Structural and serological studies of the O-antigen of Proteus mirabilis O-9

The following structure of the O-polysaccharide (O-antigen) of the lipopolysaccharide of Proteus mirabilis O-9 was determined by NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, ROESY, and 1H,(13)C HMQC experiments, along with chemical methods: [chemical structure: see text] where the degree of O-acetylation is approximately 70%. Immunochemical studies using rabbit polyclonal anti-Proteus mirabilis O-9 serum showed the importance of the O-acetyl groups in manifesting the serological specificity of the O-9 antigen. Anti-P. mirabilis O-9 cross-reacted with the lipopolysaccharides (LPS) of P. vulgaris O-25 and Proteus penneri 14, which could be accounted for by a structural similarity of their O-polysaccharides.     

Biosynthesis of Proteus mirabilis Nuclease

The culture liquid and periplasm of Proteus mirabilis contained nuclease, an enzyme with DNase and RNase activities. The nuclease was most actively synthesized in the early exponential and stationary growth phases. Nuclease synthesis was regulated by nucleic acids (induction by substrate) and inorganic phosphate (end-product inhibition). The synthesis and secretion of nuclease by P. mirabilis was induced by mitomycin C, an inducer of the SOS functions of cells. This suggests the involvement of SOS-response proteins in the regulation of nuclease synthesis.     

Structure of the O-polysaccharide of a serologically separate strain of Proteus mirabilis, TG 332, from a new proposed Proteus serogroup O50

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis TG 332 strain. The following structure of the O-polysaccharide was determined by chemical methods along with NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments: [see equation in text]. The O-polysaccharide studied has a unique structure among Proteus O-antigens. Accordingly, P. mirabilis TG 332 is serologically separate, and we propose to classify this strain into a new Proteus serogroup, O50. The nature of minor epitopes that provide a cross-reactivity of P. mirabilis TG 332 O-antiserum with the LPS of P. mirabilis O30 and Proteus penneri 34 (O60) is discussed.     

Structure of the O-polysaccharide of Proteus mirabilis OC (CCUG 10702) from a new proposed Proteus serogroup O75

A neutral O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis OC (CCUG 10702) and studied by sugar and methylation analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text]. Based on the unique structure of the O-polysaccharide and serological data, we propose classifying P. mirabilis OC (CCUG 10702) into a new separate Proteus serogroup O75. A weak cross-reaction of O-antiserum against P. mirabilis OC with the lipopolysaccharide of P. mirabilis O49 was accounted for by a similarity in the O-polysaccharide structures.     

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10701 (OB) classified into a new Proteus serogroup, O74

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus mirabilis CCUG 10701 (OB) and studied by chemical analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: --> 3)-beta-D-GlcpNAc6Ac-(1 --> 2)-beta-D-GalpA4Ac-(1--> 3)-alpha-D-GalpNAc-(1 --> 4)-alpha-D-GalpA-(1 -->, where the degree of O-acetylation at position 6 of GlcNAc is approximately 50% and at position 4 of beta-GalA approximately 60%. Based on the unique structure of the O-polysaccharide and serological data, it is proposed to classify P. mirabilis CCUG 10701 (OB) into a new Proteus serogroup, O74.     

Proteus mirabilis interkingdom swarming signals attract blow flies

Flies transport specific bacteria with their larvae that provide a wider range of nutrients for those bacteria. Our hypothesis was that this symbiotic interaction may depend on interkingdom signaling. We obtained Proteus mirabilis from the salivary glands of the blow fly Lucilia sericata; this strain swarmed significantly and produced a strong odor that attracts blow flies. To identify the putative interkingdom signals for the bacterium and flies, we reasoned that as swarming is used by this bacterium to cover the food resource and requires bacterial signaling, the same bacterial signals used for swarming may be used to communicate with blow flies. Using transposon mutagenesis, we identified six novel genes for swarming (ureR, fis, hybG, zapB, fadE and PROSTU_03490), then, confirming our hypothesis, we discovered that fly attractants, lactic acid, phenol, NaOH, KOH and ammonia, restore swarming for cells with the swarming mutations. Hence, compounds produced by the bacterium that attract flies also are utilized for swarming. In addition, bacteria with the swarming mutation rfaL attracted fewer blow flies and reduced the number of eggs laid by the flies. Therefore, we have identified several interkingdom signals between P. mirabilis and blow flies.     

Structure of a highly phosphorylated O-polysaccharide of Proteus mirabilis O41

A highly phosphorylated O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis O41 followed by GPC. The initial and dephosphorylated polysaccharides and phosphorylated products from two sequential Smith degradations were studied by (1)H, (13)C and (31)P NMR spectroscopy and ESI-MS. The O-polysaccharide was found to have a tetrasaccharide repeating unit containing one ribitol phosphate (presumably d-Rib-ol-5-P) and two ethanolamine phosphate (Etn-P) groups, one of which is present in the stoichiometric amount and the other in a nonstoichiometric amount. The following structure of the O-polysaccharide was established:     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus mirabilis 2002

The structure of the O-polysaccharide of the lipopolysaccharide of Proteus mirabilis 2002 was elucidated by chemical methods and 1H and 13C NMR spectroscopy. It was found that the polysaccharide consists of branched pentasaccharide repeating units having the following structure: [structure in text]. The O-polysaccharide of P. mirabilis 2002 has a common tetrasaccharide fragment with that of P. mirabilis 52/57 from serogroup O29, and the lipopolysaccharides of the two strains are serologically related. Therefore, based on the structural and serological data, we propose to classify P. mirabilis 2002 into the Proteus O29 serogroup as a subgroup O29a,29b.     

Structure of the neutral O-polysaccharide and biological activities of the lipopolysaccharide of Proteus mirabilis O20

Mild acid degradation of the lipopolysaccharide (LPS) of Proteus mirabilis O20 resulted in depolymerisation of the O-polysaccharide to give a repeating-unit pentasaccharide. A polysaccharide was obtained by O-deacylation of the LPS followed by nitrous acid deamination. The derived pentasaccharide and polysaccharide were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the repeating unit of the O-polysaccharide was established: [Carbohydrate structure: see text]. As opposite to most other P. mirabilis O-polysaccharides studied, that of P. mirabilis O20 is neutral. A week serological cross-reactivity was observed between anti-P. mirabilis O20 serum and LPS of a number of Proteus serogroups with known O-polysaccharide structure. The ability of LPS of P. mirabilis O20 to activate the serine protease cascade was tested in Limulus amoebocyte lysate and in human blood plasma and compared with that of P. mirabilis O14a,14c having an acidic O-polysaccharide. The LPS of P. mirabilis O20 was found to be less active in both assays than the LPS of P. mirabilis O14a,14c and, therefore, the structurally variable O-polysaccharide may influenced the biological activity of the conserved lipid A moiety of the LPS.     

Membranes of the protoplast L-form of Proteus mirabilis

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysaccharide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of d-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypetidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10), which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.     

Structure and serological characterization of the O-antigen of Proteus mirabilis O18 with a phosphocholine-containing oligosaccharide phosphate repeating unit

A phosphorylated, choline-containing polysaccharide was obtained by O-deacylation of the lipopolysaccharide (LPS) of Proteus mirabilis O18 by treatment with aqueous 12% ammonia, whereas hydrolysis with dilute acetic acid resulted in depolymerisation of the polysaccharide chain by the glycosyl phosphate linkage. Treatment of the O-deacylated LPS with aqueous 48% hydrofluoric acid cleaved the glycosyl phosphate group but, unexpectedly, did not affect the choline phosphate group. The polysaccharide and the derived oligosaccharides were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the pentasaccharide phosphate repeating unit was established: [carbohydrate structure in text] Where ChoP=Phosphocoline Immunochemical studies of the LPS, O-deacylated LPS and partially dephosphorylated pentasaccharide using rabbit polyclonal anti-P. mirabilis O18 serum showed the importance of the glycosyl phosphate group in manifesting the serological specificity of the O18-antigen.     

Structure of the O-polysaccharide of Proteus mirabilis O38 containing 2-acetamidoethyl phosphate and N-linked D-aspartic acid

The O-antigen of Proteus mirabilis O38 was found to be unique among bacterial polysaccharides and to have the following structure: [carbohydrate structure in text] where D-Qui4N(Ac-D-Asp) is 4-(N-acetyl-D-aspart-4-ylamino)-4,6-dideoxy-D-glucose and AcEtnP is 2-acetamidoethyl phosphate. Neither of these entities have been hitherto found in natural polysaccharides. Structural studies were performed using 1D and 2D NMR spectroscopy, including experiments run in an H2O/D2O mixture to reveal correlations for NH protons. In addition, dephosphorylation, carboxyl reduction and selective cleavages were applied. Solvolysis of the polysaccharide with anhydrous HF gave an alpha-D-GlcNAc-(1-->3)-D-Qui4N(Ac-D-Asp) disaccharide. Solvolysis with trifluoromethanesulfonic (triflic) acid afforded D-GlcNAc6(AcEtnP), thus showing the suitability of this reagent for the preparation of phosphorylated sugar derivatives.     

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.Abbreviation SDS sodium dodecylsulfate     

Structure of the O-polysaccharide of Proteus serogroup O34 containing 2-acetamido-2-deoxy-alpha-D-galactosyl phosphate

On mild acid degradation of the lipopolysaccharide of Proteus vulgaris O34, strain CCUG 4669, the O-polysaccharide was cleaved at a glycosyl-phosphate linkage that is present in the main chain. The resultant phosphorylated oligosaccharides and an alkali-treated lipopolysaccharide were studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, and the following structure of the branched tetrasaccharide phosphate repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text]The O-polysaccharide of Proteus mirabilis strain TG 276 was found to have the same structure and, based on the structural and serological data, this strain was proposed to be classified into the same Proteus serogroup O34.     

Effects of phorbol esters on endothelial cell microfilaments: Laser scanning confocal microscopy and quantitative morphometry of dose dependent changes

Phorbol esters are known to alter microfilaments but it is not clear if the changes correspond to modulation of the phosphoinositide turnover/protein kinase C system. The novel technique of laser scanning confocal epifluorescence was used to study fiber orientation in phorbol ester treated cells. We treated endothelial cells with control agents and agents known to stimulate protein kinase C: 4 alpha-phorbol, phorbol 12-myristate 13-acetate (PMA), phorbol dibutyrate (PDB), or lipopolysaccharide. After incubation with the test agents, the endothelial cell microfilaments were stained with rhodamine pholloidin and viewed by conventional epifluorescence and by laser scanning confocal epifluorescence microscopy. The images obtained by the confocal microscopy corresponded to a thin optical section through the cells, 300 nm or more in thickness. The microfilaments extended predominantly in the plane of focus. After exposure of the cells to phorbol esters, the stress fibers became more nearly parallel in arrangement or were shortened, but remained in the plane of focus. The modification of microfilaments in response to phorbol esters was quantitated by a single blind analysis. In order to compare the morphological changes with a biochemical action of the phorbol esters, we measured phosphoinositide turnover. The dose-dependence of morphological changes was compared and contrasted to the dose-dependent effect of phorbol esters on bradykinin-stimulated phosphoinositide turnover. PMA had about the same EC50 (1-5 nM) for both biochemical and morphological processes. PDB was less potent in inducing the disruption of microfilament structure than in inhibiting phosphoinositide turnover. Lipopolysaccharide was ineffective in inducing a morphological change under these conditions. A simple activation of protein kinase C is insufficient to explain the dose-dependent effects of phorbol esters. Thus a morphometric analysis can help distinguish the potency of cytoskeleton modulators.     

Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis

Nitrate reductase A has been solubilized from purified cytoplasmic membranes by extraction with terl-amyl alcohol. The resulting aqueous solution contained monomeric reductase which polymerized slowly to dimers and tetramers with sedimentation coefficients of respectively 10.5, 16 and 23 Svedbergunits. The polymerization could be stopped to some extent by addition of a small amount of Triton X-100. These distinct entities of nitrate reductase A were separable on electro-focusing, DEAE-column chromatography and polyacrylamide gel electrophoresis, and have been proved to consist of similar subunits with molecular weights of 104000, 63000, and 56000 daltons. The molecular weights of monomeric nitrate reductase A was found to be about 240000 daltons.Chlorate reductase C has been solubilized by a similar procedure, resulting in only monomeric enzyme. Chlorate reductase C exhibited a sedimentation coefficient of 7.7 Svedbergunits, an isoelectric point of pH=4.55 and a molecular weight of approx. 180000 daltons. It was found to consist of three subunits with molecular weights of 75000, 63000 and 56000 daltons. The latter two subunits are most probably common in nitrate reductase A and chlorate reductase C.     

Simultaneous multi-parameter observation of Harringtonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.     

The influence of growth conditions on the synthesis of molybdenum cofactor in Proteus mirabilis

Cell-free extracts of Proteus mirabilis were able to reconstitute NADPH-dependent assimilatory nitrate reductase in crude extracts of the Neurospora crassa mutant strain nit-1, lacking molybdenum cofactor. Molybdenum cofactor was formed in the cytoplasm of the bacterium even in the presence of oxygen during growth though under these conditions no molybdo enzymes are formed. As a consequence no cofactor could be released by acid treatment from membranes of cells grown aerobically. The amount of cofactor released from membranes of cells grown anaerobically under various conditions was proportional to the amount of molybdo enzymes formed. During growth in the presence of tungstate a cofactor, which lacks molybdenum, was found in the cytoplasm. For detection of this so-called demolybdo cofactor the presence of molybdate during reconstitution was essential. Moreover, the cytoplasmic cofactor pool in cells grown in the presence of tungstate appeared to be two to three times higher than in cells grown under similar conditions without tungstate. After anaerobic growth in the presence of tungstate, the inactive demolybdo reductases were shown to contain partly no cofactor and partly a demolybdo cofactor. The P. mirabilis chlorate resistant mutant S 556 did not contain molybdenum cofactor. In two other chl-mutants the cofactor activity was the same as in the wild type.     

Molybdenum cofactor from the cytoplasmic membrane of Proteus mirabilis

Molybdenum cofactor was extracted from membranes of Proteus mirabilis by three methods: acidification, heat treatment and heat treatment in the presence of sodium-dodecylsulphate (SDS). Extracts prepared by the latter method contained the highest concentration of molybdenum cofactor. In these extracts molybdenum cofactor was present in a low molecular weight form. It could not penetrate an YM-2 membrane during ultrafiltration suggesting a molecular weight above 1000. During aerobic incubation of cofactor extracts from membranes at least four fluorescent species were formed as observed in a reversed-phase high performance liquid chromatography (HPLC) system. The species in the first peak was inhomogeneous while the species in the others seem to be homogenous. In water, all fluorescent products had an excitation maximum at 380 nm and an emission maximum at 455 nm. Their absorption spectra showed maxima at around 270 nm and 400 nm. Fluorescent compounds present in the first peak could penetrate an YM-2 membrane during ultrafiltration, whereas the compounds in the other peaks hardly did. Using xanthine oxidase from milk as source of molybdenum cofactor apparently identical cofactor species were found. Cytoplasmic nor membrane extracts of the chlorate resistant mutant chl S 556 of P. mirabilis could complement nitrate reductase of Neurospora crassa nit-1 in the presence of 20 mM molybdate. However, fluorescent species with identical properties as found for the wild-type were formed during aerobic incubation of extracts from membranes of this mutant.Non-common Abbreviations HPLC high performance liquid chromatography - I.D. internal diameter - SDS sodium dodecyl sulphate