Nitrite and nitrosyl compounds in food preservation.
(17/2206)
Nitrite is consumed in the diet, through vegetables and drinking water. It is also added to meat products as a preservative. The potential risks of this practice are balanced against the unique protective effect against toxin-forming bacteria such as Clostridium botulinum. The chemistry of nitrite, and compounds derived from it, in food systems and bacterial cells are complex. It is known that the bactericidal species is not nitrite itself, but a compound or compounds derived from it during food preparation. Of a range of nitrosyl compounds tested, the anion of Roussin's black salt [Fe4S3(NO)7]- was the most inhibitory to C. sporogenes. This compound is active against both anaerobic and aerobic food-spoilage bacteria, while some other compounds are selective, indicating multiple sites of action. There are numerous possible targets for inhibition in the bacterial cells, including respiratory chains, iron-sulfur proteins and other metalloproteins, membranes and the genetic apparatus. (+info)
Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium.
(18/2206)
Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD+ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h-1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase. (+info)
Three surface layer homology domains at the N terminus of the Clostridium cellulovorans major cellulosomal subunit EngE.
(19/2206)
The gene engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been isolated and sequenced. engE is comprised of an open reading frame (ORF) of 3,090 bp and encodes a protein of 1,030 amino acids with a molecular weight of 111,796. The amino acid sequence derived from engE revealed a structure consisting of catalytic and noncatalytic domains. The N-terminal-half region of EngE consisted of a signal peptide of 31 amino acid residues and three repeated surface layer homology (SLH) domains, which were highly conserved and homologous to an S-layer protein from the gram-negative bacterium Caulobacter crescentus. The C-terminal-half region, which is necessary for the enzymatic function of EngE and for binding of EngE to the scaffolding protein CbpA, consisted of a catalytic domain homologous to that of family 5 of the glycosyl hydrolases, a domain of unknown function, and a duplicated sequence (DS or dockerin) at its C terminus. engE is located downstream of an ORF, ORF1, that is homologous to the Bacillus subtilis phosphomethylpyrimidine kinase (pmk) gene. The unique presence of three SLH domains and a DS suggests that EngE is capable of binding both to CbpA to form a CbpA-EngE cellulosome complex and to the surface layer of C. cellulovorans. (+info)
Naturally occurring Tyzzer's disease in a calf.
(20/2206)
Naturally occurring Clostridium piliforme infection (Tyzzer's disease) was found in a calf. Light microscopic examination revealed multifocal coagulative necrosis in the liver, catarrhal gastroenteritis, tracheitis and pneumonia, and thymic atrophy. Warthin-Starry staining clearly showed large filamentous bacilli in bundles or criss-cross patterns within the hepatocytes and epithelium and smooth muscle cells of the ileum and cecum. Immunohistochemistry using anti-C. piliforme RT and MSK strain antisera showed positive reaction against the bacilli. Electron microscopic examination revealed bacilli within the hepatocytes that demonstrated a characteristic vegetative form, with peritrichous flagella, and spores. The polymerase chain reaction (PCR) study using the paraffin-embedded liver sections, the 196-bp DNA fragment specific to 16S ribosomal RNA of C. piliforme was amplified. The characteristics of these bacilli are consistent with those of of C. piliforme. The PCR technique using paraffin-embedded sections should be useful for confirming C. piliforme infection in spontaneous cases. (+info)
Chemical rescue of the catalytically disabled clostridial glutamate dehydrogenase mutant D165S by fluoride ion.
(21/2206)
The catalytically disabled Asp165-->Ser mutant of clostridial glutamate dehydrogenase shows 100000-fold less activity than the wild-type (WT) enzyme in a standard glutamate oxidation assay and 1000-fold less activity in the reductive-amination reaction. The large reduction in the rate has been attributed to removal of the negative charge and the postulated proton-donor capacity of the aspartate carboxyl group. However, fluoride ion (1 M NaF) causes a 1000-fold activation of the mutant enzyme while simultaneously inhibiting WT activity by 20-fold in the forward reaction. For the reverse reaction, F- (1 M) activates the mutant 4-fold and inhibits WT activity to approx. 64%. The net result when 1 M F- is present is a decrease in the WT:mutant activity ratio from 100000 to 5 for the forward reaction. None of the other halides tested, nor NO3(-), CHCOO- or HCOO-, give comparable activation. Re-activation took 15-30 s under assay conditions, suggesting the possibility of conformational change; CD spectroscopy, however, provided no evidence of a substantial change and kinetics of modification using 5,5'-dithiobis(2-nitrobenzoic acid) suggested only subtle structural rearrangement. This phenomenon is discussed in the light of available information about the structure of the mutant enzyme. It is suggested that the F- ion provides a fixed negative charge at the position of the missing aspartate carboxyl group. Therefore, this appears to be an example of 'chemical rescue'. (+info)
Incorporation of molybdenum into the iron-molybdenum cofactor of nitrogenase.
(22/2206)
The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99Mo to follow the incorporation of Mo into precursors. 99Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis. 99Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99Mo on this protein required nucleotide. (+info)
Digestion of crystalline cellulose substrates by the clostridium thermocellum cellulosome: structural and morphological aspects.
(23/2206)
The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valonia ventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Ialpha/Ibeta composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its 'commitment' towards complete degradation. (+info)
Evidence that MgATP accelerates primary electron transfer in a Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein nitrogenase tight complex.
(24/2206)
The nitrogenase catalytic cycle involves binding of the iron (Fe) protein to the molybdenum-iron (MoFe) protein, transfer of a single electron from the Fe protein to the MoFe protein concomitant with the hydrolysis of at least two MgATP molecules, followed by dissociation of the two proteins. Earlier studies found that combining the Fe protein isolated from the bacterium Clostridium pasteurianum with the MoFe protein isolated from the bacterium Azotobacter vinelandii resulted in an inactive, nondissociating Fe protein-MoFe protein complex. In the present work, it is demonstrated that primary electron transfer occurs within this nitrogenase tight complex in the absence of MgATP (apparent first-order rate constant k = 0.007 s-1) and that MgATP accelerates this electron transfer reaction by more than 10,000-fold to rates comparable to those observed within homologous nitrogenase complexes (k = 100 s-1). Electron transfer reactions were confirmed by EPR spectroscopy. Finally, the midpoint potentials (Em) for the Fe protein [4Fe-4S]2+/+ cluster and the MoFe protein P2+/N cluster were determined for both the uncomplexed and complexed proteins and with or without MgADP. Calculations from electron transfer theory indicate that the measured changes in Em are not likely to be sufficient to account for the observed nucleotide-dependent rate accelerations for electron transfer. (+info)