Study on the relationship of protease production and luminescence in Vibrio harveyi

AIMS: To demonstrate that Vibrio harveyi produces various types of toxins and how the production of those toxins is related with luminescence. METHODS AND RESULTS: Luminescence and toxicity of eight V. harveyi were evaluated. We demonstrated that all V. harveyi emitting luminescence were isolated from marine organisms and also showed that they were highly pathogenic when compared with culture collection V. harveyi based on cytotoxic assay test. On the contrary, V. harveyi isolated from shrimp farm showed no luminescence but showed high pathogenicity based on toxicity test. The effect of protease inhibitors on pathogenicity and luminescence was also investigated. We demonstrated that light emission of pathogenic V. harveyi remarkably decreased after addition of protease inhibitor. Furthermore, extracellular proteins from cell-free culture supernatant of luminescent and nonluminescent V. harveyi were compared using SDS-PAGE analysis. Results showed that there were differences in molecular weight and amount of proteins. CONCLUSIONS: Vibrio harveyi parasiting marine organisms have both luminescence and pathogenicity. Based on this study, luminescence and protease toxin activity in V. harveyi are related. Moreover, this paper clarified that V. harveyi produces various types of toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study demonstrated that V. harveyi produces two kinds of toxins, haemolysin and protease toxin. It may be clear roots of V. harveyi toxin.     

Functional determinants of the quorum‐sensing non‐coding RNAs and their roles in target regulation

Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1–5, control quorum‐sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem‐loops: the first stem‐loop is crucial for base pairing with a subset of targets. This stem‐loop also protects the Qrr sRNAs from RNase E‐mediated degradation. The second stem‐loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem‐loop plays an accessory role in base pairing and stability. The fourth stem‐loop functions as a rho‐independent terminator. In the quorum‐sensing regulon, Qrr sRNAs‐controlled genes are the most rapid to respond to quorum‐sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing.     

The effect of copper concentration on the virulence of pathogenic Vibrio harveyi

AIM: To demonstrate the influence of copper on luminescence and toxin production in Vibrio harveyi. METHODS AND RESULTS: The effect of copper concentration on the expression of both luminescence and toxin of V. harveyi was investigated. Copper concentration of less than 40 ppm had no effect on the growth. While V. harveyi cultured with 40 ppm copper concentration showed decreased luminescence as measured by spectrofluorophotometer and as observed. LuxD gene, which is related to luminescence expression, was monitored using real-time RT-PCR. Result showed that the concentration of cDNA coding for luxD was lower in V. harveyi with copper. Toxic activity against both HeLa cells and shrimp haemocytes was also lower in the culture supernatant of V. harveyi grown with 40 ppm copper concentration. Moreover, V. harveyi extracellular proteins were analysed using SDS-PAGE. Results showed that culture supernatant from V. harveyi grown without copper had thicker band indicating a higher concentration of the putative cysteine protease, one of the major toxin of V. harveyi. CONCLUSIONS: This study proved that both luminescence and toxin were repressed by copper. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study demonstrated that copper inhibited expression of phenotype of V. harveyi. Furthermore, it may inhibit quorum sensing of V. harveyi.     

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.     

Bacterial cell‐to‐cell signaling promotes the evolution of resistance to parasitic bacteriophages

The evolution of host–parasite interactions could be affected by intraspecies variation between different host and parasite genotypes. Here we studied how bacterial host cell‐to‐cell signaling affects the interaction with parasites using two bacteria‐specific viruses (bacteriophages) and the host bacterium Pseudomonas aeruginosa that communicates by secreting and responding to quorum sensing (QS) signal molecules. We found that a QS‐signaling proficient strain was able to evolve higher levels of resistance to phages during a short‐term selection experiment. This was unlikely driven by demographic effects (mutation supply and encounter rates), as nonsignaling strains reached higher population densities in the absence of phages in our selective environment. Instead, the evolved nonsignaling strains suffered relatively higher growth reduction in the absence of the phage, which could have constrained the phage resistance evolution. Complementation experiments with synthetic signal molecules showed that the Pseudomonas quinolone signal (PQS) improved the growth of nonsignaling bacteria in the presence of a phage, while the activation of las and rhl quorum sensing systems had no effect. Together, these results suggest that QS‐signaling can promote the evolution of phage resistance and that the loss of QS‐signaling could be costly in the presence of phages. Phage–bacteria interactions could therefore indirectly shape the evolution of intraspecies social interactions and PQS‐mediated virulence in P. aeruginosa.     

Effects of slight structural distortion on the luminescence performance in (Ca1‐xEux)WO4 luminescent materials

(Ca_1‐xEu_x)WO₄ (x = 0–21 mol%) phosphors were prepared using the classical solid‐state reaction method. The influence of Eu³⁺ ion doping on lattice structure was observed using powder X‐ray diffraction and Fourier transform infrared spectroscopy. Furthermore, under this influence, the luminescence properties of all samples were analyzed. The results clearly illustrated that the element europium was successfully incorporated into the CaWO₄ lattice with a scheelite structure in the form of a Eu³⁺ ion, which introduced a slight lattice distortion into the CaWO₄ matrix. These lattice distortions had no effect on phase purity, but had regular effects on the intrinsic luminescence of the matrix and the f–f excitation transitions of Eu³⁺ activators. When the Eu³⁺ concentration was increased to 21 mol%, a local luminescence centre of [WO₄]^2? groups was detected in the matrix and manifested as the decay curves of [WO₄]^2? groups and luminescence changed from single exponential to double exponential fitting. Furthermore, the excitation transitions of Eu³⁺ between different energy levels (such as ⁷F₀→⁵L₆, ⁷F₀→⁵D₂) also produced interesting changes. Based on analysis of photoluminescence spectra and the chromaticity coordinates in this study, it could be verified that the nonreversing energy transfer of [WO₄]^2?→Eu³⁺ was efficient and incomplete.     

Resuscitation of viable but non‐culturable bacteria to enhance the cellulose‐degrading capability of bacterial community in composting

Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation‐promoting factors (Rpfs) due to entering a viable but non‐culturable (VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose‐degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl‐cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.     

Synthesis and luminescence properties of 2‐(benzylcarbamoyl)phenyl derivatives and their europium complexes

Six novel 2‐(benzylcarbamoyl)phenyl derivatives were synthesized and characterized by ¹H‐NMR, mass spectrometry, infrared spectra and elemental analysis. Their europium complexes were prepared and characterized by elemental analysis, EDTA titrimetric analysis, IR and UV spectra as well as molar conductivity measurements. The luminescence properties of these complexes were investigated and results show that 2‐(benzylcarbamoyl)phenyl derivatives possess high selectivity and good coordination with the europium ion. Complex Eu‐2‐(benzylcarbamoyl)phenyl‐2‐phenylacetate showed green luminescence that was emitted by the ligand of 2‐(benzylcarbamoyl)phenyl‐2‐phenylacetate, while other complexes showed the characteristic red luminescence of europium ion and also possessed high luminescence intensity. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and luminescence properties of novel 8‐hydroxyquinoline derivatives and their Eu(III) complexes

Six novel 8‐hydroxyquinoline derivatives were synthesized using 2‐methyl‐8‐hydroxyquinoline and para‐substituted phenol as the main starting materials, and were characterized by ¹H nuclear magnetic resonance (NMR), mass spectrometry (MS), ultraviolet (UV) light analysis and infra‐red (IR) light analysis. Their complexes with Eu(III) were also prepared and characterized by elemental analysis, molar conductivity, UV light analysis, IR light analysis, and thermogravimetric–differential thermal analysis (TG–DTA). The results showed that the ligand coordinated well with Eu(III) ions and had excellent thermal stability. The structure of the target complex was EuY^1–6(NO₃)₃.2H₂O. The luminescence properties of the target complexes were investigated, the results indicated that all target complexes had favorable luminescence properties and that the introduction of an electron‐donating group could enhance the luminescence intensity of the corresponding complexes, but the addition of an electron‐withdrawing group had the opposite effect. Among all the target complexes, the methoxy‐substituted complex (–OCH₃) had the highest fluorescence intensity and the nitro‐substituted complex (–NO₂) had the weakest fluorescence intensity. The results showed that 8‐hydroxyquinoline derivatives had good energy transfer efficiency for the Eu(III) ion. All the target complexes had a relatively high fluorescence quantum yield. The fluorescence quantum yield of the complex EuY³(NO₃)₃.2H₂O was highest among all target complexes and was up to 0.628. Because of excellent luminescence properties and thermal stabilities of the Eu(III) complexes, they could be used as promising candidate luminescent materials.     

川白芷抑菌活性及对铜绿假单胞菌群体感应的抑制作用

为寻找更加有效的抑菌杀菌药物,本研究利用琼脂平板打孔法和倍比稀释法评价川白芷不同溶剂萃取物对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌以及肺炎克雷伯氏菌的抗菌活性;特别是通过对铜绿假单胞菌毒力表型的影响研究考察该药物对病菌群体感应是否具有抑制作用。研究结果表明:川白芷提取液对四种细菌均有不同程度的抑制效果,且随提取物浓度增大抑制效果增强;进一步分离发现,三氯甲烷、乙酸乙酯和正丁醇提取物对四种细菌均有一定的抑制效果,其中乙酸乙酯萃取物的抑菌效果最佳;对铜绿假单胞菌的四种毒力表型的影响研究结果表明川白芷具有抑制铜绿假单胞菌群体感应的能力。试验表明川白芷的活性成分可以作为一种新型的群体感应抑制剂,其具有抑制多种细菌能力的同时不易产生耐药性,表明白芷这一传统中草药在现代医疗中具有较好的应用潜力。     

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.     

群体感应在生物强化功能菌定殖及降解能力增强中的作用研究进展

由于难降解有机污染物和外界环境对水处理系统的冲击干扰,污水水质常出现不达标现象。引入外源含有相关功能基因并且具有基因水平转移能力的工程菌株进行生物强化处理是提高污水处理效能的有效措施。污水处理系统中存在能够分泌信号分子的菌体,菌间具有群体感应现象,当种群密度达到感应阈值时,菌体会通过释放信号分子来触发一些群体行为,从而激活相关基因的表达(如生物膜形成、生物发光、抗生素合成和毒力因子表达等)。早期的群体感应技术研究主要集中在信号传递学、微生物社会行为学和医学微生物领域,近年来,在水处理领域也开始有相继报道,研究表明群体感应在污水生物处理中发挥重要作用,并且影响生物强化菌株的定殖和污染物降解,因此群体感应行为调控是生物强化技术成效显著与否的关键因素。本文综述了群体感应及信号分子的作用机制、信号分子释放及存在的影响因素以及群体感应对菌株定殖、微生物群落结构和污染物去除的影响,并对从群体感应角度出发研究生物强化过程进行了展望,旨在为生物强化技术的有效实施及提升污水处理效能提供一种新思路,为深入理解生物强化过程中群体感应调控行为提供理论参考。     

Effect of quinones and phenols on the triple‐enzyme bioluminescent system with protease

The study addressed the effects of redox‐active compounds on trypsin activity. Series of organic oxidizers (quinones) and reducers (phenols) were chosen as model redox‐active compounds. Trypsin activity was quanti?ed by bioluminescent technique. Interactions of these compounds with trypsin were studied by ?uorescent and light absorption methods. Luminescence intensity decay constants in the reduced nicotinamidadeninedinucleotide (NADH): ?avinmononucleotide (FMN)‐oxidoreductase (R)–luciferase (L)–trypsin (T) (R + L + T) triple‐enzyme system were calculated and compared in the presence of different concentrations of quinones and phenols. The triple‐enzyme system was shown to be sensitive to quinones and not sensitive to phenols. It has been found that the effects produced by quinones on the coupled enzyme system (R + L) and on the trypsin molecule (T) are not related. The conclusions were extrapolated to the properties of other proteases and antiproteases. Copyright © 2003 John Wiley & Sons, Ltd.     

Effect of sintering temperature on the luminescence properties of Ca0.8Ba0.2TiO3:Pr3+ red‐emitting phosphor

Nanoparticles with the nominal composition Ca_0.8Ba_0.2Ti0₃:Pr³⁺ were prepared using the sol–gel process. Barium nitrate, 4‐hydrated calcium nitrate and praseodymium oxide were used as raw materials. The structural evolution and decomposition processes of the precursors were investigated by powder X‐ray diffraction, differential thermal analysis and thermogravimetric analysis. Crystalline Ca_0.8Ba_0.2Ti0₃:Pr³⁺ could be obtained at 700°C. The photoluminescence properties of the samples were investigated using excitation and emission spectra. Ca_0.8Ba_0.2Ti0₃:Pr³⁺ nanoparticles showed strong red emission, which could be assigned to the typical ¹D₂→³H₄ transition of Pr³⁺. Furthermore, the study found that sintering temperature and the introduction of Ba²⁺ influence the decay time of persistent luminescence. Copyright © 2014 John Wiley & Sons, Ltd.     

Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the α-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.     

Synthesis and properties of a new red luminescence europium complex containing a 2‐(benzimidazol‐2‐yl)‐8‐octyloxyquinoline as the second ligand

A new Eu(III) complex, Eu(III)(DBM)₃BIOQ, has been synthesized with dibenzoylmethane (DBM) as the first ligand and 2‐(benzimidazol‐2‐yl)‐8‐octyloxyquinoline (BIOQ) as the second ligand. The stability of the complex was analysed by DSC–TG. The results show that the Eu(III) complex has a relatively high thermal stability with a melting point of 235 °C and a decomposition temperature (onset) of 252 °C. The fluorescence properties of the compound were also investigated. The fluorescence results reveal that the as‐prepared complex shows the characteristic maximum emission spectra of Eu(III) at 611 nm (λ_ex = 350 nm). In addition, the photoluminescence spectrum of the complex in the solid state exhibits a single and symmetrical emission band at 611 nm, with a full width at half‐maximum of 4.7 nm, showing high colour purity. This finding indicates the possibility for the development of brighter red luminescent materials. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of propagators and inhibitors on the ultraweak luminescence from maize roots

This study has investigated the kinetics and mechanism of ultraweak luminescence in maize roots. Mannitol induced the second maximum and enhanced the main maximum of the relative intensity of luminescence from the roots. Hydroquinone and quinone enhanced the relative intensity of the luminescence. Catalase enhanced the maximum of the luminescence and changed the kinetics of the light emission. The effect of catalase on the kinetics was abolished by superoxide dismutase. Ascorbate in the presence of catalase reduced the luminescence maximum, but did not alter the kinetics. In the presence of catalase only, or in the combination with superoxide dismutase, or ascorbate, the luminescence intensity in the stationary phase was significantly lower compared to the control. The results support the participation of superoxide-radical, singlet oxygen, electron transfer and the role of peroxidase in the reactions generating ultraweak luminescence in the roots. Ascorbate, catalase and superoxide dismutase have a protective role in the luminescent reactions.     

Influence of enteric bacteria conditioned media on recovery of Escherichia coli O157:H7 exposed to starvation and sodium hypochlorite

AIMS: To examine the effect that starvation and sodium hypochlorite stress have on virulence of Escherichia coli O157:H7 and the influence of conditioned media on recovery of stressed cells. METHODS AND RESULTS: Escherichia coli O157:H7 was starved for 5 days then exposed to 1 microg ml(-1) sodium hypochlorite, suspended in defined media Dulbecco's Modified Eagle's Medium supplemented with conditioned media, sampled over a 12-h period; and assayed for growth, production of Shiga toxin (Stx), and attachment to HCT-8 cells. During recovery, stressed and control cells grown in conditioned media exhibited greater attachment efficiencies to HCT-8 cells then cells in DMEM alone. Production of Stx by treated cells mimicked Stx production by control cells suggesting that components of conditioned media assist in recovery. Results showed that levels of autoinducer-2 fluctuate during recovery and growth suggesting involvement of a quorum sensing mechanism during the recovery of stressed E. coli O157:H7. CONCLUSIONS: The recovery of stressed E. coli O157:H7 exposed to starvation conditions and HOCl is positively affected by the presence of autoinducer-2 thereby influencing virulence factor production. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens in a stressed state prior to ingestion can rapidly recover in the presence of bacterial by-products; exhibiting virulence characteristics and presenting a microbial food safety hazard.