A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.     

Siderophore-mediated iron transport correlates with the presence of specific iron-regulated proteins in the outer membrane of Rhizobium meliloti.

A universal chemical assay used to detect the production of siderophores in a range of Rhizobium strains showed that production is strain specific. Iron nutrition bioassays carried out on Rhizobium meliloti strains to determine cross-utilization of their siderophores showed that R. meliloti 2011, 220-5, and 220-3 could each use the siderophores produced by the other two but not the siderophore produced by R. meliloti DM4 (and vice versa). Mutants of R. meliloti 2011 and 220-5 defective in siderophore production were isolated by Tn5-mob mutagenesis. The Tn5-mob-containing EcoRI fragment of mutant R. meliloti 220-5-1 was cloned into pUC19. By using this fragment as a probe, the presence of a homologous region was observed in R. meliloti 2011 and 220-3 but not in R. meliloti DM4. A complementing cosmid from a gene bank of R. meliloti 2011 was identified by using the same probe. Introduction of this cosmid into R. meliloti 102F34, a strain not producing a siderophore, resulted in the ability of this strain to produce a siderophore and also in the ability to utilize the siderophores produced by R. meliloti 2011, 220-5, and 220-3 but not the siderophore produced by R. meliloti DM4. A comparative analysis of the outer membrane proteins prepared from iron-deficient cultures of R. meliloti 102F34 and 102F34 harboring the cosmid revealed the presence, in the latter, of a low-iron-induced outer membrane protein corresponding to a low-iron-induced protein in R. meliloti 2011, 220-5, and 220-3. This protein is not present in R. meliloti DM4. The results suggest that R. meliloti 2011, 220-5, and 220-3 produce siderophores that are identical or sufficiently similar in structure to be transported by the membrane transport system of each strain while also indicating that utilization of a particular siderophore is correlated with the presence of specific outer membrane proteins.     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

Linear plasmidlike DNA in the plant pathogenic fungus Fusarium oxysporum f. sp. conglutinans.

Double-stranded, 1.9-kilobase-pair (kbp) DNA molecules were found in 18 strains representing three pathogenic races of Fusarium oxysporum f. sp. conglutinans. The DNA element (pFOXC1) from a race 1 strain and the DNA element (pFOXC2) from a race 2 strain were shown by restriction endonuclease mapping to be linear. pFOXC2 was found in mitochondrial preparations and appears to have blocked 5' termini, as it was sensitive to 3'----5' exonuclease III but insensitive to 5'----3' lambda exonuclease. The major 1.8-kbp BglII restriction endonuclease fragment of pFOXC2 was cloned in plasmid pUC12. The recombinant plasmid (pCK1) was not homologous to the mitochondrial or nuclear genomes from F. oxysporum f. sp. conglutinans. This suggests that pFOXC2 is self-replicating. pCK1 was homologous to all 1.9-kbp DNA elements of race 2 but was not homologous to those of race 1 or race 5. All race 1 and 5 elements were also shown to share common DNA sequences.     

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.     

Volatile organic compounds: a potential direct long-distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic Fusarium that lives in association with a consortium of bacteria belonging to the genera Serratia, Achromobacter , Bacillus and Stenotrophomonas in an Italian soil suppressive to Fusarium wilt. Typing experiments and virulence tests provided evidence that the F. oxysporum isolate when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms identical to those caused by F. oxysporum f. sp. lactucae . Here, we demonstrate that small volatile organic compounds (VOCs) emitted from the WT strain negatively influence the mycelial growth of different formae speciales of F. oxysporum. Furthermore, these VOCs repress gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10, a fungus against which the WT strain MSA 35 has antagonistic activity. The VOC profile of the WT and CU fungus shows different compositions. Sesquiterpenes, mainly caryophyllene, were present in the headspace only of WT MSA 35. No sesquiterpenes were found in the volatiles of ectosymbiotic Serratia sp. strain DM1 and Achromobacte r sp. strain MM1. Bacterial volatiles had no effects on the growth of the different ff. spp. of F. oxysporum examined. Hyphae grown with VOC from WT F. oxysporum f. sp. lactucae strain MSA 35 were hydrophobic whereas those grown without VOCs were not, suggesting a correlation between the presence of volatiles in the atmosphere and the phenotype of the mycelium. This is the first report of VOC production by antagonistic F. oxysporum MSA 35 and their effects on pathogenic F. oxysporum. The results obtained in this work led us to propose a new potential direct long-distance mechanism for antagonism by F. oxysporum MSA 35 mediated by VOCs . Antagonism could be the consequence of both reduction of pathogen mycelial growth and inhibition of pathogen virulence gene expression.     

高产铁载体棉田土壤细菌SS05的筛选与鉴定

【目的】研究从棉田土壤中筛选得到的高产铁载体细菌产铁载体能力、分类地位和抑菌活性。【方法】通过改良蔗糖-天冬氨酸培养基选择性筛选产铁载体细菌,通过分光光度计法测定铁载体活性,通过混菌法测定产铁载体细菌上清液对棉花枯萎病致病菌尖孢镰刀菌(Fusarium oxysporum)的抑菌效果,采用形态学、生理生化鉴定及16S rDNA序列系统发育分析对高产铁载体菌株进行鉴定。【结果】从棉田土壤中筛选到162株产铁载体细菌,30株产铁载体能力较强的细菌中21株具有较高产铁载体能力,菌株SS05的铁载体活性单位达到98.3%;在低铁条件下,SS05上清液对F.oxysporum具有显著的抑制作用;SS05与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近。【结论】SS05是高产铁载体菌株,与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近,在低铁培养条件下其上清液对F.oxysporum具有显著的抑制作用。     

A robust identification and detection assay to discriminate the cucumber pathogens Fusarium oxysporum f. sp. cucumerinum and f. sp. radicis-cucumerinum

The fungal species Fusarium oxysporum is a ubiquitous inhabitant of soils worldwide that includes pathogenic as well as non-pathogenic or even beneficial strains. Pathogenic strains are characterized by a high degree of host specificity and strains that infect the same host range are organized in so-called formae speciales. Strains for which no host plant has been identified are believed to be non-pathogenic strains. Therefore, identification below the species level is highly desired. However, the genetic basis of host specificity and virulence in F. oxysporum is so far unknown. In this study, a robust random-amplified polymorphic DNA (RAPD) marker-based assay was developed to specifically detect and identify the economically important cucumber pathogens F. oxysporum f. sp. cucumerinum and F. oxysporum f. sp. radicis-cucumerinum. While the F. oxysporum radicis-cucumerinum strains were found to cluster in a separate clade based on elongation factor-1alpha phylogeny, strains belonging to F. oxysporum f. sp. cucumerinum were found to be genetically more diverse. This is reflected in the observation that specificity testing of the identified markers using a broad collection of F. oxysporum strains with all known vegetative compatibility groups of the target formae speciales, as well as representative strains belonging to other formae speciales, resulted in two cross-reactions for the F. oxysporum f. sp. cucumerimum marker. However, no cross-reactions were observed for the F. oxysporum f. sp. radicis-cucumerimum marker. This F. oxysporum f. sp. radicis-cucumerimum marker shows homology to Folyt1, a transposable element identified in the tomato pathogen F. oxysporum f. sp. lycopersici and may possibly play a role in host-range specificity in the target forma specialis. The markers were implemented in a DNA array that enabled parallel and sensitive detection and identification of the pathogens in complex samples from diverse origins.     

Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization.

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow with toluene as the sole C-source. Tn5 mutagenesis was carried out and a mutant unable to use toluene as the sole C-source was isolated. DNA was sequenced upstream and downstream of the site where the Tn5 was inserted. Analysis of the DNA revealed 13 open reading frames (ORFs) homologous to the tod genes for the toluene dioxygenase pathway of P. putida F1, which are organized in two operons: todXFC1C2BADEGIH and todST. The Tn5 was inserted at the todH gene. The role of the todXFC1C2BADEGIH operon in toluene metabolism was further confirmed in a todC1 mutant (generated by insertional inactivation), which was unable to use toluene as the sole C-source. Primer extension analysis identified a single promoter upstream from the todX gene. The -10 and -35 regions of this promoter showed no significant homology to known promoters. Expression from the todX promoter occurred in response to toluene, ethylbenzene, styrene, xylenes and other aromatic hydrocarbons. Expression from the todS gene was constitutive. Sensitivity to toluene of the todH and todC1 mutants was similar to that of the wild-type strain. This suggests that toluene metabolism is not involved in toluene tolerance.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Alteration of surface properties in a Tn5 mutant strain of Rhizobium trifolii 0403.

A symbiotically defective mutant strain of Rhizobium trifolii, UR251, was obtained by transposon Tn5 mutagenesis of R. trifolii 0403 rif and recognized by its partially ineffective (Fix +/-) phenotype on white clover plants. UR251 had a single Tn5 insertion in plasmid DNA, a wild-type plasmid pattern, and no detectable Mu DNA sequences originally present in the vector used for Tn5 mutagenesis. Agglutination by the clover lectin trifoliin A and attachment to clover root hairs was higher with UR251 than with the wild-type strain. The capsular polysaccharide (CPS) of UR251 was altered, as shown by a slower rate of CPS depolymerization with a CPS beta-lyase, PD-I; more pyruvate and less acetate and 3-hydroxybutanoate noncarbohydrate substitutions as quantitated by 1H nuclear magnetic resonance; and a higher pyruvyl transferase activity (enzymatic pyruvylation of lipid-bound saccharides). The site of increased pyruvylation in the CPS of UR251 was on the terminal galactose of the branch of the repeating oligosaccharide unit. These results show that the level of noncarbohydrate substitutions of the CPS as well as pyruvyl transferase activity are altered in R. trifolii UR251 and that trifoliin A-binding ability and clover root hair attachment are improved in this mutant strain of R. trifolii 0403 rif.     

Pseudomonas aeruginosa (GRC1) as a strong antagonist of Macrophomina phaseolina and Fusarium oxysporum

A plant growth promotory bacterial strain, isolated from the potato rhizosphere, was characterized as Pseudomonas aeruginosa (GRC1). The isolate produced an hydroxamate type of siderophore after 48 h of incubation on tryptic soy medium under iron deficient conditions. The in vitro antifungal activity of P. aeruginosa was tested against two soil-borne plant pathogens, Macrophomina phaseolina and Fusarium oxysporum. The antagonistic behaviour of the isolate was tested by dual culture technique. The growth inhibition of M. phaseolina and F. oxysporum was 74.1% and 70.5%, respectively, after 5 days of incubation. The production of hydrocyanic acid and indole acetic acid was also recorded under normal growth conditions.     

Insertional specificity of transposon Tn5 in Acinetobacter sp.

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced from Escherichia coli 1830 into Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413. Kanamycin-resistant (Kmr) exconjugants of HO1-N and BD413, isolated on complex medium, were screened for auxotrophic requirements. Over 10,000 Kmr clones were examined, but no auxotrophs were detected. Several Kmr exconjugants of BD413 and HO1-N, obtained from independent matings, were chosen for further study. All Tn5-containing strains exhibited kanamycin phosphotransferase activity. Kmr strains lacked plasmid DNA as determined by three plasmid screening procedures, and the Kmr phenotype was not transferable by conjugal matings to kanamycin-sensitive BD413, HO1-N, or E. coli HB101. The chromosomal location of Tn5 insertions in independently isolated Kmr exconjugants of BD413 and HO1-N was characterized by restriction endonuclease mapping and hybridization studies. Results obtained from Southern hybridization studies were consistent with a single Tn5-specific insertion site in HO1-N and two such sites in BD413. Phage Mu sequences were not detected in Tn5-containing Acinetobacter sp.     

Tomatinase from Fusarium oxysporum f. sp. lycopersici defines a new class of saponinases.

Plants produce a variety of secondary metabolites, many of which have antifungal activity. Saponins are plant glycosides that may provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici and other tomato pathogens produce extracellular enzymes known as tomatinases, which deglycosylate alpha-tomatine to yield less toxic derivatives. We have cloned and characterized the cDNA and genomic DNA encoding tomatinase from the vascular pathogen of tomato F. oxysporum f. sp. lycopersici. This gene encodes a protein (FoTom1) with no amino acid sequence homology to any previously described saponinase, including tomatinase from Septoria lycopersici. Although FoTom1 is related to family 10 glycosyl hydrolases, which include mainly xylanases, it has no detectable xylanase activity. We have overexpressed and purified the protein with a bacterial heterologous system. The purified enzyme is active and cleaves alpha-tomatine into the less toxic compounds tomatidine and lycotetraose. Tomatinase from F. oxysporum f. sp. lycopersici is encoded by a single gene whose expression is induced by alpha-tomatine. This expression is fully repressed in the presence of glucose, which is consistent with the presence of two putative CREA binding sites in the promoter region of the tomatinase gene. The tomatinase gene is expressed in planta in both roots and stems throughout the entire disease cycle of F. oxysporum f. sp. lycopersici.     

Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.     

Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens

Seven bacterial isolates screened from rhizosphere of common bean growing at Uttarakhand Himalaya showed potential plant growth promoting (PGP) and antagonistic activities. Based on 16S rRNA gene sequence the isolate BPR7 was identified as Bacillus sp. BPR7. The strain BPR7 produced IAA, siderophore, phytase, organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate oxidase, and solubilized various sources of organic and inorganic phosphates as well as potassium and zinc. Strain BPR7 strongly inhibited the growth of several phytopathogens such as Macrophomina phaseolina, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Rhizoctonia solani and Colletotricum sp. in vitro. Cell-free culture filtrate of strain BPR7 also caused colony growth inhibition of all test pathogens. PGP and antifungal activities of Bacillus sp. BPR7 suggest that it may be exploited as a potential bioinoculant agent for P. vulgaris.     

Isolation of competition-defective mutants of Rhizobium fredii.

We coupled Tn5 mutagenesis with a competition assay to isolate mutants of Rhizobium fredii USDA 257 that are defective in competition for nodulation of soybeans. Two mutants with single Tn5 inserts in the chromosome showed reduced competitiveness in vermiculite but were identical to the wild-type strain in symbiotic properties when inoculated alone. Recombination of Tn5 and flanking genomic regions cloned from the mutants into the parent strain showed that Tn5 was responsible for the mutant phenotype.     

Isolation and characterization of an aminolevulinate-requiring Rhodobacter capsulatus mutant.

Using transposon Tn5 mutagenesis, we isolated a mutant strain of Rhodobacter capsulatus that requires aminolevulinate for growth. Southern blot analysis indicated that this strain has a single Tn5 insertion. The addition of 0.1 mM aminolevulinate to the medium allowed the mutant to grow either aerobically or photosynthetically with generation times similar to those of the parental strain. When grown photosynthetically, bacteriochlorophyll accumulation increased with increasing aminolevulinate concentration. The mutant strain had only 10% of the normal aminolevulinate synthase activity, but it had a normal level of porphobilinogen synthase activity. The requirement for aminolevulinate could be satisfied by porphobilinogen, hemin, or protoporphyrin. While the mutant grew well on agar plates containing any of these substrates, growth in liquid media containing hemin or protoporphyrin was poor. Introduction of an R' factor containing all the known R. capsulatus bch genes into the mutant strain did not relieve the requirement for aminolevulinate, suggesting that the Tn5 insertion is not within the bch region.