Transfer of T-DNA from agrobacteria into plant cells through cell walls and membranes

Discusses probable routes of agrobacterial penetration through the plant integumental tissues, cell wall, and plant cell plasmodesma. Analyzes the contribution of extracellular structures of agrobacteria in penetration through barriers of a plant cell, primary contact (adhesion), and during DNA transfer from bacterial (E. coli, A. tumefaciens) to recipient (bacterial or plant) cells. Discusses the relationship between donor cell adhesion to recipient cell surface and the infectious and conjugation processes. Considers the probable role of piles in conjugative transfer of agrobacterial DNA through membranes of donor and recipient (bacterial and plant) cells. Analyzes the contribution of the plant cell cytoskeleton to T-DNA transfer. Suggests a model of transport of T-DNA-VirD2 complex and VirE2 proteins through independent channels consisting of vir-coded proteins.     

Study of extracellular structures of Agrobacterium involved in bacterial and plant interactions

Agrobacterial cells produced straight microfibrils not only when in contact with wheat seedling roots, but also when in contact with each other. After 2 h of incubation, agrobacterial cells were found to form aggregates, in which the cells were in contact either directly or through thick straight microfibrils (bridges) of an unknown composition. The majority of the microfibrils were susceptible to attack by cellulase, although some of them showed resistance to this enzyme. Like the wild-type flagellated agrobacteria, their bald mutants produced long straight microfibrils. The cells surface structures of agrobacteria were examined by labeling them immunocytochemically with colloidal gold conjugated antibodies against O-specific lipopolysaccharides, Vir proteins, and cellulase. Agrobacterial cells treated with acetosyringone and brought into contact were found to contain subpolar and polar cell surface structures. Antibodies against the VirB2 protein were able to interact with a tuft of thin microfibrils located on one pole of the agrobacterial cell, whose vir genes were induced by acetosyringone, but were unable to interact with the surface structures of the agrobacterial cells aggregated in liquid medium in the absence of wheat seedlings.     

A process related to membrane potential involved in bacterial chemotaxis to galactose

Attractants, in the presence of respiration and ATPase inhibitors, stimulate a hyperpolarization in Escherichia coli [Eisenbach, M. (1982) Biochemistry 21, 6818-6825]. In order to examine whether this hyperpolarization is correlated with chemotaxis, the effect of the attractant D-galactose and its analogues on the membrane potential of wild-type E. coli strains and some of their mutants was studied. The main observations were the following: (i) Wild-type cells became hyperpolarized by either galactose or its nonmetabolizable analogues, D-fucose and L-sorbose. (ii) A mutant defective in galactose metabolism became hyperpolarized by galactose. (iii) Inhibiting the galactose permease system did not prevent the hyperpolarization, rather it facilitated the observation of the hyperpolarization. (iv) Mutants unable to transport galactose via the methyl beta-galactoside (Mgl) transport system but having normal chemotaxis to galactose became normally hyperpolarized by D-fucose. (v) Mutants which cannot bind galactose were not hyperpolarized by galactose. (vi) The hyperpolarization in flaI mutants, in which the whole chemotaxis machinery is repressed, was reduced to 12-15% of the hyperpolarization in the parent strains. (vii) Nonattractant sugars did not stimulate hyperpolarization. It is concluded that the hyperpolarization is the consequence of neither galactose metabolism nor transport but rather is correlated with galactose taxis.     

Transfer of genetic information in an organism

New knowledge in biology led us to a better understanding of organization and functioning of living organisms. Today, re-evaluation of our concept of human biology is taking place. Theoretical analysis shows that taking into account the complexity of the organism and frequency of spontaneous mutations, it is impossible to explain the real time of organismal life. Therefore, besides extant systems, other repair systems must also exist. There are three "levels" at which a cell population withstands mutational pressure. First - intracellular (repair), second - intercellular (all forms of informational flows), and third - cellular replacement. Stem cells undertake regenerative functions following damage at the level of the tissue. They are also influenced by mutations, and for stem cells, it is most important that they preserve and support their full activity.     

Peroxisomes are formed from complex membrane structures in PEX6-deficient CHO cells upon genetic complementation

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.     

Affinity driven molecular transfer from erythrocyte membrane to target cells

A wide variety of antimicrobial peptides are known to bind to - and disrupt microbial plasma membranes. Recently, derivatives of the antimicrobial peptide dermaseptin S4 were shown to selectively disrupt the plasma membrane of the intracellular parasite Plasmodium falciparum without harming that of the mammalian host cell. The resulting antimalarial activity is allegedly exerted after the harmless peptide binding to the membrane of the host cell, followed by peptide translocation across a number of intracellular membrane systems and interaction with that of the intraerythrocyte parasite. In this study, we present evidence in support of the ability of a membrane-bound peptide, the dermaseptin S4 derivative K(4)-S4(1-13)a, to transfer from red blood cells (RBCs) to another distant membrane. Binding of K(4)-S4(1-13)a to the plasma membrane of RBCs was assessed in vitro and in vivo, and found to be rapid, spontaneous and receptor independent, as was the transfer of the RBC-bound peptide to the plasma membrane of microorganisms. The present study further provides a basis for the possible use of RBCs as a transport vehicle to deliver drugs to distant targets. This drug delivery system involves the transient "loading" of RBCs with a lipophilic "hook" peptide. Such a peptide has enough affinity for the RBC's plasma membrane to bind to the membrane, but given the opportunity, the peptide will exit its position and transfer to another (target) cell for which it has a greater affinity. The efficacy of such an affinity driven transfer system was demonstrated experimentally by the transfer of K(4)-S4(1-13)a from pre-loaded RBCs to bacteria, yeast and protozoan target cells.     

Secretion and membrane recycling in plant cells: novel intermediary structures visualized in ultrarapidly frozen sycamore and carrot suspension-culture cells

Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.) suspension-culture cells. Stabilization of the cells by means of ultrarapid freezing has enabled us to preserve the cells in a turgid state and to visualize new intermediate membrane configurations related to these events. Indeed, many of the observed membrane configurations, such as flattened membrane vesicles with slit-shaped membrane fusion sites and horseshoe-shaped membrane infoldings, appear to result from the action of turgor forces on the plasma membrane. Individual cells exhibited great variations in numbers and types of membrane configurations postulated to be related to secretion and membrane-recycling events. In the majority of cells, the different membrane profiles displayed a patchy distribution, and within each patch the membrane configurations tended to be of the same stage. This result indicates that secretory events are triggered in domains measuring from 0.1 to about 10 μm in diameter. Based on an extensive analysis of the different membrane configurations seen in our samples, we have formulated the following model of vesicle-mediated secretion in plant cells: Fusion of a secretory vesicle with the plasma membrane leads to the formation of a single, narrow-necked pore that increases in diameter up to about 60 nm. During discharge, the vesicle is flattened, forming a disc-shaped structure perpendicular to the plane of the plasma membrane. As the vesicle is flattened, the pore is converted to a slit, the maximum length of which coincides with the diameter of the flattened vesicle. The flattened vesicle then tips over and concomitantly the plasma-membrane slit becomes curved into a horseshoe-shaped configuration as it extends along the outer margins of the tipped-over vesicle. Some coated pits are present interspersed between the above-mentioned structures, but their numbers appear insufficient to account for an exclusively endocytotic mechanism of membrane recycling. Instead, our micrographs are more consistent with a mixed mode of recycling of membrane components to the cortical endoplamic reticulum and to Golgi cisternae that involves both internalization of membrane by endocytosis and of individual lippid molecules by unknown mechanisms (lipid exchange proteins?). To this end, overall flattening out of the horseshoe-shaped membrane infoldings is accompanied by a retraction and reduction in size of their central, tongue-like structure.     

Cross-talk and information transfer in mammalian and bacterial signaling

In mammalian and bacterial cells simple phosphorylation circuits play an important role in signaling. Bacteria have hundreds of two-component signaling systems that involve phosphotransfer between a receptor and a response regulator. In mammalian cells a similar pathway is the TGF-beta pathway, where extracellular TGF-beta ligands activate cell surface receptors that phosphorylate Smad proteins, which in turn activate many genes. In TGF-beta signaling the multiplicity of ligands begs the question as to whether cells can distinguish signals coming from different ligands, but transduced through a small set of Smads. Here we use information theory with stochastic simulations of networks to address this question. We find that when signals are transduced through only one Smad, the cell cannot distinguish between different levels of the external ligands. Increasing the number of Smads from one to two significantly improves information transmission as well as the ability to discriminate between ligands. Surprisingly, both total information transmitted and the capacity to discriminate between ligands are quite insensitive to high levels of cross-talk between the two Smads. Robustness against cross-talk requires that the average amplitude of the signals are large. We find that smaller systems, as exemplified by some two-component systems in bacteria, are significantly much less robust against cross-talk. For such system sizes phosphotransfer is also less robust against cross-talk than phosphorylation. This suggests that mammalian signal transduction can tolerate a high amount of cross-talk without degrading information content. This may have played a role in the evolution of new functionalities from small mutations in signaling pathways, allowed for the development of cross-regulation and led to increased overall robustness due to redundancy in signaling pathways. On the other hand the lack of cross-regulation observed in many bacterial two-component systems may partly be due to the loss of information content due to cross-talk.     

Transfer of electrons across the cytoplasmic membrane by DsbD, a membrane protein involved in thiol-disulphide exchange and protein folding in the bacterial periplasm

Reduction of non-native protein disulphides in the periplasm of Escherichia coli is catalysed by three enzymes, DsbC, DsbG and DsbE, each of which harbours a catalytic Cys-X-X-Cys dithiol motif. This dithiol motif requires continuous reduction for activity. Genetic evidence suggests that the source of periplasmic reducing power resides within the cytoplasm, provided by thioredoxin (trxA) and thioredoxin reductase (trxB). Cytoplasmic electrons donated by thioredoxin are thought to be transferred into the periplasm via the DsbD membrane protein. To understand the molecular nature of electron transfer, we have analysed the membrane topology of DsbD. DsbD is exported by an N-terminal signal peptide. The N- and C-terminal domains are positioned in the periplasmic space, connected by eight transmembrane segments. Electron transfer was shown to require five cysteine sulphydryl of DsbD. Trans complementation of mutant DsbD molecules revealed intermolecular electron transfer. We discuss a model whereby the membrane-embedded disulphides of DsbD accept electrons from cytoplasmic thioredoxin and transfer them to the C-terminal periplasmic dithiol motif of DsbD.     

植物抗细菌基因工程策略与应用

植物与病原物互作分子水平的研究进展极大地促进了转基因技术在植物保护方面的应用。人们提出了多种植物抗细菌基因工程策略 ,包括利用非植物来源的抗菌蛋白 (肽 )、抑制病菌的致病 (毒性 )因子、增强植物自身的抗性、诱使细胞程序死亡。综述了应用不同策略所取得的进展 ,并分析了抗细菌基因工程研究中存在的问题。     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin     

Intercellular communication: diverse structures for exchange of genetic information

An emerging concept is that cellular communication in mammals can be mediated by the exchange of genetic information, mainly in the form of microRNAs. This can occur when extracellular vesicles, such as exosomes, secreted by a donor cell are taken up by an acceptor cell. Transfer of genetic material can also occur through intimate membrane contacts between donor and acceptor cells. Specialized cell-cell contacts, such as synapses, have the potential to combine these modes of genetic transfer.     

Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells.

The 11 VirB proteins from Agrobacterium tumefaciens are predicted to form a membrane-bound complex that mediates the movement of DNA from the bacterium into plant cells. The studies reported here on the possible VirB protein interactions in such a complex demonstrate that VirB9 and VirB10 can each form high-molecular-weight complexes after treatment with a chemical cross-linker. Analysis of nonpolar virB mutants showed that the formation of the VirB10 complexes does not occur in a virB9 mutant and that VirB9 and VirB10 are not components of the same cross-linked complex. VirB9, when stabilized by the concurrent expression of VirB7, was shown to be sufficient to permit VirB10 to cross-link into its usual high-molecular-weight forms in the absence of other Vir proteins. Randomly introduced single point mutations in virB9 resulted in Agrobacterium strains with severely attenuated virulence. Although some of the mutants contained wild-type levels of VirB9 and displayed an unaltered VirB9 cross-linking pattern, VirB10 cross-linking was drastically reduced. We conclude that specific amino acid residues in VirB9 are necessary for interaction with VirB10 resulting in the capacity of VirB10 to participate in high-molecular-weight complexes that can be visualized by chemical cross-linking.     

New magnet-sensitive structures in bacterial and archaeal cells

The objects of the investigation were: distribution of intracellular magnet-sensitive structures among different taxonomic groups of prokaryotes, localisation and organisation of the magnet-sensitive inclusions (MsI) in cells. The MsI were discovered in representatives of both prokaryotic domains (Bacteria and Archaea), 2 kingdoms and 7 orders of bacteria. They were some amorphous or non-crystalline globules with the electron-transparent centre surrounded with an electron-dense homogenous matrix. The magnetic nature of the structures was shown by attraction with an applied magnet both for the cell suspensions and for the MsI isolated and separated from the destroyed cells. The MsI were studied with transparent electron microscopy and with X-ray analyses. When the cells were grown in the iron-containing nutrient medium, the matrix was enriched with iron. It was shown also that some bacteria grown with cobalt or with chromium contained the cobalt- or chromium-enriched magnetic inclusions.     

Hypotheses on the establishment of a genetic code and transfer of information from proteinoids to nucleic acids

An hypothesis is proposed in which the specificity of interaction between an aminoacid and a nucleotide sequence of a tRNA would be enhanced by a ternary association with a specific proteinoid. These strict relations would have led to the present genetic code that we know. It is also proposed that the origin of the enzymatic activity of the primitive proteinoids would have arisen from the presence of different substrates during polymerisation, which would have favored specific sequences of aminoacids by forming more stable complexes with them, corresponding to the lowest free enthalpy. The information included in the aminoacid sequences of the proteinoids would have been transferred to messenger type RNA, according to a mechanism reverse of that for the present process for protein synthesis, and then to DNA.     

The stoicheiometry of electron transfer by bacterial and plant ferredoxins

1. The number of electrons carried by ferredoxins from spinach, the blue-green alga Anacystis nidulans, the anaerobic bacterium Clostridium welchii and the photosynthetic bacterium Chromatium was determined. 2. Ferredoxins were reduced by illuminated chloroplasts, and the stoicheiometry of the reoxidation in the dark of the ferredoxins by NADP and benzyl viologen was measured. 3. Spinach and A. nidulans ferredoxins were found to be one-electron carriers, and Cl. welchii and Chromatium ferredoxins were two-electron carriers.     

The chromophore structures of the Pr States in plant and bacterial phytochromes

The resonance Raman spectra of the Pr state of the N-terminal 65-kDa fragment of plant phytochrome phyA have been measured and analyzed in terms of the configuration and conformation of the tetrapyrroles methine bridges. Spectra were obtained from phyA adducts reconstituted with the natural chromophore phytochromobilin as well as phycocyanobilin and its isotopomers labeled at the terminal methine bridges through (13)C/(12)C and D/H substitution. Upon comparing the resonance Raman spectra of the various phyA adducts, it was possible to identify the bands that originate from normal modes dominated by the stretching coordinates of the terminal methine bridges A-B and C-D. Quantum chemical calculations of the isolated tetrapyrroles reveal that these modes are sensitive indicators for the methine bridge configuration and conformation. For all phyA adducts, the experimental spectra of Pr including this marker band region are well reproduced by the calculated spectra obtained for the ZZZasa configuration. In contrast, there are substantial discrepancies between the experimental spectra and the spectra calculated for the ZZZssa configuration, which has been previously shown to be the chromophore geometry in the Pr state of the bacterial, biliverdin-binding phytochrome from Deinococcus radiodurans (Wagner, J. R., J. S. Brunzelle, K. T. Forest, R. D. Vierstra. 2005. Nature. 438:325-331). The results of this work, therefore, suggest that plant and bacterial (biliverdin-binding) phytochromes exhibit different structures in the parent state although the mechanism of the photoinduced reaction cycle may be quite similar.     

Maintenance of genetic information in stem cells

The available data on DNA cosegregation in some stem cells are reviewed. Cairns was the first to assume cosegregation of template DNA strands for adult stem cells; i.e., all maternal DNA strands are preserved in one daughter cell, which remains a stem cell, while the newly synthesized DNA strands, which may contain errors, appear in the daughter cell that is committed to differentiation and passes to the transitory compartment of the cell population. The role of asymmetric mitosis in DNA cosegregation and maintenance of genetic information in stem cells is discussed.