Evidence of quorum sensing in the rumen ecosystem: detection of N-acyl homoserine lactone autoinducers in ruminal contents

Acyl-homoserine lactone (AHL) based quorum-sensing systems are widespread among gram-negative bacteria, particularly in association with plants and animals. As yet, there have been no reports of AHL signaling in the anaerobic rumen environment, an ecosystem of great complexity in which cell-cell signaling is likely to occur. We detected multiple AHL autoinducers in the rumen contents of 6 out of 8 cattle fed a representative selection of diets. The signals were not associated with feed. Surprisingly, no pure cultures produced AHLs in vitro when grown under the laboratory conditions we tested. Our observations suggest that either (a) a factor specific to the rumen ecosystem is required for the rumen isolates we tested to produce AHLs or (b) a strain (or strains) that we were not able to culture but which grows to a high cell density in the rumen produces the AHLs we detected.     

Acyl-Homoserine-Lactone Autoinducer in the Gastrointesinal Tract of Feedlot Cattle and Correlation to Season, <Emphasis Type="Italic">E. Coli</Emphasis> O157:H7 Prevalence,and Diet

Acyl-homoserine-lactone autoinducer (AHL) produced by nonenterohemorrhagic Escherichia coli species in cattle appears to be required for enterohemorrhagic E. coli (EHEC) colonization of the gastrointestinal tract (GIT). The current research aimed to examine the effect of season, diet, EHEC shedding, and location within the GIT on AHL prevalence in the ruminant. Luminal content samples were collected from the rumen and rectum of feedlot cattle at slaughter in the spring, summer, fall, and winter for culture of E. coli O157:H7 and AHL determination. During the spring collection, samples were additionally collected from the cecum and small intestine, but these samples all were AHL negative and therefore not examined again. To assess the influence of diet on AHL prevalence, 14 lambs were fed either 100% forage or 80% concentrate diets and experimentally inoculated with EHEC. At 8 days after infection, all the lambs were killed, and necropsies were taken, with luminal contents collected from the GIT. The collections from the feedlot cattle had AHL in 100% of the rumen content samples from the spring, summer, and fall, but not in any of the winter samples. No other GIT samples from feedlot cattle were AHL positive, and all the samples from the sheep study were AHL negative. The cattle seemed to show a weak correlation between ruminal AHL and EHEC prevalence. This research found AHL only in the rumen and not in the lower GIT of feedlot cattle. However, it is unclear whether this is because the pH of the lower gut destroys the AHL or because a lack of certain bacteria in the lower gut producing AHL. Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.     

Cross-kingdom signalling: exploitation of bacterial quorum sensing molecules by the green seaweed Ulva

The green seaweed Ulva has been shown to detect signal molecules produced by bacteria. Biofilms that release N-acylhomoserine lactones (AHLs) attract zoospores--the motile reproductive stages of Ulva. The evidence for AHL involvement is based on several independent lines of evidence, including the observation that zoospores are attracted to wild-type bacteria that produce AHLs but are not attracted to mutants that do not produce signal molecules. Synthetic AHL also attracts zoospores and the attraction is lost in the presence of autoinducer inactivation (AiiA) protein. The mechanism of attraction is not chemotactic but involves chemokinesis. When zoospores detect AHLs, the swimming rate is reduced and this results in accumulation of cells at the source of the AHL. It has been demonstrated that the detection of AHLs results in calcium influx into the zoospore. This is the first example of a calcium signalling event in a eukaryote in response to bacterial quorum sensing molecules. The role of AHLs in the ecology of Ulva is discussed. It is probable that AHLs act as cues for the settlement of zoospores, rather than being directly involved as a signalling mechanism.     

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.     

Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.     

华癸根瘤菌中自体诱导物的初步研究

群体感应 (Quorumsensing)是细菌通过产生可扩散的小分子量自体诱导物信号分子感知细胞群体密度变化 ,进行基因表达调控的生理行为。将根癌土壤杆菌 (Agrobacteriumtumefaciens)构建为超量表达群体感应调节蛋白TraR的检测菌株JZA1,试验证明该检测菌株能检测纳摩尔浓度的自体诱导物 ,利用该菌株对 3株不同华癸根瘤菌(Mesorhizobiumhuakuii)进行自体诱导物活性检测 ,发现该 3株华癸根瘤菌均能产生自体诱导物 ,其表达量与菌体密度成正相关 ,但 3株菌在相同培养条件下自体诱导物的表达量存在差异 ,结果表明自体诱导物在种内水平上存在一定的多样性 ;同时发现高pH条件能大大降低自体诱导物的稳定性 ,为进一步研究群体感应调节在共生固氮上的作用提供理论及实践依据     

N-Acylated homoserine lactone production and involvement in the biodegradation of aromatics by an environmental isolate of Pseudomonas aeruginosa

N-Acyl homoserine lactone (AHL) is a widespread quorum sensing signal molecule in Gram-negative bacteria and has an important role in many biological processes. However, it is still poorly understood whether or not AHL is present in pollutant treatment processes and further, what its role is in biodegradation processes. In this work, an environmental isolate of Pseudomonas aeruginosa CGMCC 1.860 that is an aromatic degrader and AHL producer was selected. The AHL plate bioassay indicated that AHL was produced by this strain during biodegradation of aromatic compounds including phenol, benzoate, p-hydroxy-benzoate, salicylate, and naphthalene. The AHLs were identified as N-butyryl-l-homoserine lactone (BHL) and N-hexanoyl-l-homoserine lactone (HHL) by using thin layer chromatography (TLC) and high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (HPLC–APCI-MS/MS) analyses. Furthermore, phenol biodegradation was improved by exogenously added AHL extracts or by endogenously over-produced AHLs, repressed by abolishment of AHLs production, and not affected by the addition of extracts without AHLs. The results indicated that AHL was involved in the process of biodegradation of pollutants.     

Detection of N-acyl homoserine lactones using a traI-luxCDABE-based biosensor as a high-throughput screening tool

Background

Bacteria use N-acyl homoserine lactone (AHL) molecules to regulate the expression of genes in a density-dependent manner. Several biosensors have been developed and engineered to detect the presence of all types of AHLs.

Results

In this study, we describe the usefulness of a traI-luxCDABE-based biosensor to quickly detect AHLs from previously characterized mutants of Burkholderia cenocepacia and Pseudomonas aeruginosa in both liquid and soft-agar co-culture assays in a high-throughput manner. The technique uses a co-culture system where the strain producing the AHLs is grown simultaneously with the reporter strain. Use of this assay in liquid co-culture allows the measurement of AHL activity in real time over growth. We tested this assay with Burkholderia cenocepacia and Pseudomonas aeruginosa but it should be applicable to a broad range of gram negative species that produce AHLs.

Conclusion

The co-culture assays described enable the detection of AHL production in both P. aeruginosa and B. cenocepacia and should be applicable to AHL analysis in other bacterial species. The high-throughput adaptation of the liquid co-culture assay could facilitate the screening of large libraries for the identification of mutants or compounds that block the synthesis or activity of AHLs.     

Specificity of acyl-homoserine lactone synthases examined by mass spectrometry

Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources.     

Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing

Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles. The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs. The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core. Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo. A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.     

Synthesis of N-acyl homoserine lactone analogues reveals strong activators of SdiA, the Salmonella enterica serovar Typhimurium LuxR homologue

N-Acyl homoserine lactones (AHLs) are molecules that are synthesized and detected by many gram-negative bacteria to monitor the population density, a phenomenon known as quorum sensing. Salmonella enterica serovar Typhimurium is an exceptional species since it does not synthesize its own AHLs, while it does encode a LuxR homologue, SdiA, which enables this bacterium to detect AHLs that are produced by other species. To obtain more information about the specificity of the ligand binding by SdiA, we synthesized and screened a limited library of AHL analogues. We identified two classes of analogues that are strong activators of SdiA: the N-(3-oxo-acyl)-homocysteine thiolactones (3O-AHTLs) and the N-(3-oxo-acyl)-trans-2-aminocyclohexanols. To our knowledge, this is the first report of compounds (the 3O-AHTLs) that are able to activate a LuxR homologue at concentrations that are lower than the concentrations of the most active AHLs. SdiA responds with greatest sensitivity to AHTLs that have a keto modification at the third carbon atom and an acyl chain that is seven or eight carbon atoms long. The N-(3-oxo-acyl)-trans-2-aminocyclohexanols were found to be less sensitive to deactivation by lactonase and alkaline pH than the 3O-AHTLs and the AHLs are. We also examined the activity of our library with LuxR of Vibrio fischeri and identified three new inhibitors of LuxR. Finally, we performed preliminary binding experiments which suggested that SdiA binds its activators reversibly. These results increase our understanding of the specificity of the SdiA-ligand interaction, which could have uses in the development of anti-quorum-sensing-based antimicrobials.     

Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.     

Use of a Whole-Cell Biosensor and Flow Cytometry to Detect AHL Production by an Indigenous Soil Community During Decomposition of Litter

Quorum sensing, mediated by acylated homoserine lactones (AHLs), is well described for pure culture bacteria, but few studies report detection of AHL compounds in natural bacterial habitats. In this study, we detect AHL production during a degradation process in soil by use of whole-cell biosensor technology and flow cytometry analysis. An indigenous soil bacterium, belonging to the family of Enterobacteriaceae, was isolated and transformed with a low-copy plasmid harboring a gene encoding an unstable variant of the green fluorescent protein (gfpASV) fused to the AHL-regulated P_luxI promoter originating from Vibrio fischeri. This resulted in a whole-cell biosensor, responding to the presence of AHL compounds. The biosensor was introduced to compost soil microcosms amended with nettle leaves. After 3 days of incubation, cells were extracted and analyzed by flow cytometry. All microcosms contained induced biosensors. From these microcosms, AHL producers were isolated and further identified as species previously shown to produce AHLs. The results demonstrate that AHL compounds are produced during degradation of litter in soil, indicating the presence of AHL-mediated quorum sensing in this environment.     

Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: detection of autoinducers in Mesorhizobium huakuii

An ultrasensitive bioassay system for the detection of N-acylhomoserine lactones (AHLs) was constructed in Agrobacterium tumefaciens by using the T7 expression system to overproduce the AHL receptor TraR. This strain detected many diverse AHLs, some at extremely low concentrations. We used this strain to detect for the first time AHLs made by Mesorhizobium huakuii, which symbiotically fixes nitrogen in association with the legume Astragalus sinicus, a source of green manure throughout eastern Asia.