Acyl-homoserine Lactone Quorum-sensing Signals Research Articles

A FadR-Type Regulator Activates the Biodegradation of Polycyclic Aromatic Hydrocarbons by Mediating Quorum Sensing in Croceicoccus naphthovorans Strain PQ-2.

Bacteria employ multiple transcriptional regulators to orchestrate cellular responses to adapt to constantly varying environments. The bacterial biodegradation of polycyclic aromatic hydrocarbons (PAHs) has been extensively described, and yet, the PAH-related transcriptional regulators remain elusive. In this report, we identified an FadR-type transcriptional regulator that controls phenanthrene biodegradation in Croceicoccus naphthovorans strain PQ-2. The expression of fadR in C. naphthovorans PQ-2 was induced by phenanthrene, and its deletion significantly impaired both the biodegradation of phenanthrene and the synthesis of acyl-homoserine lactones (AHLs). In the fadR deletion strain, the biodegradation of phenanthrene could be recovered by supplying either AHLs or fatty acids. Notably, FadR simultaneously activated the fatty acid biosynthesis pathway and repressed the fatty acid degradation pathway. As intracellular AHLs are synthesized with fatty acids as substrates, boosting the fatty acid supply could enhance AHL synthesis. Collectively, these findings demonstrate that FadR in C. naphthovorans PQ-2 positively regulates PAH biodegradation by controlling the formation of AHLs, which is mediated by the metabolism of fatty acids. IMPORTANCE Master transcriptional regulation of carbon catabolites is extremely important for the survival of bacteria that face changes in carbon sources. Polycyclic aromatic hydrocarbons (PAHs) can be utilized as carbon sources by some bacteria. FadR is a well-known transcriptional regulator involved in fatty acid metabolism; however, the connection between FadR regulation and PAH utilization in bacteria remains unknown. This study revealed that a FadR-type regulator in Croceicoccus naphthovorans PQ-2 stimulated PAH biodegradation by controlling the biosynthesis of the acyl-homoserine lactone quorum-sensing signals that belong to fatty acid-derived compounds. These results provide a unique perspective for understanding bacterial adaptation to PAH-containing environments.

Positive Autoregulation of an Acyl-Homoserine Lactone Quorum-Sensing Circuit Synchronizes the Population Response.

ABSTRACTMany proteobacteria utilize acyl-homoserine lactone quorum-sensing signals. At low population densities, cells produce a basal level of signal, and when sufficient signal has accumulated in the surrounding environment, it binds to its receptor, and quorum-sensing-dependent genes can be activated. A common characteristic of acyl-homoserine lactone quorum sensing is that signal production is positively autoregulated. We have examined the role of positive signal autoregulation in Pseudomonas aeruginosa. We compared population responses and individual cell responses in populations of wild-type P. aeruginosa to responses in a strain with the signal synthase gene controlled by an arabinose-inducible promoter so that signal was produced at a constant rate per cell regardless of cell population density. At a population level, responses of the wild type and the engineered strain were indistinguishable, but the responses of individual cells in a population of the wild type showed greater synchrony than the responses of the engineered strain. Although sufficient signal is required to activate expression of quorum-sensing-regulated genes, it is not sufficient for activation of certain genes, the late genes, and their expression is delayed until other conditions are met. We found that late gene responses were reduced in the engineered strain. We conclude that positive signal autoregulation is not a required element in acyl-homoserine lactone quorum sensing, but it functions to enhance synchrony of the responses of individuals in a population. Synchrony might be advantageous in some situations, whereas a less coordinated quorum-sensing response might allow bet hedging and be advantageous in other situations.

Production and Degradation ofN-Acylhomoserine Lactone Quorum Sensing Signal Molecules in Bacteria Isolated from Activated Sludge

N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n = 107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n = 46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.

Coordinated, long-range, solid substrate movement of the purple photosynthetic bacterium Rhodobacter capsulatus.

The long-range movement of Rhodobacter capsulatus cells in the glass-agar interstitial region of borosilicate Petri plates was found to be due to a subset of the cells inoculated into plates. The macroscopic appearance of plates indicated that a small group of cells moved in a coordinated manner to form a visible satellite cluster of cells. Satellite clusters were initially separated from the point of inoculation by the absence of visible cell density, but after 20 to 24 hours this space was colonized by cells apparently shed from a group of cells moving away from the point of inoculation. Cell movements consisted of flagellum-independent and flagellum-dependent motility contributions. Flagellum-independent movement occurred at an early stage, such that satellite clusters formed after 12 to 24 hours. Subsequently, after 24 to 32 hours, a flagellum-dependent dispersal of cells became visible, extending laterally outward from a line of flagellum-independent motility. These modes of taxis were found in several environmental isolates and in a variety of mutants, including a strain deficient in the production of the R. capsulatus acyl-homoserine lactone quorum-sensing signal. Although there was great variability in the direction of movement in illuminated plates, cells were predisposed to move toward broad spectrum white light. This predisposition was increased by the use of square plates, and a statistical analysis indicated that R. capsulatus is capable of genuine phototaxis. Therefore, the variability in the direction of cell movement was attributed to optical effects on light waves passing through the plate material and agar medium.

Bacillus marcorestinctum sp. nov., a Novel Soil Acylhomoserine Lactone Quorum-Sensing Signal Quenching Bacterium

A Gram-positive, facultatively anaerobic, endospore-forming and rod-shaped bacterium was isolated from soil samples and designated strain LQQ. This organism strongly quenches the acylhomoserine lactone quorum-sensing signal. The LQQ strain exhibits phenotypic characteristics consistent with its classification in the genus Bacillus. It is positive in catalase and no special growth factor is needed. It uses glucose as sole carbon source. The DNA G + C content is 39.8 mol %. The closest relatives based on the 16S rRNA gene sequence are Bacillus anthracis, Bacillus thuringiensis, and Brevibacillus brevis (syn. Bacillus brevis) with the similarity of 96.5%. The DNA–DNA hybridization data indicates a low level of genomic relatedness with the relative type strains of Bacillus thuringiensis (6.1%), Bacillus anthracis (10.5%) and Brevibacillus brevis (8.7%). On the basis of the phenotypic and phylogenetic data together with the genomic distinctiveness, the LQQ strain represents a novel species of the genus Bacillus, for which the name Bacillus marcorestinctum sp. nov. is proposed. The type strain is LQQT.

Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal

Conjugative transfer of the Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of TraR and its signal N-(3-oxo-octanoyl)-L-homoserine lactone. This system is, in turn, controlled by the conjugative opines produced by crown gall tumors induced on plants by the bacteria. Using nonpolar traI mutants, we examined the kinetics of induction of conjugative transfer in response to exogenous acyl-homoserine lactone. In the absence of the antiactivator TraM, onset of induction of transfer requires about 30 min, 15 to 20 min of which is needed for expression and construction of the conjugative apparatus. TraM delays the onset of conjugation by 30 min. While the rate of development of conjugative competence was not significantly affected by levels of TraR, maximum efficiencies of transfer were correlated with amounts of the activator in the donors. Donors harboring Ti plasmids lacking TraM were fully induced by the quormone at concentrations as low as 100 pM. TraM raised the concentration of signal required for maximum activity to 1 nM. Donors grown in batch culture retained conjugative competence following signal removal, even when in stationary phase. However, donors kept in balanced growth rapidly lost transfer ability following signal removal. Loss of transfer was mirrored by a decrease in levels of active TraR. Decreases in TraR activity and conjugative competence could be accounted for by dilution associated with cell division, suggesting that while induction of Ti plasmid conjugation is an active process, the cells lack a mechanism for disassembling the conjugative apparatus when signals become limiting.

A structurally unrelated mimic of a Pseudomonas aeruginosa acyl-homoserine lactone quorum-sensing signal.

The pathogenic bacterium Pseudomonas aeruginosa uses acyl-homoserine lactone quorum-sensing signals to coordinate the expression of a battery of virulence genes in a cascade of regulatory events. The quorum-sensing signal that triggers the cascade is N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), which interacts with two signal receptor-transcription factors, LasR and QscR. This signal is base labile, and it is degraded by mammalian PON lactonases. We have identified a structurally unrelated triphenyl mimic of 3OC12-HSL that is base-insensitive and PON-resistant. The triphenyl mimic seems to interact specifically with LasR but not with QscR. In silico analysis suggests that the mimic fits into the 3OC12-HSL-binding site of LasR and makes key contacts with LasR. The triphenyl mimic is an excellent scaffold for developing quorum-sensing inhibitors, and its stability and potency make it ideal for biotechnology uses such as heterologous gene expression.

Chemical identification of N-acyl homoserine lactone quorum-sensing signals produced by Sinorhizobium meliloti strains in defined medium.

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.

Two opines control conjugal transfer of an Agrobacterium plasmid by regulating expression of separate copies of the quorum-sensing activator gene traR.

Conjugal transfer of Ti plasmids from Agrobacterium spp. is controlled by a hierarchical regulatory system designed to sense two environmental cues. One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself. This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon. Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine. Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer. However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84. In this study we showed that this plasmid contains two copies of traR, and each is associated with a different opine-regulated operon. One copy, traR(noc), is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B. Mutating traR(noc) abolished induction of transfer by nopaline but not by the agrocinopines. A mutation in ocd, an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine. The second copy, traR(acc), is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline. Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline. pAtK84b with mutations in both traR genes was not induced for transfer by either opine. However, expression of a traR gene in trans to this plasmid resulted in opine-independent transfer. The association of traR(noc) with nox is unique, but the operon containing traR(acc) is related to the arc operons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively. We conclude that pAtK84b codes for two independently functioning copies of traR, each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types.

Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa.

The LasR-LasI and RhlR-RhlI quorum-sensing systems are global regulators of gene expression in the opportunistic pathogen Pseudomonas aeruginosa. Previous studies suggest that the RhlR-RhlI system activates expression of rpoS. We constructed merodiploid strains of P. aeruginosa containing the native rpoS gene and an rpoS-lacZ fusion. Studies of lacZ transcription in these strains indicated that rpoS was not regulated by RhlR-RhlI. We also generated an rpoS null mutant. This rpoS mutant showed elevated levels of rhlI (but not rhlR) transcription, elevated levels of the RhlI-generated acylhomoserine lactone quorum-sensing signal, and elevated levels of RhlR-RhlI-regulated gene transcription. These findings indicate that there is a relationship between RpoS and quorum sensing, but rather than the RhlR-RhlI system influencing the expression of rpoS, it appears that RpoS regulates rhlI.

AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora

N-acylhomoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many gram-negative bacteria. AIs are involved in the regulation of diverse biological functions, including expression of pathogenic genes in the plant pathogens Pseudomonas solanacearum, several Erwinia species, and the human pathogen Pseudomonas aeruginosa. A bacterial isolate, Bacillus sp. 240B1, is capable of enzymatic inactivation of AIs. The gene (aiiA) for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Sequence alignment indicates that AiiA contains a "HXHXDH" zinc-binding motif that is conserved in several groups of metallohydrolases. Site-directed mutagenesis showed that conserved aspartate and most histidine residues are required for AiiA activity. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. Our results indicate that the AI-inactivation approach represents a promising strategy for prevention of diseases in which virulence is regulated by AIs.

AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora.

N-acylhomoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many gram-negative bacteria. AIs are involved in the regulation of diverse biological functions, including expression of pathogenic genes in the plant pathogens Pseudomonas solanacearum, several Erwinia species, and the human pathogen Pseudomonas aeruginosa. A bacterial isolate, Bacillus sp. 240B1, is capable of enzymatic inactivation of AIs. The gene (aiiA) for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Sequence alignment indicates that AiiA contains a "HXHXDH" zinc-binding motif that is conserved in several groups of metallohydrolases. Site-directed mutagenesis showed that conserved aspartate and most histidine residues are required for AiiA activity. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. Our results indicate that the AI-inactivation approach represents a promising strategy for prevention of diseases in which virulence is regulated by AIs.

Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria.

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.