Taxono-genomics description of 'Lactobacillus raoultii sp. nov.', strain Marseille-P4006T, a new Lactobacillus species isolated from the female genital tract of a patient with bacterial vaginosis.

Strain Marseille-P4006T, a Gram-stain-positive, rod-shaped, non-sporulating, facultatively anaerobic bacterium, was isolated from the vaginal swab of a 45-year-old woman with recurrent bacterial vaginosis. We studied its phenotypic characteristics and sequenced its whole genome. The major fatty acids were C16:0 (48%), C19:1n9 (14%) and C18:0 (11%). The 3 070 142-bp-long genome contains 2855 protein-coding genes and 68 RNAs. Strain Marseille-P4006T exhibited 98.1% 16S rRNA similarity with Lactobacillus farraginis, the closest species phylogenetically. Thus, strain Marseille-P4006 is distinct enough to represent a new species for which we propose the name Lactobacillus raoultii sp. nov. The type strain is Marseille-P4006T.

Shape of adsorbed supercoiled plasmids: an equilibrium description.

Inspired by recent atomic force microscope (AFM) images of plasmids deposited on oppositely charged supported lipid bilayers from salt free solution, we propose a model for strongly adsorbed supercoiled cyclic stiff polyelectrolytes. We discuss how the excess linking number Lk of the deposited cycle is shared between writhe Wr and twist Tw at equilibrium and obtain the typical number of self-crossings in the deposited cycle as a function of surface charge density. The number of crossings at equilibrium is simply determined by the crossing penalty which is a local quantity and by the excess linking number. The number of crossings is well defined despite versatile plasmid shapes. For moderate numbers of crossings the loops are rather small and localized along the primary cycle, as expected from entropic loops. In the regime of many crossings, the cycle takes the shape of a regular flat ply ruled by local stiffness. The model allows for a semiquantitative comparison with the AFM images of deposited plasmids which are strongly charged.

Secondary structure of double-stranded DNA under stretching: elucidation of the stretched form.

Almost two decades ago, measurements of force versus extension on isolated double-stranded DNA molecules revealed a force plateau. This unusual stretching phenomenon in DNA suggests that the long molecules may be extended from the usual B form into a new conformation. Different models have been proposed to describe the nature of DNA in its stretched form, S-DNA. Using atomic force microscopy combined with a molecular combing method, we identified the structure of λ-phage DNA for different stretching values. We provide strong evidence for the existence of a first-order transition between B form and S form. Beyond a certain extension of the natural length, DNA molecules adopt a new double-helix conformation characterized by a diameter of 1.2 nm and a helical pitch of 18 nm.

Specific molecular interactions by force spectroscopy: from single bonds to collective properties.

Interactions involving multiple bonds occur throughout biology, and have distinct properties that are fundamentally different from those present in single bond systems. We have developed a new method to analyse the AFM force measurements in order to extract relevant information and to characterise the interactions involving from single to multiple bonds. Our study reveals a surprising behaviour in the presence of multiple bonds with a high rebinding probability: the mean binding forces increase with decreasing pulling velocity. Such behaviour is different from the force dependence on the loading rate for single bond rupture or existing models for multiple bonds rupture.

New tools for force spectroscopy.

Force curves using atomic force microscopy have been proposed as a new tool to probe the conformation of adsorbed polymers and interactions between molecules through the analysis of the rupture distributions. We describe an algorithm for the computer-assisted detection of ruptures in force curves. This program allows us to automatically detect the ruptures. The algorithm is based on an analysis of the standard deviation in a sliding window along the length of the force. Automatic detection of ruptures constitutes an important step forward because the time required to analyse the curves is significantly reduced, thus allowing the user to perform multiple experiments. In addition, using these tools we can address the problem of the interpretation of rupture distributions.

Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy.

We report the study of the dynamics of the unbinding process under a force load f of adsorbed proteins (fibrinogen) on a solid surface (hydrophilic silica) by means of atomic force microscopy spectroscopy. By varying the loading rate r(f), defined by f = r(f) t, t being the time, we find that, as for specific interactions, the mean rupture force increases with r(f). This unbinding process is analyzed in the framework of the widely used Bell model. The typical dissociation rate at zero force entering in the model lies between 0. 02 and 0.6 s(-1). Each measured rupture is characterized by a force f(0), which appears to be quantized in integer multiples of 180-200 pN.

Direct observation of the anchoring process during the adsorption of fibrinogen on a solid surface by force-spectroscopy mode atomic force microscopy.

Atomic force microscopy in a force-spectroscopy mode has been used to investigate the kinetics of the adsorption process of fibrinogen molecules on a silica surface. An original "approach/retraction" cycle of the tip/surface was used for this purpose. Fibrinogen molecules were adsorbed on the atomic force microscopy tip and were brought into contact with the silica surface for different interaction times varying from 5 to 2,000 ms. Multiple consecutive ruptures were observed. The mean number of ruptures nr per cycle increases steadily with the interaction time as well as the mean strength fr which varies from 300 pN for 5 ms to 1,400 pN for 2,000 ms. The minimal interaction time for a fibrinogen molecule to bind strongly to a silica surface during an adsorption process appears to lie between 50 and 200 ms. The histograms of the distances between two consecutive ruptures in one cycle exhibit maxima around 20-25 nm. This length is comparable to the characteristic distance between D and E globules of one fibrinogen molecule and suggests that fibrinogen molecules mainly adsorb through their D and E globules.

Agarose gel structure using atomic force microscopy: gel concentration and ionic strength effects.

Agarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the "unperturbed" gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogeneous with decreasing Tris-borate-EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.

TEM moiré patterns explain STM images of bacteriophage T5 tails.

A subtle combination of constant current and constant height modes in scanning tunnelling microscopy allowed the imaging of a non-flat uncoated biological specimen, namely the tail of the bacteriophage T5. In parallel, a reference three-dimensional structure of the T5 tail was calculated from cryo-transmission electron microscopy images, based on its helical symmetry. This three dimensional reconstruction was compared with scanning tunnelling microscopy data. The images of the tail obtained by transmission electron microscopy, as well as projections of the reconstructed model, show similar moiré patterns. Here we show that scanning tunnelling microscopy performed in an aqueous environment provides direct images which are remarkably similar to the projection of the three dimensional model obtained by transmission electron microscopy. We deduce that our scanning tunnelling microscopy images are the result of a transmission of electrons through the gap between the scanning tip and the conductive support across the biological specimen.

Pore size of agarose gels by atomic force microscopy.

The pore size of agarose gel in water at different concentrations was directly measured using atomic force microscopy (AFM). The experiment was specially designed to work under aqueous conditions and allows direct observation of the "unperturbed" gel without invasive treatment. The pore size a as a function of gel concentration C shows a power law dependence a approximately C-gamma, where gamma lies between the prediction of the Ogston model for a random array of straight chains, 0.5, and the value predicted by De Gennes for a network of flexible chains, 0.75. We confirm that gels present a wide pore size distribution and show that it narrows as the concentration increases.

Approaching microtubule structure with the scanning tunneling microscope (STM).

We demonstrate that the scanning tunneling microscope can be used to obtain information about arrangement of tubulin subunits in the microtubule wall. Long rows of subunits with a periodicity of 3.8 +/- 0.4 nm were clearly visible in the images of microtubules. The separation between the rows of subunits was 4.8 +/- 0.4 nm. Close inspection of two images revealed another periodicity of 7.8 +/- 0.4 nm in the contour levels of the protofilaments. This indicates that alpha and beta tubulin monomers can be resolved. In these areas the monomers were arranged according to a 'B-type' lattice. Scanning tunneling microscope images confirm that the lateral contacts between tubulin monomers in adjacent protofilaments are compatible with a three-start, left-handed helix model. This study demonstrates that scanning tunneling microscopy can give direct information on the structure and organization of macromolecular assemblies and can complement the classical methods of electron microscopy and X-ray scattering.