n-hexanoyl-l-homoserine-lactone and homoserine-lactone

In the phylum of Proteobacteria, quorum sensing (QS) system is widely driven by synthesis and response of N-acyl homoserine lactone (AHL) signalling molecules. AHL is synthesized by LuxI homologue and sensed by LuxR homologue. Once the AHL concentration achieves a threshold level, it triggers the regulation of target genes. In this study, QS activity of Citrobacter amalonaticus strain YG6 which was isolated from clams was investigated. In order to characterise luxI/R homologues, the genome of C. amalonaticus strain YG6 (4.95 Mbp in size) was sequenced using Illumina MiSeq sequencer. Through in silico analysis, a pair of canonical luxI/R homologues and an orphan luxR homologue were identified and designated as camI, camR, and camR2, respectively. A putative lux box was identified at the upstream of camI. The camI gene was cloned and overexpressed in E. coli BL21 (DE3)pLysS. High-resolution triple quadrupole liquid chromatography mass spectrometry (LC-MS/MS) analysis verified that the CamI is a functional AHL synthase which produced multiple AHL species, namely N‑butyryl‑l‑homoserine lactone (C4-HSL), N‑hexanoyl‑l‑homoserine lactone (C6-HSL), N‑octanoyl‑l‑homoserine lactone (C8-HSL), N‑tetradecanoyl‑l‑homoserine lactone (C14-HSL) and N‑hexadecanoyl‑l‑homoserine lactone (C16-HSL) in C. amalonaticus strain YG6 and camI gene in recombinant E. coli BL21(DE3)pLysS. To our best knowledge, this is the first functional study report of camI as well as the first report describing the production of C14-HSL by C. amalonaticus.

Topics: 4-Butyrolactone; Acyl-Butyrolactones; Bacterial Proteins; Base Sequence; Carboxylic Ester Hydrolases; Citrobacter; DNA, Bacterial; Escherichia coli; Genes, Bacterial; Homoserine; Lactones; Quorum Sensing

Several model plants are known to respond to bacterial quorum sensing molecules with altered root growth and gene expression patterns and induced resistance to plant pathogens. These compounds may represent novel elicitors that could be applied as seed primers to enhance cereal crop resistance to pathogens and abiotic stress and to improve yields. We investigated whether the acyl-homoserine lactone N-hexanoyl-L-homoserine lactone (C6-HSL) impacted winter wheat (Triticum aestivum L.) seed germination, plant development and productivity, using two Ukrainian varieties, Volodarka and Yatran 60, in both in vitro experiments and field trials. In vitro germination experiments indicated that C6-HSL seed priming had a small but significant positive impact on germination levels (1.2x increase, p < 0.0001), coleoptile and radicle development (1.4x increase, p < 0.0001). Field trials over two growing seasons (2015-16 and 2016-17) also demonstrated significant improvements in biomass at the tillering stage (1.4x increase, p < 0.0001), and crop structure and productivity at maturity including grain yield (1.4-1.5x increase, p < 0.0007) and quality (1.3x increase in good grain, p < 0.0001). In some cases variety effects were observed (p ≤ 0.05) suggesting that the effect of C6-HSL seed priming might depend on plant genetics, and some benefits of priming were also evident in F1 plants grown from seeds collected the previous season (p ≤ 0.05). These field-scale findings suggest that bacterial acyl-homoserine lactones such as C6-HSL could be used to improve cereal crop growth and yield and reduce reliance on fungicides and fertilisers to combat pathogens and stress.

Topics: 4-Butyrolactone; Acyl-Butyrolactones; Bacteria; Bacterial Proteins; Biomass; Crop Production; Germination; Plant Development; Quorum Sensing; Seasons; Seeds; Triticum

Two lab-scale nitrifying sequencing batch biofilm reactors, with (SBBR_CN) or without the addition of organics (SBBR_N), were operated to investigate potential roles of acyl homoserine lactone (AHL) based quorum sensing. AHLs of N-[(RS)-3-Hydroxybutyryl]-L-homoserine lactone, N-hexanoyl-L-homoserine lactone (C

Topics: 4-Butyrolactone; Biofilms; Carbon; Quorum Sensing

Investigating microbial interactions from an ecological perspective is a particularly fruitful approach to unveil both new chemistry and bioactivity. Microbial predator-prey interactions in particular rely on natural products as signal or defense molecules. In this context, we identified a grazing-resistant

Topics: 4-Butyrolactone; Anti-Bacterial Agents; Bacterial Proteins; Biological Products; Dictyostelium; Drug Synergism; Genome, Bacterial; Methicillin-Resistant Staphylococcus aureus; Mupirocin; Pseudomonas; Quorum Sensing; Staphylococcal Infections

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.

Topics: 4-Butyrolactone; Bacterial Proteins; Citrobacter; Dental Plaque; DNA, Bacterial; High-Throughput Nucleotide Sequencing; Homoserine; Humans; Lactones; Phylogeny; Quorum Sensing; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Thermaerobacter marianensis is an extremely thermophilic bacterium, which was isolated from the Mariana Trench, with an optimal growth temperature of approximately 75 °C. N-Acylhomoserine lactone (AHL) is a quorum-sensing signal molecule used by many gram-negative bacteria. Here, we report the identification of an AHL-degrading gene homolog (designated aiiT) in the genome of T. marianensis JCM 10246. AiiT has 59.7%, 21.2%, and 11.2% identity to AhlS from Solibacillus silvestris, AiiA from Bacillus cereus, and AidC from Chryseobacterium sp., respectively. Homologs of aiiT were also found in Thermaerobacter nagasakiensis, T. composti, and T. subterraneus. A purified AiiT-maltose binding fusion showed high AHL-degrading activity against N-hexanoyl-L-homoserine lactone, N-octanoyl-L-homoserine lactone, and N-decanoyl-L-homoserine lactone at temperatures ranging from 40 to 80 °C. HPLC analysis revealed that AiiT functions as an AHL-lactonase that catalyzes AHL ring opening by hydrolyzing lactones. AiiT displayed maximal activity at high temperatures (60-80 °C) and showed higher thermostability than other AHL lactonases.

Topics: 4-Butyrolactone; Bacillus; Bacillus cereus; Bacteria, Aerobic; Carboxylic Ester Hydrolases; Enzyme Stability; Homoserine; Hydrolysis; Lactones; Planococcaceae; Quorum Sensing; Substrate Specificity; Temperature

A number of gram-negative bacteria have a quorum-sensing system and produce the N-acylhomoserine lactone (AHL) as a signal molecule. Pseudomonas chlororaphis subsp. aurantiaca StFRB508 produces one of the phenazine derivatives, phenazine-1-carboxylic acid (PCA). Whole-genome sequencing of StFRB508 revealed the presence of two sets of AHL-synthase and AHL-receptor gene, phzIR and aurIR. The mutation of phzI drastically decreased PCA production, but the mutation of aurI did not affect PCA production. The phzI and aurI double mutant did not show any PCA production. StFRB508 produces three major AHLs, N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). As the results of TLC analysis, PhzI mainly catalyzes the biosynthesis of 3-hydroxy-C6-HSL, and AurI catalyzes the biosynthesis of C4-HSL and C6-HSL. PCA production in the phzI and aurI double mutant was restored by exogenous AHLs and the most active AHL was 3-hydroxy-C6-HSL. StFRB508 showed high inhibitory activity of the development of mycelia of plant pathogenic fungi, Fusarium oxysporum f. sp. conglutinans. However, the phzI and aurI double mutant could not inhibit the development of mycelia. These results demonstrated that the multiple quorum-sensing system play an important role in PCA production and antifungal activity in StFRB508.

Topics: 4-Butyrolactone; Anti-Bacterial Agents; Antifungal Agents; Bacterial Proteins; Computer Simulation; Fusarium; Genome, Bacterial; Phenazines; Pseudomonas; Quorum Sensing

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acyl-homoserine lactones produced by this strain (N-butanoyl-L-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.

Topics: 4-Butyrolactone; Ascomycota; Carboxylic Ester Hydrolases; Gene Expression Regulation, Bacterial; Phenazines; Plasmids; Pseudomonas; Rhizoctonia

N-acyl-L-homoserine lactone (AHL) mediated quorum sensing is a widespread communication system in gram-negative bacteria which regulates a wide range of target genes in a cell density-dependent manner. Although Escherichia coli is not capable of synthesizing AHL molecules because it lacks an AHL synthase encoding gene, it does produce a predicted AHL receptor of the LuxR family, named SdiA. In this work, we used a promoter trap library to screen for E. coli MG1655 promoters whose expression was affected by synthetic N-hexanoyl-L-homoserine lactone (C6-HSL), and we identified six upregulated and nine downregulated promoters, which also responded to synthetic 3-oxo-N-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). The AHL responsiveness of these promoters was eliminated by knock-out of sdiA, and was temperature dependent, since the identified promoters showed a response at 30 degrees C but not, or only very weakly at 37 degrees C. In addition, in line with the observed induction of gadA encoding a glutamate decarboxylase, we could demonstrate an increased acid tolerance of E. coli upon exposure to C6-HSL. In conclusion, our work shows that E. coli has the capacity to alter its pattern of gene expression and its phenotypical properties in response to AHLs by means of the AHL responsive transcriptional regulator SdiA.

Topics: 4-Butyrolactone; Acids; Cell Communication; Down-Regulation; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Library; Glutamate Decarboxylase; Green Fluorescent Proteins; Membrane Proteins; Promoter Regions, Genetic; Time Factors; Trans-Activators; Up-Regulation

A rapid, simple and selective method involving direct separation by gas chromatography (GC) with electron ionization mass spectrometry (EI-MS) was employed to determine some N-acylhomoserine lactones (AHLs). Using GC/EI-MS, simultaneous separation and characterization of AHLs were possible without prior derivatization. Informative fragmentation patterns were obtained to identify the structures of N-acyl chains of AHLs. Electron ionization resulted in a common fragmentation pattern with the most abundant ion at m/z 143 and other minor peaks at m/z 71, 57, and 43. The presence of AHLs in extracts of Burkholderia cepacia strains was achieved in selected ion monitoring mode by using the prominent fragment at m/z 143.

Topics: 4-Butyrolactone; Acylation; Burkholderia cepacia; Gas Chromatography-Mass Spectrometry; Homoserine

Cell to cell signaling systems in Gram-negative bacteria rely on small diffusible molecules such as the N-acylhomoserine lactones (AHL). These compounds are involved in the production of antibiotics, exoenzymes, virulence factors and biofilm formation. They belong to the class of furanone derivatives which are frequently found in nature as pheromones, flavor compounds or secondary metabolites. To obtain more information on the relation between molecular structure and quorum sensing, we tested a variety of natural and chemically synthesized furanones for their ability to interfere with the quorum sensing mechanism using a quantitative bioassay with Chromobacterium violaceum CV026 for antagonistic and agonistic action. We were looking at the following questions: 1) Do these compounds affect growth? 2) Do these compounds activate the quorum sensing system of C. violaceum CV026? 3) Do these compounds inhibit violacein formation induced by the addition of the natural inducer N-hexanoylhomoserine lactone (HHL)? 4) Do these compounds enhance violacein formation in presence of HHL?. The naturally produced N-acylhomoserine lactones showed a strong non-linear concentration dependent influence on violacein production in C. violaceum with a maximum at 3.7*10-8 M with HHL. Apart from the N-acylhomoserine lactones only one furanone (emoxyfurane) was found to simulate N-acylhomoserine lactone activity and induce violacein formation. The most effective substances acting negatively both on growth and quorum sensing were analogs and intermediates in synthesis of the butenolides from Streptomyces antibioticus.. As the regulation of many bacterial processes is governed by quorum sensing systems, the finding of natural and synthetic furanones acting as agonists or antagonists suggests an interesting tool to control and handle detrimental AHL induced effects. Some effects are due to general toxicity; others are explained by a competitive interaction for LuxR proteins. For further experiments it is important to be aware of the fact that quorum sensing active compounds have non-linear effects. Inducers can act as inhibitors and inhibitors might be able to activate or enhance the quorum sensing system depending on chemical structure and concentration levels.

Topics: 4-Butyrolactone; Chromobacterium; Furans; Indoles; Signal Transduction; Structure-Activity Relationship

The distribution of quorum-sensing genes among strains from seven genomovars of the Burkholderia cepacia complex was examined by PCR. cepR and cepI were amplified from B. cepacia genomovars I and III, B. stabilis, and B. vietnamiensis. cepR was also amplified from B. multivorans and B. cepacia genomovar VI. bviIR were amplified from B. vietnamiensis. All genomovars produced N-octanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. B. vietnamiensis and B. cepacia genomovar VII produced additional N-acyl-L-homoserine lactones.

Topics: 4-Butyrolactone; Amino Acid Sequence; Bacterial Proteins; Base Sequence; Burkholderia; Burkholderia cepacia; Genes, Bacterial; Homoserine; Lactones; Ligases; Molecular Sequence Data; Sequence Homology, Amino Acid

Seventy strains of the Burkholderia cepacia complex, which currently comprises six genomic species, were tested for their ability to produce N-acylhomoserine lactone (AHL) signal molecules. Using thin layer chromatography in conjunction with a range of AHL biosensors, we show that most strains primarily produce two AHLs, namely N-octanoylhomoserine lactone (C8-HSL) and N-hexanoylhomoserine lactone (C6-HSL). Furthermore, some strains belonging to B. vietnamiensis (genomovar V) produce additional long chain AHL molecules with acyl chains ranging from C10 to C14. For B. vietnamiensis R-921 the structure of the most abundant long chain AHL was confirmed as N-decanoylhomoserine lactone (C10-HSL) by liquid chromatography-mass spectrometry (LC-MS) in combination with total chemical synthesis. Interestingly, a number of strains, most notably all representatives of B. multivorans (genomovar II), did not produce AHLs at least under the growth conditions used in this study. All strains were also screened for the production of extracellular lipase, chitinase, protease, and siderophores. However, no correlation between the AHL production and the synthesis of these exoproducts was apparent. Southern blot analysis showed that all the B. cepacia complex strains investigated, including the AHL-negative strains, possess genes homologous to the C8-HSL synthase cepI and to cepR, which encodes the cognate receptor protein. The nucleotide sequence of the cepI and cepR genes from one representative strain from each of the six genomovars was determined. Furthermore, the cepI genes from the different genomovars were expressed in Escherichia coli and it is demonstrated that all genes encode functional proteins that direct the synthesis of C8-HSL and C6-HSL. Given that cepI from the B. multivorans strain encodes a functional AHL synthase, yet detectable levels of AHLs were not produced by the wild-type, this suggests that additional regulatory functions may be present in members of this genomovar that negatively affect expression of cepI.

Topics: 4-Butyrolactone; Acylation; Amino Acid Sequence; Bacterial Proteins; Burkholderia cepacia; Burkholderia Infections; Chitinases; Chromosome Mapping; Endopeptidases; Fatty Acids; Genes, Bacterial; Homoserine; Humans; Lactones; Ligases; Lipase; Molecular Sequence Data; Nuclear Localization Signals; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Siderophores

In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylhomoserine lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.

Topics: 4-Butyrolactone; Acetyl Coenzyme A; Bacterial Proteins; Cerulenin; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Kinetics; Ligases; Mass Spectrometry; Pseudomonas aeruginosa; Recombinant Proteins; S-Adenosylmethionine; Substrate Specificity; Transcription Factors

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21 degrees C. Biofilm formation was maximal ca. 20-50 h after inoculation of the liquid medium and as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spend culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C6) exogenous homoserines can trigger such response in P. fluorescens, its own signal substance is likely to possess a longer (> C8) fatty acyl chain.

Topics: 4-Butyrolactone; Bacterial Adhesion; Biofilms; Culture Media; Polysaccharide-Lyases; Polysaccharides, Bacterial; Pseudomonas fluorescens