Development and relations of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> in soil

The development of Fusarium culmorum and Pseudomonas fluorescens in soil, and the relations between them, were studied using membrane filters containing the fungus, the bacterium, or both microorganisms; the filters were incubated in soil. F. culmorum was identified by indirect immunofluorescence; the GUS-labeled strain was used to visualize P. fluorescens. It was found that F. culmorum introduced in soil can develop as a saprotroph, with the formation of mycelium, macroconidia, and a small amount of chlamydospores. Introduction of glucose and cellulose resulted in increased density of the F. culmorum mycelium and macroconidia. P. fluorescens suppressed the development of the F. culmorum mycelium in soil, but stimulated chlamydospore formation. Decreased mycelial density in the presence of P. fluorescens was more pronounced in soil without additions and less pronounced in the case of introduction of glucose or cellulose. F. culmorum had no effect on P. fluorescens growth in soil.     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].     

Expression of gene for <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactonase <Emphasis Type="Italic">AiiA</Emphasis> affects properties of rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Biotechnological potential of a rhizosphere <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">aeruginosa</Emphasis> strain producing phenazine-1-carboxylic acid and phenazine-1-carboxamide

Bacterial phenazine metabolites belong to a group of nitrogen-containing heterocyclic compounds with antimicrobial activities. In this study, a rhizosphere Pseudomonas aeruginosa strain PA1201 was isolated and identified through 16S rDNA sequence analysis and fatty acid profiling. PA1201 inhibited the growth of various pathogenic microorganisms, including Rhizotonia solani, Magnaporthe grisea, Fusarium graminearum, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Staphylococcus aureus. High Performance Liquid Chromatography showed that PA1201 produced high levels of phenazine-1-carboxylic acid (PCA), a registered green fungicide ‘Shenqinmycin’ with the fermentation titers of 81.7 mg/L in pigment producing medium (PPM) and 926.9 mg/L in SCG medium containing soybean meal, corn steep liquor and glucose. In addition, PA1201 produced another antifungal metabolite, phenazine-1-carboxaminde (PCN), a derivative of PCA, with the fermentation titers of 18.1 and 489.5 mg/L in PPM and SCG medium respectively. To the best of our knowledge, PA1201 is a rhizosphere originating P. aeruginosa strain that congenitally produces the highest levels of PCA and PCN among currently reported P. aeruginosa isolates, which endows it great biotechnological potential to be transformed to a biopesticide-producing engineering strain.     

<Emphasis Type="Italic">Metarhizium bibionidarum</Emphasis> and <Emphasis Type="Italic">M. purpureogenum</Emphasis>: new species from Japan

Two new species of Metarhizium, M. bibionidarum and M. purpureogenum are described from Japan. Metarhizium bibionidarum is the phylogenetic sister species of M. pemphigi and a member of the M. flavoviride species complex. It is distinguished morphologically from M. pemphigi by its larger conidia. The species is based on a collection of an infected March fly larva (Diptera: Bibionidae) but is also known to occur on fruit beetle (Coleoptera: Scarabaeidae) encountered in France. Metarhizium purpureogenum was isolated from soil by plating and insect baiting methods and represents a unique phylogenetic lineage placed outside the M. anisopliae and M. flavoviride species complexes. Three isolates of M. purpureogenum excreted a distinctive red-purple pigment into agar medium when co-cultured with M. robertsii or Aspergillus oryzae.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

Functional analysis of alcohol dehydrogenase (<Emphasis Type="Italic">ADH</Emphasis>) genes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To characterize the genes responsible for ethanol utilization in Pichia pastoris.

Results

ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.

Conclusion

The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

     

Characterization of phosphate-solubilizing bacteria exhibiting the potential for growth promotion and phosphorus nutrition improvement in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) in calcareous soils of Sinaloa,Mexico

Greenhouse bioassays were used to examine the ability of selected strains of the rhizobacteria Sinorhizobium meliloti, Bacillus flexus and B. megaterium to solubilize phosphorus (P) and to affect growth promotion and phosphorus nutrition in maize. These bacterial strains were found to decrease the pH and solubilize some forms of insoluble P, such as tricalcium phosphate and hydroxyapatite, as well as to exhibit acid and alkaline phosphatase enzymatic activities in culture medium, properties that are possibly involved in P solubilization. Inoculation of the strains separately and as a consortium of the three bacteria (S. meliloti, B. flexus and B. megaterium) in P-deficient soil (4.33 w/v P) fertilized without P improved plant height, shoot and root dry weight, as well as P nutrition in the maize plants. Use of the B. flexus and B. megaterium strains separately and in a consortium positively affected several growth parameters and P nutrition in plants supplemented with insoluble P. No effect was observed when pots in which the seedlings were growing were supplied with soluble fertilizer. A second assay using a P-deficient soil (6.64 w/v P) showed that inoculation with the consortium of B. flexus and B. megaterium significantly increased growth and total P content in maize plants. A dose–response P fertilization experiment using sterile P-deficient soil led us to conclude that inoculation to soil of the mixture of B. flexus and B. megaterium may improve P nutrition and growth to a level previously attained by the addition of soluble P-fertilizer at 40 w/v P. A non-sterile experiment showed a beneficial response with B. megaterium but not with B. flexus. We propose utilizing these bacteria in P-deficient alkaline soils in future field trials in order to evaluate their potential as biofertilizers.     

Reduction of soil-borne pathogen <Emphasis Type="Italic">Fusarium solani</Emphasis> reproduction in soil enriched with phenolic acids by inoculation of endophytic fungus <Emphasis Type="Italic">Phomopsis liquidambari</Emphasis>

The increase of soil-borne pathogens induced by phenolic acids that accumulate in continuous cropping soil reduces the yield and quality of crops. The aims of this study were to investigate (i) the biological control of Fusarium solani, in soil enriched with phenolic acids, by the inoculation of the endophytic fungus Phomopsis liquidambari, and (ii) the biocontrol mechanisms involved. Inoculation of P. liquidambari significantly inhibited the reproduction of F. solani. The prompt degradation of soil phenolic acids by P. liquidambari was determined, but no direct antagonism relationship was observed between P. liquidambari and F. solani, implying the alleviated stimulation of phenolic acids was a major factor in controlling F. solani. Moreover, the presence of glucose did not significantly impact the biocontrol function of P. liquidambari, and P. liquidambari inoculation significantly alleviated disease severity of peanut. Therefore, P. liquidambari could be an effective means to control F. solani in phenolic acids-rich continuous cropping soils.     

Melanin production by a yeast strain XJ5-1 of <Emphasis Type="Italic">Aureobasidium melanogenum</Emphasis> isolated from the Taklimakan desert and its role in the yeast survival in stress environments

The yeast strain XJ5-1 isolated from the Taklimakan desert soil was identified to be a strain of Aureobasdium melanogenum and could produce a large amount of melanin when it was grown in the PDA medium, but its melanin biosynthesis and expression of the PKS gene responsible for the melanin biosynthesis was significantly repressed in the presence of (NH₄)₂SO₄. However, A. melanogenum P5 strain isolated from a mangrove ecosystem grown in both the presence and the absence of (NH₄)₂SO₄ did not produce any melanin. The cell size of A. melanogenum XJ5-1 strain was much higher than that of A. melanogenum P5 strain. The melanized cells of the yeast strain XJ5-1 had higher tolerance to UV radiation, oxidation (200.0 mM H₂O₂), heat treatment (40 °C), salt shock (200.0 g/L NaCl), desiccation and strong acid hydrolysis (6.0 M HCl) at high temperature (80 °C) than the non-melanized cells of the same yeast strain XJ5-1. At the same time, the melanized cells of the yeast strain XJ5-1 also had higher tolerance to UV radiation, oxidation (200.0 mM H₂O₂), desiccation and strong acid hydrolysis (6.0 M HCl) at high temperature (80 °C) than A. melanogenum P5 strain, but had similar resistance to heat treatment (40 °C) and salt shock (200.0 g/L NaCl) compared to those of A. melanogenum P5 strain. All the results revealed that many characteristics of A. melanogenum XJ5-1 isolated from the Taklimakan desert soil was different from those of A. melanogenum P5 strain isolated from the mangrove ecosystem.     

Biocontrol of a root rot of kale by <Emphasis Type="Italic">Muscodor albus</Emphasis> strain MFC2

Pythium ultimum is an oomycetous root rot pathogen that causes significant crop production losses on many crops including kale (Brassica oleracea), an economically important vegetable in Thailand. An endophytic fungus from Thailand designated Muscodor albus MFC2 controlled P. ultimum both in vitro and on kale seedlings grown under outdoor conditions via the production of volatile antibiotics. Ten-day old M. albus MFC2 PDA cultures killed P. ultimum in vitro. Thoroughly mixing three PDA plates of 10-day old M. albus MFC2 into a 500 g mixture of commercial soil and field soil did not adversely affect kale seed germination. The same amount of M. albus MFC2 could restore seedling emergence in P. ultimum inoculated soil to a level close to that of a non-infested control. In addition, M. albus MFC2 did not cause any disease symptoms, but rather seemed to promote the growth of kale in the presence or absence of P. ultimum for up to eight weeks after planting.     

The antibacterial activity of <Emphasis Type="Italic">E. coli</Emphasis> bacteriophage lysin lysep3 is enhanced by fusing the <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> bacteriophage endolysin binding domain D8 to the C-terminal region

Bacteriophage endolysin is one of the most promising antibiotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidoglycans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in catalytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall binding domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to obtain an engineered lysin that can lyse bacteria from the outside. Our results showed that lysep3-D8 could lyse both Gramnegative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 μg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bactericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 μg/ml. The cell wall binding domain of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic domain to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.     

<Emphasis Type="Italic">Amycolatopsis vastitatis</Emphasis> sp. nov., an isolate from a high altitude subsurface soil on Cerro Chajnantor,northern Chile

The taxonomic position of a novel Amycolatopsis strain isolated from a high altitude Atacama Desert subsurface soil was established using a polyphasic approach. The strain, isolate H5^T, was shown to have chemical properties typical of members of the genus Amycolatopsis such as meso-diaminopimelic acid as the diamino acid in the cell wall peptidoglycan, arabinose and galactose as diagnostic sugars and MK-9(H₄) as the predominant isoprenologue. It also has cultural and morphological properties consistent with its classification in the genus, notably the formation of branching substrate hyphae which fragment into rod-like elements. 16S rRNA gene sequence analyses showed that the strain is closely related to the type strain of Amycolatopsis mediterranei but could be distinguished from this and other related Amycolatopsis strains using a broad range of phenotypic properties. It was separated readily from the type strain of Amycolatopsis balhymycina, its near phylogenetic neighbour, based on multi-locus sequence data, by low average nucleotide identity (92.9%) and in silico DNA/DNA relatedness values (51.3%) calculated from draft genome assemblies. Consequently, the strain is considered to represent a novel species of Amycolatopsis for which the name Amycolatopsis vastitatis sp. nov. is proposed. The type strain is H5^T (= NCIMB 14970^T = NRRL B-65279^T).     

Conjugative chromosomal gene transfer in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Conjugative transfer of 20-kb chromosomal fragment carrying genes encoding tetracycline (tet ^r ) and lincomycin (lin ^r ) resistance in the soil strain Bacillus subtilis 19 is described. Transfer was preceded by this fragment insertion into the large conjugative p19cat plasmid producing a hybrid plasmid. Insertion frequency was 10^?4?10^?5. Then genes tet ^r and lin ^r were transferred to the recipient strains. The transfer of chromosomal genes inserted into the plasmid and plasmid gene cat occurred sequentially and resembled sexduction, which represents chromosomal gene transfer by F′ and R′ plasmids during conjugation in Escherichia coli and other gram negative bacteria.     

Enhanced biosynthesis of phenazine-1-carboxamide by <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> strains using statistical experimental designs

Phenazine-1-carboxamide (PCN) is one of the major biocontrol agents produced by plant growth-promoting rhizosphere (PGPR) pseudomonads including Pseudomonas chlororaphis. In this study, a combined strategy of genetic modification and statistical experimental designs was applied to obtain mutants of P. chlororaphis strains with high-yield PCN production. To achieve this, the lon gene was knocked out in wild-type P. chlororaphis HT66 and the breeding mutant P3 strain with a non-scar deletion strategy. The resulting HT66Δlon and P3Δlon mutants produced a significantly higher PCN production in shake-flask cultures which was 5- and  9-folds greater than their native counterparts. The potential ability of strain P3Δlon for PCN production was further optimized by statistical designs. A two-level Plackett–Burman (PB) experimental design with six variables was employed to scrutinize medium components that significantly influence PCN production. Notably, glycerol, tryptone, and soy peptone were identified to be the most significant factors (p?<?0.05). Response surface methodology (RSM) based on the central composite design (CCD) was adopted to determine these factors optimal levels and their interactive effects between culture components for PCN production. The predicted maximum PCN production was 9002 mg/L, whereas an actual PCN production of 9174 mg/L was recorded in the validation experiments using the optimal medium containing glycerol 37.08 mL/L, tryptone 20.00 g/L, and soy peptone 25.03 g/L, which was nearly threefolds higher than without optimization and 20-folds higher than the wild-type strain. In conclusion, the results revealed that P. chlororaphis display a high potential for industrial-scale production for phenazine biopesticides.     

<Emphasis Type="Italic">Saccharothrix tharensis</Emphasis> sp. nov., an actinobacterium isolated from the Thar Desert,India

The taxonomic provenance of a filamentous actinobacterial strain isolated from a desert soil was established using a polyphasic approach. The strain has chemotaxonomic and morphological properties consistent with its classification in the genus Saccharothrix. It forms a distinct branch in the Saccharothrix 16S rRNA gene tree, related to the type strain of Saccharothrix saharensis (96.7%) but was distinguished readily from it using a combination of phenotypic properties. The genotypic and phenotypic data show that the strain represents a novel species in the genus Saccharothrix, for which the name Saccharothrix tharensis sp. nov. is proposed with the type strain TD-093^T (=?KCTC 39724^T?=?MCC 2832^T).     

Thidiazuron: an efficient plant growth regulator for enhancing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Petunia hybrida cv. Mitchell. Leaf explants of petunia were cultured on Murashige and Skoog (MS) medium with different concentrations of thidiazuron (TDZ) without auxin. The highest frequency of shoot regeneration (52.1%) and mean number of shoots per explant (4.1) were obtained on medium containing 2 mg l^?1 TDZ. Leaf explants inoculated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring ß-glucuronidase (uidA) and hygromycin resistance genes developed putative transformant shoots. The highest frequency of shoot regeneration (22.5%) and mean number of transformant shoots per explant (2.4) were obtained on a selection medium consisting of the above described regeneration medium and containing 25 mg l^?1 hygromycin as the selection agent. Approximately 95% of putative transformant shoots expressed the uidA gene following histochemical ß-glucuronidase (GUS) assay. These were confirmed to be transgenic by PCR analysis and Southern blot hybridization.