Simple electrospray mass spectrometry detection of acylhomoserine lactones

Simple (non‐tandem) electrospray mass spectrometry (ESMS) can detect acyl homoserine lactones (AHL) in bacteriological media in picomole amounts. The chemical reactivity of AHLs and their extraction behaviour into ethyl acetate, coupled with detection in the ESMS, has shown that these lactones can be detected as the protonated pseudomolecular ions themselves as well as solvent and ammonium adducts, and as dimers. ESMS detects and identifies these molecules, utilizing simple chemical properties of AHLs. Copyright © 2005 John Wiley & Sons, Ltd.     

Quorum-sensing system in Acinetobacter baumannii: a potential target for new drug development

Acinetobacter baumannii causes several nosocomial infections and poses major threat when it is multidrug resistant. Even pan drug-resistant strains have been reported in some countries. The intensive care unit (ICU) mortality rate ranged from 45.6% to 60.9% and it is as high as 84.3% when ventilator-associated pneumonia was caused by XDR (extensively drug resistant) A. baumannii. Acinetobacter baumannii constituted 9.4% of all Gram-negative organisms throughout the hospital and 22.6% in the ICUs according to a study carried out in an Indian hospital. One of the major factors contributing to drug resistance in A. baumannii infections is biofilm development. Quorum sensing (QS) facilitates biofilm formation and therefore the search for ‘quorum quenchers’ has increased recently. Such compounds are expected to inhibit biofilm formation and hence reduce/prevent development of drug resistance in the bacteria. Some of these compounds also target synthesis of some virulence factors (VF). Several candidate drugs have been identified and are at various stages of drug development. Since quorum quenching, inhibition of biofilm formation and inhibition of VF synthesis do not pose any threat to the DNA replication and cell division of the bacteria, chances of resistance development to such compounds is presumably rare. Thus, these compounds ideally qualify as adjunct therapeutics and could be administered along with an antibiotic to reduce chances of resistance development and also to increase the effectiveness of antimicrobial therapy. This review describes the state-of-art in QS process in Gram-negative bacteria in general and in A. baumannii in particular. This article elaborates the nature of QS mediators, their characteristics, and the methods for their detection and quantification. Various potential sites in the QS pathway have been highlighted as drug targets and the candidate quorum quenchers which inhibit the mediator’s synthesis or function are enlisted.     

Profiling acylated homoserine lactones in Yersinia ruckeri and influence of exogenous acyl homoserine lactones and known quorum-sensing inhibitors on protease production

AIMS: To profile the quorum-sensing (QS) signals in Yersinia ruckeri and to examine the possible regulatory link between QS signals and a typical QS-regulated virulence phenotype, a protease. METHODS AND RESULTS: Liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) showed that Y. ruckeri produced at least eight different acylated homoserine lactones (AHLs) with N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL) being the dominant molecule. Also, some uncommon AHL, N-(3-oxoheptanoyl)-L-homoserine lactone (3-oxo-C7-HSL) and N-(3-oxononanoyl)-L-homoserine lactone (3-oxo-C9-HSL), were produced. 3-oxo-C8-HSL was detected in organs from fish infected with Y. ruckeri. Protease production was significantly lower at temperatures above 23 degrees C than below although growth was faster at the higher temperatures. Neither addition of sterile filtered high-density Y. ruckeri culture supernatant nor the addition of pure exogenous AHLs induced protease production. Furthermore, three QS inhibitors (QSIs), sulfur-containing AHL analogues, did not inhibit protease production in Y. ruckeri. CONCLUSIONS: Exogenous AHL or sulfur-containing AHL analogues did not influence the protease production indicating that protease production may not be QS regulated in Y. ruckeri. SIGNIFICANCE AND IMPACT OF THE STUDY: The array of different AHLs produced indicates that the QS system of Y. ruckeri is complex and could involve several regulatory systems. In this case, neither AHLs nor QSI would be likely to directly affect a QS-regulated phenotype.     

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.     

Detection of quorum sensing signal molecules in the family Vibrionaceae

Aims: The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N‐acyl‐homoserine lactone (AHL), the autoinducer‐2 (AI‐2) and the cholerae autoinducer‐1‐like (CAI‐1‐like) molecules in 25 Vibrionaceae strains. Methods and Results: The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty‐one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin‐layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI‐2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI‐1‐like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. Conclusions: The results indicated that the QS signal molecules, especially AHL and AI‐2 molecules, were widespread in the family Vibrionaceae. Significance and Impact of the Study: In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.     

Sharing of quorum-sensing signals and role of interspecies communities in a bacterial plant disease

Pathogenic bacteria interact not only with the host organism but most probably also with the resident microbial flora. In the knot disease of the olive tree (Olea europaea), the causative agent is the bacterium Pseudomonas savastanoi pv. savastanoi (Psv). Two bacterial species, namely Pantoea agglomerans and Erwinia toletana, which are not pathogenic and are olive plant epiphytes and endophytes, have been found very often to be associated with the olive knot. We identified the chemical signals that are produced by strains of the three species isolated from olive knot and found that they belong to the N-acyl-homoserine lactone family of QS signals. The luxI/R family genes responsible for the production and response to these signals in all three bacterial species have been identified and characterized. Genomic knockout mutagenesis and in planta experiments showed that virulence of Psv critically depends on QS; however, the lack of signal production can be complemented by wild-type E. toletana or P. agglomerans. It is also apparent that the disease caused by Psv is aggravated by the presence of the two other bacterial species. In this paper we discuss the potential role of QS in establishing a stable consortia leading to a poly-bacterial disease.     

酰基高丝氨酸内酯(AHLs)介导群感效应在好氧颗粒污泥中的研究进展

虽然好氧颗粒污泥(Aerobic Granular Sludge,AGS)具有沉降性能好、高效脱氮除磷以及抗冲击负荷等优点,但是该技术仍然存在颗粒化进程缓慢及容易解体等技术瓶颈.因此,如何克服上述瓶颈是实现好氧颗粒污泥技术在实际污水处理推广的关键.近年来,酰基高丝氨酸内酯(Acyl Homoserine Lactone...     

Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development

Plants have evolved complex strategies to maintain phosphate (Pi) homeostasis and to maximize Pi acquisition when the macronutrient is limiting. Adjustment of root system architecture via changes in meristem initiation and activity is integral to the acclimation process. However, the mechanisms that monitor external Pi status and interpret the nutritional signal remain to be elucidated. Here, we present evidence that the Pi deficiency response, pdr2, mutation disrupts local Pi sensing. The sensitivity and amplitude of metabolic Pi-starvation responses, such as Pi-responsive gene expression or accumulation of anthocyanins and starch, are enhanced in pdr2 seedlings. However, the most conspicuous alteration of pdr2 is a conditional short-root phenotype that is specific for Pi deficiency and caused by selective inhibition of root cell division followed by cell death below a threshold concentration of about 0.1 mm external Pi. Measurements of general Pi uptake and of total phosphorus (P) in root tips exclude a defect in high-affinity Pi acquisition. Rescue of root meristem activity in Pi-starved pdr2 by phosphite (Phi), a non-metabolizable Pi analog, and divided-root experiments suggest that pdr2 disrupts sensing of low external Pi availability. Thus, PDR2 is proposed to function at a Pi-sensitive checkpoint in root development, which monitors environmental Pi status, maintains and fine-tunes meristematic activity, and finally adjusts root system architecture to maximize Pi acquisition.     

Quorum sensing regulation of gene expression: A promising target for drugs against bacterial pathogenicity

Bacteria are sensitive to an increase in population density and respond quickly and coordinately by induction of certain sets of genes. This mode of regulation, known as quorum sensing (QS), is based on the effect of low-molecular-weight signal molecules, autoinducers (AIs). When the population density is high, AIs accumulate in the medium and interact with regulatory receptor proteins. QS systems are global regulators of bacterial gene expression and play a key role in controlling many metabolic processes in the cell, including bacterial virulence. The review considers the molecular mechanisms of QS in different taxonomic groups of bacteria and discusses QS regulation as a possible target in treating bacterial infections. This is a new, alternative strategy of antibacterial therapy, which includes the construction of drugs acting directly against bacterial pathogenicity by suppressing QS (antipathogenicity drugs). This strategy makes it possible to avoid a wide distribution of antibiotic-resistant pathogenic bacteria and the formation of biofilms, which dramatically increase drug resistance.     

Regulation of root hair density by phosphorus availability in Arabidopsis thaliana

We characterized the response of root hair density to phosphorus (P) availability in Arabidopsis thaliana. Arabidopsis plants were grown aseptically in growth media with varied phosphorus concentrations, ranging from 1 mmol m⁻³ to 2000 mmol m⁻³ phosphorus. Root hair density (number of root hairs per mm of root length) was analysed starting at 7 d of growth. Root hair density was highly regulated by phosphorus availability, increasing significantly in roots exposed to low-phosphorus availability. The initial root hairs produced by the radicle were not sensitive to phosphorus availability, but began to respond after 9 d of growth. Root hair density was about five times greater in low phosphorus (1 mmol m⁻³) than in high phosphorus (1000 mmol m⁻³) media. Root hair density decreased logarithmically in response to increasing phosphorus concentrations within that range. Root hair density also increased in response to deficiencies of several other nutrients, but not as strongly as to low phosphorus. Indoleacetic acid (IAA), the auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid (CMPA), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the ethylene synthesis inhibitor amino-oxyacetic acid (AOA) all increased root hair density under high phosphorus but had very little effect under low phosphorus. Low phosphorus significantly changed root anatomy, causing a 9% increase in root diameter, a 31% decrease in the cross-sectional area of individual trichoblasts, a 40% decrease in the cross-sectional area of individual atrichoblasts, and 45% more cortical cells in cross-section. The larger number of cortical cells and smaller epidermal cell size in low phosphorus roots increased the number of trichoblast files from eight to 12. Two-thirds of increased root hair density in low phosphorus roots was caused by increased likelihood of trichoblasts to form hairs, and 33% of the increase was accounted for by changes in low phosphorus root anatomy resulting in an increased number of trichoblast files. These results show that phosphorus availability can fundamentally alter root anatomy, leading to changes in root hair density, which are presumably important for phosphorus acquisition.     

Auxin-induced inhibition of lateral root initiation contributes to root system shaping in Arabidopsis thaliana

The hormone auxin is known to inhibit root elongation and to promote initiation of lateral roots. Here we report complex effects of auxin on lateral root initiation in roots showing reduced cell elongation after auxin treatment. In Arabidopsis thaliana, the promotion of lateral root initiation by indole-3-acetic acid (IAA) was reduced as the IAA concentration was increased in the nanomolar range, and IAA became inhibitory at 25 nM. Detection of this unexpected inhibitory effect required evaluation of root portions that had newly formed during treatment, separately from root portions that existed prior to treatment. Lateral root initiation was also reduced in the iaaM-OX Arabidopsis line, which has an endogenously increased IAA level. The ethylene signaling mutants ein2-5 and etr1-3, the auxin transport mutants aux1-7 and eir1/pin2, and the auxin perception/response mutant tir1-1 were resistant to the inhibitory effect of IAA on lateral root initiation, consistent with a requirement for intact ethylene signaling, auxin transport and auxin perception/response for this effect. The pericycle cell length was less dramatically reduced than cortical cell length, suggesting that a reduction in the pericycle cell number relative to the cortex could occur with the increase of the IAA level. Expression of the DR5:GUS auxin reporter was also less effectively induced, and the AXR3 auxin repressor protein was less effectively eliminated in such root portions, suggesting that decreased auxin responsiveness may accompany the inhibition. Our study highlights a connection between auxin-regulated inhibition of parent root elongation and a decrease in lateral root initiation. This may be required to regulate the spacing of lateral roots and optimize root architecture to environmental demands.     

Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability

Low phosphorus availability stimulates root hair elongation in many plants, which may have adaptive significance in soil phosphorus acquisition. We investigated the effect of low phosphorus on the elongation of Arabidopsis thaliana root hairs. Arabidopsis thaliana plants were grown in plant culture containing high (1000 mmol m^?3) or low (1 mmol m^?3) phosphorus concentrations, and root hair elongation was analysed by image analysis. After 15d of growth, low-phosphorus plants developed root hairs averaging 0.9 mm in length while high-phosphorus plants of the same age developed root hairs averaging 0.3 mm in length. Increased root hair length in low-phosphorus plants was a result of both increased growth duration and increased growth rate. Root hair length decreased logarithmically in response to increasing phosphorus concentration. Local changes in phosphorus availability influenced root hair growth regardless of the phosphorus status of the plant. Low phosphorus stimulated root hair elongation in the hairless axr2 mutant, exogenously applied IAA stimulated root hair elongation in wild-type high-phosphorus plants and the auxin antagonist CM PA inhibited root hair elongation in low-phosphorus plants. These results indicate that auxin may be involved in the low-phosphorus response in root hairs.     

Development of a sensitive bioassay method for quorum sensing inhibitor screening using a recombinantAgrobacterium tumefaciens

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the β-galactosidase activity (β-GAL) of a recombinantAgrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing β-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration-dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of β-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.     

Detection of different quorum-sensing signal molecules in a virulent Edwardsiella tarda strain LTB-4

Aims:  The aim of this study was to elucidate the potential quorum-sensing (QS) signal molecules of an emerging pathogen ( Edwardsiella tarda strain LTB-4) of cultured turbot ( Scophthalmus maximus ).
Methods and Results:  A sensitive and rapid double-layer plate method using biosensor strain Agrobacterium tumefaciens KYC55 was developed to detect the N-acylhomoserine lactone (AHL)-related compounds in bacteria. LTB-4 was found to have two QS systems, one was based on the AHLs and the other was based on the autoinducer-2 (AI-2). The AI-2 activity produced by LTB-4 was growth phase dependent and topped at OD₆₀₀ of 1·0. The protocol to detect cholerae autoinducer 1 (CAI-1) activity in bacteria was modified, lowering the background luminescence of biosensor strain Vibrio harveyi JAF375. CAI-1 activity could not be detected in LTB-4.
Conclusion:  Edwardsiella tarda LTB-4 produced at least four kinds of AHLs during its whole growth phase. In comparison with the AHL-inducing QS, AI-2 may be the first predominant signal, functioning at early exponential phase. LTB-4 did not produce any CAI-1 activity.
Significance and Impact of the Study:  Different QS signal molecules of Edw. tarda LTB-4 were clarified by improved bioassays. In contrast to earlier studies detecting two types of AHLs, strain LTB-4 produced at least four kinds of AHLs, which seemed to be C₄-HSL, C₆-HSL, 3-oxo-C₆-HSL and an uncharacterized AHL molecule.     

低磷供应对拟南芥根系构型的影响

在人工气候箱中,采用Johnson培养基对拟南芥在低磷供应条件下根系构型的变化进行了研究,结果表明：拟南芥在磷饥饿诱导下,主根缩短,侧根密度、根毛的数量和长度显著增加,并且,根尖到第一侧根和第一根毛的距离也大大缩短。这些改变增加了根系比表面积,并且使得根系分布更加靠近土壤表层,有利于提高植物吸收土壤中有机磷的效率。低磷胁迫还导致拟南芥根系分生组织区细胞形状变异,柱细胞数量减少;主根生长和细胞伸长的动力学分析显示,磷饥饿促使拟南芥主根生长变缓,细胞长度随磷饥饿程度的加深迅速缩小。CycB1;1:GUS染色分析结果表明,低磷破坏拟南芥根系分生组织细胞分裂能力,这些结果说明磷胁迫同时抑制了细胞的伸长和分裂,从而引起拟南芥主根的缩短。     

Interactions between bacterial diazotrophs and non-legume dicots: Arabidopsis thaliana as a model plant

When interactions between diazotrophic bacteria and non-legume plants are studied within the context of trying to extend biological nitrogen fixation to non-legume crops, an important first step is to establish reproducible internal colonization at high frequency of these plants. Using Azorhizobium caulinodans ORS571 (which induces stem and root nodules on the tropical legume Sesbania rostrata), tagged with a constitutively expressed lacZ reporter gene, we have studied the possibilities of internal colonization of the root system of the model dicot Arabidopsis thaliana. ORS571 was found to be able to enter A. thaliana roots after first colonizing lateral root cracks (LRCs), at the points of emergence of lateral roots. Cytological studies showed that after LRC colonization, bacteria moved into the intercellular space between the cortical and endodermal cell layers of roots. In our experimental conditions, this LRC and intercellular colonization are reproducible and occur at high frequency, although the level of colonization at each site is low. The flavonoids naringenin and daidzein, at low concentrations, were found to significantly stimulate (at the p=0.01 level) the frequency of LRC and intercellular colonization of A. thaliana roots by A. caulinodans. The role in colonization of the structural nodABC genes, as well as the regulatory gene nodD, was studied and it was found that both colonization and flavonoid stimulation of colonization are nod gene-independent. These systems should now enable the various genetic and physiological factors which are limiting both for rhizobial colonization and for endophytic nitrogen fixation in non-legumes, to be investigated. In particular, the use of A. thaliana, which has many advantages over other plants for molecular genetic studies, to study interactions between diazotrophic bacteria and non-legume dicots, should provide the means of identifying and understanding the mechanisms by which plant genes are involved in these interactions.