Regulation of LuxPQ receptor activity by the quorum-sensing signal autoinducer-2

The extracellular signaling molecule autoinducer-2 (AI-2) mediates quorum-sensing communication in diverse bacterial species. In marine vibrios, binding of AI-2 to the periplasmic receptor LuxP modulates the activity of the inner membrane sensor kinase LuxQ, transducing the AI-2 information into the cytoplasm. Here, we show that Vibrio harveyi LuxP associates with LuxQ in both the presence and absence of AI-2. The 1.9 A X-ray crystal structure of apoLuxP, complexed with the periplasmic domain of LuxQ, reveals that the latter contains two tandem Per/ARNT/Simple-minded (PAS) folds. Thus, although many prokaryotic PAS folds themselves bind ligands, the LuxQ periplasmic PAS folds instead bind LuxP, monitoring its AI-2 occupancy. Mutations that disrupt the apoLuxP:LuxQ interface sensitize V. harveyi to AI-2, implying that AI-2 binding causes the replacement of one set of LuxP:LuxQ contacts with another. These conformational changes switch LuxQ between two opposing enzymatic activities, each of which conveys information to the cytoplasm about the cell density of the surrounding environment.     

Stationary-phase quorum-sensing signals affect autoinducer-2 and gene expression in Escherichia coli

Quorum sensing via autoinducer-2 (AI-2) has been identified in different strains, including those from Escherichia, Vibrio, Streptococcus, and Bacillus species, and previous studies have suggested the existence of additional quorum-sensing signals working in the stationary phase of Escherichia coli cultures. To investigate the presence and global effect of these possible quorum-sensing signals other than AI-2, DNA microarrays were used to study the effect of stationary-phase signals on the gene expression of early exponential-phase cells of the AI-2-deficient strain E. coli DH5alpha. For statistically significant differential gene expression (P < 0.05), 14 genes were induced by supernatants from a stationary culture and 6 genes were repressed, suggesting the involvement of indole (induction of tnaA and tnaL) and phosphate (repression of phoA, phoB, and phoU). To study the stability of the signals, the stationary-phase supernatant was autoclaved and was used to study its effect on E. coli gene expression. Three genes were induced by autoclaved stationary-phase supernatant, and 34 genes were repressed. In total, three genes (ompC, ptsA, and btuB) were induced and five genes (nupC, phoB, phoU, argT, and ompF) were repressed by both fresh and autoclaved stationary-phase supernatants. Furthermore, supernatant from E. coli DH5alpha stationary culture was found to repress E. coli K-12 AI-2 concentrations by 4.8-fold +/- 0.4-fold, suggesting that an additional quorum-sensing system in E. coli exists and that gene expression is controlled as a network with different signals working at different growth stages.     

Magnetic nanofactories: localized synthesis and delivery of quorum-sensing signaling molecule autoinducer-2 to bacterial cell surfaces

Magnetic 'nanofactories', for localized manufacture and signal-guided delivery of small molecules to targeted cell surfaces, are demonstrated. They recruit nearby raw materials for synthesis, employ magnetic mobility for capture and localization of target cells, and deliver molecules to cells triggering their native phenotypic response, but with user-specified control. Our nanofactories, which synthesize and deliver the "universal" bacterial quorum-sensing signal molecule, autoinducer AI-2, to the surface of Escherichia coli, are assembled by first co-precipitating nanoparticles of iron salts and the biopolymer chitosan. E. coli AI-2 synthases, Pfs and LuxS, constructed with enzymatically activatable "pro-tags", are then covalently tethered onto the chitosan. These enzymes synthesize AI-2 from metabolite S-adenosylhomocysteine. Chitosan serves as a molecular scaffold and provides cell capture ability; magnetite provides stimuli responsiveness. These magnetic nanofactories are shown to modulate the natural progression of quorum-sensing activity. New prospects for small molecule delivery, based on localized synthesis, are envisioned.     

Mutational analysis of TraR. Correlating function with molecular structure of a quorum-sensing transcriptional activator

TraR, the quorum-sensing activator of the Agrobacterium tumefaciens Ti plasmid conjugation system, induces gene expression in response to its quormone, N-(3-oxooctanoyl)-L-homoserine lactone. Ligand binding results in dimerization of TraR and is required for its activity. Analysis of N- and C-terminal deletion mutants of TraR localized the quormone-binding domain to a region between residues 39 and 140 and the primary dimerization domain to a region between residues 119 and 156. The dominant-negative properties of these mutants predicted a second dimerization domain at the C terminus of the protein. Analysis of fusions of N-terminal fragments of TraR to lambda cI' confirmed the dimerization activity of these two domains. Fifteen single amino acid substitution mutants of TraR defective in dimerization were isolated. According to the analysis of these mutants, Asp-70 and Gly-113 are essential for quormone binding, whereas Ala-38 and Ala-105 are important, but not essential. Additional residues located within the N-terminal half of TraR, including three located in alpha-helix 9, contribute to dimerization, but are not required for ligand binding. These results and the recently reported crystal structure of TraR are consistent with and complement each other and together define some of the structural and functional relationships of this quorum-sensing activator.     

Mutational analysis of cell wall biosynthesis in Mycobacterium avium

The cell wall of the environmental pathogen Mycobacterium avium is important to its virulence and intrinsic antimicrobial resistance. To identify genes involved in cell wall biosynthesis, "transposome" insertion libraries were screened for mutants with altered colony morphology on medium containing the lipoprotein stain Congo red. Nineteen such mutants were isolated and mapped, including 10 with insertions in a functional island of cell wall biosynthetic genes that spans approximately 40 kb of the M. avium genome.     

Mutational analysis of serine-glycine biosynthesis in Rhodopseudomonas capsulata.

Rhodopseudomonas capsulata possesses the enzymes of both the "phosphorylated" and the "non-phosphorylated" pathways of serine biosynthesis. Certain mutants with lesions in the phosphorylated pathway are serine-glycine auxotrophs, though they still produce enzymes of the non-phosphorylated sequence. These results indicate that the phosphorylated pathway is essential for the synthesis of serine and glycine in R. capsulata under the condtions tested.     

安丝菌素聚酮合酶模块2中脱水酶结构域的生化功能

[目的]I型聚酮合酶(Polyketide synthase,PKS)模块中不同的修饰是聚酮类化合物结构多样性的重要原因之一.抗癌药物安丝菌素化学结构中C11-C14区域存在特殊的双键迁移结构,可能与聚酮合酶模块2或者3中脱水酶结构域(Dehydratase,DH)的催化密切相关,本研究通过探究聚酮合酶模块2中DH结构...     

铜绿假单胞菌水解弹性蛋白能力相关基因的筛选与鉴定

【目的】研究铜绿假单胞菌弹性蛋白水解能力相关基因。【方法】应用人工Mu转座技术构建铜绿假单胞菌野生型菌株PA68的转座突变文库,从2000多个突变子中筛选得到4株弹性蛋白水解能力改变的突变子,并通过克隆及测序获得转座子插入位点侧翼的序列。将铜绿假单胞菌弹性蛋白酶结构基因lasB的转录启始区序列整合入载体pDN19lacΩ并将该重组质粒电转化入野生型菌株PA68及4个突变株中,对报告基因在不同菌株中的表达水平进行测定。【结果】发现4个突变株中Mu转座子分别插入lasA、galU、xcpZ和ptsP 4个基因。ptsP基因失活的突变株中,lasB基因的转录水平是野生型菌株的7%,xcpZ和lasA基因的失活使lasB基因的转录水平分别降低为野生株的54%和75%,galU基因的插入失活使lasB基因的转录上升了1倍。【结论】推测ptsP和galU基因很可能直接或间接地调控着弹性蛋白酶的生物合成。     

Functional analysis of PDX2 from Arabidopsis, a glutaminase involved in vitamin B6 biosynthesis

Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5'-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1.     

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。     

Mutational analysis of exopolysaccharide biosynthesis by Lactobacillus sakei 0-1

Lactobacillus sakei strain 0-1 produces an exopolysaccharide (EPS) consisting of glucose and rhamnose in a ratio of 3:2. As part of a biochemical and molecular analysis of the EPS biosynthetic pathway in L. sakei strain 0-1, we have isolated a random set of EPS-negative mutants. Following treatment of cells with the mutagen ethylmethane sulfonic acid, a total of 10 mutants were identified that lacked the clear ropy appearance of wild-type colonies on agar plates. Their characterization revealed that eight mutants had completely lost the ability to synthesize the normal EPS. Six of these mutants lacked activities of enzymes involved in the biosynthesis of dTDP-rhamnose, required for EPS production. Only mutant strains 12 and 20 were directly affected in EPS synthesis. Strain 12 synthesized EPS with a different sugar composition, however. Interestingly, strain 12 showed temperature-dependent EPS synthesis, with the highest amounts synthesized at 12°C, and low amounts at the optimal temperature for growth (30°C). Two mutants were in fact EPS-positive, producing the normal EPS, but displayed a different cell morphology (elongated cells), indicating a modification in cell wall synthesis.     

Mutational analysis of chicken interleukin 2

Chicken interleukin 2 (chIL-2) has low, but significant, homology to both mammalian IL-2 and mammalian IL-15. In view of its unique phylogenetic position and potential use as a vaccine adjuvant, a detailed mutational analysis for critical functional sites was undertaken. It was found that Asp17 is a critical N terminal contact site for binding to the putative chIL-2 receptor, which is similar to results obtained for mammalian IL-2 and IL-15. Analysis of the C terminus did not reveal a single critical amino acid. However, deletion mutant studies demonstrated that removal of C terminal amino acids yielded proteins with decreased bioactivity and that this decrease was a function of the number and kind of amino acids removed. This study is the first non-mammalian IL-2 mutational analysis and proposes a model for the interaction between chIL-2 and its receptor.     

Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence

We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei (RmLIP) and Humicola lanuginose (HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward alpha-naphthylbutyrate (C4) and alpha-naphthylcaprylate (C8), respectively, was due to a lower K(m) value. The higher catalytic efficiency of D77N toward C4 substrate was due to a combination of decreased K(m) and considerably increased k(cat). The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively.     

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。     

Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding.

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.     

Characterization of a methyltransferase involved in herboxidiene biosynthesis

The herboxidiene biosynthetic gene cluster contains a regulatory gene and six biosynthetic genes that encode three polyketide synthases (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG). Through single crossover recombination, an integrative plasmid was inserted into the genome of Streptomyces chromofuscus ATCC 49982 between herE and herF, resulting in low-level expression of herF and the downstream herG. The mutant strain produced two new compounds, 18-deoxy-25-demethyl-herboxidiene and 25-demethyl-herboxidiene. HerF was expressed in Escherichia coli and biochemically characterized as the dedicated methyltransferase in herboxidiene biosynthesis. It prefers 25-demethyl-herboxidiene to 18-deoxy-25-demethyl-herboxidiene, suggesting that C-25 methylation is the last tailoring step.     

Genetic analysis of genes involved in melanin biosynthesis of Cochliobolus miyabeanus

Color mutants of Cochliobolus miyabeanus defective in melanin biosynthesis were isolated. Although the wild-type strain KU-13 formed dark green colonies, color mutants formed white, brown, and gray colonies or white colonies with red pigment secretion. From the white mutant which secreted red pigment, designated scy, a melanin precursor which restored melanization of albino mutants alm-1 was isolated and identified as scytalone. This indicated that scy mutant was defective in the conversion of scytalone to 1,3,8-trihydroxynaphthalene and that melanin of this fungus is of pentaketide origin formed from oxidation of 1,8-dihydroxynaphthalene. Albino mutants alm-1 were considered to be defective in pentaketide cyclization and brown mutants brm were considered to be defective in the conversion of 1,3,8-trihydroxynaphthalene to vermelone. Albino mutants alm-2 whose coloration was not restored by application of scytalone were also isolated. The alm-2 gene was believed to be a gene transactively regulating the pentaketide cyclization and conversion of scytalone. From crossing experiments among the color mutants, it was indicated that alm-1, alm-2, and brm were linked and that scy segregates independently of these three mutant loci. Crossing of a methionine requiring mutant with alm and scy indicated that the three loci segregate independently of each other.