The quorum-hindered transcription factor YenR of Yersinia enterocolitica inhibits pheromone production and promotes motility via a small non-coding RNA

The YenR and YenI proteins of Yersinia enterocolitica resemble the quorum sensing proteins LuxR and LuxI of Vibrio fischeri. Apo-YenR activated a gene, designated yenS, that lies adjacent to and divergent from yenR. YenR-dependent expression of yenS was inhibited by endogenous or exogenous 3-oxohexanoylhomoserine lactone (OHHL) a pheromone made by YenI. Purified apo-YenR bound non-cooperatively to two 20-nucleotide sites that lie upstream of yenS. Binding occurred in the absence of (OHHL), and YenR was largely released from the DNA by this pheromone. yenS encoded two non-translated RNAs 169 and 105 nucleotides long that share the same 5' end but have different 3' ends. One or both RNAs inhibited the translation and accumulation of the yenI mRNA by binding to a region that overlaps the YenI start codon. A mutation in yenI strongly stimulated swarming motility on the surface of semi-solid agar, while exogenous OHHL completely suppressed this phenotype. Hypermotility in yenI mutants was also suppressed by mutations in yenR or yenS, suggesting that YenS plays a direct, stimulatory role in swarming motility.     

PpoR is a conserved unpaired LuxR solo of Pseudomonas putida which binds N-acyl homoserine lactones

Background  

Only a small number of Pseudomonas putida strains possess the typical N-acyl homoserine lactone quorum sensing system (AHL QS) that consists of a modular LuxR family protein and its cognate LuxI homolog that produces the AHL signal. Moreover, AHL QS systems in P. putida strains are diverse in the type of AHLs they produce and the phenotypes that they regulate.     

N‐ACYL HOMOSERINE LACTONe LACTONASE,AiiA, INACTIVATION OF QUORUM‐SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE1

Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N‐acyl homoserine lactone (AHL) bacterial quorum‐sensing (QS) signals and alter QS‐regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal‐mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR‐stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal‐mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL‐producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.     

Non‐native acylated homoserine lactones reveal that LuxIR quorum sensing promotes symbiont stability

Quorum sensing, a group behaviour coordinated by a diffusible pheromone signal and a cognate receptor, is typical of bacteria that form symbioses with plants and animals. LuxIR‐type N‐acyl L‐homoserine (AHL) quorum sensing is common in Gram‐negative Proteobacteria, and many members of this group have additional quorum‐sensing networks. The bioluminescent symbiont Vibrio fischeri encodes two AHL signal synthases: AinS and LuxI. AinS‐dependent quorum sensing converges with LuxI‐dependent quorum sensing at the LuxR regulatory element. Both AinS‐ and LuxI‐mediated signalling are required for efficient and persistent colonization of the squid host, Euprymna scolopes. The basis of the mutualism is symbiont bioluminescence, which is regulated by both LuxI‐ and AinS‐dependent quorum sensing, and is essential for maintaining a colonization of the host. Here, we used chemical and genetic approaches to probe the dynamics of LuxI‐ and AinS‐mediated regulation of bioluminescence during symbiosis. We demonstrate that both native AHLs and non‐native AHL analogues can be used to non‐invasively and specifically modulate induction of symbiotic bioluminescence via LuxI‐dependent quorum sensing. Our data suggest that the first day of colonization, during which symbiont bioluminescence is induced by LuxIR, is a critical period that determines the stability of the V. fischeri population once symbiosis is established.     

Robust and sensitive control of a quorum‐sensing circuit by two interlocked feedback loops

The quorum‐sensing (QS) response of Vibrio fischeri involves a rapid switch between low and high induction states of the lux operon over a narrow concentration range of the autoinducer (AI) 3‐oxo‐hexanoyl‐L ‐homoserine lactone. In this system, LuxR is an AI‐dependent positive regulator of the lux operon, which encodes the AI synthase. This creates a positive feedback loop common in many bacterial species that exhibit QS‐controlled gene expression. Applying a combination of modeling and experimental analyses, we provide evidence for a LuxR autoregulatory feedback loop that allows LuxR to increase its concentration in the cell during the switch to full lux activation. Using synthetic lux gene fragments, with or without the AI synthase gene, we show that the buildup of LuxR provides more sensitivity to increasing AI, and promotes the induction process. Elevated LuxR levels buffer against spurious variations in AI levels ensuring a robust response that endows the system with enhanced hysteresis. LuxR autoregulation also allows for two distinct responses within the same cell population.     

Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics

In the process of evaluating the role of acylated homoserine lactones (AHLs) in food-spoiling Gram-negative bacteria, we have combined a range of bacterial AHL monitor systems to determine the AHL-profile and the kinetics of AHL-production. AHL production from 148 strains of Enterobacteriaceae isolated from foods was tested using Escherichia coli pSB403 (LuxR), Agrobacterium tumefaciens A136 (TraR) and both induction and inhibition of Chromobacterium violaceum CV026 (CviR). All strains except one was found to produce AHL(s). In no case could a single monitor system identify more than 64% of the Enterobacteriaceae as AHL-producers, showing that the simultaneous use of monitor strains is required in the process of screening bacterial populations for AHL-production. AHLs from 20 selected strains were profiled by thin layer chromatography. Most strains produced more than one AHL with 3-N-oxo-hexanoyl homoserine lactone being the most prominent. It was found that the simultaneous use of monitor strains in the top-layer was necessary for the detection of (presumably) all the AHLs. An agar well-diffusion assay based on A. tumefaciens pDZLR4 was used for quantifying AHLs from bacterial supernatants and enabled an assessment of the kinetics of AHL-production of 3 strains (Serratia proteamaculans strain B5a, Erwinia carotovora ATCC 39048 and V. fischeri strain MJ-1). As expected, the production of AHL (OHHL) and luminescence in Vibrio fischeri strain MJ-1 increased faster than growth indicating up-regulation of the AHL regulated phenotype and auto-induction of AHL production. In contrast, production kinetics of AHL (OHHL) in the two Enterobacteriaceae indicated lack of auto-induction.     

Inhibitors of the Pseudomonas aeruginosa quorum‐sensing regulator,QscR

QscR is a quorum‐sensing (QS) signal receptor that controls expression of virulence genes in the prevalent opportunistic pathogen, Pseudomonas aeruginosa. Unlike the previously reported LuxR‐type QS receptor proteins, that is, LasR and TraR, QscR can be obtained as an apo‐protein that can reversibly form an active complex in vitro with its cognate signal molecule, 3‐oxododecanoyl‐homoserine lactone (3OC12‐HSL), and subsequently bind to target promoter DNA sequences. To search for potential QS inhibitors, an in vitro gel retardation assay was developed using the purified QscR. Both the in vitro assay and the in vivo cell‐based assay using QscR‐overproducing recombinant strains were applied in the screening process. Furanones were chosen for testing the activity as QS inhibitors because they have been reported to strongly inhibit expression of QS‐related genes in Agrobacterium tumefaciens. Among more than a hundred furanones tested, three compounds showed strong and dose‐dependent inhibitory effects on QscR in both assays. One compound in particular, designated as F2, could completely inhibit the 3OC12‐HSL‐dependent QscR activity in vitro at a concentration of 50‐fold molar excess over 3OC12‐HSL. However, with the furanones F3 and F4, which are structurally similar to F2 but with a nitro group instead of the amine moiety, significantly decreased activities were observed. These results suggest that (i) the in vitro assay is a sensitive and reliable tool for screening QS inhibitors, and (ii) furanones are potentially important QS inhibitors for many LuxR‐type receptor proteins. Biotechnol. Bioeng. 2010; 106: 119–126. © 2010 Wiley Periodicals, Inc.     

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.     

Homoserine and quorum‐sensing acyl homoserine lactones as alternative sources of threonine: a potential role for homoserine kinase in insect‐stage Trypanosoma brucei

De novo synthesis of threonine from aspartate occurs via the β‐aspartyl phosphate pathway in plants, bacteria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: homoserine kinase (HSK) and threonine synthase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK specifically phosphorylates L‐homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, homoserine, homoserine lactone and certain acyl homoserine lactones (AHLs) were found to substitute for threonine in growth media for wild‐type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinformatic studies predict that tsetse endosymbionts possess part (up to homoserine in Wigglesworthia glossinidia) or all of the β‐aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3‐oxohexanoylhomoserine lactone which also supports trypanosome growth. We propose that T. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.     

Exogenous N‐acyl‐homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants

In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N‐acyl‐homoserine lactone (AHL)‐mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL‐producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL‐producing plants compared with wild‐type plants. The present data indicate that plant‐produced AHLs enhance disease resistance against this pathogen. Subsequent RNA‐sequencing analysis showed that the exogenous addition of AHLs up‐regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL‐producing and wild‐type plants were determined by quantitative real‐time polymerase chain reaction. These data showed that plant‐produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant‐produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.     

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.     

The LuxR family protein SpnR functions as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing in Serratia marcescens

Serratia marcescens SS-1 produces at least four N-acylhomoserine lactones (AHLs) which were identified using high-resolution mass spectrometry and chemical synthesis, as N-(3-oxohexanoyl) homo-serine lactone (3-oxo-C6-HSL), N-hexanoyl- (C6-HSL), N-heptanoyl (C7-HSL) and N-octanoyl- (C8-HSL) homoserine lactone. These AHLs are synthesized via the LuxI homologue SpnI, and regulate via the LuxR homologue SpnR, the production of the red pigment, prodigiosin, the nuclease, NucA, and a biosurfactant which facilitates surface translocation. spnR overexpression and spnR gene deletion show that SpnR, in contrast to most LuxR homologues, acts as a negative regulator. spnI overexpression, the provision of exogenous AHLs and spnI gene deletion suggest that SpnR is de-repressed by 3-oxo-C6-HSL. In addition, long chain AHLs antagonize the biosurfactant-mediated surface translocation of S. marcescens SS-1. Upstream of spnI there is a gene which we have termed spnT. spnI and spnT form an operon and although database searches failed to reveal any spnT homologues, overexpression of this novel gene negatively affected both sliding motility and prodigiosin production.     

Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.