHL-producing or -quenching activity; QS signal molecules stimulating pathogen control and plant growth promotion will also be presented.

Quorum Sensing Inhibitory Potential and Molecular Docking Studies of Phyllanthus emblica Phytochemicals Against Pseudomonas aeruginosa.

Phyllanthus emblica is a traditional medicinal plant that is endowed with curative properties including anti-bacterial, anti-fungal, anti-viral, and analgesic properties. Bacteria make use of cell-cell signaling system known as quorum sensing (QS) and respond to their own population. In most gram-negative bacteria, the transcriptional regulators belonging to the Lux R protein play a crucial role in the QS mechanism by detecting the presence of signaling molecules known as N-acyl homoserine lactones (AHLs). In this present work, the anti-quorum sensing activity of Phyllanthus emblica was evaluated against Pseudomonas aeruginosa. Anti-quorum sensing efficacy of Phyllanthus emblica was estimated with reference to QS bio-monitoring strain Chromobacterium violaceum. The binding efficacy of the phytochemicals of Phyllanthus emblica against CviR protein from Chromobacterium violaceum and LasR protein from Phyllanthus emblica were studied.

Synthesis of new 1,4‐ and 1,5‐disubstituted N‐ethyl acetate and N‐α‐butyro‐γ‐lactone alkylimidazole derivatives as N‐acylhomoserine lactone analogs

AbstractNew alkylimidazoles functionalized with a homoserine lactone or an alkyloxycarbonyl moiety have been synthesized as N‐acylhomoserine lactones (AHLs) analogs. The 1,4‐disubstituted imidazole derivatives were prepared by alkylation of 4(5)‐alkylimidazoles with α‐bromo‐γ‐butyrolactone or ethyl α‐bromoacetate. An alternative route was preferred for the synthesis of their 1,5‐disubstituted counterparts based on the use of a N1‐protected alkylimidazole, its alkylation to an N3‐imidazolyl‐α‐acetate and deprotection to the desired 1,5‐disubstituted esters and subsequent alkylation of the acetate moiety with cyclic ethylene sulfate followed by acid‐catalyzed cyclization. The ability to modulate bacterial quorum sensing of all new compounds was compared to that of previously reported AHL analogs in which the amide bond is replaced by a heterocyclic group.

Acylase enzymes disrupting quorum sensing alter the transcriptome and phenotype of Pseudomonas aeruginosa, and the composition of bacterial biofilms from wastewater treatment plants

Biofilms represent an essential way of life and colonization of new environments for microorganisms. This feature is regulated by quorum sensing (QS), a microbial communication system based on autoinducer molecules, such as N-acyl-homoserine lactones (AHLs) in Gram negative bacteria. In artificial ecosystems, like Wastewater Treatment Plants (WWTPs), biofilm attachment in filtration membranes produces biofouling. In this environment, the microbial communities are mostly composed of Gram-negative phyla. Thus, we used two AHLs-degrading enzymes, obtained from Actinoplanes utahensis (namely AuAAC and AuAHLA) to determine the effects of degradation of QS signals in the biofilm formation, among other virulence factors, of a Pseudomonas aeruginosa strain isolated from a WWTP, assessing molecular mechanisms through transcriptomics. Besides, we studied the possible effects on community composition in biofilms from activated sludge samples. Although the studied enzymes only degraded the AHLs involved in one of the four QS systems of P. aeruginosa, these activities produced the deregulation of the complete QS network. In fact, AuAAC -the enzyme with higher catalytic efficiency- deregulated all the four QS systems. However, both enzymes reduced the biofilm formation and pyocyanin and protease production. The transcriptomic response of P. aeruginosa affected QS related genes, moreover, transcriptomic response to AuAAC affected mainly to QS related genes. Regarding community composition of biofilms, as expected, the abundance of Gram-negative phyla was significantly decreased after enzymatic treatment. These results support the potential use of such AHLs-degrading enzymes as a method to reduce biofilm formation in WWTP membranes and ameliorate bacterial virulence.

Butea monosperma seed extract mediated biosynthesis of ZnO NPs and their antibacterial, antibiofilm and anti-quorum sensing potentialities

Nanoparticles (NPs) prepared through safer, eco-friendly and natural-based products can be used various applications including antimicrobial and biomedical applications. Quorum sensing (QS) is a complex system for intercellular communication that regulates the expression of many virulence determinants, in the opportunistic Pseudomonas aeruginosa. Thus, the disruption of QS-mediated communication system could be an alternative strategy to combat infection caused by drug resistant P. aeruginosa. In this study, zinc oxide nanoparticles (ZnO NPs) were synthesized by using seed extract of Butea monosperma and further characterized by XRD, SEM and TEM. The biosynthesized ZnO NPs were then subjected to the investigation of their antibacterial, antibiofilm activities as well as the anti-quorum sensing potentialities for biomedical applications. The minimum inhibitory concentration (MIC) value against P. aeruginosa was found 1600 µg/ml, and it was found that ZnO NPs effectively reduced virulence factors Viz. swarming and swimming of P. aeruginosa by 62.8 and 45%, respectively. Exopolysaccharides production was also affected by ZnO NPs which indicate the disruption of biofilm formation. Further, the in silico docking analysis displayed that ZnO NPs efficiently interacted within QS (Las/Rhl) mechanism of P. aeruginosa by binding to the catalytic cleft of LasI synthase (Gly-116-ZnO = 2.8 Å), RhlI synthase (Gly-180-ZnO = 3.7 Å) and transcription receptor protein LasR receptor (Asn-141-ZnO = 2.9 Å), RhlR receptor (Tyr-72-ZnO = 2.6 Å). Thus, it was concluded that the effective interaction of ZnO NPs with Las and Rhl system led to the inactivation of autoinducers such as N-acyl homoserine lactones (AHL) molecules which finally led to the inactivation of QS and down regulation of virulence determinants. Consequently, the present in vitro and in silico results obtained suggest that the synthesized ZnO NPs can be efficiently used as potent antimicrobial agents to prevent the colonization, biofilm formation as well as anti-QS-mediated determinants caused by pathogenic drug resistant bacteria.

Quorum-Quenching Bacteria Isolated From Red Sea Sediments Reduce Biofilm Formation by Pseudomonas aeruginosa.

Quorum sensing (QS) is the process by which bacteria communicate with each other through small signaling molecules such as N-acylhomoserine lactones (AHLs). Certain bacteria can degrade AHL molecules by a process called quorum quenching (QQ); therefore, QQ can be used to control bacterial infections and biofilm formation. In this study, we aimed to identify new species of bacteria with QQ activity. Red Sea sediments were collected either from the close vicinity of seagrass or from areas with no vegetation. We isolated 72 bacterial strains, which were tested for their ability to degrade/inactivate AHL molecules. Chromobacterium violaceum CV026-based bioassay was used for the initial screening of isolates with QQ activity. QQ activity was further quantified using high-performance liquid chromatography-tandem mass spectrometry. We found that these isolates could degrade AHL molecules of different acyl chain lengths as well as modifications. 16S-rRNA sequencing of positive QQ isolates showed that they belonged to three different genera. Specifically, two isolates belonged to the genus Erythrobacter; four, Labrenzia; and one, Bacterioplanes. The genome of one representative isolate from each genus was sequenced, and potential QQ enzymes, namely, lactonases and acylases, were identified. The ability of these isolates to degrade the 3OXOC12-AHLs produced by Pseudomonas aeruginosa PAO1 and hence inhibit biofilm formation was investigated. Our results showed that the isolate VG12 (genus Labrenzia) is better than other isolates at controlling biofilm formation by PAO1 and degradation of different AHL molecules. Time-course experiments to study AHL degradation showed that VG1 (genus Erythrobacter) could degrade AHLs faster than other isolates. Thus, QQ bacteria or enzymes can be used in combination with an antibacterial to overcome antibiotic resistance.

Cloning and characterization of short-chain N-acyl homoserine lactone-producing Enterobacter asburiae strain L1 from lettuce leaves.

In gram‐negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N‐acyl homoserine lactones (AHL). We have previously reported the genome of AHL‐producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL‐type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild‐type E. asburiae. The mass spectrometry analysis showed the production of N‐butanoyl homoserine lactone and N–hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm‐forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.

AHL-lactonase expression in three marine emerging pathogenic Vibrio spp. reduces virulence and mortality in brine shrimp (Artemia salina) and Manila clam (Venerupis philippinarum)

Bacterial infectious diseases produced by Vibrio are the main cause of economic losses in aquaculture. During recent years it has been shown that the expression of virulence genes in some Vibrio species is controlled by a population-density dependent gene-expression mechanism known as quorum sensing (QS), which is mediated by the diffusion of signal molecules such as N-acylhomoserine lactones (AHLs). QS disruption, especially the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), is one of the novel therapeutic strategies for the treatment of bacterial infections. In this study, we present the detection of AHLs in 34 marine Vibrionaceae strains. Three aquaculture-related pathogenic Vibrio strains, V. mediterranei VibC-Oc-097, V. owensii VibC-Oc-106 and V. coralliilyticus VibC-Oc-193 were selected for further studies based on their virulence and high production of AHLs. This is the first report where the signal molecules have been characterized in these emerging marine pathogens and correlated to the expression of virulence factors. Moreover, the results of AHL inactivation in the three selected strains have been confirmed in vivo against brine shrimps (Artemia salina) and Manila clams (Venerupis philippinarum). This research contributes to the development of future therapies based on AHL disruption, the most promising alternatives for fighting infectious diseases in aquaculture.

Detection of Diverse N-Acyl-Homoserine Lactones in Vibrio alginolyticus and Regulation of Biofilm Formation by N-(3-Oxodecanoyl) Homoserine Lactone In vitro.

Quorum sensing (QS) is a cell-to-cell communication system based on the exchange of small intercellular signal molecules, such as N-Acyl homoserine lactones (AHLs), which act as cell-density mediators of QS gene expression, and are highly variable both in types and amounts in most Gram-negative Proteobacteria. Understanding the regulation of AHLs may contribute to the elucidation of cell density-dependent phenomena, such as biofilm formation. Vibrio alginolyticus is among the most frequently observed marine opportunistic Vibrio pathogens. However, AHL production of this species and its effects on biofilm formation remain to be understood. Here, our study reported the diverse AHL profiles of 47 marine-isolated V. alginolyticus strains and the effects of exogenous 3-oxo-C10-HSL on biofilm formation under different temperature conditions (16°C and 28°C). A total of 11 detected AHLs were produced by the isolates, of which 3-OH-C4-HSL, 3-oxo-C10-HSL and 3-oxo-C14-HSL comprised the largest proportions. We also observed that moderate levels of exogenous 3-oxo-C10-HSL (10 and 20 μM) could induce or enhance biofilm formation and alter its structure, while high levels (40 and 100 μM) did not significantly improve and even inhibited biofilm formation in V. alginolyticus. Further, regulation by exogenous 3-oxo-C10-HSL was both concentration- and temperature-dependent in V. alginolyticus.

Acinetobacterbaumannii quorum-sensing signalling molecule induces the expression of drug-resistance genes.

Quorum-sensing signalling molecules such as N-acyl homoserine lactones (AHLs) enable certain Gram-negative bacteria to respond to environmental changes through behaviours, such as biofilm formation and flagellar movement. The present study aimed to identify Acinetobacter baumannii AHLs and assess their influence on antibiotic resistance. A clinical isolate of A. baumannii strain S (AbS) was collected from the wound of a burn patient and high-performance liquid chromatography and tandem quadrupole or quadrupole time-of-flight high-resolution mass spectrometry was used to identify AbS AHLs. Antibiotic sensitivity was assessed in an AHL-deficient AbS mutant (AbS-M), and the expression of drug-resistance genes in the presence of meropenem in AbS, AbS-M and AbS-M treated with the AHL N-3-hydroxy-dodecanoyl-homoserine lactone (N-3-OH-C12-HSL). AbS-M was more sensitive to meropenem and piperacillin than wild-type AbS, but resistance was restored by supplementation with N-3-OH-C12-HSL. In addition, meropenem-treated AbS-M expressed lower levels of the drug-resistance genes oxacillinase 51, AmpC, AdeA and AdeB; treatment with N-3-OH-C12-HSL also restored the expression of these genes. Overall, the results of the present study indicate that N-3-OH-C12-HSL may be involved in regulating the expression of drug-resistance genes in A. baumannii. Therefore, this quorum-sensing signalling molecule may be an important target for treating multidrug-resistant A. baumannii infections.

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching.

Many pathogenic bacteria utilize quorum sensing (QS) systems to regulate the expression of their virulence genes and promote the formation of biofilm, which renders pathogens with extreme resistance to conventional antibiotic treatments. As a novel approach for attenuating antibiotic resistance and in turn fighting chronic infections, enzymatic inactivation of QS signaling molecules, such as N-acyl homoserine lactones (AHLs), holds great promises. Instead of using bacterial lactonases that can evoke immune response when administered, we focus on the human paraoxonase 2 (huPON2). However, insolubility when heterologously overexpressed hinders its application as anti-infection therapeutics. In this study, huPON2 was engineered for soluble expression with minimal introduction of foreign sequences. On the basis of structure modeling, degenerate linkers were exploited for the removal of hydrophobic helices of huPON2 without disrupting its folding structure and thus retaining its enzymatic function. High soluble expression levels were achieved with a yield of 76 mg of fully human PON2 variants per liter of culture media. Particularly, two clones, D2 and E3, showed significant quorum quenching (QQ) bioactivities and effectively impeded Pseudomonas aeruginosa swimming and swarming motilities, signs of an early stage of biofilm formation. In addition, by correlating QQ with luminescence signal readouts, quantitative analysis of QQ toward natural or non-natural AHL-regulator combinations suggested that D2 and E3 exhibited strong lactone hydrolysis activities toward five AHLs of different side chain lengths and modifications widely utilized by a variety of biomedically important pathogens.

Novel linear polymers able to inhibit bacterial quorum sensing.

Bacterial phenotypes, such as biofilm formation, antibiotic resistance and virulence expression, are associated with quorum sensing. Quorum sensing is a density-dependent regulatory system of gene expression controlled by specific signal molecules, such as N-acyl homoserine lactones (AHLs), produced and released by bacteria. This study reports the development of linear polymers capable to attenuate quorum sensing by adsorption of AHLs. Linear polymers were synthesized using MMA as backbone monomer and methacrylic acid and itaconic acid as functional monomers. Two different quorum sensing-controlled phenotypes, Vibrio fischeri bioluminescence and Aeromonas hydrophila biofilm formation, were evaluated to test the polymers' efficiency. Results showed that both phenotypes were significantly affected by the polymers, with the itaconic acid-containing material being more effective than the methacrylic acid one. The polymer inhibitory effects were reverted by the addition of lactones, confirming attenuation of quorum sensing through sequestration of signal molecules. The polymers also showed no cytotoxicity when tested using a mammalian cell line.

Effect of Dietary N‐acyl Homoserin Lactonase on the Immune Response and the Gut Microbiota of Zebrafish, Danio rerio, Infected with Aeromonas hydrophila

AbstractQuorum sensing interference is a novel strategy in bacterial disease control. Degradation of quorum sensing signal molecules, such as N‐acyl homoserine lactones (AHLs), by enzymes is a potential interfering method. In this study, the in vivo effect of an N‐acyl homoserine lactonase from Bacillus sp. B546, AiiAB546, on the protection of zebrafish, Danio rerio, against Aeromonas hydrophila infection was first investigated by co‐feeding. The short‐term immune responses and the long‐term effects on the gut microbiota of zebrafish were analyzed by quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The results showed that the expression levels of some anti‐infectious immunity‐related factors, including interleukin 10 (IL‐10), toll‐like receptor 5b (TLR5b), and inducible nitric oxide synthase 2a (iNOS2a) that were related to A. hydrophila virulent factors under regulation of quorum sensing, were more susceptible to the addition of AiiAB546. It indicated that AiiAB546 may attenuate the infection of A. hydrophila by interfering with quorum sensing. A. hydrophila was not detected in the enzyme co‐feeding diet group, which might be due to its inability to adhere to the gut with enzyme interference. This study revealed that AiiAB546 as a feed additive might represent a novel biocontrol agent in aquaculture; however, the topic merits further investigations using fish species of commercial interest.

The role of N-acyl homoserine lactones in maintaining the stability of aerobic granules

In this study, porcine kidney acylase, as N-acyl homoserine lactones (AHLs)-degradation enzyme, was employed for the first time to directly investigate the role of AHLs in the structure stability of aerobic granules. Results clearly showed that inactivation of AHLs by AHLs-acylase could weaken the stability of aerobic granule. In the presence of AHLs-acylase, AHLs were degraded by hydrolyzing the amide linkage, which resulted in aerobic granular attachment potential and activity of AHLs-based quorum sensing significantly reduced. In addition, it was also found that inactivation of AHLs led to reduction of extracellular polysaccharides and protein (PN), especially PN, and induced extracellular polymeric substances matrix damaged, which was hostile to stability of aerobic granules. This study provided direct evidence that AHLs played a key role in improving aerobic granular stability, and a potential way to enhance long-term stability of aerobic granules.

Phosphate concentration alters the effective bacterial quorum in the symbiosis of Medicago truncatula-Sinorhizobium meliloti

The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of free-living and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expression of sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 105, 107 and 109 CFU ml−1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations.

N‐Acylated Alanine Methyl Esters (NAMEs) from Roseovarius tolerans, Structural Analogs of Quorum‐Sensing Autoinducers, N‐Acylhomoserine Lactones

The Roseobacter clade is one of the most important bacteria group living in the ocean. Liquid cultures of Roseovarius tolerans EL 164 were investigated for the production of autoinducers such as N-acylhomoserine lactones (AHLs) and other secondary metabolites. The XAD extracts were analyzed by GC/MS. Two AHLs, Z7-C14 : 1-homoserine lactone (HSL) and C15 : 1-HSL, were identified. Additionally, the extract contained five compounds with molecular-ion peaks at m/z 104, 145, and 158, thus exhibiting mass spectra similar to those of AHLs with corresponding peaks at m/z 102, 143, and 156. Isolation of the main compound by column chromatography, NMR analysis, dimethyl disulfide derivatization for the determination of the location of the CC bond and finally synthesis of the compound with the proposed structure confirmed the compound to be (Z)-N-(hexadec-9-enoyl)alanine methyl ester. Four additional minor compounds were identified as C14 : 0-, C15 : 0-, C16 : 0-, and C17 : 1-N-acylated alanine methyl esters (NAMEs). All NAMEs have not been described from natural sources before. A BLASTp search showed the presence of AHL-producing luxI genes, but no homologous genes potentially responsible for the structurally closely related NAMEs were found. The involvement of the NAMEs in chemical communication processes of the bacteria is discussed.

Bacterial Quorum Sensing and Quorum Quenching for the Inhibition of Biofilm Formation

Quorum sensing (QS) is a cell-to-cell communication system, which is used by many bacteria to regulate diverse gene expression in response to changes in population density. Bacteria recognize the differences in cell density by sensing the concentration of signal molecules such as N-acyl-homoserine lactones (AHL) and autoinducer-2 (AI-2). In particular, QS plays a key role in biofilm formation, which is a specific bacterial group behavior. Biofilms are dense aggregates of packed microbial communities that grow on surfaces, and are embedded in a self-produced matrix of extracellular polymeric substances (EPS). QS regulates biofilm dispersal as well as the production of EPS. In some bacteria, biofilm formations are regulated by c-di-GMP-mediated signaling as well as QS, thus the two signaling systems are mutually connected. Biofilms are one of the major virulence factors in pathogenic bacteria. In addition, they cause numerous problems in industrial fields, such as the biofouling of pipes, tanks and membrane bioreactors (MBR). Therefore, the interference of QS, referred to as quorum quenching (QQ) has received a great deal of attention. To inhibit biofilm formation, several strategies to disrupt bacterial QS have been reported, and many enzymes which can degrade or modify the signal molecule AHL have been studied. QQ enzymes, such as AHL-lactonase, AHL-acylase, and oxidoreductases may offer great potential for the effective control of biofilm formation and membrane biofouling in the future. This review describes the process of bacterial QS, biofilm formation, and the close relationship between them. Finally, QQ enzymes and their applications for the reduction of biofouling are also discussed.

The hanR/hanI quorum-sensing system of Halomonas anticariensis, a moderately halophilic bacterium

Quorum sensing is a cell density-dependent gene expression mechanism found in many Gram-negative bacteria which involves the production of signal molecules such as N-acylhomoserine lactones (AHLs). One significant group of micro-organisms in which quorum sensing has not been previously studied, however, are the moderate halophiles. We describe here the results of our studies of the quorum-sensing system in Halomonas anticariensis FP35(T), which is composed of luxR/luxI homologues: hanR (the putative transcriptional regulator gene) and hanI (the autoinducer synthase gene). To understand how the hanR/hanI system is organized and regulated we conducted RT-PCR and quantitative real-time PCR assays. Transcriptional analysis indicated that the hanR and hanI genes are on the same transcript and that their transcription is growth phase-dependent. HanI seems to be the only autoinducer synthase responsible for the synthesis of AHLs by the bacterium, since the inactivation of hanI resulted in the complete loss of its AHLs. We also found that the hanI gene appears to be transcribed from its own promoter and that its expression does not depend upon HanR. This finding was supported by the fact that the FP35hanR mutant showed AHL-producing activity and hanI expression similar to that of the wild-type strain, the latter being measured by RT-PCR. Moreover, hanR is expressed from its own promoter and appears to be independent of the AHL signalling molecules produced by HanI.