Morphological changes of <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic">oxysporum</Emphasis> induced by CF66I,an antifungal compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The morphological effects of CF66I, an antifungal compound produced by Burkholderia cepacia, on growing hyphae of Fusarium oxysporum were studied by fluorescence microscopy (FM) and transmission electron microscopy (TEM). At 20 μg/ml, CF66I strongly inhibited growth and induced significant changes of the hyphal morphology. These changes included swelling of hyphae with considerable thickening cell wall and abnormal chitin deposition, which was indicative of the alterations in cell wall structure. Furthermore, fluorescein diacetate (FDA) staining indicated the loss of intracellular esterase activity. CF66I probably inhibits fungal growth by interfering with the cell metabolic pathways. At 120 μg/ml, CF66I killed F. oxysporum (accompanied by propidium iodide permeation, intracellular cytoplasm leakage and crushing of hyphal tips), probably by direct damage to the cell membrane. Thus, there are two different antifungal mechanisms of CF66I, depending on its concentration, and further studies on this compound might be useful for us to develop a new class of antifungal agents.     

Recyclable chaperone-conjugated magnetic beads for in vitro refolding of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> lipase

Polymer-coated magnetic beads have become widely used in biological applications because of their facile recovery and easily modifiable surface. Herein, we report the application of magnetic beads to in vitro refolding of B. cepacia lipase. Magnetic particles (Fe₃O₄) prepared by co-precipitation of Fe²⁺ and Fe³⁺ ions under basic conditions were subsequently coated with carboxylic acid-containing polystyrene by emulsion polymerization. The polymer-coated magnetic beads were then conjugated with molecular chaperone proteins to assist with refolding. The chaperone-conjugated magnetic beads efficiently refolded B. cepacia lipase and were easily reused. The beads showed comparable refolding activity to the soluble chaperone, and retained more than 95% of their refolding activity after five cycles of refolding B. cepacia lipase.     

Diversity of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> Complex from the Moso Bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>) Rhizhosphere Soil

The purpose of this study was to determine the existence of Burkholderia cepacia complex (Bcc) at species level and the predominant species in the environment of moso bamboo plantations in Hangzhou, China. A total of 423 isolates were recovered from moso bamboo rhizhosphere soil samples of three sites on the selective medium during 2007–2008. Isolates were identified by Bcc-specific PCR assays, followed by recA-restriction fragment length polymorphism assays, species-specific PCR analysis, recA gene sequencing, multilocus sequence typing (MLST) scheme, and BOX-PCR fingerprinting for genomic diversity. Out of 423 isolates, 278 isolates were assigned to the following Bcc species, eight B. stabilis, 26 B. anthina, 193 B. pyrrocinia, and 51 B. arboris, which indicated B. pyrrocinia as the most dominant species followed by B. arboris. Moreover, false positives were observed in certain isolates of B. arboris while performing species-specific PCR test. Furthermore, the results of recA gene sequence similarity and MLST data demonstrated that nine isolates formed a single discrete cluster but were PCR negative to species-specific primers representing novel species may exist within the Bcc. In addition, BOX-PCR fingerprinting for all the Bcc isolates also showed the strain diversity. It is the first report of the existence of B. arboris and predominance of B. pyrrocinia in the moso bamboo environment.     

Metabolism-mediated induction of zinc tolerance in <Emphasis Type="Italic">Brassica rapa</Emphasis> by <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> CS2-1

Brassica rapa (Chinese cabbage) is an essential component of traditional Korean food. However, the crop is often subject to zinc (Zn⁺) toxicity from contaminated irrigation water, which, as a result, compromises plant growth and production, as well as the health of human consumers. The present study investigated the bioaccumulation of Zn⁺ by Burkholderia cepacia CS2-1 and its effect on the heavy metal tolerance of Chinese cabbage. Strain CS2-1 was identified and characterized on the basis of 16S rRNA sequences and phylogenetic analysis. The strain actively produced indole-3-acetic acid (3.08 ± 0.21 μg/ml) and was also able to produce siderophore, solubilize minerals, and tolerate various concentrations of Zn⁺. The heavy metal tolerance of B. rapa plants was enhanced by CS2-1 inoculation, as indicated by growth attributes, Zn⁺ uptake, amino acid synthesis, antioxidant levels, and endogenous hormone (ABA and SA) synthesis. Without inoculation, the application of Zn⁺ negatively affected the growth and physiology of B. rapa plants. However, CS2-1 inoculation improved plant growth, lowered Zn⁺ uptake, altered both amino acid regulation and levels of flavonoids and phenolics, and significantly decreased levels of superoxide dismutase, endogenous abscisic acid, and salicylic acid. These findings indicate that B. cepacia CS2-1 is suitable for bioremediation against Zn⁺-induced oxidative stress.     

Essential role of the small subunit of thermostable glucose dehydrogenase from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The co-expression in Escherichia coli of the -subunit and the catalytic -subunit of the thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 produced 12.7 U GDH activity mg^–1 protein. A 47-amino acid, twin-arginine translocase signal peptide was identified at the amino terminus of the -subunit. The expression of the -subunit in the absence of the -subunit or the -subunit signal peptide failed to produce any detectable GDH protein or activity. The -subunit may be a chaperone-like component that assists folding of the -subunit polypeptide to the active form and its translocation to the periplasm.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Colonization of <Emphasis Type="Italic">Morus alba</Emphasis> L. by the plant-growth-promoting and antagonistic bacterium <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> strain Lu10-1

Background  

Anthracnose, caused by Colletotrichum dematium, is a serious threat to the production and quality of mulberry leaves in susceptible varieties. Control of the disease has been a major problem in mulberry cultivation. Some strains of Burkholderia cepacia were reported to be useful antagonists of plant pests and could increase the yields of several crop plants. Although B. cepacia Lu10-1 is an endophytic bacterium obtained from mulberry leaves, it has not been deployed to control C. dematium infection in mulberry nor its colonization patterns in mulberry have been studied using GFP reporter or other reporters. The present study sought to evaluate the antifungal and plant-growth-promoting properties of strain Lu10-1, to clarify its specific localization within a mulberry plant, and to better understand its potential as a biocontrol and growth-promoting agent.     

<Emphasis Type="Italic">xylA</Emphasis> and <Emphasis Type="Italic">xylB</Emphasis> overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in <Emphasis Type="Italic">Burkholderia sacchari</Emphasis>

Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (Y_P3HB/Xil = 0.35 g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.     

Production of polyhydroxyalkanoates by <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> ATCC 17759 using a detoxified sugar maple hemicellulosic hydrolysate

Sugar maple hemicellulosic hydrolysate containing 71.9 g/l of xylose was used as an inexpensive feedstock to produce polyhydroxyalkanoates (PHAs) by Burkholderia cepacia ATCC 17759. Several inhibitory compounds present in wood hydrolysate were analyzed for effects on cell growth and PHA production with strong inhibition observed at concentrations of 1 g/l furfural, 2 g/l vanillin, 7 g/l levulinic acid, and 1 M acetic acid. Gradual catabolism of lower concentrations of these inhibitors was observed in this study. To increase the fermentability of wood hydrolysate, several detoxification methods were tested. Overliming combined with low-temperature sterilization resulted in the highest removal of total inhibitory phenolics (65%). A fed-batch fermentation exhibited maximum PHA production after 96 h (8.72 g PHA/L broth and 51.4% of dry cell weight). Compositional analysis by NMR and physical–chemical characterization showed that PHA produced from wood hydrolysate was composed of polyhydroxybutyrate (PHB) with a molecular mass (M _N) of 450.8 kDa, a melting temperature (T _m) of 174.4°C, a glass transition temperature (T _g) of 7.31°C, and a decomposition temperature (T _decomp) of 268.6°C.     

Enhanced biocatalytic activity of immobilized <Emphasis Type="Italic">Pseudomonas cepacia</Emphasis> lipase under sonicated condition

The present work reports the use of biocatalyst and ultrasound for greener synthesis of cinnamyl propionate. The lipase Pseudomonas cepacia was immobilized on a copolymer of hydroxypropyl methyl cellulose and polyvinyl alcohol. This biocatalyst was used for ultrasound-assisted synthesis of cinnamyl propionate with the detailed optimization of various reaction parameters. Besides this, protocol was extended to synthesize various industrially important propionate esters. In addition to this, different enzyme-kinetic parameters such as r _max and K _{m(vinyl propionate),} K _{m(cinnamyl alcohol)} and K _{i(cinnamyl alcohol)} were studied which presented ordered bi–bi mechanism with an inhibition by cinnamyl alcohol. The developed biocatalyst demonstrated enhancement in catalytic activity and recyclability up to five recycles. Moreover, the biocatalyst was tested to investigate the effects of sonication via various characterization techniques such as scanning electron microscopy, thermogravimetry, and water content analysis.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Expression of a lipid-inducible,self-regulating form of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> lipase <Emphasis Type="Italic">LIP2</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion of low-value lipids to value-added TAGs and other novel lipid products.     

Identification of <Emphasis Type="Italic">Burkholderia mallei</Emphasis> and <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> adhesins for human respiratory epithelial cells

Background  

Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms.     

Early perception responses of<Emphasis Type="Italic"> Nicotiana tabacum</Emphasis> cells in response to lipopolysaccharides from<Emphasis Type="Italic"> Burkholderia cepacia</Emphasis>

Lipopolysaccharides (LPS) are cell surface components of Gram-negative bacteria and, as microbe- / pathogen-associated molecular patterns, have diverse roles in plant–microbe interactions, e.g. LPS are able to promote plant disease tolerance through activation of induced or acquired resistance. However, little is known about the mechanisms of signal perception and transduction in response to elicitation by these bio-active lipoglycans. The present study focused on the involvement of LPS isolated from the outer cell wall of the Gram-negative bacterium Burkholderia cepacia (strain ASP B 2D) in the molecular mechanisms and components involved in signal perception and transduction and defense-associated responses in suspension-cultured tobacco (Nicotiana tabacum L.) cells. The purified LPS_B.cep. was found to trigger a rapid influx of Ca²⁺ into the cytoplasm of aequorin-transformed tobacco cells. An oxidative burst, concomitant with the production of reactive oxygen and nitrogen species was measured by chemiluminescence and fluorescence. These early perception responses were accompanied by K⁺/H⁺ exchange and alkalinization of the extracellular medium. Through the use of various inhibitors of the oxidative burst reaction, as well as scavengers of produced radicals, the biochemical basis of the cellular response to LPS_B.cep. elicitation was dissected, elucidated and compared to that induced by a yeast elicitor. These results suggest that LPS_B.cep. interacts with tobacco cells in a manner different from the response elicited by yeast elicitor.Abbreviations DDC Diethyldithiocarbamate - DMSO Dimethyl sulfoxide - DPI Diphenylene iodonium - H ₂ DCF-DA 2,7-Dihydrodichlorofluorescein-diacetate - LPS Lipopolysaccharides - NAC N-Acetyl-l-cysteine - PTIO 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide - ROS Reactive oxygen species - YE Yeast elicitor     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.