Glycosylation and production characteristics of epothilones in alkali-tolerant <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> strain So0157-2

3-O-α-D-ribofuranosyl epothilone A (epothiloneoside A) is a major component of glycosylated epothilones in Sorangium cellulosum strain So0157-2. The production and glycosylation ratios of epothiloneoside A in both solid and liquid culture conditions with various pH values and carbon sources were studied. The results showed that glycosylation occurs whenever epothilones are produced, regardless of changes in pH values, production time curves, and different carbon sources. We suggest that glycosylation is a stable process, paralleling the biosynthesis of epothilones in the So0157-2 strain.     

Characterisation,genome size and genetic manipulation of the myxobacterium <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> So ce56

In this study, Sorangium cellulosum So ce56 was phenotypically and genotypically analysed in order to evaluate whether this strain can be used in a comprehensive genome project as a representative of the secondary metabolite-producing myxobacteria. In contrast to many other strains of S. cellulosum, strain So ce56 was found to have various advantageous features, including fast and homogeneous growth in submerged cultures and the ability to complete its morphological differentiation cycle on agar, even when the inoculant originates from a liquid culture. Two groups of secondary metabolites isolated from the culture broth were identified, the polyketides etnangien and chivosazole. The presence of polyketide synthase-encoding genes in the genome of strain So ce56 was demonstrated via PCR. The phenotypic classification was confirmed by comparison of 16S rDNA sequences which showed that S. cellulosum So ce56 clusters within a separate lineage together with S. cellulosum ATCC 25531 and the epothilone producer S. cellulosum So ce90. The genome of S. cellulosum So ce56 belongs to the largest bacterial genomes described so far. It is estimated to be 12.2 Mb in size, by pulsed-field gel electrophoresis. In order to demonstrate that S. cellulosum So ce56 is a convenient strain for molecular biological studies, a genetic manipulation system was developed. Using triparental mating, polyketide synthase-encoding genes were inactivated, leading to chivosazole-negative mutants.     

Damage of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> biofilms by carolacton,a secondary metabolite from the myxobacterium <Emphasis Type="Italic">Sorangium cellulosum</Emphasis>

Background  

Streptococcus mutans is a major pathogen in human dental caries. One of its important virulence properties is the ability to form biofilms (dental plaque) on tooth surfaces. Eradication of such biofilms is extremely difficult. We therefore screened a library of secondary metabolites from myxobacteria for their ability to damage biofilms of S. mutans.     

Isolation and characterisation of an <Emphasis Type="Italic">HpSHP</Emphasis> gene from <Emphasis Type="Italic">Hosta plantaginea</Emphasis>

Based on genetic and molecular analyses, the ABC model has been proposed to explain the genetic control of floral development. C-class MADS-box genes play crucial roles in Arabidopsis thaliana development by regulating the organ identities of stamens and gynoecium. The present research reports for the first time the cloning of an HpSHP gene from Hosta plantaginea (Lam.) Aschers. Phylogenetic analysis shows that HpSHP is a member of the C-class MADS-box genes that is closely related to C-lineage SHP homologues from monocot species. Semi-quantitative and real-time polymerase chain reaction analyses show that HpSHP expression is stamen and gynoecium specific. HpSHP also has spatial and temporal expression patterns in the reproductive organs of H. plantaginea. A functional analysis is carried out in Arabidopsis by overexpression of HpSHP. Homeotic transformations of sepals into carpelloid organs, bent ovaries, and prematurely shattering fruits are observed in 35S::HpSHP transgenic plants. All these results show that HpSHP plays a crucial role in gynoecium development.     

<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.     

Characterization and Engineering of a Novel Pyrroloquinoline Quinone Dependent Glucose Dehydrogenase from <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> So ce56

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized. The recombinant protein has a relative molecular mass of 63 kDa and shows 43% homology to PQQ GDH-B from Acinetobacter calcoaceticus. In the presence of PQQ and CaCl₂ the enzyme has dehydrogenase activity with the substrate glucose as well as with other mono- and disaccharides. The thermal stability and its pH activity profile mark the enzyme as a potential glucose biosensor enzyme. In order to decrease the activity on maltose, which is unwanted for a potential application in biosensors, the protein was rationally modified at three specified positions. The best variant showed a 59% reduction in activity on maltose compared to the wild type enzyme. The catalytic efficiency (k _cat/K _M) was reduced fivefold but the specific activity still amounted to 63% of the wild type activity.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Improving conjugation efficacy of <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> by the addition of dual selection antibiotics

The conjugation protocols in myxobacterium Sorangium cellulosum are often inapplicable due to the strain-specific sensitivity to the presence of Escherichia coli cells or the resistances to many antibiotics. Here we report that the conjugative transfer of the mobilizable plasmid pCVD442 from E. coli DH5alpha (lambda pir) to Sorangium strains could be greatly increased by the presence of low doses of dual selection antibiotics in the mating medium. The improvement was efficient in either E. coli-tolerant or sensitive Sorangium strains. For those phleomycin and hygromycin tolerant Sorangium strains, chloramphenicol-resistance gene was developed as a new selectable marker by driving the resistance gene with the aphII promoter. Using the improved protocol, the epothilone biosynthetic pathway was inactivated by an insertion mutation in the biosynthetic genes of the producing Sorangium strains.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

<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.     

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">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.