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 1 mM N-(3-oxo-dodecanoyl)-l-homoserine lactone and 2 mM 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).     

LasR Receptor for Detection of Long-Chain Quorum-Sensing Signals: Identification of <Emphasis Type="Italic">N</Emphasis>-Acyl-homoserine Lactones Encoded by the <Emphasis Type="Italic">avsI</Emphasis> Locus of <Emphasis Type="Italic">Agrobacterium vitis</Emphasis>

Bacterial biosensor strains have greatly facilitated the rapid discovery, isolation, and study of quorum-sensing systems. In this study, we determined the relative sensitivity of a LasR-based E. coli bacterial bioluminescence biosensor JM109 (pSB1075) for 13 diverse long-chain N-acyl-homoserine lactones (AHLs) including oxygen-substituted and -unsubstituted AHLs containing 14, 16, and 18 carbons and with and without double bonds. Furthermore, we show by bioassay, HPLC, and GC/MS that four long-chain AHLs of the C16-HSL family are encoded by the avsI gene of Agrobacterium vitis strain F2/5, a non-tumorigenic strain that inhibits pathogenic strains of A. vitis from causing crown gall on grape. The four C16-HSLs include: C16-HSL, N-hexadecanoyl homoserine lactone; 3-oxo-C16-HSL, N-(3-oxohexadecanoyl)homoserine lactone; C16:1-HSL, N-(cis-9-octadecenoyl)homoserine lactone; and 3-oxo-C16:1-HSL, N-(3-oxo-cis-11-hexadecenoyl)homoserine lactone. Thus, the LasR-based bioluminescent biosensor tested in this study should serve as a useful tool for the detection of various long-chain AHLs with and without double bonds as well as those oxylated at the third carbon from uninvestigated species.     

Quorum-sensing molecules: Sampling,identification and characterization of N-acyl-homoserine lactone in Vibrio sp

Quorum sensing (QS) is a mechanism by which gram-negative bacteria regulate their gene expression by making use of cell density. QS is triggered by a small molecule known as an autoinducer. Typically, gram-negative bacteria such as Vibrio produce signaling molecules called acyl homoserine lactones (AHLs). However, their levels are very low, making them difficult to detect. We used thin layer chromatography (TLC) to examine AHLs in different Vibrio species, such as Vibrio alginolyticus, Vibrio parahemolyticus, and Vibrio cholerae, against a standard- Chromobacterium violaceum. Further, AHLs were characterised by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC–MS). C4-HSL (N- butanoyl- L- homoserine lactone), C6-HSL (N- hexanoyl- L- homoserine lactone), 3-oxo-C8-HSL (N-(3-Oxooctanoyl)-DL-homoserine lactone), C8-HSL (N- octanoyl- L- homoserine lactone), C₁10-HSL (N- decanoyl- L- homoserine lactone), C12-HSL (N- dodecanoyl- L- homoserine lactone) and C14-HSL (N- tetradecanoyl- L- homoserine lactone) were identified from Vibrio. These results may provide a basis for blocking the AHL molecules of Vibrio, thereby reducing their pathogenicity and eliminating the need for antimicrobials.     

Pseudomonas as a frequent and important quorum quenching bacterium with biocontrol capability against many phytopathogens

The aim of the present study was to isolate a variety of quorum quenching bacteria (QB) from the rhizosphere and phyllosphere of three agricultural plants using minimal medium (MM)- and non-minimal medium (NM)-based methods. The members of the Pseudomonas genus constituted the most abundant QB genus, particularly in the rhizospheres of all plant samples and showed the highest quorum quenching (QQ) activity according to a screening assay using a biosensor and 3-oxo-C6-HSL (as an important quorum sensing signal in many phytopathogenic bacteria). In addition, QQ-Pseudomonas were recognised as versatile biocontrol agents against non-bacterial and bacterial plant pathogens, such as Pectobacterium carotovorum subsp. carotovorum (Pcc). Three types of quenching activities, including intracellular and extracellular enzymatic and non-enzymatic activities, were observed in QQ-Pseudomonas. Pseudomonas strains, particularly NM-isolated strains with extracellular activity, are the strongest QQ-based biocontrol agents.     

Halogenated furanones from the red alga, Delisea pulchra, inhibit carbapenem antibiotic synthesis and exoenzyme virulence factor production in the phytopathogen Erwinia carotovora

The plant pathogen Erwinia carotovora regulates expression of virulence factors and antibiotic production via an N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL) dependent quorum sensing mechanism. The marine alga Delisea pulchra produces halogenated furanones known to antagonise 3-oxo-C6-HSL activity. We have tested the effects of a halogenated furanone on the production of carbapenem, cellulase and protease in E. carotovora. Despite differences in the regulatory mechanisms controlling carbapenem and exoenzyme production each was inhibited by the algal metabolite. We present evidence to suggest that the furanone dependent inhibition of carbapenem production is a result of the disruption of the 3-oxo-C6-HSL dependent expression of the carABCDEFGH operon.     

Isolation and characterization of sulphate-reducing bacteria <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> from Vajreshwari thermal springs in Maharashtra,India

Sulphate-reducing bacteria (SRB) in the thermal springs of Vajreshwari were investigated with combined microbiological and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 25 to 55 °C and could use pyruvate, lactate and ethanol as electron donors. Desulfoviridin was detected in all the isolates. The partial 16S rRNA and dissimilatory sulphite reductase (DSR) gene sequences of five representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris.     

Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species

Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.     

N-acylhomoserine lactone-degrading bacteria isolated from hatchery bivalve larval cultures

Quorum sensing (QS) systems, which depend on N-acylhomoserine lactone (AHL) signal molecules, mediate the production of virulence factors in many pathogenic microorganisms. One hundred and forty-six bacterial strains, isolated from a bivalve hatchery, were screened for their capacity to degrade five synthetic AHLs [N-butyryl-dl-homoserine lactone (C₄-HSL), N-hexanoyl-dl-homoserine lactone (C₆-HSL), N-octanoyl-dl-homoserine lactone (C₈-HSL), N-decanoyl-dl-homoserine lactone (C₁₀-HSL) and N-dodecanoyl-dl-homoserine lactone (C₁₂-HSL)] using well diffusion agar-plate assays with three biosensors, Chromobacterium violaceum CV026, C. violaceum VIR07 and Agrobacterium tumefaciens NTL4 (pZLR4). The results of these assays led to our choosing four strains (PP2-67, PP2-459, PP2-644 and PP2-663) that were able to degrade all five synthetic AHLs, thus showing a wide spectrum of quorum quenching (QQ) activity. We subsequently confirmed and measured the QQ activity of the four strains by high-performance liquid chromatography plus mass-spectrometry analysis (HPLC–MS). One of the strains which showed the highest AHL-degrading activity, PP2-459, identified as being a member of the genus Thalassomonas was chosen for further study. Finally, using thin-layer chromatography (TLC), we went on to confirm this strain's capacity to degrade the AHLs produced by other non-pathogenic and pathogenic bacteria not taxonomically related.     

Characterization of wetland quorum quenching Pseudomonas aeruginosa strain 2SW8 and its 2-heptyl-3-hydroxy-4-quinolone production

Most Proteobacteria produce N-acylhomoserine lactones for bacterial cell-to-cell communication, a process called quorum sensing. Interference of quorum sensing, commonly known as quorum quenching, represents an important way to control quorum sensing. This work reports the isolation of quorum quenching bacterium strain 2WS8 from Malaysia tropical wetland water (2°11'8"N, 102°15'2"E, in 2007) by using a modified version of a previously reported KG medium. Strain 2WS8 was isolated based on its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) as the sole source of energy. This bacterium clustered closely to Pseudomonas aeruginosa PAO1. Strain 2SW8 possesses both quiP and pvdQ homologue acylase genes. Rapid Resolution Liquid Chromatography analysis confirmed that strain 2SW8 preferentially degraded N-acylhomoserine lactones with 3-oxo group substitution but not those with unsubstituted groups at C3 position in the acyl side chain. Strain 2SW8 also showed 2-heptyl-3-hydroxy-4-quinolone production.     

<Emphasis Type="Italic">N</Emphasis>-acyl homoserine lactones are degraded via an amidolytic activity in <Emphasis Type="Italic">Comamonas</Emphasis> sp. strain D1

Abtsract Comamonas strain D1 enzymatically inactivates quorum-sensing (QS) signal molecules of the N-acyl homoserine lactone (N-AHSL) family, and exhibits the broadest inactivation range of known bacteria. It degrades N-AHSL with acyl-side chains ranging from 4 to 16 carbons, with or without 3-oxo or 3-hydroxy substitutions. N-AHSL degradation yields HSL but not N-acyl homoserine: strain D1 therefore harbors an amidohydrolase activity. Strain D1 is the fifth bacterium species in which an N-AHSL amidohydrolase is described. Consistent with its N-AHSL degradation ability, strain D1 efficiently quenches various QS-dependent functions in other bacteria, such as violacein production by Chromobacterium violaceum and pathogenicity and antibiotic production in Pectobacterium.     

Presence of Acyl-Homoserine Lactone in Subtidal Biofilm and the Implication in Larval Behavioral Response in the Polychaete <Emphasis Type="Italic">Hydroides elegans</Emphasis>

Quorum sensing (QS) signals have been considered to play important roles in biofilm development and in the attractiveness of biofilms to higher organisms in marine ecosystem. In this study, bacterial QS signalsacylated homoserine lactone derivatives (AHLs) were detected in 2-, 4-, and 6-day-old subtidal biofilms by using AHLs reporter strains. N-dodecanoyl-homoserine lactone (C12-HSL) was identified in 6-day-old biofilm at a concentration of 9.04 μg cm^−minus;2 (3.36 mmol l^−minus;1). To investigate the possible role of AHLs in the consequent eventlarval settlement of the polychaete Hydroides elegans onto subtidal biofilmsseven biofilm-derived bacteria that effectively induced larval settlement of H. elegans, were screened for AHL production. One of them, the Vibrio sp. UST950701-007, produced N-hexanoyl-homoserine lactone (C6-HSL). Larval settlement bioassay showed that C6-HSL, C12-HSL, and 3-oxo-octanoyl-homoserine lactone (3-oxo-C8-HLS) at certain concentrations induced some initial larval settlement behaviors such as reducing swimming speed, crawling on the bottom. However, these AHLs did not effectively induce larval settlement in comparison to the effective settlement inducer 3-isobutyl-1-methylxanthine. The possible chemokinetic mechanism and indirect effects of AHLs on larval settlement are suggested.     

凡纳滨对虾源不动杆菌群体感应信号分子分离鉴定及其调控

【目的】鉴定凡纳滨对虾源不动杆菌(Acinetobacter spp.M1)分泌的N-酰基高丝氨酸内酯(AHLs)类型,探究细菌生长阶段及环境因素对其分泌信号分子的影响。【方法】报告菌株平板法检测M1的AHLs的活性;采用报告平板与薄层层析(TLC)相结合法对M1分泌的AHLs类型进行鉴定。【结果】菌株M1分泌N-3-氧代-己酰基-高丝氨酸内酯和N-3-氧代-辛酰基-高丝氨酸内酯两种信号分子。在适宜条件下AHLs活性随着培养时间的延长先升高后降低,在对数末期(30 h)达到最大。弱酸和弱碱环境能够降低M1分泌AHLs的能力,p H 7.0是M1分泌AHLs的最适p H。较高浓度的Na Cl促进了个体M1分泌AHLs的能力,但是Na Cl浓度对M1总体分泌AHLs没有显著的影响。菌株M1分泌AHLs的最佳温度为30°C,温度过高或过低都会影响其分泌。【结论】菌株M1主要产生N-3-氧代-己酰基-高丝氨酸内酯和N-3-氧代-辛酰基-高丝氨酸内酯两种类型信号分子。M1的QS系统受菌体密度和环境因素的双重调控。     

Presence of Quorum-Sensing-Mediated Gene Regulation in Pathogenic Ice-Nucleation-Active (INA) Bacteria

Nine out of a total of 20 pathogenic ice-nucleation-active bacteria, with different levels of inducible INA, were tested and found positive for their ability to synthesize quorum-sensing (QS) signals. The bacteria were isolated from willow plants and belonged to the genera Bacillus, Erwinia, Pseudomonas and Sphingomonas. As reporter bacteria, to detect the homoserine lactone (HSL) autoinducer, Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeroginosa, Aeromonas hydrophila and Vibrio fischeri strains were used. We thus provide evidence that pathogenic ice-nucleation bacteria with inducible INA produce QS signals that in other bacteria have been shown to be in the control of genes of importance for pathogenicity.     

Redirecting Carbon Flux in Torulopsis glabrata from Pyruvate to α-Ketoglutaric Acid by Changing Metabolic Co-factors

The nutrition conditions needed to redirect the carbon flux in Torulopsis glabrata, a pyruvate hyper-production yeast, from pyruvate to α-ketoglutaric acid (KG) were investigated in a stirred fermentor. A minor amount of KG (1.3 gl⁻¹) was produced when NaOH was used to control the pH, while 12 g KG l⁻¹ was produced when CaCO₃ was used instead. When thiamine and biotin were included in the medium, 13 g KG l⁻¹ and 68 g pyruvate l⁻¹ were produced after 48 h when glucose was nearly consumed (approximately 5 gl⁻¹). With fermentation continuing for a further 16 h, the concentration of pyruvate decreased to 31 gl⁻¹, and KG increased to 30 gl⁻¹. KG thus accumulated at the expense of pyruvate consumption. Received 2 June 2005; Revisions requested 30 June 2005 and 1 September 2005; Revisions received 1 September 2005 and 28 October 2005; Accepted 28 October 2005     

Rapid degradation of <Emphasis Type="Italic">N</Emphasis>-3-oxo-acylhomoserine lactones by a <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">cereus</Emphasis> isolate from Malaysian rainforest soil

A bacterial strain, KM1S, was isolated from a Malaysian rainforest soil sample by using a defined enrichment medium that specifically facilitates selection of quorum quenching bacteria. KM1S was clustered closely to Bacillus cereus by 16S ribosomal DNA sequence analysis. It degraded N-3-oxo-hexanoyl homoserine lactone and N-3-oxo-octanoyl homoserine lactone in vitro rapidly at 4.98 and 6.56 μg AHL h⁻¹ per 10⁹ CFU/ml, respectively, as determined by the Rapid Resolution Liquid Chromatography. The aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of N-acylhomoserine lactones, of KM1S was amplified and cloned. Sequence analysis indicated the presence of the motif ₁₀₆HXDH-59 amino acids-H₁₆₉-21 amino acids-D₁₉₁ for N-acylhomoserine lactone lactonases.     

The plant pathogen Pantoea ananatis produces N-acylhomoserine lactone and causes center rot disease of onion by quorum sensing

A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-l-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that P. ananatis SK-1 produced two AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in P. ananatis SK-1.     

肉类腐败性假单胞菌群体感应信号分子的研究

【目的】研究两株假单胞菌的标准菌株荧光假单胞菌(Pseudomonas fluorescens)和铜绿假单胞菌(Pseudomonas aeruginosa)在纯培养条件下所释放的AHLs类信号分子种类、量和变化规律。【方法】利用乙酸乙酯等有机溶剂萃取菌种纯培养液的AHLs类信号分子, 检测手段利用HPLC-MS-MS。【结果】荧光假单胞菌释放信号分子的种类为: C4-HSL、C6-HSL、C8-HSL、3-oxo-C10-HSL、3-oxo-C12-HSL、3-oxo-C14-HSL。铜绿假单胞菌释放信号分子的种类为: C4-HSL、C6-HSL、C8-HSL、C10-SL、C12-HSL、C14-HSL、3-oxo-C8-HSL、3-oxo-C10-HSL、3-oxo-C12-HSL、3-oxo-C14-HSL。【结论】两株菌所释放各类信号分子的量均随时间变化, 当菌落数达到109?1010时信号分子的量达到峰值, 两株菌所释放各类信号分子含量差异较大。     

Glyphosate degradation byAgrobacterium radiobacter isolated from activated sludge

Summary Two species of bacteria capable of growth onN-phosphonomethylglycine (glyphosate) were isolated from a bench scale sequencing batch reactor degrading a waste stream containing glyphosate. The enrichment and isolation medium contained defined salts and glyphosate as the sole carbon and energy source. Glyphosate was stoichiometrically degraded to aminomethylphosphonic acid (AMPA). The bacteria have been identified asAgrobacterium radiobacter andAchromobacter Group V D.