Functional Analysis of the Cytoskeleton Protein MreB from Chlamydophila pneumoniae

In rod-shaped bacteria, the bacterial actin ortholog MreB is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homologue FtsZ is known to tether incorporation of cell wall building blocks at the developing septum. For intracellular bacteria, there is no need to compensate osmotic pressure by means of a cell wall, and peptidoglycan has not been reliably detected in Chlamydiaceae. Surprisingly, a nearly complete pathway for the biosynthesis of the cell wall building block lipid II has been found in the genomes of Chlamydiaceae. In a previous study, we discussed the hypothesis that conservation of lipid II biosynthesis in cell wall-lacking bacteria may reflect the intimate molecular linkage of cell wall biosynthesis and cell division and thus an essential role of the precursor in cell division. Here, we investigate why spherical-shaped chlamydiae harbor MreB which is almost exclusively found in elongated bacteria (i.e. rods, vibrios, spirilla) whereas they lack the otherwise essential division protein FtsZ. We demonstrate that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from Bacillus subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from Chlamydophila (Chlamydia) pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. Our findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining a functional divisome machinery in Chlamydiaceae

A Laser Interferometric Method for Small- and Finite-Amplitude Ultrasonic Waves’ Detection in Transparent Media

The acousto-optic interaction affords a convenient way of optically probing ultrasonic waves in medical diagnosis and nondestructive evaluation. The effects of ultrasonic waves on the light transmitting through transparent media arise from the refractive index variations produced by ultrasonic waves. The index variations may be detected by optical deflection, diffraction or interference methods [1–4]. In Raman-Nath regime, the acoustic waves act as a moving phase grating and diffract the light into different orders. Schlieren visualisation derived from this mechanism has been extensively used to ultrasonic measurements in liquids. In solid media, the acousto-optic effects become more complicated because of the induced optical birefringence. The usual photoelastic method consists in detecting the change in the polarization state of the light caused by ultrasonic waves [5]. Both of the methods are only amplitude-sensitive to ultrasonic waves.</p

Efficient Algorithms for Incremental Updates of Frequent Sequences

Surface acoustic waves propagating at the interface between two solids are very important for Non Destructive Evaluation of layered media [1]. Dispersion features and transmission losses of such waves strongly depend upon the elastic constants of the solids as well as the mechanical boundary conditions at the interface between the solids. One particular kind of interface waves is the Stoneley wave, which can exist at the interface between certain specifically combined elastic half-spaces in rigid contact [2]. The acoustic field of this wave, travelling with no loss along the interface, decays away from the interface in each medium (Fig. 1a). Murty studied interface waves in another extreme case, i.e. slip contact [3]. For such type of contact, only the normal stress and displacement components are continuous but shear stresses are cancelled at the interface. In the slip contact case, Murty’s study revealed also that the conditions of existence of the Stoneley-like wave are much less rigorous. When material combinations and/or boundary conditions are not satisfied to support the Stoneley-like wave, interface waves may become lossy [3] and radiate acoustic energy through mode conversion into bulk waves (Fig. 1b), like the case of the leaky Rayleigh wave propagating at a solid-liquid interface. Besides, if the presence of a coupling layer between the two half-spaces is taken into account as in most practical conditions, the features of interface waves depends also on the viscoelastic parameters and thickness of the coupling layer[4,5]. Up till now, most of the investigations involving interface waves are indirect ones using ultrasonic reflection and transmission measurements [6], and some others are related to dispersion measurements using interdigital or wedge transducers [4,5]. Few experiments were reported illustrating the field distribution of interface waves. Claus and Palmer observed the Stoneley wave [7] using an optical interferometer. The wave displacements were only detected by the probe beam focused at the Nickel-Pyrex interface

Identification of cryptic sites of DNA sequence amplification in human breast cancer by chromosome microdissection

We have performed microdissection of 16 putative homogeneously staining regions (hsrs) from nine different breast cancer cell lines in order to determine their chromosomal origin and composition. As expected, the most commonly amplified chromosomal band-region was 17q12 (containing ERBB2). However, regions not containing known oncogenes were also identified, including 13q31 (5/9 cases) and 20q12-13.2 (4/9 cases). The chromosomal composition of the integrated amplified DNA within each hsr was determined and in 13/16 cases (81%), hsrs were shown to be composed of two or more chromosomal regions. These studies shed light on the mechanism of formation of hsrs, and identify chromosomal regions likely to harbour genes amplified in breast cancer.link_to_subscribed_fulltex

Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan

International audienceThe maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance to date no structure of a protein in complex with an intact bacterial peptidoglycan has been re-solved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spec-troscopy to derive for the first time an atomic model of an L,D-transpeptidase from Bacillussubtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains – the catalytic domain as well as the proposed peptidoglycan recognition domain – are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the impli-cation of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidogly -can complex paves the way for the design of new antibiotic drugs targeting L,D-transpeptidases. The strategy devel-oped here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis

SEDS proteins are a widespread family of bacterial cell wall polymerases

Summary Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan synthetic machinery called the Rod complex. We report that in Bacillus subtilis this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS family of proteins that play essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a new family of peptidoglycan polymerases. Thus, B. subtilis and likely most bacteria use two distinct classes of polymerases to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development