Detection of viral proteins during synthesis of defective phage of Proteus mirabilis D52 using the immunocolloid gold method

We have applied the method of immunocolloidal gold in order to label the ribonuclear proteins of prokaryotic cells on isolated bacterial chromatosomes. In the process of protein synthesis it was possible to visualize a definite protein of defective phage D52 of Proteus mirabilis.     

The ribitol-phosphate-containing lipopolysaccharide from Proteus mirabilis, strain D52. Investigations on the structure of O-specific chains.

A soluble hydrophilic lipopolysaccharide, termed lipopolysaccharide II, isolated from Proteus mirabilis, strain D52 contained N-acetylglucosamine, glucose, galactose, ribitol phosphate and ethanolamine phosphate as constituents of the O-specific polysaccharide. Periodate oxidation studies were carried out on the polymer before and after dephosphorylation with hydrofluoric acid and on oligosaccharides derived from the polymer by partial acid hydrolysis. The results obtained indicate that the polysaccharide chain consists of the chemical repeating unit Gal-1,3(4)-GlcNAc-1,3-Glc-1,3-GlcNAc-, where GlcNAc stands for N-acetylglucosamine. Whereas the galactose residue is substituted at C-3 by ribitol phosphate, the glucose is substituted by ethanolamine phosphate at C-6.     

Electron transport and cytochromes in aerobically grown Proteus mirabilis

The function of the cytochromes in electron transport from NADH to oxygen in aerobically grown Proteus mirabilis has been determined. 77K-Spectra of cytoplasmic membrane suspensions, frozen while catalyzing electron transport from NADH to oxygen, in the presence as well as in the absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide, have been recorded. Analysis of these 77K-spectra revealed that cytochrome b-563 (E'0 = +140 mV), cytochrome b-556 (E'0 = +140 mV) [or alternatively cytochrome b-563/556 (E'0 = +140 mV)] and cytochrome b-557 (E'0 = +50 mV) may function in a Q or b-cycle. The function of cytochrome c-549 (E'0 = +75 mV), which seems to be present only in a very low concentration, and cytochrome b-556 (E'0 = -105 mV), which reacts very slowly to the addition of NADH and oxygen, remains unclear. Cytochrome o, the main oxidase of aerobically grown P. mirabilis cells, can not be detected by the methods described above. Only when the reduced form of cytochrome o is liganded with carbon monoxide a specific alpha-band can be detected at 569 nm at 25 degrees C and 565 nm at 77K.     

Structure of the O-specific polysaccharide of Proteus mirabilis D52 and typing of this strain to Proteus serogroup O33.

The acidic O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Proteus mirabilis strain D52 was studied using chemical analyses along with 1H-NMR and 13C-NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C and 1H,31P HMQC experiments. The polysaccharide was found to contain D-ribitol 5-phosphate (D-Rib-ol-5-P) and ethanolamine phosphate (Etn-P) and has the following structure: D-Rib-ol-5-P (3) approximately 75% EtnP(6)-->2)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-GlcpNAc-(1-->). This structure is identical with that of the O-polysaccharide of P. mirabilis O33 strain 59/57, and, hence, P. mirabilis D52 belongs to the same Proteus serogroup O33. Serological studies with O-antiserum against P. mirabilis D52 confirmed this but showed that the LPS species of P. mirabilis 59/57 and D52 are not identical, having different epitopes in the core region. A serological cross-reactivity of P. mirabilis D52 O-antiserum was observed with LPS of two other Proteus strains, P. mirabilis O16 and P. penneri 103, which have structurally different O-polysaccharides. The role of charged groups, Rib-ol-5-P and Etn-P in the immunospecificity is discussed.     

Genetic analysis of Proteus mirabilis mutants defective in swarmer cell elongation.

Swarmer cell differentiation is a complex process involving the activity of many gene products. In this report, we characterized the genetic locus of Tn5 insertion in each of 12 mutants defective in swarmer cell elongation. The mutations fell into four categories affecting either flagellar biosynthesis or energetics, lipopolysaccharide and cell wall biosynthesis, cellular division, or proteolysis of peptides.     

Ultrastructural characteristics of Proteus vulgaris and Proteus mirabilis cells differing in the capacity for swarming

Specific differences in the structure of colonies and the location of microbial cells in colonies, characteristic for aggregating and nonaggregating genetically related pairs of P. vulgaris and P. mirabilis strains, have been demonstrated by means of transmission and scanning electron microscopy. In calculating the number of flagellae per 100 outlines of microbial bodies revealed in negatively stained preparations, the fact that both aggregating and nonaggregating bacteria possess practically the same number of flagellae, on the average 4-8 flagellae per microbial cell outline, has been established. This fact indicates that the presence of flagellae in microbial cells is unrelated to their capacity for swarming.     

Alterations in the cell envelope composition of Proteus mirabilis during the development of swarmer cells.

Long, swarming cells of Proteus mirabilis had different proportions of some lipopolysaccharide components when compared to short cells, either agar grown or broth grown. Fluorescence spectrophotometry of antibody binding, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the change was in the proportion of lipopolysaccharide with long O-antigenic sidechains, swarmer lipopolysaccharide relative to short sidechain lipopolysaccharide than the non-swarming cells. The proteins and phospholipids of the envelop remained the same during swarmer development. The results are discussed in relation to the increase in flagella synthesis and permeability to some antibacterial agents during swarmer development.     

Application of 1D and 2D NMR techniques to the structure elucidation of the O-polysaccharide from Proteus mirabilis O: 57

Summary The LPS O-polysaccharide (O-PS) produced by Proteus mirabilis serotype O: 57 (ATCC 49995) was shown by NMR spectroscopy and chemical analysis to be a high-molecular-weight acidic branched polymer of pentasaccharide repeating units, composed of d-glucose, d-galactose, 2-acetamido-2-deoxy-d-galactose and glycerophosphate residues (1:2:2:2:1). Application of one-and two-dimensional NMR methods allowed the complete assignment of notoriously crowded ¹H and ¹³C spectra of the O-PS, leading to the determination of its structure. Several of the NMR techniques used were applied for the first time to the structure elucidation of polysaccharides. The resonances of galactose H5, H6 and H6 were identified by a 1D analog of 3D NOESY-TOCSY and 2D {¹H, ¹H} triple-quantum experiments. The position and the nature of the phosphate group were determined from 2D ³¹P (₁)-half-filtered COSY and 2D ³¹P-relayed COSY spectra. 2D HMQC-TOCSY and 2D single-quantum proton-carbon long-range correlation techniques were used to overcome the difficulties of severe overlap and higher order effects in the ¹H NMR spectrum of the O-PS. The latter technique, together with 2D NOESY, enabled us to identify the substitution positions, the anomeric configurations and the sequence of the component glycose residues in the O-PS.     

Electron microscopic study on the structure of septate and comb desmosomes

Summary An electron microscopic study of the desmosomes of Pelmatohydra oligactis and the silkworm (Bombyx mori L.) demonstrates that these two species of invertebrates have two different kinds of desmosomes. The term septate desmosome is applicable only in the case of the Pelmatohydra desmosomes, while a new term, comb desmosome (Danilova, 1968a, b), is suggested for the insect type. Differences in the structure of these two desmosome varieties can be detected only in tangential sections.In Pelmatohydra, two patterns of septate desmosomes are to be observed in ultrathin sections, yet this difference is due to different directions of the section planes. A scheme to show the structure of the septate desmosome of Pelmatohydra and that of the comb desmosome of insects is presented, as are some variant structures of these desmosomes in sections made in various directions. Globular subunits 30–40 Å in diameter have been detected in the hydroid septa.Fixation with potassium permanganate has failed to reveal any desmosome septa or combs. This observation supports the opinion that septate and comb desmosomes are of non-membranous nature.