<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.     

Insecticidal genes of <Emphasis Type="Italic">Yersinia</Emphasis> spp.: taxonomical distribution,contribution to toxicity towards <Emphasis Type="Italic">Manduca sexta</Emphasis> and <Emphasis Type="Italic">Galleria mellonella</Emphasis>, and evolution

Background  

Toxin complex (Tc) proteins termed TcaABC, TcdAB, and TccABC with insecticidal activity are present in a variety of bacteria including the yersiniae.     

<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.     

Identification of metabolites produced from <Emphasis Type="Italic">N</Emphasis>-phenylpiperazine by <Emphasis Type="Italic">Mycobacterium</Emphasis> spp

Mycobacterium sp. 7E1B1W and seven other mycobacterial strains known to degrade hydrocarbons were investigated to determine their ability to metabolize the piperazine ring, a substructure found in many drugs. Cultures were grown at 30°C in tryptic soy broth and dosed with 3.1 mM N-phenylpiperazine hydrochloride; samples were removed at intervals and extracted with ethyl acetate. Two metabolites were purified from each of the extracts by high-performance liquid chromatography; they were identified by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy as N-(2-anilinoethyl)acetamide and N-acetyl-N′-phenylpiperazine. The results show that mycobacteria have the ability to acetylate piperazine rings and cleave carbon-nitrogen bonds.     

<Emphasis Type="Italic">Megaviridae</Emphasis>-like particles associated with <Emphasis Type="Italic">Symbiodinium</Emphasis> spp. from the endemic coral <Emphasis Type="Italic">Mussismilia braziliensis</Emphasis>

Coral reefs are one of the most dynamic and productive marine ecosystems. The coral holobiont consists of the coral animal and a variety of associated microorganisms that include symbiotic dinoflagellates of the genus Symbiodinium, bacteria, archaea, fungi and viruses. The interactions among these components are crucial for coral health and, consequently, to the coral reef resilience to disturbance. Environmental stressors such as elevated temperature, high irradiance and ultraviolet (UV) radiation can lead to the breakdown of the coral-Symbiodinium symbiosis in a phenomenon known as “coral bleaching”. The present study provides evidence for virus-like particles (VLPs) induced in UV-irradiated Symbiodinium spp. cultures (clades A and C) that were isolated from the coral Mussismilia braziliensis, suggesting a latent viral infection in these strains. Scanning and transmission electron microscopy images of the UV stressed cultures revealed the presence of giant (ca. 450 nm) and small (ca. 40 nm) VLPs. Morphological features link the giant VLPs to the family Megaviridae. Symbiodinium spp. Megaviridae giant viruses and other associated viruses may represent dynamic forces driving and influencing health of the coral holobiont.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

In vitro selection of resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella</Emphasis> spp. at <Emphasis Type="Italic">in vivo</Emphasis> fluoroquinolone concentrations

Background  

Fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. However, resistance to fluoroquinolones in Enterobacteriaceae is increasingly reported. Studies assessing the ability of fluoroquinolones to select for resistance have often used antimicrobial concentrations quite different from those actually acquired at the site of infection. The present study compared the ability to select for resistance of levofloxacin, ciprofloxacin and prulifloxacin at concentrations observed in vivo in twenty strains of Escherichia coli and Klebsiella spp. isolated from patients with respiratory and urinary infections. The frequencies of spontaneous single-step mutations at plasma peak and trough antibiotic concentrations were calculated. Multi-step selection of resistance was evaluated by performing 10 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 10 subcultures on antibiotic-free agar. E. coli resistant strains selected after multi-step selection were characterized for DNA mutations by sequencing gyrA, gyrB, parC and parE genes.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Molecular Characteristics of OmpF-Like Porins from Pathogenic <Emphasis Type="Italic">Yersinia</Emphasis>

Nonspecific pore-forming proteins (porins) are the major proteins of the outer membrane of Gram-negative bacteria responsible for diffusion of low-molecular-weight compounds. Nucleotide sequences of the OmpF-like porins from the pathogenic bacteria Yersinia pseudotuberculosis (YPS) and Yersinia enterocolitica (YE) were cloned and determined. Values of molecular weights (MW) and isoelectric points (IEP) calculated for these proteins (for OmpF-YPS: MW 37.7 kD, IEP 4.45; for OmpF-YE: MW 39.5 kD, IEP 4.34) are in good agreement with experimental data. The OmpF-like Yersinia porins are highly homologous to each other (83-92%) and also to the OmpF protein from Serratia marcescens (70%); the homology to the OmpF porin from E. coli is significantly lower (52-58%). Multiple alignment of the amino acid sequences of mature OmpF proteins provided the distribution of conservative amino acid residues typical for porins. Moreover, the OmpF-like porins from Yersinia are characterized by the presence of extended regions with high and low homologies, which coincide with the transmembrane domains and "external" loops, respectively, of the topological model of the OmpF porin from E. coli. By predictive methods, the secondary structure of the OmpF-like porins from Yersinia was obtained. This structure is represented by 16 beta-strands connected by short "periplasmic" and longer "external" loops with unordered structure.     

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.     

Gene transfer into <Emphasis Type="Italic">Solanum tuberosum</Emphasis> via <Emphasis Type="Italic">Rhizobium</Emphasis> spp.

Agrobacterium tumefaciens-mediated transformation (ATMT) is the preferred technique for gene transfer into crops. A major disadvantage of the technology remains the complexity of the patent landscape that surrounds ATMT which restricts its use for commercial applications. An alternative system has been described (Broothaerts et al. in Nature 433:629-633, 2005) detailing the propensity of three rhizobia to transform the model crop Arabidopsis thaliana, the non-food crop Nicotiana tabacum and, at a very low frequency, the monocotyledonous crop Oryza sativa. In this report we describe for the first time the genetic transformation of Solanum tuberosum using the non-Agrobacterium species Sinorhizobium meliloti, Rhizobium sp. NGR234 and Mesorhizobium loti. This was achieved by combining an optimal bacterium and host co-cultivation period with a low antibiotic regime during the callus and shoot induction stages. Using this optimized protocol the transformation frequency (calculated as % of shoots equipped with root systems with the ability to grow in rooting media supplemented with 25 μg/ml hygromycin) of the rhizobia strains was calculated at 4.72, 5.85 and 1.86% for S. meliloti, R. sp. NGR234 and M. loti respectively, compared to 47.6% for the A. tumefaciens control. Stable transgene integration and expression was confirmed via southern hybridisation, quantitative PCR analysis and histochemical screening of both leaf and/or tuber tissue. In light of the rapid advances in potato genomics, combined with the sequencing of the potato genome, the ability of alternative bacteria species to genetically transform this major food crop will provide a novel resource to the Solanaceae community as it continues to develop potato as both a food and non-food crop.     

Phosphonate catabolism by <Emphasis Type="Italic">Campylobacter</Emphasis> spp.

The catabolism of phosphonates (Phn) by Campylobacter spp. was investigated employing nuclear magnetic resonance spectroscopy and cell culture techniques. The bacteria were capable of cleaving the Phn bonds of different compounds, including -aminomethylphosphonate, phosphonoacetate and phenylphosphonate (PhePhn). The kinetic parameters of these activities were determined in vivo in intact cells and in situ in whole-cell lysates. Cleavage of Phn-bearing compounds was associated with the cell-wall and cytosolic fractions. Results from substrate competition experiments suggested that at least two enzyme activities appeared to be involved in the cleavage of carbon–phosphate (C–P) bonds. In silico analyses indicated that no genes orthologous to those encoding C–P bond-cleaving enzymes in other bacteria were present in the Campylobacter jejuni genome. In most bacteria studied, Phn catabolism is induced under conditions of phosphate limitation; however, in Campylobacter spp. these activities were expressed in cells grown in media rich in phosphate. In chemically defined media, PhePhn supported bacterial growth and proliferation at concentrations above 100 M in the absence of phosphate. Thus, Phn utilisation may be a survival mechanism of Campylobacter spp. in milieux lacking sufficient phosphate. The expression of these enzyme activities in media abundant in phosphate suggested also that they may have other physiological roles.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Mineralization of chlorpyrifos by co-culture of <Emphasis Type="Italic">Serratia</Emphasis> and <Emphasis Type="Italic">Trichosporon</Emphasis> spp.

A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).     

Control of fusarium wilt of <Emphasis Type="Italic">Solanum melongena</Emphasis> by <Emphasis Type="Italic">Trichoderma</Emphasis> spp.

Biological control of wilt of egg plant (Solanum melongena L.) caused by Fusarium solani was made with the application of five Trichoderma species, T. harzianum, T. viride, T. lignorum, T. hamatum and T. reesei. The effect of volatile and non-volatile antibiotics of Trichoderma origin on growth inhibition of the wilt pathogen was studied. T. harzianum showed maximum growth inhibition (86.44 %) of the pathogen through mycoparasitism. The non-volatiles produced by the Trichoderma species exhibited 100 % growth inhibition of the pathogen under in vitro condition. Production of siderophores and fungal cell wall degrading enzymes, chitinase and β-1,3-glucanase were found. Treatments with two most efficient Trichoderma species, T. harzianum and T. viride resulted in the decreasing population of Fusarium solani in soil thereby deterring disease incidence in field condition.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).