Dirk Rothenstein

Sida micrantha mosaic is associated with a complex infection of begomoviruses different from Abutilon mosaic virus

Summary.We report on the nucleotide sequences of geminiviruses of the genus Bemogovirus infecting Sida micrantha Schr., a common weed in Brazil. For decades, the mosaic frequently associated with Sida plants was considered to be caused by a Brazilian strain of Abutilon mosaic virus (AbMV). By infection studies and sequence comparisons, we demonstrate that it is associated with a complex of at least two begomoviruses as different from AbMV as most South American geminiviruses. Two molecules of DNA A (A1, A2) and three of DNA B (B1, B2, B3) were cloned and sequenced. According to the high homology in their common regions, DNA A1 and DNA B3, as well as DNA A2 and DNA B2, are cognate components of two begomoviruses, which were infectious in Nicotiana benthamiana plants. No trans-replication was found for any other A/B combination. The intergenic region of DNA B2 appears to be the product of the recombination between DNA B1 and DNA A2. These results show that a coinfection of begomoviruses can persist over decades, producing a reservoir of partially recombined but distinct geminiviruses.

Virus-templated synthesis of ZnO nanostructures and formation of field-effect transistors.

The search for novel methods for the synthesis of nanostructured materials is an important step towards the miniaturization of multifunctional devices, which requires careful and appropriate integration of various materials into a single unit. However, most of the conventional synthesis methods for multicomponent systems involve harsh reaction conditions and thereby introduce limitations in the choice of materials to be combined. For instance, in ceramic synthesis methods, extreme heating and/or pressure are often used, which may be inapplicable to certain components of a device structure. Further factors critical to the miniaturization are the size of the obtained powder particles and their tendency to agglomerate. Hence, the integration of different materials is still a challenging goal and can hardly be achieved by conventional processing. Biomineralization is a process used by organisms to generate composite materials composed of organic and inorganic phases, which often exhibit exceptional properties. [ 1 ] Organic molecules, such as peptides, proteins, or polysaccharides, guide the crystal growth at ambient conditions that eventually determine the morphology and the functional properties of the materials. [ 2 ] The integration of biomolecules as templates or structure-directing agents, on the other hand, offers the opportunity to explore alternative low-temperature methods in the synthesis of bioinorganic hybrid materials with novel tailored functionalities. [ 3 , 4 ] For some applications, however, the adaptation of bionic mineralization approaches to the synthesis of artifi cial composite materials is not possible, since no interactions between the inorganic phase

Isolation of ZnO-binding 12-mer peptides and determination of their binding epitopes by NMR spectroscopy.

Inorganic-binding peptides are in the focus of research fields such as materials science, nanotechnology, and biotechnology. Applications concern surface functionalization by the specific coupling to inorganic target substrates, the binding of soluble molecules for sensing applications, or biomineralization approaches for the controlled formation of inorganic materials. The specific molecular recognition of inorganic surfaces by peptides is of major importance for such applications. Zinc oxide (ZnO) is an important semiconductor material which is applied in various devices. In this study the molecular fundamentals for a ZnO-binding epitope was determined. 12-mer peptides, which specifically bind to the zinc- or/and the oxygen-terminated sides of single-crystalline ZnO (0001) and (000-1) substrates, were selected from a random peptide library using the phage display technique. For two ZnO-binding peptides the mandatory amino acid residues, which are of crucial importance for the specific binding were determined with a label-free nuclear magnetic resonance (NMR) approach. NMR spectroscopy allows the identification of pH dependent interaction sites on the atomic level of 12-mer peptides and ZnO nanoparticles. Here, ionic and polar interaction forces were determined. For the oxygen-terminated side the consensus peptide-binding sequence (HSXXH) was predicted in silico and confirmed by the NMR approach.

Biodiversity and recombination of cassava-infecting begomoviruses from southern India

Summary.Cassava mosaic disease (CMD) is caused by various begomoviruses of the family Geminiviridae leading to considerable crop losses in Africa and Asia. Recombination between their genomic components has generated new pathotypes with enhanced virulence in Africa. Here, we report about a survey on the biodiversity of begomoviruses in cassava from southern India (Tamil Nadu and Kerala states) performed in 2001 and 2002. Viral DNA A components from stem cuttings were analysed using polymerase chain reaction and restriction fragment length polymorphism. Eight representative examples were completely sequenced. The majority of DNA sequences (7 of 8) obtained were more closely related to that of Sri Lankan cassava mosaic virus (SLCMV) than of Indian cassava mosaic virus (ICMV). Only one sequence collected in Kerala was related to ICMV. The diversity of the SLCMV-like sequences was rather low compared to the variability of African viruses associated with cassava mosaic disease. Based on DNA A sequence data, all of these isolates should be classified as variants of SLCMV or ICMV. Phylogenetic analysis revealed mosaic structures within the DNA sequences which may indicate footprints of recombination events between ancestors of SLCMV and ICMV.

Peptide-equipped tobacco mosaic virus templates for selective and controllable biomineral deposition

Summary The coating of regular-shaped, readily available nanorod biotemplates with inorganic compounds has attracted increasing interest during recent years. The goal is an effective, bioinspired fabrication of fiber-reinforced composites and robust, miniaturized technical devices. Major challenges in the synthesis of applicable mineralized nanorods lie in selectivity and adjustability of the inorganic material deposited on the biological, rod-shaped backbones, with respect to thickness and surface profile of the resulting coating, as well as the avoidance of aggregation into extended superstructures. Nanotubular tobacco mosaic virus (TMV) templates have proved particularly suitable towards this goal: Their multivalent protein coating can be modified by high-surface-density conjugation of peptides, inducing and governing silica deposition from precursor solutions in vitro. In this study, TMV has been equipped with mineralization-directing peptides designed to yield silica coatings in a reliable and predictable manner via precipitation from tetraethoxysilane (TEOS) precursors. Three peptide groups were compared regarding their influence on silica polymerization: (i) two peptide variants with alternating basic and acidic residues, i.e. lysine–aspartic acid (KD)x motifs expected to act as charge-relay systems promoting TEOS hydrolysis and silica polymerization; (ii) a tetrahistidine-exposing polypeptide (CA4H4) known to induce silicification due to the positive charge of its clustered imidazole side chains; and (iii) two peptides with high ZnO binding affinity. Differential effects on the mineralization of the TMV surface were demonstrated, where a (KD)x charge-relay peptide (designed in this study) led to the most reproducible and selective silica deposition. A homogenous coating of the biotemplate and tight control of shell thickness were achieved.

Biolistic infection of cassava using cloned components of Indian cassava mosaic virus

Summary.Cassava mosaic disease (CMD) is a major constraint to cassava production in Africa and Asia. Of the two begomoviruses associated with CMD on the Indian subcontinent, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus, only the latter has been successfully reintroduced into cassava to resolve the aetiology of the disease. Here, we report the complete nucleotide sequence of an ICMV isolate from Maharashtra (ICMV-[Mah2]), central India. Biolistic inoculation of the cloned components produced a systemic infection and typical mosaic symptoms in cassava, thereby fulfilling Koch’s postulates. The availability of infectious clones will provide a valuable tool to screen new cassava cultivars for disease resistance under defined conditions.

Adsorption and Self-Assembly of M13 Phage into Directionally Organized Structures on C and SiO2 Films

A versatile method for the directional assembly of M13 phage using amorphous carbon and SiO2 thin films was demonstrated. A high affinity of the M13 phage macromolecules for incorporation into aligned structures on an amorphous carbon surface was observed at the concentration range, in which the viral nanofibers tend to disorder. In contrast, the viral particles showed less freedom to adopt an aligned orientation on SiO2 films when deposited in close vicinity. Here an interpretation of the role of the carbon surface in significant enhancement of adsorption and generation of viral arrays with a high orientational order was proposed in terms of surface chemistry and competitive electrostatic interactions. This study suggests the use of amorphous carbon substrates as a template for directional organization of a closely-packed and two-dimensional M13 viral film, which can be a promising route to mineralize a variety of smooth and homogeneous inorganic nanostructure layers.

Generation of Multishell Magnetic Hybrid Nanoparticles by Encapsulation of Genetically Engineered and Fluorescent Bacterial Magnetosomes with ZnO and SiO2

Magnetic nanoparticles (MNPs) have great potential in biomedical applications, but the chemical synthesis of size-controlled and functionalized core-shell MNPs remain challenging. Magnetosomes produced by the magnetotactic bacterium Magnetospirillum gryphiswaldense are naturally uniform and chemically pure magnetite MNPs with superior magnetic characteristics. Here, additional functionalities are made possible by the incorporation of biomolecules on the magnetosome surface; the magnetosome system is then chemically encapsulated with an inorganic coating. The novel multishell nanoparticles consist of the magnetosome core-which includes the magnetite crystal, the magnetosome membrane, and additional moieties, such as the enhanced green fluorescent protein (EGFP) and peptides-and an outer shell, comprising either silica or zinc oxide. Coating the functionalized magnetosomes with silica improves their colloidal stability and preserves the EGFP fluorescence in the presence of proteases and detergents. In addition, the surface charge of magnetosomes can be adjusted by varying the coating. This method will be useful for the versatile generation of new, multifunctional, multishell, and magnetic hybrid nanomaterials with potential applications in various biotechnological fields.

Influence of zinc on the calcium carbonate biomineralization of Halomonas halophila.

BackgroundThe salt tolerance of halophilic bacteria make them promising candidates for technical applications, like isolation of salt tolerant enzymes or remediation of contaminated saline soils and waters. Furthermore, some halophilic bacteria synthesize inorganic solids resulting in organic–inorganic hybrids. This process is known as biomineralization, which is induced and/or controlled by the organism. The adaption of the soft and eco-friendly reaction conditions of this formation process to technical syntheses of inorganic nano materials is desirable. In addition, environmental contaminations can be entrapped in biomineralization products which facilitate the subsequent removal from waste waters. The moderately halophilic bacteria Halomonas halophila mineralize calcium carbonate in the calcite polymorph. The biomineralization process was investigated in the presence of zinc ions as a toxic model contaminant. In particular, the time course of the mineralization process and the influence of zinc on the mineralized inorganic materials have been focused in this study.ResultsH. halophila can adapt to zinc contaminated medium, maintaining the ability for biomineralization of calcium carbonate. Adapted cultures show only a low influence of zinc on the growth rate. In the time course of cultivation, zinc ions accumulated on the bacterial surface while the medium depleted in the zinc contamination. Intracellular zinc concentrations were below the detection limit, suggesting that zinc was mainly bound extracellular. Zinc ions influence the biomineralization process. In the presence of zinc, the polymorphs monohydrocalcite and vaterite were mineralized, instead of calcite which is synthesized in zinc-free medium.ConclusionsWe have demonstrated that the bacterial mineralization process can be influenced by zinc ions resulting in the modification of the synthesized calcium carbonate polymorph. In addition, the shape of the mineralized inorganic material is chancing through the presence of zinc ions. Furthermore, the moderately halophilic bacterium H. halophila can be applied for the decontamination of zinc from aqueous solutions.

Phage-assisted assembly of organic–inorganic hybrid bilayers

Abstract Protein-based bottom-up synthesis of functional nanomaterials and devices is one of the most promising areas in bio-nanotechnology. Here, we demonstrate that organic assemblies can serve as biologically controllable scaffolds for the deposition of inorganic nanoparticles. In this work, wild-type M13 phages were employed for controlled mineralization of zinc oxide particles. Our aim was to construct layered structures of organic and inorganic materials which contain alternating layers on a smooth substrate. The structure, elemental composition, and also the integrity of the organic and the biologically-templated inorganic layers were studied. A uniform nano-hybrid structure without significant thickness fluctuations was fabricated by using a high concentration of M13 phages and a carbon-coated substrate. The current study gives insight into the combination of organic–inorganic materials to form a multilayered structure, which in turn sets the stage for the fabrication of electronic devices, e. g. actuators or capacitors.