Investigation of structure and antigenic capacities of Thermococcales cell envelopes and reclassification of ``Caldococcus litoralis'' Z-1301 as Thermococcus litoralis Z-1301

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis. Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates

Tropomodulins and tropomyosins: working as a team

Actin filaments are major components of the cytoskeleton in eukaryotic cells and are involved in vital cellular functions such as cell motility and muscle contraction. Tropomyosin is an alpha-helical, coiled coil protein that covers the grooves of actin filaments and stabilizes them. Actin filament length is optimized by tropomodulin, which caps the slow growing (pointed end) of thin filaments to inhibit polymerization or depolymerization. Tropomodulin consists of two structurally distinct regions: the N-terminal and the C-terminal domains. The N-terminal domain contains two tropomyosin-binding sites and one tropomyosin-dependent actin-binding site, whereas the C-terminal domain contains a tropomyosin-independent actin-binding site. Tropomodulin binds to two tropomyosin molecules and at least one actin molecule during capping. The interaction of tropomodulin with tropomyosin is a key regulatory factor for actin filament organization. The binding efficacy of tropomodulin to tropomyosin is isoform-dependent. The affinities of tropomodulin/tropomyosin binding influence the proper localization and capping efficiency of tropomodulin at the pointed end of actin filaments in cells. Tropomodulin and tropomyosin are crucial constituents of the actin filament network, making their presence indispensable in living cells. Here we describe how a small difference in the sequence of the tropomyosin-binding sites of tropomodulin may result in dramatic change in localization of Tmod in muscle cells or morphology of non-muscle cells. We also suggest most promising directions to study and elucidate the role of Tmod-TM interaction in formation and maintenance of sarcomeric and cytoskeletal structure

Function of FlhB, a Membrane Protein Implicated in the Bacterial Flagellar Type III Secretion System

The membrane protein FlhB is a highly conserved component of the flagellar secretion system, and it plays an active role in the regulation of protein export. In this study conserved properties of FlhB that are important for its function were investigated. Replacing the flhB gene (or part of the gene) in Salmonella typhimurium with the flhB gene of the distantly related bacterium Aquifex aeolicus greatly reduces motility. However, motility can be restored to some extent by spontaneous mutations in the part of flhB gene coding for the cytoplasmic domain of Aquifex FlhB. Structural analysis suggests that these mutations destabilize the structure. The secondary structure and stability of the mutated cytoplasmic fragments of FlhB have been studied by circular dichroism spectroscopy. The results suggest that conformational flexibility could be important for FlhB function. An extragenic suppressor mutation in the fliS gene, which decreases the affinity of FliS to FliC, partially restores motility of the FlhB substitution mutants