Analysis of the cell wall and lipopolysaccharide of Spirillum serpens.

Isolated walls of Spirillum serpens VHA contained lipid, lipopolysaccharide, and protein in amounts similar to those of other gram-negative organisms. The loosely bound lipids consisted mainly of phosphatidylethanolamine, lyso-phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. Lipopolysaccharide was tightly bound to the wall and could only be removed in a substantial amount after digestion of the wall with Pronase. The lipopolysaccharide contained L-glycero-D-mannoheptose, rhamnose, glucosamine, ethanolamine, and phosphate in common with many of the lipopolysaccharides isolated from the Enterobacteriaceae. However, 2-keto-3-deoxyoctonic acid was not detected. Several unidentified sugars were present. The fatty acid composition resembled that found in lipopolysaccharides isolated from various pseudomonads. Two major regions were identified in the polysaccharide moiety, one apparently corresponding to the core polysaccharide and the other corresponding to the side-chain polysaccharide as in enterobacterial and pseudomonad lipopolysaccharides. The side chains were obtained as low-molecular-weight material and their structure was partially elucidated by the isolation and partial characterization of N-acetylglucosaminyl-(1 leads to 4)-rhamnose.     

Outer cell envelope membrane and shape of Spirillum serpens.

“Ghosts” have been isolated from Spirillum serpens that are free of murein, are surrounded by a unit membrane (derived from the outer membrane of the cell envelope), have lost all intracellular material (except for some poly-β-hydroxybutyrate), and still maintain Spirillum's shape.The ghost membrane contains about 50% protein which is resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into three bands corresponding to apparent molecular weights between 21,000 and 40,000, and the major protein band I (40,000) consists of at least two (Ia and Ib) but not many more polypeptide chains. HP-layer protein (hexagonally packed surface protein) is absent. At least one of the latter polypeptide chains is required for the establishment of the long-range order apparent in ghosts since proteases degrade band I proteins and concomitantly destroy the ghost. The other polypeptides (II and III) do not appear to be required for maintenance of shape of the Spirillum ghost since their amounts can vary widely from preparation to preparation. Ghosts as well as cells can be cross-linked with dimethyl diimidoesters. Such ghosts proved to be cross-linked over their entire surface, and a covalently closed macromolecule of the size of the cell had been created. Under certain conditions of cross-linking these ghosts upon extraction with hot sodium dodecyl sulphate were pure protein. Ammonolysis of this material liberated band I protein.These findings strongly suggest that there is a rather dense packing of the protein in the ghost membrane, and proteins Ia and Ib may be arranged as repeating subunits in the sense that protein-protein interaction exists along the whole membrane. Several observations also suggest that the ghost membrane concerning the arrangement of these proteins does not represent a gross artifact regarding the outer cell envelope membrane. The possibility exists that the assembly of polypeptides Ia and Ib participates in the determination of cellular shape.     

Substructure and in vitro assembly of the outer, structured layer of Spirillum serpens.

Electron micrographs of disintegrating units of the outer, structured (HP) layer of Spirillum serpens and of the isolated protein obtained from the HP layer revealed V- and Y-shaped and linear profiles. Interpretation of these forms, influenced by the seemingly trimeric form of the isolated protein and by biochemical data, suggested that the protein subunits were identical and Y shaped. A model is proposed for the assembly of the Y-shaped subunits to form a hexagon composed of two triads (three Y-shaped subunits each). The isolated protein adsorbed to a template of wall fragments (basal layer) to the same degree (over 90%) in high concentrations of Na+, K+ (5 X 10(-2) M), Ca2+, Sr2+, and Mg2+ (10(-2) M). At a lower concentration (4 x 10(-5) M) of the cations there was differential adsorption of the protein. Adsorption to the template in the presence of each cation, followed by dilution, also led to differential release of the protein. The adsorption of the protein to the basal layer was correlated with reassembly of the HP layer on the template. The mechanisms seem to be: (i) an ionic strength-dependent reassembly, which results in an HP layer loosely attached to the template (this layer is easily dissociated by decreasing the ionic strength); and (ii) a cation-specific (Ca2+ or Sr2+, but not Mg2+, Na+, or K+) mechanism independent of ionic strength. In this latter case, the specific cations presumably form strong noncovalent "salt" linkages between triads and the basal layer, enabling stable hexagons and the HP layer to be formed.     

Alterations in the cell wall of Spirillum serpens VHL early in its association with Bdellovibrio bacteriovorus 109D

In both freeze-etched and critical-point dried preparations examined by transmission and scanning electron microscopy, respectively, the outer surfaces of the cells of Spirillum serpens VHL assume a wrinkled appearance 10–15 min after challenge by Bdellovibrion bacteriovorus 109D. This wrinkling effect is believed (on circumstantial evidence) to be caused by the bdellovibrio's disruption of the cell wall lipoprotein of the Spirillum. With the exception of those topological changes caused by wrinkling, the outer membrane of the Spirillum cell wall retains a normal appearance as viewed in freeze-etched preparations, even after the Spirillum cell has been converted into a bdelloplast. Although the peptidoglycan layer of the Spirillum cell presumably is weakened somewhat by the invading Bdellovibrio, evidence obtained from freeze-fractured preparations of Spirillum bdelloplasts suggests that the peptidoglycan remains as a discrete cell wall layer, even though the Spirillum cell wall apparently has lost much of its rigidity. That the peptidoglycan backbone remains essentially intact, even after the Spirillum cell has been entered by the Bdellovibrio, is supported by the observation that the soluble amino sugar content of the culture medium, as determined by chemical analysis, does not rise even 5.0 h after the association of the Bdellovibrio with the Spirillum has begun.     

Isolation and Chemical Structure of the Peptidoglycan of Spirillum serpens Cell Walls

The peptidoglycan layer of Spirillum serpens cell walls was isolated from intact cells after treatment with sodium dodecylsulfate and digestion with Pronase. The isolated peptidoglycan contained glucosamine, muramic acid, alanine, glutamic acid, and meso-diaminopimelic acid in the approximate molar ratio of 1:1:2:1:1. Aspartic acid and glycine were the only other amino acids found in significant quantities. N-terminal amino acid analyses of the tetrapeptide amino acids in the peptidoglycan revealed that 54% of the diaminopimelic acid molecules are involved in cross-linkage between tetrapeptides. This amount of cross-linkage is greater than that found in the peptidoglycan of previously studied cell walls of gram-negative bacteria. The polysaccharide backbone was isolated, after myxobacter AL-1 enzyme digestion of the peptidoglycan, by fractionation with ECTEOLA-cellulose and Sephadex G-100. An average length of 99 hexosamines for the polysaccharide chains was found (ratio of total hexosamines to reducing end groups).     

Ultrastructural aspects of localized membrane damage in Spirillum serpens VHL early in its association with Bdellovibrio bacteriovorus 109D

The freeze-fracture technique and electron microscopy have been used to demonstrate that localized damage is inflicted upon the cytoplasmic membrane of Spirillum serpens VHL within 20 to 30 min after the start of its association with Bdellovibrio bacteriovorus 109D. This damage is not observed in uninfected Spirillum cells, nor in infected cells within the first 10 min. This damage takes the form of a “blister” which, when viewed stereoscopically in electron micrographs, is seen to project toward the interior of the Spirillum cell. Shortly after its formation, the blister becomes elaborated into a series of ridges which may assume forms ranging from an elaborate spiral to a series of loops or knots. The formation of a blister is shown to involve both the inner and outer leaves of the membrane bilayer, and evidence is presented to indicate that the blister site corresponds to the site of attachment of the Bdellovibrio cell. The hypothesis is proposed that this ultrastructural damage is the cytological basis for the controlled and localized leakage through the cytoplasmic membrane into the periplasmic space of the Spirillum cell at locations adjacent to the Bdellovibrio cell. It is suggested that this localized membrane damage may be the ultrastructural basis for the high efficiency with which bdellowvibrios are known to incorporate cytoplasmic materials from the other bacteria in whose periplasmic spaces they develop.