群体感应系统对紫色杆菌CV31532积累PHA的影响

紫色杆菌CV31532中群体感应系统产生的信号分子C6-HSL是由cviI基因编码合成的。在限氮条件下,以D-葡萄糖酸钠、果糖、葡萄糖为唯一碳源时,紫色杆菌CV31532均产生聚-3-羟基丁酸,且以D-葡萄糖酸钠为唯一碳源时积累量最高。利用气相色谱分析发现,紫色杆菌CV31532培养48 h的PHA积累量显著高于其群体感应合成酶突变株CV026 PHA的积累量;通过薄层层析分析发现紫色杆菌CV31532合成信号分子的量在48 h显著提高;外源添加C6-HSL信号分子可显著提高突变体CV026 PHA的积累量。由此推测,紫色杆菌群体感应系统参与调控胞内PHA的合成。     

Signal mimics derived from a metagenomic analysis of the gypsy moth gut microbiota

Bacterial signaling is an important part of community life, but little is known about the signal transduction pathways of the as-yet-uncultured members of microbial communities. To address this gap, we aimed to identify genes directing the synthesis of signals in uncultured bacteria associated with the midguts of gypsy moth larvae. We constructed a metagenomic library consisting of DNA extracted directly from the midgut microbiota and analyzed it using an intracellular screen designated METREX, which detects inducers of quorum sensing. In this screen, the metagenomic DNA and a biosensor reside in the same cell. The biosensor consists of a quorum-sensing promoter, which requires an acylhomoserine lactone or other small molecule ligand for activation, driving the expression of the reporter gene gfp. We identified an active metagenomic clone encoding a monooxygenase homologue that mediates a pathway of indole oxidation that leads to the production of a quorum-sensing inducing compound. The signal from this clone induces the activities of LuxR from Vibrio fischeri and CviR from Chromobacterium violaceum. This study is the first to identify a new structural class of quorum-sensing inducer from uncultured bacteria.     

raiIR genes are part of a quorum-sensing network controlled by cinI and cinR in Rhizobium leguminosarum

Analysis of N-acyl-L-homoserine lactones (AHLs) produced by Rhizobium leguminosarum bv. viciae indicated that there may be a network of quorum-sensing regulatory systems producing multiple AHLs in this species. Using a strain lacking a symbiosis plasmid, which carries some of the quorum-sensing genes, we isolated mutations in two genes (raiI and raiR) that are required for production of AHLs. The raiIR genes are located adjacent to dad genes (involved in D-alanine catabolism) on a large indigenous plasmid. RaiR is predicted to be a typical LuxR-type quorum-sensing regulator and is required for raiI expression. The raiR gene was expressed at a low level, possibly from a constitutive promoter, and its expression was increased under the influence of the upstream raiI promoter. Using gene fusions and analysis of AHLs produced, we showed that expression of raiI is strongly reduced in strains carrying mutations in cinI or cinR, genes which determine a higher-level quorum-sensing system that is required for normal expression of raiIR. The product of CinI, N-(3-hydroxy-7-cis tetradecenoyl) homoserine lactone, can induce raiR-dependent raiI expression, although higher levels of expression are induced by other AHLs. Expression of raiI in a strain of Agrobacterium that makes no AHLs resulted in the identification of N-(3-hydroxyoctanoyl)-L-homoserine lactone (3OH,C(8)-HSL) as the major product of RaiI, although other AHLs that comigrate with N-hexanoyl-, N-heptanoyl-, and N-octanoyl-homoserine lactones were also made at low levels. The raiI gene was strongly induced by 3OH,C(8)-HSL (the product of RaiI) but could also be induced by other AHLs, suggesting that the raiI promoter can be activated by other quorum-sensing systems within a network of regulation which also involves AHLs determined by genes on the symbiotic plasmid. Thus, the raiIR and cinIR genes are part of a complex regulatory network that influences AHL biosynthesis in R. leguminosarum.     

The plant pathogen Pantoea ananatis produces N-acylhomoserine lactone and causes center rot disease of onion by quorum sensing

A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-l-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that P. ananatis SK-1 produced two AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in P. ananatis SK-1.     

The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).     

The Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)Homoserine Lactone Contributes to Virulence and Induces Inflammation In Vivo

Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl. These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI. LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone. There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells. In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P. aeruginosa had on colonization of the lung. We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P. aeruginosa to colonize the lung. To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice. In this model, 3O-C(12)-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1alpha (IL-1alpha) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1beta, inducible protein 10, and T-cell activation gene 3. Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression. The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain. We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment. Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections. Therefore, the quorum-sensing systems of P. aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

Characterization of a Transcriptional Activator Controlling Trichothecene Toxin Biosynthesis

Trichothecene biosynthetic pathway genes are localized within a gene cluster in Fusarium sporotrichioides and require the zinc-finger containing protein, TRI6, for expression. We show here that TRI6 is able to bind within the promoter regions of nine different pathway genes and that TRI6 binding is involved in pathway gene activation. TRI6 binding occurs at three distinct sites in the TRI5 promoter, all of which contain the sequence TNAGGCCT. DNA fragments from the promoter regions of six other pathway genes containing this sequence are also substrates for TRI6 binding. Specific nucleotide changes in the TNAGGCCT sequence dramatically reduced TRI6 binding. Analysis of TRI6 binding within the TRI3 and TRI11 promoters and the TRI4-TRI6 intergenic region which do not contain the TNAGGCCT motif suggests that the minimum sequence required for TRI6 binding is YNAGGCC. Two potential TRI6 binding sites, T4A and T4B, were identified within the intergenic region for the divergently transcribed TRI4 and TRI6 genes. Alteration or deletion of the T4A site resulted in the loss of nearly all in vitro TRI6 binding and was correlated with the loss of promoter activity in vivo as measured by the expression of mutant TRI4(p)/GUS fusions. This establishes a physiological role for TRI6 binding and demonstrates that TRI6 is directly involved in the regulation of pathway gene expression. To determine if a predicted Cys2His2 zinc-finger motif at the C-terminus of TRI6 is involved in DNA binding, a C187A mutant was constructed in TRI6 using site-directed mutagenesis. The C187A mutant did not bind promoter DNA fragments, supporting the role of C187 in DNA binding. In addition, a TRI6 homologue in the distantly related macrocyclic trichothecene pathway of Myrothecium roridum (MRTRI6) was also shown to bind to the same TRI5 and TRI4 promoter fragments bound by TRI6. Together, these data confirm our previous proposal that TRI6 is an activator of trichothecene pathway gene expression and that DNA binding employs the C-terminal region of TRI6 containing three predicted Cys2His2 zinc fingers.