N-acyl homoserine lactone production by Klebsiella pneumoniae isolated from human tongue surface.
ABSTRACT: Bacteria communicate by producing quorum sensing molecules called autoinducers, which include autoinducer-1, an N-hexanoyl homoserine lactone (AHL), and autoinducer-2. Bacteria present in the human oral cavity have been shown to produce autoinducer-2, but not AHL. Here, we report the isolation of two AHL-producing Klebsiella pneumoniae strains from the posterior dorsal surface of the tongue of a healthy individual. Spent culture supernatant extracts from K. pneumoniae activated the biosensors Agrobacterium tumefaciens NTL4(pZLR4) and Escherichia coli [pSB401], suggesting the presence of both long and short chain AHLs. High resolution mass spectrometry analyses of these extracts confirmed that both K. pneumoniae isolates produced N-octanoylhomoserine lactone and N-3-dodecanoyl-L-homoserine lactone. To the best of our knowledge, this is the first report of the isolation of K. pneumoniae from the posterior dorsal surface of the human tongue and the production of these AHLs by this bacterium.
Project description:We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium.
Project description:Erwinia amylovora causes fire blight disease of apple, pear, and other members of the Rosaceae. Here we present the first evidence for autoinduction in E. amylovora and a role for an N-acyl-homoserine lactone (AHL)-type signal. Two major plant virulence traits, production of extracellular polysaccharides (amylovoran and levan) and tolerance to free oxygen radicals, were controlled in a bacterial-cell-density-dependent manner. Two standard autoinducer biosensors, Agrobacterium tumefaciens NTL4 and Vibrio harveyi BB886, detected AHL in stationary-phase cultures of E. amylovora. A putative AHL synthase gene, eamI, was partially sequenced, which revealed homology with autoinducer genes from other bacterial pathogens (e.g., carI, esaI, expI, hsII, yenI, and luxI). E. amylovora was also found to carry eamR, a convergently transcribed gene with homology to luxR AHL activator genes in pathogens such as Erwinia carotovora. Heterologous expression of the Bacillus sp. strain A24 acyl-homoserine lactonase gene aiiA in E. amylovora abolished induction of AHL biosensors, impaired extracellular polysaccharide production and tolerance to hydrogen peroxide, and reduced virulence on apple leaves.
Project description:Cell-to-cell communication, or quorum-sensing (QS), systems are employed by bacteria for promoting collective behaviour within a population. An analysis to detect QS signal molecules in 43 species of the Halomonadaceae family revealed that they produced N-acyl homoserine lactones (AHLs), which suggests that the QS system is widespread throughout this group of bacteria. Thin-layer chromatography (TLC) analysis of crude AHL extracts, using Agrobacterium tumefaciens NTL4 (pZLR4) as biosensor strain, resulted in different profiles, which were not related to the various habitats of the species in question. To confirm AHL production in the Halomonadaceae species, PCR and DNA sequencing approaches were used to study the distribution of the luxI-type synthase gene. Phylogenetic analysis using sequence data revealed that 29 of the species studied contained a LuxI homolog. Phylogenetic analysis showed that sequences from Halomonadaceae species grouped together and were distinct from other members of the Gammaproteobacteria and also from species belonging to the Alphaproteobacteria and Betaproteobacteria.
Project description:Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.
Project description:In many species of bacteria, the quorum sensing mechanism is used as a unique communication system which allows them to regulate gene expression and behavior in accordance with their population density. N-Acylhomoserine lactones (AHLs) are known as diffusible autoinducer molecules involved in this communication network. This finding aimed to characterize the production of AHL of a bacterial strain ND04 isolated from a Malaysian waterfall. Strain ND04 was identified as Kluyvera sp. as confirmed by molecular analysis of its 16S ribosomal RNA gene sequence. Kluyvera sp. is closely related to the Enterobacteriaceae family. Chromobacterium violaceum CV026 was used as a biosensor to detect the production of AHL by strain ND04. High resolution triple quadrupole liquid chromatography-mass spectrometry analysis of strain ND04 showed our isolate produced two AHLs which are N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6 HSL) and N-3-oxo-octanoyl-L-homoserine lactone (3-oxo-C8 HSL).
Project description:Myriad proteobacteria use N-acyl homoserine lactone (AHL) molecules as quorum sensing (QS) signals to regulate different physiological functions, including virulence, antibiotic production, and biofilm formation. Many of these proteobacteria possess LuxI/LuxR system as the QS mechanism. Recently, we reported the 3.89 Mb genome of Acinetobacter sp. strain GG2. In this work, the genome of this long chain AHL-producing bacterium was unravelled which led to the molecular characterization of luxI homologue, designated as aciI. This 552 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was ?20.5 kDa and is highly similar to several autoinducer proteins of LuxI family among Acinetobacter species. To verify the AHL synthesis activity of this protein, high-resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-dodecanoyl-homoserine lactone and 3-hydroxy-dodecanoyl-homoserine lactone from induced E. coli harboring the recombinant AciI. Our data show for the first time, the cloning and characterization of the luxI homologue from Acinetobacter sp. strain GG2, and confirmation of its AHLs production. These data are of great significance as the annotated genome of strain GG2 has provided a valuable insight in the study of autoinducer molecules and its roles in QS mechanism of the bacterium.
Project description:We have investigated the quorum sensing control in Aeromonas veronii MTCC 3249, originally isolated as A. culicicola from the midgut of Culex quinquefasciatus. Based on biosensor assays, the bacterium showed constant production of multiple acyl-homoserine lactones (AHLs) with increasing cell-density. The luxRI gene homologs, acuR (A. culicicola transcriptional Regulator) and acuI (A. culicicola autoInducer) were successfully amplified by inverse-PCR. Sequence analysis indicated acuRI were divergent from all known quorum sensing gene homologs in Aeromonas. Two localized regions in the C-terminal autoinducer binding domain of acuR showed indels suggesting variations in autoinducer specificity. Further, only a single copy of the quorum sensing genes was detected, suggesting a tight regulation of mechanisms under its control. Chromatography and further chemical analysis identified two AHLs in the culture supernatant: 6-carboxy-HHL (homoadipyl homoserine lactone), a novel AHL, and N-tetradecanoylhomoserine lactone. The existence of a potentially variant quorum sensing system might therefore, reflect in some way the ecological strategies adopted by this bacterium in the mosquito midgut.
Project description:Quorum sensing is a unique bacterial communication system which permits bacteria to synchronize their behaviour in accordance with the population density. The operation of this communication network involves the use of diffusible autoinducer molecules, termed N-acylhomoserine lactones (AHLs). Serratia spp. are well known for their use of quorum sensing to regulate the expression of various genes. In this study, we aimed to characterized the AHL production of a bacterium designated as strain RB-25 isolated from a former domestic waste landfill site. It was identified as Serratia fonticola using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis and this was confirmed by 16S ribosomal DNA sequencing. High resolution triple quadrupole liquid chromatography-mass spectrometry analysis of S. fonticola strain RB-25 spent culture supernatant indicated the existence of three AHLs namely: N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl) homoserine-lactone (3-oxo-C6 HSL). This is the first report of the production of these AHLs in S. fonticola.
Project description:An N-acylhomoserine lactone (AHL)-degrading bacterial strain, L62, was isolated from a sample of fermentation brine of Chinese soya sauce by using rich medium agar supplemented with soya sauce (10% v/v). L62, a rod-shaped Gram positive bacterium with amylolytic activity, was phylogentically related to Bacillus sonorensis by 16S ribosomal DNA and rpoB sequence analyses. B. sonorensis L62 efficiently degraded N-3-oxohexanoyl homoserine lactone and N-octanoylhomoserine lactone. However, the aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of AHLs, was not detected in L62, suggesting the presence of a different AHL-degrading gene in L62. To the best of our knowledge, this is the first report of AHL-degrading B. sonorensis from soya sauce liquid state fermentation.
Project description:A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of beta-galactosidase upon addition of exogenous AHLs. We have shown that beta-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (-80 degrees C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.