The plant pathogen Erwinia amylovora produces acyl-homoserine lactone signal molecules in vitro and in planta

We report for the first time the production of acyl homoserine lactones (AHLs) by Erwina amylovora, an important quarantine bacterial pathogen that causes fire blight in plants. E. amylovora produces one N-acyl homoserine lactone [a N-(3-oxo-hexanoyl)-homoserine lactone or a N-(3-hydroxy-hexanoyl)-homoserine lactone] quorum sensing signal molecule both in vitro and in planta (pear plant). Given the involvement of AHLs in plant pathogenesis, we speculate that AHL-dependent quorum sensing could play an important role in the regulation of E. amylovora virulence.     

A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

Biotechnology Letters - To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by...     

Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing

SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K(m) values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k(cat)/K(m) = 5.5 × 10(3) M(-1)·s(-1)), but SsoPox can also degrade N-butyryl homoserine lactone (C(4)-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C(12)-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C(4)-HSL- and oxo-C(12)-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.     

Oryza sativa rice plants contain molecules that activate different quorum-sensing N-acyl homoserine lactone biosensors and are sensitive to the specific AiiA lactonase

Gram-negative bacteria most often use N-acyl homoserine lactones (AHLs) as intercellular quorum-sensing signal molecules. In this study, it was demonstrated that rice plants contain AHL mimic molecules that are very sensitive to the highly specific AiiA lactonase enzyme and can activate three different AHL bacterial biosensors, indicating that the compounds have a homoserine lactone structure and could be AHLs. The possible source and biological significance of this finding are discussed.     

Identification of virulence markers in clinically relevant strains of Acinetobacter genospecies

Nine Acinetobacter strains from patients and hospital environment were analyzed for virulence markers, quorum sensing signal production, and the presence of luxI and luxR genes. The strains had several properties in common: growth in iron limited condition, biofilm formation, and no active protease secretion. Significantly higher catechol production was determined in patient isolates (P < 0.03), but other invasiveness markers, such as lipase secretion, amount of biofilm, cell motility, antibiotic resistance, and hemolysin production, showed large variability. Notably, all members of the so-called A. calcoaceticus-A. baumannii complex, regardless of whether the source was a patient or environmental, secreted mediumto long-chain N-acyl homoserine lactones (AHL) and showed blue light inhibition of cell motility. In these strains, a luxI homologue with a homoserine lactone synthase domain and a luxR putative regulator displaying the typical AHL binding domain were identified.     

Sensitive whole-cell biosensor suitable for detecting a variety of N-acyl homoserine lactones in intact rhizosphere microbial communities

To investigate quorum sensing in rhizosphere soil, a whole-cell biosensor, Agrobacterium tumefaciens(pAHL-Ice), was constructed. The biosensor responded to all N-acyl homoserine lactones (AHLs) tested, except C(4) homoserine lactone, with a minimum detection limit of 10(-12) M, as well as to both exogenously added AHLs and AHL-producing bacterial strains in soil. This highly sensitive biosensor reveals for the first time the increased AHL availability in intact rhizosphere microbial communities compared to that in bulk soil.     

Identification of quorum-sensing-regulated genes of Burkholderia cepacia

Quorum sensing is a regulatory mechanism (operating in response to cell density) which in gram-negative bacteria usually involves the production of N-acyl homoserine lactones (HSL). Quorum sensing in Burkholderia cepacia has been associated with the regulation of expression of extracellular proteins and siderophores and also with the regulation of swarming and biofilm formation. In the present study, several quorum-sensing-controlled gene promoters of B. cepacia ATCC 25416 were identified and characterized. A total of 28 putative gene promoters show CepR-C(8)-HSL-dependent expression, suggesting that quorum sensing in B. cepacia is a global regulatory system.     

Bacterial populations in the rhizosphere of tobacco plants producing the quorum-sensing signals hexanoyl-homoserine lactone and 3-oxo-hexanoyl-homoserine lactone

A tobacco line genetically modified to produce two N-acyl homoserine lactones and its non-transformed parental line were grown in non-sterile soil. Microbial populations inhabiting the bulk soil, and those colonizing the root system of the two tobacco lines, were analyzed using cultivation-independent (phospholipid fatty acid and denaturing gradient gel electrophoresis) and cultivation-based assays. The cell density of total cultivable bacteria, fluorescent pseudomonads, sporulated, and thermotolerant bacteria was also determined in a time-course experiment (15 weeks). A possible "rhizosphere effect" related to the development of the plant was seen. However, no dissimilarities in cell population densities or population ratios of the microbial groups were detected in the rhizosphere of the two plant lines. Similarly, bacterial communities that either produced N-acyl homoserine lactone or degraded the signal hexanoyl homoserine lactone were enumerated from the two plant lines. No noticeable differences were evidenced from one plant genotype to the other. Whilst the transgenic plants released detectable amounts of the quorum-sensing signal molecules and efficiently cross-talked with the surrounding microbial populations, the bias generated by these signals in the reported experimental conditions therefore appears to remain weak, if not non-existent.     

The presence, type and role of N-acyl homoserine lactone quorum sensing in fluorescent Pseudomonas originally isolated from rice rhizospheres are unpredictable

In Gram-negative bacteria, a typical quorum-sensing (QS) system involves the production and response to N-acyl homoserine lactones (AHLs). It still remains unclear as to how pivotal and conserved AHL QS is in root-colonizing rhizosphere Pseudomonas. We, therefore, performed a systematic study of AHL QS on a set of 50 rice rhizosphere Pseudomonas isolates. We also isolated the AHL QS genes in two representative strains and analyzed the role of AHL QS regulation of various phenotypes. Our results are discussed with the current knowledge of AHL QS of rhizosphere Pseudomonas, implicating a lack of conservation and an unpredictable role played by AHL QS in this group of bacteria.     

A marine Mesorhizobium sp. produces structurally novel long-chain N-acyl-L-homoserine lactones

Our study focused on a Mesorhizobium sp. that is phylogenetically affiliated by 16S rRNA gene sequence to other marine and saline bacteria of this genus. Liquid chromatography-mass spectrometry investigations of the extract obtained from solid-phase extraction of cultures of this bacterium indicated the presence of several N-acyl homoserine lactones (AHLs), with chain lengths of C(10) to C(16). Chromatographic separation of the active bacterial extract yielded extraordinarily large amounts of two unprecedented acylated homoserine lactones, 5-cis-3-oxo-C(12)-homoserine lactone (5-cis-3-oxo-C(12)-HSL) (compound 1) and 5-cis-C(12)-HSL (compound 2). Quorum-sensing activity of compounds 1 and 2 was shown in two different biosensor systems [Escherichia coli MT102(pSB403) and Pseudomonas putida F117(pKR-C12)]. Furthermore, it was shown that both compounds can restore protease and pyoverdin production of an AHL-deficient Pseudomonas aeruginosa PAO1 lasI rhlI double mutant, suggesting that these signal molecules maybe used for intergenus signaling. In conclusion, these data indicate that the quorum-sensing activity of compounds 1 and 2 is modulated by the chain length and functional groups of the acyl moiety. Additionally, compound 1 showed antibacterial and cytotoxic activities.     

Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors

Many Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum-sensing (QS) signal molecules. AHL QS has been the subject of extensive investigation in the last decade and has become a paradigm for bacterial intercellular signaling. Research in AHL QS has been considerably aided by simple methods devised to detect AHLs using bacterial biosensors that phenotypically respond when exposed to exogenous AHLs. This article reviews and discusses the currently available bacterial biosensors which can be used in detecting and studying the different AHLs.     

Degradation of N-acyl homoserine lactone quorum sensing signal molecules by forest root-associated fungi

A collection of mycorrhizal and nonmycorrhizal root-associated fungi coming from forest environments was screened for their ability to degrade N-acyl homoserine lactones (AHL) or to prevent AHL recognition by producing quorum sensing inhibitors (QSI). No production of QS-inhibitors or -activators was detected using the two biosensors Chromobacterium violaceum CV026 and Agrobacterium tumefaciens in the culture supernatant of these fungi. However, the ability to degrade C6- and 3O,C6-HSL was detected for three fungal isolates. Acidification assay revealed that the AHL were degraded by a lactonase activity for two of these isolates. These results demonstrated for the first time that the forest root-associated fungi are capable of degrading the AHL signal molecules.     

Crystal structures of a quorum-quenching antibody

A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing.     

Suppressing Erwinia carotovora pathogenicity by projecting N-acyl homoserine lactonase onto the surface of Pseudomonas putida cells

N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/ aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect.     

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.     

Processing and export of peptide pheromones and bacteriocins in Gram-negative bacteria

Cell-density-dependent gene expression is widespread in bacteria and is mediated by extracellular communication molecules. Gram-negative bacteria often use N-acyl homoserine lactones, whereas cell-cell signaling in Gram-positive bacteria is accomplished using post-translationally processed peptide pheromones. In many Gram-positive bacteria, export of these peptides requires the activity of a dedicated ATP-binding cassette (ABC) transporter, which cleaves off a typical leader peptide termed the double-glycine leader sequence concomitant with translocation across the membrane. Inspection of bacterial genome sequences has revealed the presence of similar ABC transporters, as well as genes encoding peptides with double-glycine-type leader sequences in Gram-negative bacteria, and it is suggested that the postulated transported peptides could perform a signaling function.     

群体感应(QS)在牙菌斑形成中的研究进展

形成牙菌斑的生物膜中存在大量微生物,各种微生物通过高丝氨酸内酯(AHL)或寡肽等不同的信号分子产生群体感应(QS),形成的群体感应使各种微生物建立了区系平衡,对龋齿及牙周炎等口腔疾病的治疗产生严重影响。因组成生物膜的各种微生物对抗生素的敏感性和耐受性有显著差异,因此在治疗中增加了对宿主免疫的相应要求。为进一步探讨QS系统在牙菌斑形成中的作用特点,本文就牙菌斑的形成与抗药机制、群体感应系统及其信号分子、群体感应系统的抑制因子和研究展望进行综述。