Jianuan Zhou

A Novel Multidomain Polyketide Synthase Is Essential for Zeamine Production and the Virulence of Dickeya zeae

Dickeya zeae is the causal agent of the rice foot rot disease, but its mechanism of infection remains largely unknown. In this study, we identified and characterized a novel gene designated as zmsA. The gene encodes a large protein of 2,346 amino acids in length, which consists of multidomains arranged in the order of N-terminus, β-ketoacyl synthase, acyl transferase, acyl carrier protein, β-ketoacyl reductase, dehydratase. This multidomain structure and sequence alignment analysis suggest that ZmsA is a member of the polyketide synthase family. Mutation of zmsA abolished antimicrobial activity and attenuated the virulence of D. zeae. To determine the relationship between antimicrobial activity and virulence, active compounds were purified from D. zeae EC1 and were structurally characterized. This led to identification of two polyamino compounds, i.e., zeamine and zeamine II, that were phytotoxins and potent antibiotics. These results have established the essential role of ZmsA in zeamine biosynthesis and presented a new insight on the molecular mechanisms of D. zeae pathogenicity.

Fenpropathrin Biodegradation Pathway in Bacillus sp. DG-02 and Its Potential for Bioremediation of Pyrethroid-Contaminated Soils

The widely used insecticide fenpropathrin in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals, yet little is known about the kinetic and metabolic behaviors of this pesticide. This study reports the degradation kinetics and metabolic pathway of fenpropathrin in Bacillus sp. DG-02, previously isolated from the pyrethroid-manufacturing wastewater treatment system. Up to 93.3% of 50 mg L(-1) fenpropathrin was degraded by Bacillus sp. DG-02 within 72 h, and the degradation rate parameters qmax, Ks, and Ki were determined to be 0.05 h(-1), 9.0 mg L(-1), and 694.8 mg L(-1), respectively. Analysis of the degradation products by gas chromatography-mass spectrometry led to identification of seven metabolites of fenpropathrin, which suggest that fenpropathrin could be degraded first by cleavage of its carboxylester linkage and diaryl bond, followed by degradation of the aromatic ring and subsequent metabolism. In addition to degradation of fenpropathrin, this strain was also found to be capable of degrading a wide range of synthetic pyrethroids including deltamethrin, λ-cyhalothrin, β-cypermethrin, β-cyfluthrin, bifenthrin, and permethrin, which are also widely used insecticides with environmental contamination problems with the degradation process following the first-order kinetic model. Bioaugmentation of fenpropathrin-contaminated soils with strain DG-02 significantly enhanced the disappearance rate of fenpropathrin, and its half-life was sharply reduced in the soils. Taken together, these results depict the biodegradation mechanisms of fenpropathrin and also highlight the promising potentials of Bacillus sp. DG-02 in bioremediation of pyrethroid-contaminated soils.

Pathway and kinetics of cyhalothrin biodegradation by Bacillus thuringiensis strain ZS-19

Cyhalothrin is a common environmental pollutant which poses increased risks to non-target organisms including human beings. This study reported for the first time a newly isolated strain, Bacillus thuringiensis ZS-19 completely degraded cyhalothrin in minimal medium within 72 h. The bacterium transformed cyhalothrin by cleavage of both the ester linkage and diaryl bond to yield six intermediate products. Moreover, a novel degradation pathway of cyhalothrin in strain ZS-19 was proposed on the basis of the identified metabolites. In addition to degradation of cyhalothrin, this strain was found to be capable of degrading 3-phenoxybenzoic acid, a common metabolite of pyrethroids. Furthermore, strain ZS-19 participated in efficient degradation of a wide range of pyrethroids including cyhalothrin, fenpropathrinn, deltamethrin, beta-cypermethrin, cyfluthrin and bifenthrin. Taken together, our results provide insights into the mechanism of cyhalothrin degradation and also highlight the promising potentials of B.thuringiensis ZS-19 in bioremediation of pyrethroid-contaminated environment. This is the first report of (i) degradation of cyhalothrin and other pyrethroids by B.thuringiensis, (ii) identification of 3-phenoxyphenyl acetonitrile and N-(2-isoproxy-phenyl)-4-phenoxy-benzamide as the metabolites in the degradation pathway of pyrethroids, and (iii) a pathway of degradation of cyhalothrin by cleavage of both the ester linkage and diaryl bond in a microorganism.

A Nonribosomal Peptide Synthase Containing a Stand-Alone Condensation Domain Is Essential for Phytotoxin Zeamine Biosynthesis

Dickeya zeae is the causal agent of rice foot rot and maize stalk rot diseases, which could cause severe economic losses. The pathogen is known to produce two phytotoxins known as zeamine and zeamine II which are also potent antibiotics against both gram-positive and gram-negative bacteria pathogens. Zeamine II is a long-chain aminated polyketide and zeamine shares the same polyketide structure as zeamine II, with an extra valine derivative moiety conjugated to the primary amino group of zeamine II. In this study, we have identified a gene designated as zmsK encoding a putative nonribosomal peptide synthase (NRPS) by screening of the transposon mutants defective in zeamine production. Different from most known NRPS enzymes, which are commonly multidomain proteins, ZmsK contains only a condensation domain. High-performance liquid chromatography and mass spectrometry analyses showed that the ZmsK deletion mutant produced only zeamine II but not zeamine, suggesting that ZmsK catalyzes the amide bond formation by using zeamine II as a substrate to generate zeamine. We also present evidence that a partially conserved catalytic motif within the condensation domain is critical for zeamine production. Furthermore, we show that deletion of zmsK substantially decreased the total antimicrobial activity and virulence of D. zeae. Our findings provide a new insight into the biosynthesis pathway of zeamines and the virulence mechanisms of the bacterial pathogen D. zeae.

Control of litchi downy blight by zeamines produced by Dickeya zeae

Zeamines (ZMS), a class of polyamine-polyketide-nonribosomal peptide produced by bacterial isolate Dickeya zeae, were shown recently to be potent antibiotics against some bacterial pathogens. In this study, the results indicated that ZMS showed antifungal activity against Peronophythora litchii and other fungal pathogens. The activity of ZMS against the oomycete pathogen P. litchi, which causes the devastating litchi downy blight, was further investigated under in vitro and in vivo conditions. ZMS displayed potent inhibitory activity against the mycelial growth and sporangia germination of P. litchii. At a concentration of 2 μg/mL, about 99% of the sporangia germination was inhibited. Scanning electron microscopy and transmission electron microscopy analyses showed that treatment with ZMS could cause substantial damages to the oomycete endomembrane system. Furthermore, treatment of litchi fruits with ZMS solution significantly (P < 0.05) reduced the fruits decay and peel browning caused by P. litchii infection during storage at 28 °C. Taken together, our results provide useful clues on the antifungal mechanisms of ZMS, and highlight the promising potentials of ZMS as a fungicide, which in particular, may be useful for prevention and control of litchi fruits decay and browning caused by P. litchii infection during storage and transportation.

Production of Novel Antibiotics Zeamines through Optimizing Dickeya zeae Fermentation Conditions

Dickeya zeae strain EC1 was recently shown to produce a new type of phytotoxins designated as zeamine and zeamine II, which are potent wide-spectrum antibiotics against Gram-positive and Gram-negative bacterial pathogens, suggesting their promising potential as clinical medicines. In this study, the optimized medium composition and culture conditions for biosynthesis of novel antibiotics zeamines have been established by using response surface methodology, largely increasing the yield of zeamines from original about 7.35 µg·mL−1 in minimal medium to about 150 µg·mL−1 in LS5 medium. The study identified the major factors contributing to zeamines production, which include nitrate, sucrose, asparaginate, mineral elements Mg2+ and K+, and optimized amount of phosphate. In addition, the results showed that overexpression of zmsK in D. zeae strain EC1 could further increase zeamines yield to about 180 µg·mL−1 in LS5 medium. The findings from this study could facilitate further characterization and utilization of these two novel antibiotics, and also provide useful clues for understanding the regulatory mechanisms that govern D. zeae virulence.

Design and characterization of a polyamine derivative inhibiting the expression of type III secretion system in Pseudomonas aeruginosa

The type III secretion system (TTSS) of Pseudomonas aeruginosa is a key virulence determinant for infection of eukaryotic hosts. Based on the findings that spermidine-mediated host-pathogen signalling is important for activation of type III secretion systems (TTSS), in this study, we designed, synthesized and evaluated a series of polyamine derivatives for their potentials in inhibiting the expression TTSS in P. aeruginosa. In vitro assay of 15 compounds synthesized in this study unveiled stringent structural requirements for TTSS-inhibitory activity. Among them, R101SPM, a conjugate between rhodamine 101 and spermine, showed a potent activity in inhibition of the TTSS gene expression and in attenuation of the TTSS-mediated cytotoxicity on human cells. In vivo analysis demonstrated that R101SPM could rescue mice from the lethal infection by P. aeruginosa. Moreover, genetic analysis showed that the full TTSS-inhibitory activity of R101SPM required a functional spermidine transporter. Taken together, our results present a new class of lead molecules for developing anti-virulence drugs and demonstrate that the spermidine transporter SpuDEGHF of P. aeruginosa is a promising drug target.

Genomic Analysis of Phylotype I Strain EP1 Reveals Substantial Divergence from Other Strains in the Ralstonia solanacearum Species Complex

Ralstonia solanacearum species complex is a devastating group of phytopathogens with an unusually wide host range and broad geographical distribution. R. solanacearum isolates may differ considerably in various properties including host range and pathogenicity, but the underlying genetic bases remain vague. Here, we conducted the genome sequencing of strain EP1 isolated from Guangdong Province of China, which belongs to phylotype I and is highly virulent to a range of solanaceous crops. Its complete genome contains a 3.95-Mb chromosome and a 2.05-Mb mega-plasmid, which is considerably bigger than reported genomes of other R. solanacearum strains. Both the chromosome and the mega-plasmid have essential house-keeping genes and many virulence genes. Comparative analysis of strain EP1 with other 3 phylotype I and 3 phylotype II, III, IV strains unveiled substantial genome rearrangements, insertions and deletions. Genome sequences are relatively conserved among the 4 phylotype I strains, but more divergent among strains of different phylotypes. Moreover, the strains exhibited considerable variations in their key virulence genes, including those encoding secretion systems and type III effectors. Our results provide valuable information for further elucidation of the genetic basis of diversified virulences and host range of R. solanacearum species.

Biocontrol of Sugarcane Smut Disease by Interference of Fungal Sexual Mating and Hyphal Growth Using a Bacterial Isolate

Sugarcane smut is a fungal disease caused by Sporisorium scitamineum, which can cause severe economic losses in sugarcane industry. The infection depends on the mating of bipolar sporida to form a dikaryon and develops into hyphae to penetrate the meristematic tissue of sugarcane. In this study, we set to isolate bacterial strains capable of blocking the fungal mating and evaluate their potential in control of sugarcane smut disease. A bacterial isolate ST4 from rhizosphere displayed potent inhibitory activity against the mating of S. scitamineum bipolar sporida and was selected for further study. Phylogenetic analyses and biochemical characterization showed that the isolate was most similar to Pseudomonas guariconensis. Methanol extracts from minimum and potato dextrose agar (PDA) agar medium, on which strain ST4 has grown, showed strong inhibitory activity on the sexual mating of S. scitamineum sporida, without killing the haploid cells MAT-1 or MAT-2. Further analysis showed that only glucose, but not sucrose, maltose, and fructose, could support strain ST4 to produce antagonistic chemicals. Consistent with the above findings, greenhouse trials showed that addition of 2% glucose to the bacterial inoculum significantly increased the strain ST4 biocontrol efficiency against sugarcane smut disease by 77% than the inoculum without glucose. The results from this study depict a new strategy to screen for biocontrol agents for control and prevention of the sugarcane smut disease.

Modulation of Inter-kingdom Communication by PhcBSR Quorum Sensing System in Ralstonia solanacearum Phylotype I Strain GMI1000

Ralstonia solanacearum is a ubiquitous soil-borne plant pathogenic bacterium, which frequently encounters and interacts with other soil cohabitants in competition for environmental niches. Ralsolamycin, which is encoded by the rmy genes, has been characterized as a novel inter-kingdom interaction signal that induces chlamydospore development in fungi. In this study, we provide the first genetic evidence that the rmy gene expression is controlled by the PhcBSR quorum sensing (QS) system in strain GMI1000. Mutation of phcB could lead to significant reduction of the expression levels of the genes involved in ralsolamycin biosynthesis. In addition, both the phcB and rmy mutants were attenuated in induction of chlamydospore formation in Fusarium oxysporum f. cubense and diminished in the ability to compete with the sugarcane pathogen Sporisorium scitamineum. Agreeable with the pattern of QS regulation, transcriptional expression analysis showed that the transcripts of the rmy genes were increased along with the increment of the bacterial population density. Taken together, the above findings provide new insights into the regulatory mechanisms that the QS system involves in governing the ralsolamycin inter-kingdom signaling system.