Cryo-EM structures of pentameric autoinducer-2 exporter from Escherichia coli reveal its transport mechanism.

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer-2 (AI-2), a universal molecule for both intra- and inter-species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI-2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI-2 exporter superfamily, has been shown to export AI-2. Here, we report the cryogenic electron microscopic structures of two AI-2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI-2 exporter exists as a homo-pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI-2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator-type transport mechanism.

Increasement of O-acetylhomoserine production in Escherichia coli by modification of glycerol-oxidative pathway coupled with optimization of fermentation.

OBJECTIVE: O-acetylhomoserine (OAH) is an important platform chemical to produce high-valuable chemicals. To improve the production of O-acetylhomoserine from glycerol, the glycerol-oxidative pathway was investigated and the optimization of fermentation with crude glycerol was carried out. RESULTS: The glycerol-uptake system and glycerol-oxidative pathway were modified and O-acetyltransferase from Corynebacterium glutamicum was introduced into the engineered strain to produce O-acetylhomoserine. It was found that overexpression of glycerol 3-phosphate dehydrogenase improved the OAH production to 6.79 and 4.21 g/L from pure and crude glycerol, respectively. And the higher OAH production depending on higher level of transcription of glpD. Two-step statistical approach was employed to optimize the fermentation conditions. The significant effects of glycerol, ammonium chloride and yeast extract were screened applying Plackett-Burman design and were optimized further by employing the Response Surface Methodology. Under optimized conditions, the OAH production was up to 9.42 and 7.01 g/L when pure and crude glycerol were used in shake flask cultivations, respectively. CONCLUSIONS: The enzymatic step catalyzing the oxidation of glycerol through GlpD was the key step for OAH production, which served the foundation for realization of a consistent OAH production from crude glycerol in the future.

Enhanced O-succinyl-l-homoserine production by recombinant Escherichia coli ΔIJBB*TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH via multilevel fermentation optimization.

AIMS: Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization. METHODS AND RESULTS: OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70 g l-1 in 500 ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5 g l-1 , which is 5·6 times higher than before (15·6 g l-1 ). CONCLUSIONS: O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization.

The use of multifunctional yeast-lactobacilli starter cultures improves fermentation performance of Spanish-style green table olives.

In this work, Lactobacillus pentosus LPG1, Lactobacillus pentosus Lp13, Lactobacillus plantarum Lpl15, and Wickerhanomyces anomalous Y12, all of them previously isolated from fermented table olive biofilms, were used (alone or in combination) as multifunctional starters for Manzanilla Spanish-style green table olive fermentations. Their performances were evaluated through the changes in the key physico-chemical and microbiological parameters, correlation between AI-2 production and biofilm formation, inoculum imposition, metataxonomic analysis and sensory characteristics of the finished products. Inoculation only with lactic acid bacteria (LAB) strains led to higher titratable acidities and lower pH values than the spontaneous fermentation (non-inoculated control), mainly during the first steps of processing. However, the sequential inoculation of the yeast and then the combination of the 3 LAB strains showed the most favourable evolution. LPG1 strain and, particularly Lp13, were excellent biofilms former and showed the major imposition on the fruit epidermis, as corroborated by rep-PCR analysis. Production of AI-2 was lower in the treatment inoculated exclusively with yeast Y12 but had the highest presence in the sequential yeast-LAB inoculum, with its maximum concentration and maximum LAB population on fruits (19th days) strongly related. Metataxonomic analysis of the biofilms at the end of the fermentation revealed, in addition to Lactobacillus, high proportions of sequences from genera Marinilactobacillus, Alkalibacterium, Halolactobacillus, and low levels of Halomonas and Aerococcus. Compositional data analysis of the omics data revealed that Lpl15 was scarcely efficient for controlling the spontaneous microbiota since its treatment presented the highest proportions of Aerococcus genus. Finally, the sensory analysis showed similar characteristics for the treatment inoculated with LPG1 and the spontaneous process, with olives inoculated with the yeast (alone or in combination with Lactobacillus strains) showing attractive scores. Then, inoculation of Spanish-style table olive fermentations with a sequential yeast and LAB combination could be an advisable practice.

Determination of quorum-sensing signal substances in water and solid phases of activated sludge systems using liquid chromatography-mass spectrometry.

The detection of acyl homoserine lactones (AHLs) in activated sludge is essential for clarifying their function in wastewater treatment processes. An LC-MS/MS method was developed for the detection of AHLs in both the aqueous and solid phases of activated sludge. In addition, the effects of proteases and extracellular polymeric substances (EPS) on the detection of AHLs were evaluated by adding protease inhibitors and extracting EPS, respectively. Recoveries of each AHL were improved by adding 50µL of protease inhibitor, and recoveries were also improved from 0 to 56.9% to 24.2%-105.8% by EPS extraction. Applying the developed method to determine the type and concentration of AHLs showed that C4-HSL, C6-HSL, C8-HSL and 3-oxo-C8-HSL were widely detected in a suspended activated sludge system. The dominant AHL was C8-HSL, with a highest concentration of 304.3ng/L. C4-HSL was mainly distributed in the aqueous phase, whereas C6-HSL, C8-HSL and 3-oxo-C8-HSL were preferentially distributed in the sludge phase.

Production of N-acyl homoserine lactones by Chromobacterium haemolyticum KM2 isolated from the river water in Malaysia.

Quorum sensing (QS) is a term used to describe cell-to-cell communication that enables bacteria to orchestrate group behaviours according to density of bacterial cells. In Gram-negative bacteria, this signalling system is widely known to regulate a variety of different phenotypes such as antibiotic production and biofilm formation. In this study, we report the production of N-acyl homoserine lactones produced by Chromobacterium haemolyticum strain KM2, a bacterium isolated from a river water of a reserved tropical national park. Preliminary screening of QS activity using biosensor reporter assays indicated that C. haemolyticum strain KM2 produces both short- and long-chain AHLs. Analysis with high-resolution liquid chromatography-mass spectrometry (LC-MS/MS) analysis revealed the production of three AHLs by strain KM2: N-octanoyl-L-homoserine lactone (C8-HSL), N-dodecanoyl-L-homoserine lactone (C12-HSL), and N-3-oxo-dodecanoyl-L-homoserine lactone (OC12-HSL). This bacterial isolate also exhibited strong ß-haemolytic activity. To the best of our knowledge, this is the first documentation of QS activity and multiple AHLs production by C. haemolyticum strain KM2.

Evaluation of two autoinducer-2 quantification methods for application in marine environments.

AIM: This study evaluated two methods, namely high performance liquid chromatography with fluorescence detection (HPLC-FLD) and Vibrio harveyi BB170 bioassay, for autoinducer-2 (AI-2) quantification in marine samples. Using both methods, the study also investigated the stability of AI-2 in varying pH, temperature and media, as well as quantified the amount of AI-2 signals in marine samples. METHODS AND RESULTS: HPLC-FLD method showed a higher level of reproducibility and precision compared to V. harveyi BB170 bioassay. Alkaline pH (>8) and high temperature (>37°C) increased the instability of AI-2. The AI-2 concentrations in seawater were low, c. 3·2-27·6 pmol l-1 , whereas 8-week-old marine biofilm grew on an 18·8 cm2 substratum accumulated c. 0·207 nmol of AI-2. CONCLUSION: Both methods have pros and cons for AI-2 quantification in marine samples. Regardless, both methods reported a ubiquitous presence of AI-2 in both planktonic and biomass fractions of seawater, as well as in marine biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, AI-2 signals were for the first time enumerated in marine samples to reveal the ubiquitous presence of AI-2 in this environment. The findings suggest a possible role of AI-2 in biofilm formation in marine environment, and the contribution of AI-2 in biofilm-associated problems such as biofouling and biocorrosion.

Estimation of spatial distribution of quorum sensing signaling in sequencing batch biofilm reactor (SBBR) biofilms.

Quorum sensing (QS) signaling, plays a significant role in regulating formation of biofilms in the nature; however, little information about the occurrence and distribution of quorum sensing molecular in the biofilm of carriers has been reported. In this study, distribution of QS signaling molecules (the acylated homoserine lactones-AHLs, and AI-2), extracellular polymeric substances (EPS) and the mechanical properties in sequencing batch biofilm reactor (SBBR) biofilms have been investigated. Using increased centrifugal force, the biofilms were detached into different fractions. The AHLs ranged from 5.2ng/g to 98.3ng/g in different fractions of biofilms, and N-decanoyl-dl-homoserine lactone (C10-HSL) and N-dodecanoyl-dl-homoserine lactone (C12-HSL) in the biofilms obtained at various centrifugal forces displayed significant differences (p<0.01). Interspecies communication signal autoinducer-2(AI-2) in the biofilms ranged from 79.2ng/g to 98.3ng/g. Soluble EPS and loosely bound EPS content in the different fractions of biofilms displayed significant positive relationship with the distribution of C12-HSL (r=0.86, p<0.05). Furthermore, 49.62% of bacteria in the biofilms were positively related with AHLs with 22.76% was significantly positively (p<0.05) related with AHLs. Biofilm adhesion and compliance was the strongest in the tightly-bound biofilm, the weakest in the supernatant/surface biofilm, which was in accordance with the distribution of C12 HSL(r=0.77, p<0.05) and C10-HSL(r=0.75, p<0.05), respectively. This study addressed on better understanding of possible methods for the improvement of wastewater bio-treatment through biofilm application.

Detection of the Bacterial Quorum-Sensing Signaling Molecules N-Acyl-Homoserine Lactones (HSL) and N-Acyl-Homoserine (HS) with an Enzyme-Linked Immunosorbent Assay (ELISA) and via Ultrahigh-Performance Liquid Chromatography Coupled to Mass Spectrometry (UHPLC-MS).

Quick and reliable quantitative methods requiring low amounts of sample volume are needed for the detection of N-acyl-homoserine lactones (HSL) and their degradation products N-acyl-homoserines (HS) in order to elucidate the occurrence and dynamics of these prevalent quorum-sensing molecules of Gram-negative bacteria in natural samples and laboratory model experiments. A combination of ELISA and UHPLC-MS is presented here which has proven to meet these requirements. Both methods can not only precisely detect and quantify HSLs but also their degradation products HS and thereby enable studying signaling dynamics in quorum sensing, which have been identified to play an essential role in bacterial communication.

Biosensors for the Detection and Quantification of AI-2 Class Quorum-Sensing Compounds.

Intercellular small-molecular-weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum-sensing molecules. These molecules mediate a variety of population-dependent responses including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules have important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused to the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. Ligand-insensitive LuxP mutant FRET protein sensors were also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2 compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of BAI-2.

Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells".

BACKGROUND: Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population. RESULTS: For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control. CONCLUSIONS: The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.

Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition.

High-resolution mass spectrometry (HRMS)-based metabolomics approaches have made significant advances. However, metabolite identification is still a major challenge with significant bottleneck in translating metabolomics data into biological context. In the current study, a liquid chromatography (LC)-HRMS metabolomics method was developed using an all ion fragmentation (AIF) acquisition approach. To increase the specificity in metabolite annotation, four criteria were considered: (i) accurate mass (AM), (ii) retention time (RT), (iii) MS/MS spectrum, and (iv) product/precursor ion intensity ratios. We constructed an in-house mass spectral library of 408 metabolites containing AMRT and MS/MS spectra information at four collision energies. The percent relative standard deviations between ion ratios of a metabolite in an analytical standard vs sample matrix were used as an additional metric for establishing metabolite identity. A data processing method for targeted metabolite screening was then created, merging m/z, RT, MS/MS, and ion ratio information for each of the 413 metabolites. In the data processing method, the precursor ion and product ion were considered as the quantifier and qualifier ion, respectively. We also included a scheme to distinguish coeluting isobaric compounds by selecting a specific product ion as the quantifier ion instead of the precursor ion. An advantage of the current AIF approach is the concurrent collection of full scan data, enabling identification of metabolites not included in the database. Our data acquisition strategy enables a simultaneous mixture of database-dependent targeted and nontargeted metabolomics in combination with improved accuracy in metabolite identification, increasing the quality of the biological information acquired in a metabolomics experiment.

Quantitative determination of AI-2 quorum-sensing signal of bacteria using high performance liquid chromatography-tandem mass spectrometry.

Autoinducer 2 (AI-2), an important bioactive by-product of the LuxS-catalyzed S-ribosylhomocysteine cleavage reaction in the activated-methyl-cycle, has been suggested to serve as a universal intra- and inter-species signaling molecule. The development of reliable and sensitive methods for quantitative determination of AI-2 is highly desired. However, the chemical properties of AI-2 cause difficulty in its quantitative analysis. Herein, we report a high performance liquid chromatography-tandem mass spectrometric method that enables reproducible and sensitive measurement of AI-2 concentrations in complex matrixes. 4,5-Dimethylbenzene-1,2-diamine (DMBDM), an easy-to-obtain commercial reagent, was used for the derivatization treatment. The assay was linear in the concentration range of 1.0-1000ng/mL (R2=0.999) and had a lower limit of quantification of 0.58ng/mL. The method exhibited several advantages, e.g., high selectivity, wide linear response range, and good sensitivity. Furthermore, the effectiveness of the method was further validated through measuring AI-2 concentrations in the cell-free culture supernatant from Escherichia coli wild type.

Involvement of Acylated Homoserine Lactones (AHLs) of Aeromonas sobria in Spoilage of Refrigerated Turbot (Scophthalmus maximus L.).

One quorum sensing strain was isolated from spoiled turbot. The species was determined by 16S rRNA gene analysis and classical tests, named Aeromonas sobria AS7. Quorum-sensing (QS) signals (N-acyl homoserine lactones (AHLs)) were detected by report strains and their structures were further determined by GC-MS. The activity changes of AHLs on strain growth stage as well as the influence of different culture conditions on secretion activity of AHLs were studied by the punch method. The result indicated that strain AS7 could induce report strains to produce typical phenotypic response. N-butanoyl-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), N-dodecanoyl-dl-homoserine lactone (C12-HSL) could be detected. The activities of AHLs were density-dependent and the max secretion level was at pH 8, sucrose culture, 1% NaCl and 32 h, respectively. The production of siderophore in strain AS7 was regulated by exogenous C8-HSL, rather than C6-HSL. Exogenous C4-HSL and C8-HSL accelerated the growth rate and population density of AS7 in turbot samples under refrigerated storage. However, according to the total viable counts and total volatile basic nitrogen (TVB-N) values of the fish samples, exogenous C6-HSL did not cause spoilage of the turbot fillets. In conclusion, our results suggested that QS was involved in the spoilage of refrigerated turbot.

Autoinducer-2 detection among commensal oral streptococci is dependent on pH and boric acid.

Autoinducer-2, considered a universal signaling molecule, is produced by many species of bacteria; including oral strains. Structurally, autoinducer-2 can exist bound to boron (borated autoinducer-2). Functionally, autoinducer-2 has been linked to important bacterial processes such as virulence and biofilm formation. In order to test production of autoinducer-2 by a given bacterial strain, a bioassay using marine bioluminescent bacteria Vibrio harveyi as a reporter for autoinducer-2 has been designed. We hypothesize that pH adjustment and addition of boron are required for optimal bioluminescence and accurate autoinducer-2 detection. Using this reporter strain we tested autoinducer-2 activity from two oral commensal species, Streptococcus gordonii DL1 and Streptococcus oralis 34. Spent broth was collected and adjusted to pH 7.5 and supplemented with boric acid prior to measuring autoinducer- 2 activity. Results show that low pH inhibits bioluminescence of the reporter strain, but pH 7.5 allows for bioluminescence induction and proper readings of autoinducer-2 activity. Addition of boric acid also has a positive effect on bioluminescence allowing for a more sensitive detection of autoinducer-2 activity. Our data suggests that although autoinducer-2 is present in spent broth, low pH and/or low levels of boric acid become an obstacle for proper autoinducer-2 detection. For proper autoinducer-2 detection, we propose a protocol using this bioassay to include pH adjustment and boric acid addition to spent broth. Studies on autoinducer-2 activity in several bacteria species represent an important area of study as this universal signaling molecule is involved in critical bacterial phenotypes such as virulence and biofilm formation.

Quorum sensing activity of Citrobacter amalonaticus L8A, a bacterium isolated from dental plaque.

Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium.

The role of autoinducer-2 in aerobic granulation using alternating feed loadings strategy.

Quorum sensing (QS) plays an important role in aerobic granulation while how QS system regulates the formation of aerobic granules needs further discussion. This study cultivated activated sludge in two identical sequencing batch reactors (R1 and R2) at different influent organic loading rate (OLR) strategies: R1 was operated using constant OLR (around 8.0kg/m(3)d), while R2 was operated at alternating OLR (4.0-17.0kg/m(3)d). Microbial aggregates appeared in R2 on day 19, while the morphology of sludge in R1 changed little compared with the initial sludge. The concentration of autoinducer-2 (AI-2) in R2 showed an ascending trend, along with the increase of cell adhesiveness. The total extracellular polymeric substances (EPS) amount and large molecular weight EPS of R2 rose steadily, which was different from R1. Some bacteria able to self-aggregate and promote EPS secretion were exclusive in R2. A mechanism about aerobic granulation at alternating OLR was proposed.

Bacterial autoinducer-2 detection via an engineered quorum sensing protein.

Autoinducer-2 (AI-2) is a Quorum Sensing (QS) molecule utilized by bacteria in interspecies communication. More recently, it is identified to be vital in regulating QS pathways in a number of human and foodborne pathogens. Methods to detect AI-2 in a rapid and highly sensitive manner can help in the early detection of bacterial infections. Herein, we describe a rapid, selective, and highly sensitive protein based biosensing system employing the Fluorescence Resonance Energy Transfer (FRET) between a protein fusion LuxP-EGFP and 7-diethylamino-3-[N-(2-maleimidoethyl)carbamoyl]coumarin (MDCC). The developed biosensing system, which can detect AI-2 at subnanomolar levels, was successfully applied to detect AI-2 in clinical samples such as saliva and blood serum.

Determination of autoinducer-2 in biological samples by high-performance liquid chromatography with fluorescence detection using pre-column derivatization.

Autoinducer-2 (AI-2), as a small-molecular-weight organic molecule secreted and perceived by various bacteria, enables intra- and inter-species communications. Quantitative determination of AI-2 is essential for exploring the bacterial AI-2-related physiological and biochemical processes. However, current strategies for sensitive detection of AI-2 require sophisticated instruments and complicated procedures. In this work, on the basis of the derivatization of AI-2 with 2,3-diaminonaphthalene, a simple, sensitive and cost-effective high-performance liquid chromatography with fluorescence detector (HPLC-FLD) method is developed for the quantitative detection of AI-2. Under the optimized conditions, this method had a broad linear range of 10-14,000 ng/ml (R(2)=0.9999), and a low detection limit of 1.0 ng/ml. Furthermore, the effectiveness of this approach was further validated through measuring the AI-2 concentrations in the cell-free culture supernatants of both Escherichia coli and Vibrio harveyi.

Quorum sensing activity in Pandoraea pnomenusa RB38.

Strain RB38 was recovered from a former dumping area in Malaysia. MALDI-TOF mass spectrometry and genomic analysis identified strain RB-38 as Pandoraea pnomenusa. Various biosensors confirmed its quorum sensing properties. High resolution triple quadrupole liquid chromatography-mass spectrometry analysis was subsequently used to characterize the N-acyl homoserine lactone production profile of P. pnomenusa strain RB38, which validated that this isolate produced N-octanoyl homoserine lactone as a quorum sensing molecule. This is the first report of the production of N-octanoyl homoserine lactone by P. pnomenusa strain RB38.