Lactone Production Research Articles

In vitro Callus Culture of Cynara cardunculus subsp. scolymus: A Biosystem for Production of Caffeoylquinic Acid Derivatives, Sesquiterpene Lactones, and Flavonoids

In vitro culture of Cynara cardunculus subsp. scolymus was induced from bracts and leaves, using different phytohormone combinations. The most suitable one was 1-naphthaleneacetic acid: 6-benzylaminopurine (NAA-BAP), 3:1 mg/L. Modification of different phytohormone combinations altered the profile of C. cardunculus subsp. scolymus secondary metabolites as revealed by High Performance Liquid Chromatography. Rutin was recorded at 1.77 mg/g DW in leaf callus (not detected in leaves). After addition of 100 µM of methyl jasmonate (MeJA) as elicitor, 1.20 mg/g DW of cynarin was recorded in bract-derived callus and 1.18 mg/g DW in leaf-derived callus (not detected in leaves and bracts). Leaf callus enriched with NAA-BAP (3:1 mg/L) showed more elevated levels of rutin, cynarin, cynaropicrin, and pinoresinol (1.43, 1.2, 1.14, and 1.48 mg/g DW, respectively) as compared to the cultivated artichoke (0.0, 0.0, 0.40, and 0.0 mg/g DW, respectively). Moreover, elicitation of this callus by addition of 50 µM MeJA elevated luteolin, quercetin, cynarin, and chlorogenic acid levels relative to the initial callus. Higher Total Phenolics Content (TPC) was recorded in leaf and bract calluses (106 ± 2.69 and 55.19 ± 0.34 mg GAE/g DW, respectively) compared to leaves and bracts of cultivated plants (69.04 ± 0.05 and 19.42 ± 0.34 mg GAE/g DW, respectively).

Media and strain studies for the scaled production of cis-enone resorcylic acid lactones as feedstocks for semisynthesis

Resorcylic acid lactones (RALs) with a cis-enone moiety, represented by hypothemycin (1) and (5Z)-7-oxozeaenol (2), are fungal secondary metabolites with irreversible inhibitory activity against protein kinases, with particularly selective activity for inhibition of TAK1 (transforming growth factor beta-activated kinase 1). Gram-scale quantities of these compounds were needed as feedstock for semi-synthesizing RAL-analogues in a step-economical fashion. To do so, this study had three primary goals: identifying fungi that biosynthesized 1 and 2, enhancing their production by optimizing the fermentation conditions on the lab scale, and developing straight forward purification processes. After evaluating 536 fungal extracts via an in-house dereplication protocol, three strains were identified as producing cis-enone RALs (i.e., MSX78495, MSX63935, MSX45109). Screening these fungal strains on three grain-based media revealed enhanced production of 1 by strain MSX78495 on oatmeal medium, while rice medium increased the biosynthesis of 2 by strain MSX63935. Furthermore, the purification processes were improved, moving away from HPLC purification to utilizing two to four cycles of resuspension and centrifugation in small volumes of organic solvents, generating gram-scale quantities of these metabolites readily. In addition, studying the chemistry profiles of strains MSX78495 and MSX63935 resulted in the isolation of ten other RALs (3-12), two radicinin analogues (13-14), and six benzopyranones (15-20), with 19 and 20 being newly described chlorinated benzopyranones.

A novel anti-infective molecule nesfactin identified from sponge associated bacteria Nesterenkonia sp. MSA31 against multidrug resistant Pseudomonas aeruginosa

Overuse of antibiotics coupled with biofilm-forming ability has led to the emergence of multi-drug P. aeruginosa strains worldwide. Quorum sensing is a bacterial cell-cell communication system that regulates the expression of genes, including virulence factors, through production of acyl-homoserine lactones (AHLs) in Pseudomonas aeruginosa. The phenotypic expression of virulence factors in P. aeruginosa is mediated by quorum sensing systems (las and rhl). In this study an anti-infective molecule produced by a marine actinomycetes Nesterenkonia sp. MSA31 was elucidated as lipopeptide by NMR and LC-MS/MS analysis. The new lipopeptide molecule was named Nesfactin. This molecule effectively inhibited virulence phenotypes including production of hemolysin, protease, lipase, phospholipase, esterase, elastase, rhamnolipid, alginate, and pyocyanin, as well as motility and biofilm formation in P. aeruginosa. The high-performance thin layer chromatography (HPTLC) analysis revealed that the lipopeptide (50 μg/mL) inhibited production of the AHLs produced by the las and rhl quorum sensing systems (3-oxo-C12-HSL and C4-HSL, respectively). Docking analysis showed the binding affinity of the ligand towards the quorum sensing receptor molecules. The confocal laser scanning microscopy images showed the anti-biofilm effect of lipopeptide against P. aeruginosa. Nesfactin based hydrogel showed a significant antibiofilm effect on the catheter. This study suggests that the lipopeptide may be an effective anti-virulence treatment for Pseudomonas aeruginosa infections.

Chelativorans alearense sp. nov., A Novel Bacterial Species Isolated From Soil in Alear, China.

A novel Gram-strain-negative, rod-shaped, non-flagellated, non-gliding, beige-pigmented and aerobic bacterium, designated strain UJN715T, was isolated from rhizosphere soil of Alhagi sparsifolia obtained from Alear city, located in Xinjiang province, PR China. Growth optimally occurred at 37°C, pH 6.5-7.5, and 0-3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain UJN715T belonged to the genus Chelativorans, with the highest sequence similarity to Chelativorans multitrophicus DSM 9103T (97.7%). Genome sequencing revealed a genome size of 5 702 301bp and a G + C content of 64.1mol%. The ANI, POCP and the dDDH between strain UJN715T and C. multitrophicus DSM 9103T were 76.2%, 49.3%, and 20.5%, respectively. The prediction result of secondary metabolites based on genome showed that the strain UJN715T contained one cluster of ectoine production, one cluster of non-ribosomal peptide synthetase (NRPS), one cluster of type I polyketide synthases (TIPKS), one cluster of bacteriocin, one cluster of TfuA-related, one cluster of N-acetylglutaminylglutamine amide (NAGGN) production, one cluster of terpene production, two clusters of homoserine lactone (Hserlactone) production. The major respiratory quinone was Q-10. The major fatty acids were iso-C17:0, C18:0 and C19:0 cyclo ω8c and its polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phospholipids, unknown lipids, diphosphatidylglycerol, aminoglycolipid, unidentified aminophospholipids. On the basis of these data, strain UJN715T is considered to represent a novel species of the genus Chelativorans, for which the name Chelativorans alearense sp. nov. is proposed. The type strain is UJN715T (= KCTC 72856T = CCTCC AB2019378T).

Naphthoquinones inhibit formation and viability of Yersinia enterocolitica biofilm.

The capacity of different naphthoquinones to inhibit and eradicate Yersinia enterocolitica biofilm was investigated and possible mechanisms of action were evaluated. Inhibition of biofilm formation and cell viability, quorum sensing (QS) inhibition and oxidative stress generation of 23 naphthoquinones were assayed against Yersinia enterocolitica. The best anti-biofilm agents at 100µmoll-1 were compounds 3, 11 and 13, which showed biofilm inhibition higher than 75%. Compound 3 was the most effective against biofilm forming capacity of Y. enterocolitica WAP 314 with a minimum biofilm inhibitory concentration (MBIC) of 25µmoll-1; while against Y. enterocolitica CLC001, the lowest MBIC was 6.1µmoll-1 for compound 11. Acyl-homoserine lactones production was decreased with compound 13. We showed that the oxidative stress influence biofilm growth, by means of ROS and RNI increment. All treatments increased ROS and RNI values in the biofilm of both strains; while in planktonic cells, the increase was lesser. Additionally, Y. enterocolitica WAP 314 biofilm treated with compounds 11 and 13 showed above 80% of SOD consumption. In Y. enterocolitica CLC001 biofilm all compounds induced above 90% of SOD consumption. The SOD activity was higher in biofilm than in planktonic cells. In conclusion, this is the first study to demonstrate that naphthoquinones are able to inhibit biofilm formation of Y. enterocolitica without critical disturbing its planktonic growth. Naphthoquinones as anti-biofilm agents might potentially be useful in the treatment of biofilm-associated infections in the future.

Chemical inhibitors of the conserved bacterial transcriptional regulator DksA1 suppressed quorum sensing-mediated virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen whose virulence is dependent on quorum sensing (QS). DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in determining a number of phenotypes, including QS-mediated virulence. We therefore envisioned that DksA1 inhibitors may help to control P. aeruginosa infection. Here, we screened a library of 6970 chemical compounds and identified two compounds (henceforth termed Dkstatins) that specifically suppressed DksA1 activity. Treatment with these two compounds also substantially decreased the production of elastase and pyocyanin, dominant virulence determinants of P. aeruginosa, and protected murine hosts from lethal infection from a prototype strain of P. aeruginosa, PAO1. The Dkstatins also suppressed production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced by Dkstatin-treated wildtype PAO1 closely resembled that of the ΔdksA1 mutant. RNA-Seq analysis showed that transcription levels of QS- and virulence-associated genes were markedly reduced in Dkstatin-treated PAO1 cells, indicating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased the antibiotic susceptibilities of PAO1, particularly to protein synthesis inhibitors, such as tobramycin and tetracycline. Co-immunoprecipitation assays demonstrated that these Dkstatins interfered with DksA1 binding to the β subunit of RNA polymerase, pointing to a potential mechanism of action. Collectively, our results illustrate that inhibition of P. aeruginosa QS may be achieved via DksA1 inhibitors and that Dkstatins may serve as potential lead compounds to control infection.

Chelativorans xinjiangense sp. nov., a novel bacterial species isolated from soil in Xinjiang, China

A novel Gram-strain-negative, beige-pigmented, aerobic, rod-shaped, non-flagellated and non-gliding bacterium, designated strain lm93T, was isolated from rhizosphere soil of Alhagi sparsifolia obtained from Alar city, located in Xinjiang province, China. Growth optimally occurred at 30°C, pH 6.5-7.5, and 0-2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain lm93T belonged to the genus Chelativorans, with highest sequence similarity to Chelativorans multitrophicus DSM 9103T (96.9%). Genome sequencing revealed a genome size of 5 689 708bp and a G + C content of 64.3mol%. The ANI, POCP and the dDDH between strain lm93T and C. multitrophicus DSM 9103T were 76.4%, 54.8% and 0.8%, respectively. The prediction result of secondary metabolites based on genome showed that the strain lm93T contained one cluster of bacteriocin, one cluster of terpene production, two clusters of ectoine production, one cluster of non-ribosomal peptide synthetase, one cluster of type I polyketide synthases, three clusters of homoserine lactone production, one cluster of N-acetylglutaminylglutamine amide production and one cluster of phosphonate production. The major respiratory quinone was Q-10. The major fatty acids were C19:0 cyclo ω8c, iso-C17:0 and summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and its polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, aminoglycolipid, three unknown lipids and diphosphatidylglycerol. On the basis of these data, strain lm93T is considered to represent a novel species of the genus Chelativorans, for which the name Chelativorans xinjiangense sp. nov. is proposed. The type strain is lm93T (= KCTC 72857T = CCTCC AB2019376T).

Developing a broad-range promoter set for metabolic engineering in the thermotolerant yeast Kluyveromyces marxianus

Kluyveromyces marxianus is an emerging host for metabolic engineering. This thermotolerant yeast is the fastest growing eukaryote, has high flux through the TCA cycle, and can metabolize a broad range of C5, C6, and C12 carbon sources. In comparison to the common host Saccharomyces cerevisiae, this non-conventional yeast suffers from a lack of metabolic engineering tools to control gene expression over a wide transcriptional range. To address this issue, we designed a library of 25 native-derived promoters from K. marxanius CBS6556 that spans 87-fold transcriptional strength under glucose metabolism. Six promoters from the library were further characterized in both glucose and xylose as well as across various temperatures from 30 to 45 ​°C. The temperature study revealed that in most cases EGFP expression decreased with elevating temperature; however, two promoters, PSSA3 and PADH1, increased expression above 40 ​°C in both xylose and glucose. The six-promoter set was also validated in xylose for triacetic acid lactone (TAL) production. By controlling the expression level of heterologous 2-pyrone synthase (2-PS), the specific TAL titer increased over 8-fold at 37 ​°C. Cultures at 41 ​°C exhibited a similar TAL biosynthesis capability, while at 30 ​°C TAL levels were lower. Taken together, these results advance the metabolic engineering tool set in K. marxianus and further develop this new host for chemical biosynthesis.

Terminalia chebula and Ficus racemosa principles mediated repression of novel drug target Las R – the transcriptional regulator and its controlled virulence factors produced by multiple drug resistant Pseudomonas aeruginosa - Biocompatible formulation against drug resistant bacteria

Pseudomonas aeruginosa– major group of an aerobic bacteria associated with nosocomial and other life threatening infections. Diverse virulence factors produced by P. aeruginosa is due to distinct molecular cell signaling mechanism termed as quorum sensing (QS). Interfering with normal QS mechanism by active biomolecules is an effective strategy for attenuating its virulence. With this objective, the present study is undertaken to evaluate the inhibition of quorum sensing of clinical isolate of P. aeruginosa by repression of Las R-a transcriptional regulator for QS by ethanol extract of Terminalia chebula and Ficus racemosa. Las R repression by the plant extracts was measured in inhibition of various virulence factors like biofilm, pyocyanin production, total proteolytic activity, swarming and twisting motility. Fabrication of the extracted metabolites on the wound dressing and its effect on anti bacterial activity was also investigated. Compatibility of plant extracts on zebra fish development and blood cells was further studied. P. aeruginosa was isolated from the post operative patient and the isolated pure culture was identified by cultural, biochemical, molecular characteristics. Active principles of both the plants were readily extracted in ethanol and effectively repressed the expression of Las R. Both the tested plant extracts effectively repressed Las R expression which in turn affect the production of various virulence factors like biofilm formation, pyocyanin production, swarming motility, twisting motility, total proteolytic activity, cell adhesion and signaling molecule acyl honoserine lactone (AHL) production. Plant extract treatment brought about drastic reduction of all the tested virulence factors and AHL production. Extracted metabolites were fabricated on the wound dressing material adopting simple dip or immersion method reveals uniform coating, effective embedding of phytochemicals with the fibers and retained the anti bacterial activity against P. aeruginosa. Biocompatibility studies with zebra fish model shows both the tested plant extracts treatment was not exhibited any sign of toxicity on the developmental stages of Zebra fish. Hemolysis and changes in anti oxidative enzymes were not recorded in the plant extracts treated blood which demonstrated the best biocompatibility of the tested plant extracts. These results shows that the presence of potential phytochemicals in the ethanolic extract of Terminalia chebula and Ficus racemosa effectively represses the Las R followed by inhibition of quorum sensing mediated virulence factors production may be useful in the lead of anti bacterial drugs.

Secondary metabolites from plant-associated Pseudomonas are overproduced in biofilm.

SummaryPlant rhizosphere soil houses complex microbial communities in which microorganisms are often involved in intraspecies as well as interspecies and inter‐kingdom signalling networks. Some members of these networks can improve plant health thanks to an important diversity of bioactive secondary metabolites. In this competitive environment, the ability to form biofilms may provide major advantages to microorganisms. With the aim of highlighting the impact of bacterial lifestyle on secondary metabolites production, we performed a metabolomic analysis on four fluorescent Pseudomonas strains cultivated in planktonic and biofilm colony conditions. The untargeted metabolomic analysis led to the detection of hundreds of secondary metabolites in culture extracts. Comparison between biofilm and planktonic conditions showed that bacterial lifestyle is a key factor influencing Pseudomonas metabolome. More than 50% of the detected metabolites were differentially produced according to planktonic or biofilm lifestyles, with the four Pseudomonas strains overproducing several secondary metabolites in biofilm conditions. In parallel, metabolomic analysis associated with genomic prediction and a molecular networking approach enabled us to evaluate the impact of bacterial lifestyle on chemically identified secondary metabolites, more precisely involved in microbial interactions and plant‐growth promotion. Notably, this work highlights the major effect of biofilm lifestyle on acyl‐homoserine lactone and phenazine production in P. chlororaphis strains.

Bacterial Biotransformation of Oleic Acid: New Findings on the Formation of γ-Dodecalactone and 10-Ketostearic Acid in the Culture of Micrococcus luteus.

Microbial conversion of oleic acid (1) to form value-added industrial products has gained increasing scientific and economic interest. So far, the production of natural lactones with flavor and fragrance properties from fatty acids by non-genetically modified organisms (non-GMO) involves whole cells of bacteria catalyzing the hydration of unsaturated fatty acids as well as yeast strains responsible for further β-oxidation processes. Development of a non-GMO process, involving a sole strain possessing both enzymatic activities, significantly lowers the costs of the process and constitutes a better method from the customers’ point of view regarding biosafety issues. Twenty bacteria from the genus of Bacillus, Comamonas, Dietzia, Gordonia, Micrococcus, Pseudomonas, Rhodococcus and Streptomyces were screened for oxidative functionalization of oleic acid (1). Micrococcus luteus PCM525 was selected as the sole strain catalyzing the one-pot transformation of oleic acid (1) into natural valuable peach and strawberry-flavored γ-dodecalactone (6) used in the food, beverage, cosmetics and pharmaceutical industries. Based on the identified products formed during the process of biotransformation, we clearly established a pathway showing that oleic acid (1) is hydrated to 10-hydroxystearic acid (2), then oxidized to 10-ketostearic acid (3), giving 4-ketolauric acid (4) after three cycles of β-oxidation, which is subsequently reduced and cyclized to γ-dodecalactone (6) (Scheme 1). Moreover, three other strains (Rhodococcus erythropolis DSM44534, Rhodococcus ruber PCM2166, Dietzia sp. DSM44016), with high concomitant activities of oleate hydratase and alcohol dehydrogenase, were identified as efficient producers of 10-ketostearic acid (3), which can be used in lubricant and detergent formulations. Considering the prevalence of γ-dodecalactone (6) and 10-ketostearic acid (3) applications and the economic benefits of sustainable management, microbial bioconversion of oleic acid (1) is an undeniably attractive approach.

Chemoinformatic-Guided Engineering of Polyketide Synthases

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKSmodule to produce a saturated β-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structuresimilarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced bythe hostStreptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

Bacterial Cellulose-Alginate Composite Beads as Yarrowia lipolytica Cell Carriers for Lactone Production.

The demand for natural lactone gamma-decalactone (GDL) has increased in the fields of food and cosmetic products. However, low productivity during bioprocessing limits its industrial production. In this study, a novel composite porous cell carrier, bacterial cellulose-alginate (BC-ALG), was used for long-term biotransformation and production of GDL. The effects of this carrier on biotransformation and related mechanisms were investigated. BC-ALG carriers showed improved mechanical strength over ALG carriers, with their internal embedded cell pattern changed to an interconnected porous structure. In five repeated-batch biotransformation experiments, the maximum concentration of GDL obtained in culture with BC-ALG carriers was 8.37 g/L, approximately 3.7 times higher than that from the medium with an ALG carrier alone. The result indicated that multiple hydrogen bonding interactions at the interface between BC and ALG contributed to the compatibility and stability of BC-ALG carriers. On the basis of the above results, the BC-ALG composite carrier can be considered ideal for immobilisation of cells for the production of GDL on an industrial scale, and has the potential to be utilised in other biological processes.

Stimulatory effects on bacteria induced by chemical cleaning cause severe biofouling of membranes

Abstract Chemical cleaning with hypochlorite is routinely used in membrane-based processes. However, a high-transient cleaning efficiency does not guarantee a low biofouling rate when filtration is restarted, with the physiological mechanisms largely remaining unknown. Herein, we investigated the microbial regrowth and surface colonization on membrane surfaces after NaOCl cleaning had been completed. Results of this study showed that the regrowth of model bacteria, Pseudomonas aeruginosa, was initially subject to inhibition due to the damage of key enzymes' activity and the accumulation of intracellular reactive oxygen species although the oxidative stress induced by NaOCl had been removed. However, with the resuscitation ongoing, the stimulatory effects became obvious, which was associated with the enhanced production of N-acyl homoserine lactones and the secretion of eDNA that ultimately led to more severe biofouling on the membrane surface. This study elucidates the inhibition–stimulation mechanisms involved in biofilm reformation (membrane biofouling) after membrane chemical cleaning, which is of particular significance to the improvement of cleaning efficiency and application of membrane technologies.

Genomic, metabolic and phenotypic variability shapes ecological differentiation and intraspecies interactions of Alteromonas macleodii

Ecological differentiation between strains of bacterial species is shaped by genomic and metabolic variability. However, connecting genotypes to ecological niches remains a major challenge. Here, we linked bacterial geno- and phenotypes by contextualizing pangenomic, exometabolomic and physiological evidence in twelve strains of the marine bacterium Alteromonas macleodii, illuminating adaptive strategies of carbon metabolism, microbial interactions, cellular communication and iron acquisition. In A. macleodii strain MIT1002, secretion of amino acids and the unique capacity for phenol degradation may promote associations with Prochlorococcus cyanobacteria. Strain 83-1 and three novel Pacific isolates, featuring clonal genomes despite originating from distant locations, have profound abilities for algal polysaccharide utilization but without detrimental implications for Ecklonia macroalgae. Degradation of toluene and xylene, mediated via a plasmid syntenic to terrestrial Pseudomonas, was unique to strain EZ55. Benzoate degradation by strain EC673 related to a chromosomal gene cluster shared with the plasmid of A. mediterranea EC615, underlining that mobile genetic elements drive adaptations. Furthermore, we revealed strain-specific production of siderophores and homoserine lactones, with implications for nutrient acquisition and cellular communication. Phenotypic variability corresponded to different competitiveness in co-culture and geographic distribution, indicating linkages between intraspecific diversity, microbial interactions and biogeography. The finding of “ecological microdiversity” helps understanding the widespread occurrence of A. macleodii and contributes to the interpretation of bacterial niche specialization, population ecology and biogeochemical roles.

The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms.

Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection.IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

Antibacterial Activity and Anti-Quorum Sensing Mediated Phenotype in Response to Essential Oil from Melaleuca bracteata Leaves.

The prominent antibacterial and quorum sensing (QS) inhibition activity of aromatic plants can be used as a novel intervention strategy for attenuating bacterial pathogenicity. In the present work, a total of 29 chemical components were identified in the essential oil (EO) of Melaleuca bracteata leaves by gas chromatography-mass spectrometry (GC-MS). The principal component was methyleugenol, followed by methyl trans-cinnamate, with relative contents of 90.46% and 4.25%, respectively. Meanwhile, the antibacterial activity and the QS inhibitory activity of M. bracteata EO were first evaluated here. Antibacterial activity assay and MIC detection against seven pathogens (Dickeya dadantii Onc5, Staphylococcus aureus ATCC25933, Pseudomonas spp., Escherichia coli ATCC25922, Serratia marcescens MG1, Pseudomonas aeruginosa PAO1 and Chromobacterium violaceum ATCC31532) demonstrated that S. aureus ATCC25933 and S. marcescens MG1 had the higher sensitivity to M. bracteata EO, while P. aeruginosa PAO1 displayed the strongest resistance to M. bracteata EO. An anti-QS (anti-quorum sensing) assay revealed that at sub-minimal inhibitory concentrations (sub-MICs), M. bracteata EO strongly interfered with the phenotype, including violacein production, biofilm biomass, and swarming motility, as well as N-hexanoyl-L-homoserine lactone (C6-HSL) production (i.e., a signaling molecule in C. violaceum ATCC31532) of C. violaceum. Detection of C6-HSL indicated that M. bracteata EO was capable of not only inhibiting C6-HSL production in C. violaceum, but also degrading the C6-HSL. Importantly, changes of exogenous C6-HSL production in C. violaceum CV026 revealed a possible interaction between M. bracteata EO and a regulatory protein (cviR). Additionally, quantitative real-time polymerase chain reaction (RT-qPCR) analysis demonstrated that the expression of QS-related genes (cviI, cviR, vioABCDE, hmsNR, lasA-B, pilE1, pilE3, and hcnB) was significantly suppressed. Conclusively, these results indicated that M. bracteata EO can act as a potential antibacterial agent and QS inhibitor (QSI) against pathogens, preventing and controlling bacterial contamination.

Methyl anthranilate: A novel quorum sensing inhibitor and anti-biofilm agent against Aeromonas sobria

Quorum sensing (QS), bacterial cell-to-cell communication, is a gene regulatory mechanism that regulates virulence potential and biofilm formation in many pathogens. Aeromonas sobria, a common aquaculture pathogen, was isolated and identified by our laboratory from the deteriorated turbot, and its potential for virulence factors and biofilm production was regulated by QS system. In view of the interference with QS system, this study was aimed to investigate the effect of methyl anthranilate at sub-Minimum Inhibitory Concentrations (sub-MICs) on QS-regulated phenotypes in A. sobria. The results suggested that 0.5 μL/mL of methyl anthranilate evidently reduced biofilm formation (51.44%), swinging motility (74.86%), swarming motility (71.63%), protease activity (43.08%), and acyl-homoserine lactone (AHL) production. Furthermore, the real-time quantitative PCR (RT-qPCR) and in silico analysis showed that methyl anthranilate might inhibit QS system in A. sobria by interfering with the biosynthesis of AHL, as well as competitively binding with receptor protein. Therefore, our data indicated the feasibility of methyl anthranilate as a promising QS inhibitor and anti-biofilm agent for improving food safety.

Functional Characterization of a Global Virulence Regulator Hfq and Identification of Hfq-Dependent sRNAs in the Plant Pathogen Pantoea ananatis.

To successfully infect plant hosts, the collective regulation of virulence factors in a bacterial pathogen is crucial. Hfq is an RNA chaperone protein that facilitates the small RNA (sRNA) regulation of global gene expression at the post-transcriptional level. In this study, the functional role of Hfq in a broad host range phytopathogen Pantoea ananatis was determined. Inactivation of the hfq gene in P. ananatis LMG 2665T resulted in the loss of pathogenicity and motility. In addition, there was a significant reduction of quorum sensing signal molecule acyl-homoserine lactone (AHL) production and biofilm formation. Differential sRNA expression analysis between the hfq mutant and wild-type strains of P. ananatis revealed 276 sRNAs affected in their abundance by the loss of hfq at low (OD600 = 0.2) and high cell (OD600 = 0.6) densities. Further analysis identified 25 Hfq-dependent sRNAs, all showing a predicted Rho-independent terminator of transcription and mapping within intergenic regions of the P. ananatis genome. These included known sRNAs such as ArcZ, FnrS, GlmZ, RprA, RyeB, RyhB, RyhB2, Spot42, and SsrA, and 16 novel P. ananatis sRNAs. The current study demonstrated that Hfq is an important component of the collective regulation of virulence factors and sets a foundation for understanding Hfq-sRNA mediated regulation in the phytopathogen P. ananatis.

Lipid Transfer Proteins (AaLTP3 and AaLTP4) Are Involved in Sesquiterpene Lactone Secretion from Glandular Trichomes in Artemisia annua.

In Artemisia annua plants, glandular trichomes (GTs) are responsible for the biosynthesis and secretion of sesquiterpene lactones including artemisinin/arteannuin B. Nonspecific lipid transfer proteins (LTPs) in plants bind and carry lipid molecules across the cell membrane and are also known as secretary proteins. Interestingly, the transcripts of LTP genes are exceptionally abundant in the GTs of A. annua. In the present study, we isolated two trichome-specific LTP genes (AaLTP3 and AaLTP4) from a Korean ecotype of A. annua. AaLTP3 was expressed abundantly in shoots, whereas AaLTP4 was expressed in flowers. The GUS signal driven by the AaLTP3 or AaLTP4 promoter in transgenic A. annua plants revealed that the AaLTP3 promoter was active on hair-like non-GTs and that the AaLTP4 promoter was active on GTs. Analysis of enhanced cyan fluorescent protein (ECFP) fluorescence fused with the AaLTP3 or AaLTP4 protein in transgenic tobacco revealed that ECFP florescence was very bright on secreted lipids of long GTs. Moreover, the florescence was also bright on the head cells of short trichomes and their secreted granules. Immunoblotting analysis of GT exudates in petioles of A. annua revealed a strong positive signal against the AaLTP4 antibody. Overexpression of AaLTP3 or AaLTP4 in transgenic A. annua plants resulted in enhanced production of sesquiterpene lactones (arteannuin B, artemisinin, dihydroartemisinic acid and artemisinic acid) compared with those of wild type. The present study shows that LTP genes (AaLTP3 or AaLTP4) play important roles in the sequestration and secretion of lipids in GTs of A. annua, which is useful for the enhanced production of sesquiterpene lactones by genetic engineering.