Isolation and characterization of Zymomonas mobilis mutants resistant to octadecyltrimethylammonium chloride,a detergent acting on hopanoid-producing bacteria

Growth of the hopanoid-producing bacterium Zymomonas mobilis was inhibited at low concentrations of the cationic detergent octadecyltrimethylammoniumchloride (OTAC). A relationship between sensitivity of Zymomonas mobilis to OTAC, presence of hopanoids and ethanol tolerance was postulated. Mutants resistant to OTAC were isolated from strains ZM1 and ZM4. They did not present any alteration of the hopanoid content and their squalene cyclases showed the same sensitity to OTAC as the parent enzymes. Resistance to OTAC paralleled pleiotropic effects including, enhanced accessibility of the membrane-bound alkaline phosphatase, important release of proteins from cells by Tris/HCl treatment, increased resistance to antibiotics and increased sensitivity to ethanol. In addition, OTAC^R mutants were also characterized by the synthesis or the overproduction of an outer membrane protein (F53) not detected on 2D-PAGE maps of parent strains and by a normal heat shock response. The role of hopanoids, heat shock proteins, protein F53 and membrane organization in ethanol tolerance is discussed.Abbreviations OTAC octadecyltrimethylammoniumchloride - SLS sodium lauryl sarcosinate     

Morphological and histological changes of dermal scales during the fish‐to‐tetrapod transition

Witzmann F. (2011). Morphological and histological changes of dermal scales during the fish‐to‐tetrapod transition. —Acta Zoologica (Stockholm) 92 : 281–302. The gastral scales of limbed tetrapodomorphs evolved from the ‘elpistostegid’‐type of scale by an enlargement and differentiation of the articulation facets and a shortening and broadening of the keel. These changes caused a tighter connection between gastral scales within a scale row and a greater overlap between the rows. Dorsal round scales of limbed tetrapodomorphs developed from a gastral scale‐type by an alteration of the ontogenetic pathway. The posterolateral direction of scale rows in ‘elpistostegids’ was retained in the gastral scalation of most limbed tetrapodomorphs, whereas the arrangement of round dorsal scales is modified to a transverse orientation. Both gastral and dorsal scales of limbed tetrapodomorphs consist solely of parallel‐fibred bone with circumferential growth marks. The proportionally larger overlap surfaces of gastral scales and their mode of articulation in the ventral midline indicate that the body of limbed tetrapodomorphs might have been more flexible than that of their finned relatives. The alteration of dermal scales was one of the most rapid morphological changes during the fish‐to‐tetrapod transition. Once established, gastral and dorsal scales were retained as a conservative character in different lineages of basal tetrapods, in both the amphibian and the amniote lineages.     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

The role of the carboxy-terminal membrane-spanning fragment in the biogenesis of Escherichia coli K12 outer membrane protein PhoE

Summary PhoE protein of Escherichia coli K12 is an outer membrane protein which is supposed to span the membrane sixteen times. By creating a deletion which removes the last membrane-spanning fragment and studying the localization of the truncated PhoE, we show that this fragment is indispensable for trimerization and outer membrane localization. In addition, circumstantial evidence for the proposed topology model of the protein was obtained. An insertion mutation in a region supposed to be cell surface-exposed, interferes with the binding of a monoclonal antibody which recognizes a cell surface-exposed epitope of the protein.     

Probing FhuA''-''PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12

Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.     

The integumental ultrastructure of Lamippe rubra Bruzelius and Enalcyonium rubicundum Olsson (Copepoda,Poecilostomatoida)

The integument of Lamippe rubra Bruzelius and of Enalcyonium rubicundum Olsson has been studied with the electron microscope.Most of the cuticle covering the body of Lamippe is represented by the epicuticle, which shows an average thickness of about 2.0 µm, but in sclerified zones it consists of a thin epicuticle (0.2 µm) and a stratified laminated procuticle (0.5–1.5 µm) without bow-shaped structure. A complex system of epithelial microvilli or a well-developed system of membranes running parallel to the cuticle is also present.The cuticle of Enalcyonium consists of a thin procuticle (0.4–0.5 µm) covered with a uniform fibrillar coat (0.5 µm), whereas in sclerotized areas it is composed of a stratified procuticle (0.7–3.5 µm) with bow-shaped structures.In both species, cuticular hairs and gland vents occur at the dorsal and ventral surfaces. Some of the hairs are considered to be sensory in nature.The cuticular ultrastructure of L. rubra and of E. rubicundum is compared with that of some other copepods.     

Serological properties and processing in Escherichia coli K12 of OmpV fusion proteins of Vibrio cholerae

Summary Fusion proteins comprising the amino-terminal 99 amino acids of the bacteriophage MS2 replicase and various portions of OmpV a major outer membrane protein of Vibrio cholerae were expressed in Escherichia coli K12. These fusions were expressed under the control of the P_L promoter of bacteriophage , and expression was controlled using a cI_ts repressor. Fusions occurring within the secretory signal sequence of OmpV gave rise to the production of mature OmpV. The efficiency, however, decreased with progressive deletion of the signal sequence within the fusions. The reactivity of various OmpV fusions with antisera raised against purified OmpV and whole bacteria demonstrated the existence of two antigenic domains: one present in the denatured form and another in the membrane-associated form of OmpV. These domains correspond to markedly hydrophilic regions of the protein as would be predicted for surface-exposed epitopes.     

A new cell wall structure in a symbiotic and a free-living strain of the blue-green alga genus Chroococcidiopsis (Pleurocapsales)

Freeze etching studies in a symbiotic and a freeliving strain of Chroococcidiopsis revealed a specific layer in the outer cell wall not described so far from Cyanophyta. The layer showed a complex organisation: The main unit are ribbons, 2–3 nm thick, striated at right angle to the longitudinal axis. They are interwoven to a patchwork-like leaflet. The ribbons are virtually composed of globular particles associated in parallel rows. The cytoplasmic membrane and the cell walls of the symbiotic and the free-living strain were compared.Abbreviations cm cytoplasmic membrane - CW 1,2,3 cell wall layer 1,2,3 - EF exoplasmic fracture face - PF protoplasmic fracture face