Margaret J. Thornley

The fine structure of micrococcus radiodurans

SummaryThe fine structure of the radiation-resistant bacterium, Micrococcus radiodurans, isolated by Anderson, was studied by electron microscopy of intact and disrupted cells using thin sectioning and negative staining techniques. The cytoplasm and nuclear structures are normal, but the cell wall and sheath are more complex than any so far described for a bacterium. The surface consists of four distinct layers, each having a characteristic fine structure, one of which has been tentatively identified as that responsible for maintaining the rigidity of the cells. Striations with a periodicity of 175 to 200 Å are visible in thin sections of this layer, and a pseudo-hexagonal array of dark “holes” is seen in surface view of negatively-stained fragments. It is concluded that this layer is the main structural element of the cell wall of M. radiodurans. The other three layers of the surface have not been clearly located in thin sections; one of these layers has a well-defined pattern of hexagonally arranged units similar to that observed in Spirillum serpens by Murray but with different dimensions.

Properties of monoclonal antibodies to the genus-specific antigen of Chlamydia and their use for antigen detection by reverse passive haemagglutination.

The chlamydial genus-specific antigen was extracted with phenol/chloroform/petroleum ether (PCP) from preparations of Chlamydia trachomatis and C. psittaci, and quantities measured using an assay for lipopolysaccharide (LPS). The LPS from C. trachomatis contained 2.2% (w/w) of ketodeoxyoctanoic acid. Five IgG monoclonal antibodies reacted in an ELISA with LPS from both species, the antigen being periodate-sensitive and heat-resistant, confirming that all antibodies were against the genus-specific antigen. All the antibodies bound to the PCP extract of C. trachomatis on an immunoblot, at a position corresponding to the periodate-Schiff-stained bands of both C. trachomatis extract and Salmonella Re-LPS. When linked to trypsin-treated sheep erthrocytes and used in reverse passive haemagglutination tests, all antibodies gave indicator cells capable of detecting chlamydial LPS or crude preparations of chlamydiae grown in McCoy cells, the sensitivity varying with the antibody used. The antibodies varied in IgG subclass (either IgG2a or IgG3), and in ability to precipitate in immunodiffusion tests. Two antibodies cross-reacted with one strain of Acinetobacter in ELISA and with Salmonella Re-LPS in both ELISA and immunodiffusion tests. The other three did not react in ELISA with Acinetobacter strains or Salmonella Re-LPS, and none of the five reacted with LPS of E. coli or Pseudomonas morsprunorum.

The nature of the attachment of a regularly arranged surface protein to the outer membrane of an Acinetobacter sp

Acinetobacter 199A carries on the outer surface of its outer membrane a layer of regularly arranged protein subunits. The isolated surface protein assembles into the same regular array even in the absence of the underlying outer membrane. Cl- minus is required for this self-assembly. Evidence is presented that the interaction of the surface protein with the outer membrane involves the linking of a carboxyl group in the surface protein to a negatively charged group in the outer membrane protein, via a divalent cation. The surface protein could be detached from the outer membrane by the protein perturbant urea, by the chelating agent EDTA and by replacing Mg-2+ with Na+. It could not be detached by treatment with phospholipases A anc D or the detergents Tween 80 and sodium deoxycholate. The conditions favourable for reattachment of surface protein to the cell wall were the presence of divalent cations and a pH of 3-5. Conversion of carboxyl groups in the surface protein to amine with carbodiimide and ethylene diamine interfered with reattachment. The surface protein did not attach to isolated cell wall lipid or lipopolysaccharide.

A study by freeze-etching of the fine structure of Micrococcus radiodurans

Summary1.This study of the fine structure of Micrococcus radiodurans by freeze-etching confirmed and extended earlier results obtained with thin sections and negatively-stained preparations. The cell wall consists of three layers, and its outer surface shows a characteristic hexagonal array of hollow rings with a centre-to-centre spacing of about 18 nm. In dividing cells, septum formation begins at several points on the periphery of the cell and often proceeds asymmetrically until the septum is complete.2.These results, together with other studies, show that the structure of the cell wall of M. radiodurans is not that typical of Gram-positive cocci, and that it has some features in common with the walls of Gram-negative bacteria, and some unique features. M. radiodurans should therefore be reclassified into a new taxon.

Comparison of Red-Cell Linked Anti-IgE and 125I-Labelled Anti-IgE in a Solid-Phase System for the Measurement of IgE Specific for Castor Bean Allergen

The report described the development of a mixed reverse (solid-phase) passive antiglobulin haemadsorption (MRsPAH) test for specific IgE antibody to castor bean allergen. The allergen is immobilised by formalin fixation in the wells of polyvinyl chloride microtitre plates. After allowing allergen-specific antibodies in the test serum to bind to the allergen, plates are washed thoroughly, and red cells coupled by chromic chloride to sheep IgG anti-human IgE are used to detect specifically bound IgE. This system was compared with a solid-phase radioimmunoassay in which 125I-labelled anti-IgE was substituted for the antiglobulin-linked red cells of the MRsPAH test; the earlier stages of both tests being the same. 12 sera, 10 from patients with allergic asthma to castor bean allergen and two from non-allergic controls, were tested for castor-bean-specific serum IgE by both methods and the results showed high correlation. The MRsPAH tst for allergen-specific serum IgE provides a useful alternative to the RAST system, being free of the disadvantages inherent in the use of radio-labelled materials.

Freeze-etching of the outer membranes of Pseudomonas and Acinetobacter

The location and surface structure of the fracture faces revealed by freeze-etching within the cell walls of Pseudomonas fluorescens NCTC 10038 and Acinetobacter sp. strain MJT/F5/5 have been compared.Treatment of intact cells with lysozyme and ethylenediaminetetraacetic acid, followed by stabilization with MgCl2, detaches the outer membranes of both organisms from underlying layers of the envelope. In the Acinetobacter, the detached outer membrane (om) retains the a-layer of hexagonally-arranged subunits on its outer surface, whereas the Pseudomonas has no a-layer.Freeze-etching of these detached structures reveals the etched outer and inner surfaces of the outer membranes (in the Pseudomonas) or the om + a-layers (in the Acinetobacter). In addition, the detached structures in both organisms fractured along an internal plane.Since the structures of these internal fracture faces are identical with those of fracture faces produced within the cell walls of intact cells, freeze-etched in the presence of glycerol, it is clear that, under these conditions also, the main fracture plane occurs within the outer membranes.The technique used also reveals the etched outer surface of the plasma membranes, as well as the internal fracture faces which are normally seen, and these are illustrated.

Stimulation by X-radiation of enzyme induction and growth in Escherichia coli

Escherichia coliB has been grown in a liquid medium containing, besides inorganic salts, glucose and ammonium chloride as sole sources of carbon and nitrogen. The micro-organisms do not grow if maltose is substituted for glucose. Similarly, washed suspensions of Esch. coli which oxidize glucose vigorously have an insignificant O2-uptake with maltose. However, maltose is utilized for growth if NH4Cl is replaced by glutamic acid. Apparently the bacteria cannot form the enzyme needed to utilize maltose from their existing protein equipment but can if given a suitable organic nitrogen source. X-radiation of 4-8 kr (190 kVp, 10 mA, dose rate 8 kr/min) changes these conditions fundamentally: (i) Washed suspensions of irradiated Esch. coli oxidize glucose as fast as controls, but—unlike the controls—develop an increasing respiration in presence of maltose, the maximal rate (after 4 to 8 kr) being attained within 3 h and approaching that for glucose. (ii) This response to maltose is suppressed by chloramphenicol. (iii) Micro-organisms viable after irradiation can grow on maltose in the liquid medium with NH4Cl, although slowly. (iv) The viable irradiated micro-organisms multiply with maltose and glutamate much faster than unirradiated controls for most of the logarithmic growth phase. This has been confirmed by turbidity measurements and plate counts. (v) The response to maltose is lost on incubation of the bacteria with glucose and NH4Cl. (vi) Both controls and irradiated Esch. coli contain maltase. This has been demonstrated with ultrasonically disintegrated bacteria where the addition of maltose and glucose oxidase (notatin) caused an O2-uptake at equal rates in both samples. These results and further evidence justify the conclusion that the observed irradiation effects are due to the induction of permease for maltose in irradiated micro-organisms.