Tomato root colonization by fluorescent-tagged pathogenic and protective strains of Fusarium oxysporum in hydroponic culture differs from root colonization in soil

The colonization process of tomato roots inoculated separately or/and simultaneously by a pathogenic Fusarium oxysporum f. sp. lycopersici strain Fol8 and the protective F. oxysporum strain Fo47, genetically tagged with the red and green fluorescent protein genes, respectively, was studied in a hydroponic culture. Plants were coinoculated with Fol8 and Fo47 at two conidial concentration ratios of 1/1 and 1/100, in which biological control was not effective or effective, respectively. First observation of fungi on root was possible 48 h after inoculation at a high inoculum level and 5 days post inoculation at the lower concentration of inoculum. The pattern of root colonization was similar for both strains with the initial development of hyphal network on the upper part of taproot, followed by the growth of hyphae towards the elongation zone, lateral roots and root apices. Finally, the whole elongation zone and root apex were invaded by both strains but no specific infection sites were observed. When coinoculated, both strains could grow very closely or even at the same spot on the root surface. At the nonprotective ratio, Fol8 was the successful colonizer, but application of Fo47 at a concentration 100 times >Fol8 delayed vessel colonization by the pathogen.     

The GUS gene fusion system (Escherichia coli beta-D-glucuronidase gene), a useful tool in studies of root colonization by Fusarium oxysporum.

The plant-pathogenic fungus Fusarium oxysporum was successfully transformed with the beta-D-glucuronidase gene from Escherichia coli (gusA) (GUS system) in combination with the gene for nitrate reductase (niaD) as the selectable marker. The frequency of cotransformation, as determined by GUS expression on plates containing medium supplemented with 5-bromo-4-chloro-3-indolyl glucuronide (GUS+), was very high (up to 75%). Southern hybridization analyses of GUS+ transformants revealed that single or multiple copies of the gusA gene were integrated into the genomes. High levels of GUS activity are expressed in some transformants, but activity in F. oxysporum does not appear to be correlated with the copy number of the gusA gene. Since the highest activity was found in a transformant with a single copy, it can be assumed that sequence elements of F. oxysporum integrated upstream of the gene can act as a promoter or enhancer. Expression of the gusA gene was also detected during growth of the fungus in plants, indicating that the GUS system can be used as a sensitive and easy reporter gene assay in F. oxysporum.     

Plasmid size can affect the ability of Escherichia coli to produce high-quality plasmids

Large molecular weight plasmids are often used in gene therapy and DNA vaccines. To investigate the effect of plasmid size on the performance of Escherichia coli host strains during plasmid preparation, we employed E. coli JM109 and TOP10 cells to prepare four plasmids ranging from 4.7 to 16.8?kb in size. Each plasmid was extracted from JM109 and TOP10 cells using an alkaline lysis mini-preparation method. However, when commercial kits were used to extract the same plasmids from JM109 cells, the large molecular weight plasmids substantially degraded, compared with their smaller counterparts. No degradation was observed when the four plasmids were extracted from E. coli TOP10 cells using the same commercial kit. We conclude, therefore, that the performance of E. coli in high quality plasmid preparations can be affected by plasmid size.     

An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics

An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-terminal sequence. Using this vector and the E. coli DH5α, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 × 10³ units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60°C. Storage during 1 year at ?20°C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 ± 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.     

Growth substrates control the ability of Fusarium oxysporum to solubilize low-rank coal

The ability of the fungus Fusarium oxysporum to solubilize lignite was found to depend on the presence of a specific carbon source. When grown on glucose or another carbohydrate, the fungus is unable to solubilize coal but it produces the red dye bikaverin. In the coal-solubilizing state, which can be induced by cultivation in the presence of glutamate or gluconate, the fungus does not produce bikaverin. The presence or absence of the pigment can therefore be taken as an indicator of the ability of the fungus to solubilize coal. Addition of extracted and purified bikaverin to F. oxysporum growing on glutamate or gluconate inhibits coal solubilization. Hence, F. oxysporum offers a suitable system for investigating the mechanism of microbial coal degradation by comparing the two growth-substrate-controlled physiological states.     

Production and secretion of a recombinant Vibrio parahaemolyticus chitinase by Escherichia coli and its purification from the culture medium

An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.     

Synthetic chitinase gene driven by root-specific LjNRT2 and AtNRT2.1 promoters confers resistance to Fusarium oxysporum in transgenic tobacco and tomato

Fusarium wilt is a soil-borne disease causing substantial yield losses in various crops and vegetables. We have previously reported the synthetic chitinase (NIC) gene (1.2 kb), in which codon usage of fungus, replaced with that of plant, conferred resistance against Botrytis cinerea. In this study, the NIC or GUS gene was linked to two root-specific promoters, LjNRT2 or AtNRT2.1 (nitrate transporter 2), derived from Lotus japonica and Arabidopsis thaliana, respectively. Transgenic tobacco lines expressing LjNRT2-GUS and LjNRT2-NIC, and tomato lines expressing AtNRT2.1-NIC, were produced by Agrobacterium-mediated transformation. GUS histochemical staining was observed in vascular regions of the roots but was conspicuously absent in the leaves of transgenic plants. Western blot analysis showed the production of NIC proteins in the roots but not in the leaves of transgenic tobacco and tomato lines. These results indicate that LjNRT2 and AtNRT2.1 promoters expressed transgenes in a root-specific manner. When in vitro whole plant resistance assay against Fusarium oxysporum was conducted, transgenic plants showed increased levels of resistance compared to non-transgenic plants. Antifungal activities of the root extract against spore germination of F. oxysporum showed lower CFU (colony-forming unit) than those of the leaf extract. Root colonization assay against F. oxysporum showed much lower CFU values in the roots of transgenic plants than in those of non-transgenic plants. These results suggest that NIC gene triggered by the root-specific promoters successfully expressed only in the roots and conferred increased levels of resistance against the root pathogen, F. oxysporum.     

A balanced DO-stat and its application to the control of acetic acid excretion by recombinant Escherichia coli

During fed-batch cultivation of a recombinant Escherichia coli AT2471 harboring plasmid pSY130-14 for phenylalanine production, a large amount of acetic acid was excreted by the cells and accumulated in the culture medium. Acetic acid concentration reached 30-35 g/L at the end of a process conducted without special precautions for the reduction of this excretion. Cell growth stopped when acetic acid concentration was about 15 g/L, resulting in poor growth, 16 g/L cell concentration, and poor production - 8 g/L phenylalanine. A novel control strategy, called a balanced DO-stat. was developed to prevent acetic acid excretion. It represents a model-independent two-loop control structure, which is simple, reliable, and convenient for computer application. Using the balanced DO-stat, implemented in a computer control system, acetic acid concentration was kept at zero during the entire cultivation period. As a result, the cell concentration increased to 36 g/L and phenylalanine concentration reached 24 g/L. Aside from the phenylalanine fermentation, the proposed control approach might be applied to cultivation of other bacterial and yeast strains which have similar mechanism of the excretion of fermentative by-products.     

Wilting of date palm branches by Fusarium oxysporum in south of Iran and its control managements with soil solarization method

Wilting of some branches in nurseries and orchards of date palm were studied in south of Iran including Ahvaz and Abadan cities in 2005-2006 years. Different infected plants were visited and samples showing symptoms including wilting or death of branches collected from various areas and transferred to laboratory. Samples were cultured in common media (PDA) and different fungi were studied and identified. The most frequently isolated pathogen was Fusarium oxysporum which caused wilting of some branches of date palm seedling or trees in studied areas. Results showed that the disease caused main losses where date palm cuttings were cultured in infected soils, previously cropped to susceptible plants. Since chemical control was not managed the disease, soil disinfestations by soil solarization method was carried in Ahvaz as the warmer climate in studied areas to control the pathogen. Application of this method reduced population density of the pathogen from 1800 CFU -g/soil to 600 after 5 week. This method was simple, effective, non negative side and economic which can be used in nearly warm areas.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

Active,soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U‐87 MG glioma cells and inhibits growth of the gram‐positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of ～0.5–1 mg/L of E. coli culture. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1150–1157, 2013     

Generic model control of the specific growth rate in recombinant Escherichia coli cultivations

Generic model control is shown to be a powerful tool for keeping a microbial cultivation process close to its predetermined (optimized) control profile. This is demonstrated at the example of the green fluorescent protein expressed in genetically modified Escherichia coli host cells. It is shown that the process can be run very closely to a predefined complex profile of the specific cell growth rate mu(t). Controlling the experiments at many different growth conditions is a straightforward way of effectively collecting the data necessary for optimization of recombinant protein production systems. Although the process dynamics is rather complex, the model for the controller can be kept quite simple. The control technique, used here for specific growth rate control, is quite universal and can be applied for different biotechnological processes as well.     

Purification and antifungal activity of recombinant chitinase from Escherichia coli carrying the family 19 chitinase gene of Streptomyces sp. J-13-3

A recombinant chitinase was purified from the cell extract of Escherichia coli JM109 transformed by plasmid pUC19 carrying the gene encoding family 19 chitinase of Streptomyces sp. J-13-3 by column chromatography on DEAE-Sepharose, CM-Sepharose, and Bio-Gel P-100. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 32,000. The recombinant chitinase hydrolyzed the trimer to hexamer of N-acetylglucosamine and had the identical N-terminal amino acid sequence of the mature protein, indicating removal of the signal sequence by E. coli signal peptidase. The fungal growth in well (200 microl of medium) of microplate by measurement of absorbance at 595 nm indicated that the chitinase (10 microg) completely and half inhibited growth of Trichoderma reesei and Aspergillus niger respectively.     

Immunolocalization of a class III chitinase in two muskmelon cultivars reacting differently to Fusarium oxysporum f. sp. melonis

Fusarium oxysporum f. sp. melonis is a highly specialized fungus that attacks the root system of melon (Cucumis melo L.). In this work the presence of a class III chitinase was examined by immunological techniques in the root and stem base of a susceptible (cv. Galia) and a resistant (cv. Bredor) melon during the infection process. By immunolocalization it was not possible to detect the constitutive presence of class III chitinase in any of the cultivars. However, the immunolabelling appeared in the root tissues of both cultivars as a consequence of wounding and of infection by F. oxysporum f. sp. melonis. Distinct patterns of chitinase detection were observed in the roots of the two cultivars as the infection progressed. Furthermore, by western blotting distinct class III chitinase isoforms were detected, which responded differently to the F. oxysporum f. sp. melonis infection. Our results strongly indicate that a relationship exists between class III chitinase and melon resistance to Fusarium infection, and that the resistance is associated with certain isoforms of this enzyme.     

Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli

Applied Microbiology and Biotechnology - It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent...     

Plant colonization by the vascular wilt fungus Fusarium oxysporum requires FOW1, a gene encoding a mitochondrial protein

The soil-borne fungus Fusarium oxysporum causes vascular wilts of a wide variety of plant species by directly penetrating roots and colonizing the vascular tissue. The pathogenicity mutant B60 of the melon wilt pathogen F. oxysporum f. sp. melonis was isolated previously by restriction enzyme-mediated DNA integration mutagenesis. Molecular analysis of B60 identified the affected gene, designated FOW1, which encodes a protein with strong similarity to mitochondrial carrier proteins of yeast. Although the FOW1 insertional mutant and gene-targeted mutants showed normal growth and conidiation in culture, they showed markedly reduced virulence as a result of a defect in the ability to colonize the plant tissue. Mitochondrial import of Fow1 was verified using strains expressing the Fow1-green fluorescent protein fusion proteins. The FOW1-targeted mutants of the tomato wilt pathogen F. oxysporum f. sp. lycopersici also showed reduced virulence. These data strongly suggest that FOW1 encodes a mitochondrial carrier protein that is required specifically for colonization in the plant tissue by F. oxysporum.     

Vermicompost can suppress Fusarium oxysporum f. sp. lycopersici via generation of beneficial bacteria in a long-term tomato monoculture soil

Plant and Soil - Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol) has severely decreased global tomato production. Organic amendments are widely applied to suppress Fol all over...