Synthesis and applications of 8-azido photoaffinity analogs of P1,P3-bis(5'-adenosyl)triphosphate and P1,P4-bis(5'-adenosyl)tetraphosphate.

32P-labeled photoaffinity analogs of bis(5'-adenosyl)-tetraphosphate and bis(5'-adenosyl)triphosphate which contain a single photoreactive 8-azidoadenosine group distal to the radiolabel have been synthesized from commercially available components using a combination of chemical and enzymatic procedures including a water-soluble carbodiimide. The method is simple, rapid, and produces yields of high specific activity products of around 60%. The analog of bis(5'-adenosyl)-tetraphosphate is very similar to the parent compound in its inhibition of rat liver adenosine kinase and its efficiency as a substrate for the bis(5'-nucleosidyl)tetraphosphate pyrophosphohydrolase from Artemia embryos. In the latter case, ATP and 8-azidoAMP are the preferred products. As would be expected, this analog is a much more effective photoprobe for both adenosine and adenylate kinases than the corresponding analog of bis(5'-adenosyl)triphosphate. Both compounds have been used to photoaffinity label crude extracts of Artemia, Vero cells, and Clostridium acetobutylicum and preferential specific labeling of different polypeptides by each analog has been shown. In extracts of C. acetobutylicum, the labeling of a polypeptide of Mr 48,500 by the bis(5'-adenosyl)tetraphosphate analog was totally dependent on the presence of Co2+ ions. These compounds should therefore prove valuable both for the active site labeling of purified binding proteins and for the detection and identification of new target proteins for these nucleotides.

Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes.

Effects of Clostridium difficile toxins A and B on cytoskeleton organization in HEp-2 cells: a comparative morphological study.

A comparative study on the effects of toxin A and toxin B from Clostridium difficile on HEp-2 cells was carried out. Both toxins caused cell retraction and rounding and seemed to exert their effect on cell morphology via a rearrangement of actin and alpha-actinin microfilaments. Such a rearrangement occurred at an early stage, when no change in microtubular and cytokeratin systems was detectable. Nevertheless, several structural modifications accompanying the cytopathological process induced by toxins A and B appeared to be quite different. In particular, toxin B-treated cells showed an arborized phenotype as a result of cell retraction and rounding, whereas toxin A caused cell rounding without arborization. Moreover, nuclear polarization following disorganization of the microfilament system was only observed in toxin A-treated cells. The structural features distinguishing intoxication processes induced by the two toxins probably reflect a different mechanism of action and suggest the presence of a distinct subcellular component as a primary target for each toxin.

Characterization of some Clostridium species by gas-liquid chromatography using numerical analysis.

We studied 42 strains of Clostridia belonging to 20 different species. All the strains were examined for morphological characters, biochemical reactions, and analyzed by means of gas-liquid chromatography (GLC) to determine metabolic patterns of short chain fatty acids and alcohols. To increase the number of criteria for the differentiation, specimens were grown on Peptone Yeast Extract medium (PY) with the addition of 13 different carbohydrates. The strains were compared using numerical taxonomic techniques based upon 20 unit qualitative and 224 quantitative characters. Data were examined using the simple matching coefficient (SSM) for qualitative characters, and degree of overlap between superimposed trace (So) for qualitative characters, and clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) technique. DNA base composition was also determined. Numerical analysis showed remarkable difference between phenograms derived from SSM and So coefficients. The phenogram (SSM) is formed by 11 clusters and eight of these include strains from only one species. Only three clusters contained strains from different species. The cluster variability range of G + C base composition was never higher than 4 mol% except for one cluster where it reached 7 mol% G + C. In the phenogram (So) instead, there are 8 clusters and in only one case are strains from one species aggregated. In the remaining 7 clusters strains belonging to two or more species aggregated. The range values of base composition are over 4 mol% in three of the eight clusters.

Isolation and characterization of a novel four-chain ether lipid from Clostridium butyricum: the phosphatidylglycerol acetal of plasmenylethanolamine.

We describe the isolation and characterization of a novel four-chain ether phospholipid in Clostridium butyricum grown on petroselinic acid in the absence of biotin. The results of quantitative analyses of hydrolytic products, selective hydrolysis by mild acid or phospholipase C, 1H-, 13C-, and 31P-NMR, and fast atom bombardment-mass spectroscopy (FABMS) have resulted in the determination of the structure of this lipid as a phosphatidylglycerol acetal of plasmenylethanolamine. Smaller amounts of this lipid have been found in cells grown under similar conditions in the presence of oleic, cis-vaccenic, elaidic or dihydrosterculic acids. It also appears to be present in small amounts in cells grown with biotin. This lipid is structurally related to the more plentiful glycerol acetal of plasmenylethanolamine found in these cells.

Electrophoretic characterization of Clostridium difficile strains isolated from antibiotic-associated colitis and other conditions.

Clostridium difficile has been recognized as the cause of antibiotic-associated pseudomembranous colitis and of less severe diarrheal diseases associated with the use of antimicrobial agents. However, healthy carriers of this microorganism have been found, particularly healthy neonates and small children. Various typing systems have been used to clarify the epidemiology of C. difficile. We used the electrophoretic patterns of EDTA-extracted proteins to characterize C. difficile strains from various sources. Altogether, 110 strains were studied, including 2 reference strains, and 21 different protein profiles were obtained. However, two patterns were the most common: the group 2 pattern, characterized by a major 35-kilodalton polypeptide band, and the group 5 pattern, identified by principal bands of 37 and 56 kilodaltons. The group 2 pattern was characteristic of strains isolated during hospital outbreaks and from sporadic cases of pseudomembranous colitis and antibiotic-associated diarrhea. The group 5 pattern was obtained only from isolates from healthy neonates and children. A correlation between electrophoretic characteristics and virulence can be hypothesized, namely that group 2 strains are more prone to induce diseases and cause outbreaks. It is noteworthy that strains isolated from children with diarrhea of unknown etiology, not related to antibiotic use, belong to the "virulent" group 2; strains from leukemic patients showed a variety of different patterns, and only two belong to group 2. This characterization can be used to aid studies on the virulence and clinical significance of C. difficile.

Lipid shape as a determinant of lipid composition in Clostridium butyricum. The effects of incorporation of various fatty acids on the ratios of the major ether lipids.

The lipid composition of Clostridium butyricum is strongly influenced by the aliphatic chain compositions of the membrane lipids. Growth on cis-monounsaturated fatty acids in the absence of biotin was shown to affect the relative proportions of phosphatidylethanolamine, plasmenylethanolamine, and the glycerol acetal of plasmenylethanolamine most strongly, with smaller effects on the acidic lipids, phosphatidylglycerol and cardiolipin. The ratio of the glycerol acetal of plasmenylethanolamine to total phosphatidylethanolamine in cells grown on a series of fatty acids is shown to decrease in the following order; cis-vaccenic acid greater than or equal to oleic acid = C19-cyclopropane fatty acid greater than linoleic acid greater than petroselinic acid greater than elaidic acid greater than 14-methylhexadecanoic acid (anteiso-C17) greater than 12-methyltridecanoic acid (iso-C14). All fatty acids were extensively incorporated into the lipid acyl, alkenyl, and alkyl chains. There was considerable chain-elongation of the iso-C14 to iso-C16. The results are consistent with the hypothesis that the membrane lipid composition is strongly influenced by lipid shape and that the observed changes in lipid composition serve to stabilize the bilayer arrangement of the cell membrane.

2H-NMR studies on ether lipid-rich bacterial membranes: deuterium order profile of Clostridium butyricum.

Palmitic acid specifically deuterated at different carbon atoms, has been incorporated biosynthetically into the membrane lipids of Clostridium butyricum. The lipids of this organism are rich in plasmalogens and their glycerol acetals and exhibit an unusual fatty acyl and alkenyl chain distribution with saturated chains mainly at the sn-2 position and unsaturated chains at the sn-1 position. The ordering of the deuterated hydrocarbon chains in whole cells was measured with deuterium nuclear magnetic resonance and was compared to the order profiles of isolated cell membranes and membranes formed from the total phospholipid extract. The shape of the order profiles was similar for all three membranes, but the absolute values of the order profiles in whole cells and isolated membranes were lower than those of the liposomal lipids. The order profiles have the same characteristic shape as those found for the lamellar liquid-crystalline phases of synthetic diacylphospholipids.

Purification and immunochemical properties of a wall protein antigen from Clostridium difficile ATCC 11011.

A wall-surface protein antigen, designated 32K antigen, was extracted from whole cells of Clostridium difficile strain ATCC 11011 with phosphate buffered saline and purified by ion-exchange chromatography, gel filtration, and chromatofocusing. The 32K antigen preparation was determined to be highly homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the antigen was characteristic in the predominance of the acidic amino acids, the very low contents of methionine and histidine, and the lack of cysteine. A monomeric molecular weight of the 32K antigen was estimated to be 32,000 by SDS-PAGE and 30,200 by sedimentation equilibrium. The antigen exhibited two isoelectric forms (IP, 4.12 and 3.96). Neither carbohydrate nor phosphorus was detectable in the antigen. The antigen was relatively resistant to trypsin but sensitive to pepsin. Immunoblot analysis of the wall proteins isolated from other strains of C. difficile probed with monospecific antiserum against the antigen from ATCC 11011 showed that the antigenicity of 32K wall protein was common among some of the strains containing 32K wall proteins.

Comparison of beef liver and Penicillium vitale catalases.

The structures of Penicillium vitale and beef liver catalase have been determined to atomic resolution. Both catalases are tetrameric proteins with deeply buried heme groups. The amino acid sequence of beef liver catalase is known and contains (at least) 506 amino acid residues. Although the sequence of P. vitale catalase has not yet been determined chemically, 670 residues have been built into the 2 A resolution electron density map and have been given tentative assignments. A large portion of each catalase molecule (91% of residues in beef liver catalase and 68% of residues in P. vitale catalase) shows structural homology. The root-mean-square deviation between 458 equivalenced C alpha atoms is 1.17 A. The dissimilar parts include a small fragment of the N-terminal arm and an additional "flavodoxin-like" domain at the carboxy end of the polypeptide chain of P. vitale catalase. In contrast, beef liver catalase contains one bound NADP molecule per subunit in a position equivalent to the chain region, leading to the flavodoxin-like domain, of P. vitale catalase. The position and orientation of the buried heme group in the two catalases, relative to the mutually perpendicular molecular dyad axes, are identical within experimental error. A mostly hydrophobic channel leads to the buried heme group. The surface opening to the channel differs due to the different disposition of the amino-terminal arm and the presence of the additional flavodoxin-like domain in P. vitale catalase. Possible functional implications of these comparisons are discussed.

Headspace gas chromatographic-mass spectrometric analysis of light hydrocarbons and volatile organosulphur compounds in reduced-pressure cultures of Clostridium.

A static headspace gas chromatographic method for the simultaneous separation of trace light hydrocarbons and volatile organosulphur compounds in gases of nineteen Clostridium cultures at reduced pressure is described. The separation was achieved on n-octane-Porasil C after sampling of the gaseous compounds in a PTFE loop without any pretreatment. Most peaks were identified by gas chromatography-mass spectrometry. The presence of methane and ethylene sulphide among Clostridium volatiles is confirmed and 3-methyl-1-butene, 2-methyl-2-butene, dimethyl trisulphide and S-methyl thioacetate are reported for the first time in the Clostridium group.

[Differentiation of the main species of Clostridium by gas chromatography]. / Differentsiatsiia osnovnykh vidov klostridii metodom gazovoi khromatografii.

For the first time in the USSR the properties of microorganisms of the genus Clostridium have been studied with the use of the gas-chromatographic techniques. The analysis of the quantitative and qualitative composition of extracellular alcohols and carboxylic acids in 99 museum and newly isolated strains of 18 Clostridium species has made it possible to classify these microorganisms with 7 sharply differing groups. The above techniques permit the classification of clostridia with one of the groups within 2 hours if the microbial cultures have been grown in glucose-containing peptone yeast medium.

Gas chromatography/mass spectrometry of bacterial amines.

Bacterial amines were examined by gas chromatography/mass spectrometry. Under electron impact all trifluoroacetamides exhibited peaks at m/z 69 due to [CF3]+. Many trifluoroacetamides also showed peaks at m/z 97 corresponding to the [COCF3]+ ion fragment. The spectra of n-alkyl and aralkyl trifluoroacetamides were consistent with the spectra and their interpretations in the earlier literature. Molecular ions were of low abundance for all alkyl trifluoroacetamides having alkyl chains longer than two carbon atoms. Chemical ionization gave molecular weight information in all cases. Most peaks observed were molecular addition products, e.g. [M + H]+ and [M + NH4]+. Application of chemical ionization mass spectrometry to analysis of bacterial amines revealed the production of beta-phenylethylamine, n-decylamine, 1,4-diaminobutane and 1,5-diaminopentane by Clostridium histolyticum; whereas both Clostridium bifermentans and Clostridium oedematiens produced beta-phenylethylamine. The latter organism also produced a peak with a retention time similar to that of an authentic amylamine derivative.

Chromatographic separation of extruded iron-sulfur cores from the apoproteins of Clostridium pasteurianum and spinach ferredoxins in aqueous Triton X-100/urea.

Iron-sulfur core extrusions from spinach [( 2Fe-2S]) and Clostridium pasteurianum (2[4Fe-4S]) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively [FenSn(SPh)4]2- with n = 2 and n = 4, respectively. The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose. Essentially quantitative recovery of [FenSn (SPh)4]2- is achieved in the eluate. The apoprotein remaining on the column can be eluted with 0.5 M NaCl. Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent. Apoprotein recovery is comparable to that obtained by other chromatographic methods. At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted. The procedures developed in this work are potentially most applicable to selective removal of [2Fe-2S] and [4Fe-4S] centers from a multicenter enzyme without irreversible denaturation.

Characterization of [4Fe-4S]2+, [4Fe-4Se]2+ and hybrid (S, Se) clusters in Clostridium pasteurianum ferredoxin. A resonance Raman study.

Resonance Raman spectra of native 2[4Fe-4S] ferredoxin from Clostridium pasteurianum and of its selenium-substituted analogue 2[4Fe-4Se] are compared. The experimental conditions used in this study included low temperature (approx. 25K) and the absence of a glass sample cell, thus ensuring high signal-to-noise ratios. The spectra of the 2[4Fe-4S] and 2[4Fe-4Se] ferredoxins display similar numbers of bands, but the resonance Raman patterns differ largely, except for two bands observed at 353 cm-1 and 365 cm-1 in spectra of the native ferredoxin, which are only moderately shifted upon S----Se substitution and are attributed to Fe-S(cysteine) stretching modes. The activities of the latter modes, enhanced by 457.9 nm excitation, are nearly equal in both ferredoxins. These data were used to demonstrate the presence of hybrid clusters [4Fe-(4-n)S-nSe] (n = 1, 2, 3) in a ferredoxin the active sites of which had been reconstituted in the presence of both S and Se.

Polyacrylamide gel electrophoresis patterns produced by Clostridium difficile.

Clostridium difficile is a major cause of pseudomembranous colitis following antimicrobial therapy. There is evidence to suggest that this organism may be hospital acquired. Polyacrylamide gel electrophoretic (PAGE) analysis of protein profiles of C. difficile cell extracts was examined for possible usefulness in epidemiologic studies. At least 50 bands could be distinguished in soluble cell extracts of C. difficile. Freeze-thawing of extracts and/or length of storage time did not affect the protein profiles. While all strains tested were nearly identical, several strains were unique in their lack of a 34,000-dalton polypeptide. Protein patterns of C. difficile could easily be distinguished from those of Clostridium sordellii. Additionally, surface antigens extracted from several strains of C. difficile and from a few strains of C. sordellii revealed marked differences. Ethylenediaminetetraacetate (EDTA) extracts from C. difficile showed two or three major bands in PAGE analysis; strains could be divided into two major subgroups on the basis of band distribution. EDTA extracts from C. sordellii bore no similarity to those of C. difficile.

Structural differences between [2Fe-2S] clusters in spinach ferredoxin and in the "red paramagnetic protein" from Clostridium pasteurianum. A resonance Raman study.

The [2Fe-2S] ferredoxin ("Red paramagnetic protein", RPP) from C. pasteurianum has been found to be composed of two identical subunits of 10,000 +/- 2 000 daltons, each containing a [2Fe-2S] cluster. Resonance Raman (RR) spectra of RPP have been obtained at 23 degrees K, and compared to those of spinach ferredoxin (Sp Fd). Ten modes of the [2Fe-2S] chromophore were observed in the 100-450 cm-1 range. Assignments of non fundamental modes in the 500-900 cm-1 range allowed correlations between fundamental stretching modes of RPP and Sp Fd. Although assuming a [2Fe-2S] structure, the chromophore of RPP differs from that of Sp Fd by its conformation and by a slight weakening of Fe-S bonds, involving both the inorganic core and the cysteine ligands.