Cell wall of an extremely halophilic coccus

Summary Amino acid and amino sugar analyses have been carried out on hydrolyzed wall preparations from an extremely halophilic coccus. A limited number of ninhydrin-positive compounds were found. The amino acids glycine and glutamic acid or glutamine were present, as well as the amino sugars glucosamine and galactosamine. In addition, three unidentified compounds were detected. Other peptidoglycan components, as found in non-halophilic Gram-positive cocci, were absent.     

Morphological and structural aspects of the extremely halophilic archaeon Haloquadratum walsbyi

Ultrathin square cell Haloquadratum walsbyi from the Archaea domain are the most abundant microorganisms in the hypersaline water of coastal salterns and continental salt lakes. In this work, we explore the cell surface of these microorganisms using amplitude-modulation atomic-force microscopy in nearly physiological conditions. We demonstrate the presence of a regular corrugation with a periodicity of 16-20 nm attributed to the surface layer (S-layer) protein lattice, striped domains asymmetrically distributed on the cell faces and peculiar bulges correlated with the presence of intracellular granules. Besides, subsequent images of cell evolution during the drying process indicate the presence of an external capsule that might correspond to the giant protein halomucin, predicted by the genome but never before observed by other microscopy studies.     

Biodegradation of hydrocarbons by an extremely halophilic archaebacterium

An archaebacterium (strain EH4) able to biodegrade saturated and aromatic hydrocarbons has been isolated from a sail-marsh. Maximum growth on eicosane (62% of biodegradation, 10 h generation time) was reached in a medium prepared with a natural hypersaline water collected from a salt-marsh (3.5 mol/1 NaCl concentration). No growth on hydrocarbons was observed for NaCl concentration lower than 1.8 mol/1.     

Cryo-electron microscopy as an investigative tool: the ribosome as an example

Cryo-electron microscopy allows the visualization of macromolecules in their native state. Combined with techniques of three-dimensional reconstruction, cryo-EM images of single molecules can be used to study macromolecular interactions. The ribosome, a large RNA-protein complex with multiple binding interactions, is an excellent test case illustrating the power of these new techniques. Conformational changes during the binding of tRNA and protein factors to the ribosome can now be studied without the interference of crystal packing. Now that the first X-ray structures of ribosomal subunits have become available, conformational changes observed by cryo-EM in different functional states can be traced back to internal rearrangements of the underlying structural framework. Electron microscopy, X-ray crystallography, and modeling should be used together in the endeavor to understand the functioning of the translational machinery.     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

Cryo-electron microscopy of GDP-tubulin rings

Rings of guanosine diphosphate (GDP)-tubulin formed in the presence of divalent cations have been studied using conventional negative stain and cryo-electron microscopy. The structure of such rings resembles that of depolymerizing microtubule ends and corresponds to an “unconstrained” conformation of tubulin in its GDP state. The use of cryo-techniques has allowed us to image the ring polymers free from dehydration and flattening artifacts. Preparations of frozenhydrated GDP-tubulin rings are generally heterogeneous and contain a mixture of double, triple, and incomplete rings, as well as spirals and some rare single rings. Images of different polymer types can be identified and classified into groups that are then amenable for averaging and single particle reconstruction methods. Identifying the differences in tubulin structure, between straight and curve protofilaments, will be important to understand the molecular bases of dynamic instability in microtubules.     

冷冻透射电镜:从分子到系统

单颗粒电镜结合其他方法能够在(近)原子水平提供结构模型,已经成为一种研究大蛋白复合物的有效方法。该文将以两个大的蛋白裂解复合物——tripeptidyl peptidaseII(6MDa)和26S蛋白酶体(2.5MDa)举例说明。低温电子层析能进行非重复的超分子结构分析,如多核糖体和全细胞;能够为超分子组织提供前所未有的信息(可视化蛋白质组学)。     

Lack of detectable polyamines in an extremely halophilic bacterium

Polyamines (putrescine, spermidine, spermine and other analogs) were not detectable by the dansylation procedure coupled with HPLC analysis in an extremely halophilic bacterium, Halobacterium halobium. Based on the detection limit of this analytical method, we estimated that the polyamine content in H. halobium, if present, was less than 0.06% of that of E. coli. Putrescine uptake and the metabolic conversion of ornithine or arginine to polyamines were negligible in this bacterium. In a H. halobium cell-free extract, a saturated amount of KC1 was needed for poly(U) directed polyphenylalanine synthesis; neither putrescine nor spermidine could replace KC1. These results suggest that polyamines may play an insignificant role in the growth of this halophilic bacterium.     

Diversity of extremely halophilic bacteria

In this review, the history of the classification of the family Halobacteriaceae, the extremely halophilic aerobic Archaea, is reviewed with some emphasis on the recently described new genera Halobaculum, Halorubrum, Natrialba, Natronomonas, and "Haloterrigena." Speculation is made about the evolutionary relationship between members of the Halobacteriaceae and the extremely halophilic, anaerobic methanogens of the genera Methanohalobium and Methanohalophilus. Efforts to find missing links between the two groups are also reviewed. Received: January 22, 1998 / Accepted: February 16, 1998     

Cryo-electron microscopy of the giant Mimivirus

Mimivirus is the largest known virus. Using cryo-electron microscopy, the virus was shown to be icosahedral, covered by long fibers, and appears to have at least two lipid membranes within its protein capsid. A unique vertex, presumably for attachment and infection of the host, can be seen for particles that have a suitable orientation on the micrographs.     

Cryo-electron microscopy of artificial biological membranes

Vitrified synthetic phosphatidycholine liposome suspensions were studied by cryo-electron microscopy. The bilayer structure is resolved on vitrified liposome images. The packing of the aliphatic chains of the lipid within vitrified liposomes can be determined by the analysis of electron diffraction patterns. Images and electron diffraction patterns show that the structure of vitrified liposomes is related to the structure that liposomes have before vitrification. In fact, vitrified liposomes have a different structure, depending whether they are maintained before cooling at a temperature higher or lower than that corresponding to the ‘melting’ of the hydrocarbon chain of the lipids. Below the melting temperature, liposomes are formed by small domains.     

Cell walls from Actinopolyspora halophila,an extremely halophilic actinomycete

Cell wall components were prepared from Actinopolyspora halophila (strain wt), an extremely halophilic actinomycete requiring a minimum 12% NaCl concentration for growth, and from an erythromycin-resistant strain of A. halophila (strain ER) that required only 6% NaCl for growth. Both cell wall preparations contained glutamic acid, alanine, and diaminopimelic acid in a 1:2:1 molar ratio. On the basis of muramic acid content, peptidoglycans from the wt and ER strains contained 255 and 245 disaccharide units per mg dry weight respectively. In addition, both cell wall preparations contained from 10 to 20% more glucosamine than muramic acid, and equimolar amounts of d-galactose and d-arabinose. Analysis of cell walls before and after digestion with Myxobacter AL-1 protease indicated that nearly all glycan disaccharide units were peptide-substituted and that peptide cross-bridging was facilitated by direct peptide linkages between N-diaminopimelic acid and C-terminal alanine. While the peptidoglycan of A. halophila wt was 50% peptide cross-linked, that from A. halophila ER was approximately 67% peptide cross-linked. Chemical modifications involving substitution of non-N-acetylated hexosamines of the cell walls greatly enhanced their sensitivity to lysozyme. Although differences in peptidoglycan structure between the two strains of A. halophila were observed, these probably do not account for the reduced salt requirement for growth of the erythromycin-resistant strain.Issued as NRCC 25165     

Cryo-electron microscopy of myelin treated with detergents

After detergent extraction, myelin lamellae disaggregate into diverse globular fragments that correspond to the globular structures observed in normal cryofixed myelin. The interaction of these structural units in the bilayer is probably the morphological basis that provides the stability of the myelin lamellae and their lamination.     

Cryo-electron microscopy reconstruction of a poliovirus-receptor-membrane complex

To study non-enveloped virus cell entry, a versatile in vitro model system was developed in which liposomes containing nickel-chelating lipids were decorated with His-tagged poliovirus receptors and bound to virus. This system provides an exciting opportunity for structural characterization of the early steps in cell entry in the context of a membrane. Here we report the three-dimensional structure of a poliovirus-receptor-membrane complex solved by cryo-electron microscopy (cryo-EM) at a resolution of 32 A. Methods were developed to establish the symmetry of the complex objectively. This reconstruction demonstrates that receptor binding brings a viral five-fold axis close to the membrane. Density is clearly defined for the icosahedral virus, for receptors (including known glycosylation sites) and for the membrane bilayer. Apparent perturbations of the bilayer close to the viral five-fold axis may function in subsequent steps of cell entry.