Fengquan Liu

A novel immunochromatographic electrochemical biosensor for highly sensitive and selective detection of trichloropyridinol, a biomarker of exposure to chlorpyrifos.

We present a novel portable immunochromatographic electrochemical biosensor (IEB) for simple, rapid, and sensitive biomonitoring of trichloropyridinol (TCP), a metabolite biomarker of exposure to organophosphorus insecticides. Our new approach takes the advantage of immunochromatographic test strip for a rapid competitive immunoreaction and a disposable screen-printed carbon electrode for a rapid and sensitive electrochemical analysis of captured HRP labeling. Several key experimental parameters (e.g. immunoreaction time, the amount of HRP labeled TCP, concentration of the substrate for electrochemical measurements, and the blocking agents for the nitrocellulose membrane) were optimized to achieve a high sensitivity, selectivity and stability. Under optimal conditions, the IEB has demonstrated a wide linear range (0.1-100 ng/ml) with a detection limit as low as 0.1 ng/ml TCP. Furthermore, the IEB has been successfully applied for biomonitoring of TCP in the rat plasma samples with in vivo exposure to organophosphorus insecticides like Chlorpyrifos-oxon (CPF-oxon). The IEB thus opens up new pathways for designing a simple, rapid, clinically accurate, and quantitative tool for TCP detection, as well as holds a great promise for in-field screening of metabolite biomarkers, e.g., TCP, for humans exposed to organophosphorus insecticides.

Lysobacter enzymogenes Uses Two Distinct Cell-Cell Signaling Systems for Differential Regulation of Secondary-Metabolite Biosynthesis and Colony Morphology

ABSTRACT Lysobacter enzymogenes is a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown in L. enzymogenes. In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time from L. enzymogenes. The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis in L. enzymogenes. Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology of L. enzymgenes. Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions in L. enzymogenes, and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis of L. enzymogenes.

Proteomic Analysis Reveals Novel Extracellular Virulence-Associated Proteins and Functions Regulated by the Diffusible Signal Factor (DSF) in Xanthomonas oryzae pv. oryzicola

Quorum sensing (QS) in Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is mediated by the diffusible signal factor (DSF). DSF-mediating QS has been shown to control virulence and a set of virulence-related functions; however, the expression profiles and functions of extracellular proteins controlled by DSF signal remain largely unclear. In the present study, 33 DSF-regulated extracellular proteins, whose functions include small-protein mediating QS, oxidative adaptation, macromolecule metabolism, cell structure, biosynthesis of small molecules, intermediary metabolism, cellular process, protein catabolism, and hypothetical function, were identified by proteomics in Xoc. Of these, 15 protein encoding genes were in-frame deleted, and 4 of them, including three genes encoding type II secretion system (T2SS)-dependent proteins and one gene encoding an Ax21 (activator of XA21-mediated immunity)-like protein (a novel small-protein type QS signal) were determined to be required for full virulence in Xoc. The contributions of these four genes to important virulence-associated functions, including bacterial colonization, extracellular polysaccharide, cell motility, biofilm formation, and antioxidative ability, are presented. To our knowledge, our analysis is the first complete list of DSF-regulated extracellular proteins and functions in a Xanthomonas species. Our results show that DSF-type QS played critical roles in regulation of T2SS and Ax21-mediating QS, which sheds light on the role of DSF signaling in Xanthomonas.

Transcriptomic analysis reveals new regulatory roles of Clp signaling in secondary metabolite biosynthesis and surface motility in Lysobacter enzymogenes OH11

Lysobacter enzymogenes is a bacterial biological control agent emerging as a new source of antibiotic metabolites, such as heat-stable antifungal factor (HSAF) and the antibacterial factor WAP-8294A2. The regulatory mechanism(s) for antibiotic metabolite biosynthesis remains largely unknown in L. enzymogenes. Clp, a cyclic adenosine monophosphate (cAMP)-receptor-like protein, is shown to function as a global regulator in modulating biocontrol-associated traits in L. enzymogenes. However, the genetic basis of Clp signaling remains unclear. Here, we utilized transcriptome/microarray analysis to determine the Clp regulon in L. enzymogenes. We showed that Clp is a global regulator in gene expression, as the transcription of 775 genes belonging to 19 functional groups was differentially controlled by Clp signaling. Analysis of the Clp regulon detected previously characterized Clp-modulated functions as well as novel loci. These include novel loci involved in antibiotic metabolite biosynthesis and surface motility in L. enzymogenes. We further showed experimentally that Clp signaling played a positive role in regulating the biosynthesis of HSAF and WAP-8294A2, as well as surface motility which is a type-IV-pilus-dependent trait. The regulation by Clp signaling of antibiotic (HSAF and WAP-8294A2) biosynthesis and surface motility was found to be independent. Importantly, we identified a factor Lysobacter acetyltransferase (Lat), a homologue of histone acetyltransferase Hpa2, which was regulated by Clp and involved in HSAF biosynthesis, but not associated with WAP-8294A2 production and surface motility. Overall, our study provided new insights into the regulatory role and molecular mechanism of Clp signaling in L. enzymogenes.

Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb.

Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I50 of the assay ranged from 0.64 to 20.98 microg mL(-1) for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I50 and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL(-1), was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.

Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11

Here, three different suicide vectors were evaluated for the possibility of performing gene mutagenesis in strain OH11 using the chiA gene (accession number: DQ888611) as a new reporter. Suicide vector pEX18GM was selected, and it was successfully applied for disruption and in-frame deletions in the chiA gene in strain OH11, which was confirmed by PCR amplification and Southern hybridization. The chiA-deletion mutant OH11-3 did not have the ability to produce chitinase on chitine selection medium. Interestingly, the chiA-deletion mutants displayed wild-type antimicrobial activity against Saccharomyces cerevisiae, Magnaporthe grisea, Phytophthoracapsici, Rhizoctonia solani, Sclerotinia sclerotiorum and Pythium ultimum. Our data suggest that chitinase might not be a unique lytic enzyme in controlling S. cerevisiae, M. grisea, P. capsici, and P. ultimum. R. solani, S. sclerotiorum. Also, suicide vector pEX18GM might be explored as a potential tool for gene deletions in L. enzymogenes, which will facilitate the molecular study of mechanisms of biological control in L. enzymogenes.

The type VI protein secretion system contributes to biofilm formation and seed-to-seedling transmission of Acidovorax citrulli on melon.

The type VI protein secretion system (T6SS) is essential for the virulence of several Gram-negative bacteria. In this study, we identified a T6SS gene cluster in Acidovorax citrulli, a plant-pathogenic bacterium that causes bacterial fruit blotch (BFB) of cucurbits. One T6SS cluster, of approximately 25 kb in length and comprising 17 genes, was found in the A. citrulli AAC00-1 genome. Seventeen A. citrulli mutants were generated, each with a deletion of a single T6SS core gene. There were significant differences in BFB seed-to-seedling transmission between wild-type A. citrulli strain, xjl12, and ΔvasD, ΔimpK, ΔimpJ and ΔimpF mutants (71.71%, 9.83%, 8.41%, 7.15% and 5.99% BFB disease index, respectively). In addition, we observed that these four mutants were reduced in melon seed colonization and biofilm formation; however, they were not affected in virulence when infiltrated into melon seedling tissues. There were no significant differences in BFB seed-to-seedling transmission, melon tissue colonization and biofilm formation between xjl12 and the other 13 T6SS mutants. Overall, our results indicate that T6SS plays a role in seed-to-seedling transmission of BFB on melon.

Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.

The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody.

Roles of a Solo LuxR in the Biological Control Agent Lysobacter enzymogenes Strain OH11

Lysobacter enzymogenes is a ubiquitous plant-associated and environmentally friendly bacterium emerging as a novel biological control agent of plant disease. This bacterium produces diverse antifungal factors, such as lytic enzymes and a secondary metabolite (heat-stable antifungal factor [HSAF]) having antifungal activity with a novel structure and mode of action. The regulatory mechanisms for biosynthesis of antifungal factors is largely unknown in L. enzymogenes. The solo LuxR proteins have been shown to be widespread, playing important roles in plant-associated bacteria. Here, we cloned and studied a solo LuxR protein, LesR, from L. enzymogenes strain OH11. Overexpression but not deletion of lesR significantly impaired HSAF biosynthesis levels and antimicrobial activities but did not show visible effect on production of major lytic enzymes. Overexpression of lesR also led to remarkably accelerated cell aggregation and induced production of a melanin-like pigment in L. enzymogenes; these two phenotypes are mediated by the diffusible factor cell-to-cell signaling system of L. enzymogenes. The C-terminus helix-turn-helix domain was shown to be critical for several lesR-controlled functions. Overall, our study provides the first example of the roles and mechanisms of a solo LuxR protein in a plant-associated L. enzymogenes.

Development of an Enzyme Linked Immunosorbent Assay and an Immunochromatographic Assay for Detection of Organophosphorus Pesticides in Different Agricultural Products

Objective Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. Methods A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively. Results Under the optimized conditions, the IC50 values of the ELISA ranged from 3.7 to 162.2 ng mL–1 for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC50 values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL−1. The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides. Conclusions The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.