Complementary Methodologies To Identify Specific Agrobacterium Strains

Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field.     

Probe diffusion in aqueous poly(vinyl alcohol) solutions studied by fluorescence correlation spectroscopy

We report fluorescence correlation spectroscopy measurements of the translational diffusion coefficient of various probe particles in dilute and semidilute aqueous poly(vinyl alcohol) solutions. The range of sizes of the particles (fluorescent molecules, proteins, and polymers) was chosen to explore various length scales of the polymer solutions as defined by the polymer-polymer correlation length. For particles larger than the correlation length, we find that the diffusion coefficient, D, decreases exponentially with the polymer concentration. This can be explained by an exponential increase in the solution viscosity, consistent with the Stokes-Einstein equation. For probes on the order of the correlation length, the decrease of the diffusion coefficient cannot be accounted for by the Stokes-Einstein equation, but can be fit by a stretched exponential, D approximately exp(-alphacn), where we find n = 0.73-0.84 and alpha is related to the probe size. These results are in accord with a diffusion model of Langevin and Rondelez (Polymer 1978, 19, 1875), where these values of n indicate a good solvent quality.     

Conformation of a clathrin triskelion in solution

A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triskelion) that assembles into polyhedral cages principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends upon the pH, with cages forming more readily at low pH and less readily at high pH. We have developed procedures, on the basis of static and dynamic light scattering, to determine the radius of gyration, R(g), and hydrodynamic radius, R(H), of isolated triskelia, under conditions where cage assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending the legs and a puckering at the vertex. We also show that the values of R(g) and R(H) measured for clathrin triskelia in solution are qualitatively consistent with the conformation of a triskelion in a "D6 barrel" cage assembly measured by cryoelectron microscopy.     

Isolation and characterization of halophilic Archaea able to produce biosurfactants

Halotolerant microorganisms able to live in saline environments offer a multitude of actual or potential applications in various fields of biotechnology. This is why some strains of Halobacteria from an Algerian culture collection were screened for biosurfactant production in a standard medium using the qualitative drop-collapse test and emulsification activity assay. Five of the Halobacteria strains reduced the growth medium surface tension below 40 mN m⁻¹, and two of them exhibited high emulsion-stabilizing capacity. Diesel oil-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 35% sodium chloride or up to 25% ethanol in the aqueous phase. Emulsions were stable to three cycles of freezing and thawing. The components of the biosurfactant were determined; it contained sugar, protein and lipid. The two Halobacteria strains with enhanced biosurfactant producers, designated strain A21 and strain D21, were selected to identify by phenotypic, biochemical characteristics and by partial 16S rRNA gene sequencing. The strains have Mg²⁺, and salt growth requirements are always above 15% (w/v) salts with an optimal concentration of 15–25%. Analyses of partial 16S rRNA gene sequences of the two strains suggested that they were halophiles belonging to genera of the family Halobacteriaceae, Halovivax (strain A21) and Haloarcula (strain D21). To our knowledge, this is the first report of biosurfactant production at such a high salt concentration.     

Bax Activates Endophilin B1 Oligomerization and Lipid Membrane Vesiculation

Endophilins participate in membrane scission events that occur during endocytosis and intracellular organelle biogenesis through the combined activity of an N-terminal BAR domain that interacts with membranes and a C-terminal SH3 domain that mediates protein binding. Endophilin B1 (Endo B1) was identified to bind Bax, a Bcl-2 family member that promotes apoptosis, through yeast two-hybrid protein screens. Although Endo B1 does not bind Bax in healthy cells, during apoptosis, Endo B1 interacts transiently with Bax and promotes cytochrome c release from mitochondria. To explore the molecular mechanism of action of Endo B1, we have analyzed its interaction with Bax in cell-free systems. Purified recombinant Endo B1 in solution displays a Stokes radius indicating a tetrameric quarternary structure. However, when incubated with purified Bax, it assembles into oligomers more than 4-fold greater in molecular weight. Although Endo B1 oligomerization is induced by Bax, Bax does not stably associate with the high molecular weight Endo B1 complex. Endo B1 oligomerization requires its C-terminal Src homology 3 domain and is not induced by Bcl-xL. Endo B1 combined with Bax reduces the size and changes the morphology of giant unilamellar vesicles by inducing massive vesiculation of liposomes. This activity of purified Bax protein to induce cell-free assembly of Endo B1 may reflect its activity in cells that regulates apoptosis and/or mitochondrial fusion.     

Stability of drug-induced tubulin rings by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) was applied to investigate the stability of tubulin rings that result from the interaction of alpha beta-tubulin dimers with three vinca domain-binding peptides--cryptophycin 1, hemiasterlin, and dolastatin 10. These peptides inhibit tubulin polymerization into microtubules and, instead, induce the formation of single-walled tubulin rings of 23.8 nm mean diameter for cryptophycin and 44.6 nm mean diameter for hemiasterlin and dolastatin, as revealed by electron microscopy on micromolar drug-tubulin samples. However, the hydrodynamic diameter and the apparent number of fluorescent particles, determined from analysis of FCS measurements obtained from nanomolar drug-tubulin samples, indicate variation in the stability of the rings depending on the drug and the tubulin concentration. Cryptophycin-tubulin rings appear to be the most stable even with tubulin concentration as low as 1 nM, whereas hemiasterlin-tubulin rings are the least, depolymerizing even at relatively high concentrations (100 nM). In contrast, the dolastatin-tubulin rings demonstrate an intermediate level of stability, depolymerizing significantly only at tubulin concentrations below 10 nM. We also compare the stability results with those of cytotoxicity measurements taken on several cell lines and note a rough correlation between the cytotoxicity of the drugs in cell cultures and the stability of the corresponding drug-induced rings.     

Biodegradation of diesel oil and production of fatty acid esters by a newly isolated Pseudomonas citronellolis KHA

The ability of a newly isolated Pseudomonas citronellolis KHA to degrade diesel oil and to synthesize fatty acid esters has been screened in aerobic batch cultures. The microorganism was able to grow with diesel oil at initial concentrations up to 126 g/l, with optimal growth at 25 g/l. Strain KHA has produced compounds showing strong emulsifying properties (E₂₄ = 75% at the end of the exponential growth phase). The crude extract reduces the surface tension of water from 72 mN m⁻¹ down to 35 mN m⁻¹ with a corresponding minimal concentration value of 60 mg/l. GC and GC–MS analysis of crude product show that the major components are those of hexadecanoic acid propyl ester and octadecanoic acid propyl ester, which have potential for applications in cosmetics, pharmaceutical and foods industries. In addition, strain KHA represents a valuable source of compounds with surface-active properties and potential for the application in clean up of the sites contaminated with hydrocarbons.     

Histone deacetylase 8 is required for centrosome cohesion and influenza A virus entry

Influenza A virus (IAV) enters host cells by endocytosis followed by acid-activated penetration from late endosomes (LEs). Using siRNA silencing, we found that histone deacetylase 8 (HDAC8), a cytoplasmic enzyme, efficiently promoted productive entry of IAV into tissue culture cells, whereas HDAC1 suppressed it. HDAC8 enhanced endocytosis, acidification, and penetration of the incoming virus. In contrast, HDAC1 inhibited acidification and penetration. The effects were connected with dramatic alterations in the organization of the microtubule system, and, as a consequence, a change in the behavior of LEs and lysosomes (LYs). Depletion of HDAC8 caused loss of centrosome-associated microtubules and loss of directed centripetal movement of LEs, dispersing LE/LYs to the cell periphery. For HDAC1, the picture was the opposite. To explain these changes, centrosome cohesion emerged as the critical factor. Depletion of HDAC8 caused centrosome splitting, which could also be induced by depleting a centriole-linker protein, rootletin. In both cases, IAV infection was inhibited. HDAC1 depletion reduced the splitting of centrosomes, and enhanced infection. The longer the distance between centrosomes, the lower the level of infection. HDAC8 depletion was also found to inhibit infection of Uukuniemi virus (a bunyavirus) suggesting common requirements among late penetrating enveloped viruses. The results established class I HDACs as powerful regulators of microtubule organization, centrosome function, endosome maturation, and infection by IAV and other late penetrating viruses.     

Single-walled tubulin ring polymers

An unusual class of nanoscopic, ring-shaped, single-walled biopolymers arises when alphabeta-tubulin is mixed with certain small peptides obtained from various marine organisms and cyanobacteria. The single-ring structures, whose mean molecular weight depends on the specific peptide added to the reaction mixture, usually have sharp mass distributions corresponding, e.g., to rings containing eight tubulin dimers (when the added peptide is cryptophycin) and 14 dimers (e.g., with dolastatin). Although the ring-forming peptides have been shown to possess antimitotic properties when tested with cultured eukaryotic cells (and thus have generated considerable interest as possible agents to be used in the treatment of cancer), it is not our intention to extensively discuss the potential pharmacological properties of the peptides. Rather, we will review the polymeric structures that form and illustrate how certain physical techniques can be used to characterize their properties and interactions. The nanoscopic size and particular geometry of the individual rings make them appropriate targets for scattering and hydrodynamic techniques that provide details about their structure in solution, but it is necessary to relate measured data to postulated structures by nontrivial, albeit straight-forward, mathematical, and computational means. We will discuss how this is done when one uses such methods as small angle neutron scattering, dynamic light scattering, fluorescence correlation spectroscopy, and sedimentation velocity measurements. Moreover, we show that, by using several techniques, one can eliminate degeneracy to provide better discrimination between model structures.