Clostridium Specificity

Acid producers. Many bacteria produce acids. Acids may be organic or inorganic depending on the specific bacterium. In either case, the acids produced lower the pH, usually accelerating attack. Although many kinds of bacteria may generate acids, Thiobacillus thiooxidans and Clostridium species have most often been linked to accelerated corrosion on steel. 

Small GTPases of the Rho family are ADP-ribosylated (e.g., at Asn4l of RhoA) and inactivated by C3-like toxins from Clostridium botulinum, Clostridium limosum, and Staphylococcus aureus. These proteins have a molecular mass of 23-30 kDa and consist only of the enzyme domain. Specific inhibition of Rho functions (Rho but not Rac or Cdc42 are targets) is the reason why C3 is widely used as a pharmacological tool [2]. 

Important members of this toxin family are Clostridium difficile toxins A and B, which are implicated in antibiotics-associated diarrhea and pseudomembranous colitis. The large clostridial cytotoxins are single-chain toxins with molecular masses of 250-308 kDa. The enzyme domain is located at the N terminus. The toxins are taken up from an acidic endosomal compartment. They glucosylate RhoA at Thr37 also, Rac and Cdc42 are substrates. Other members of this toxin family such as Clostridium sordellii lethal toxin possess a different substrate specificity and modify Rac but not Rho. In addition, Ras subfamily proteins (e.g., Ras, Ral, and Rap) are modified. As for C3, they are widely used as tools to study Rho functions [2] [4]. 

The most ingenious exocytosis toxins, however, come from the anaerobic bacteria Clostridium botulinum and Clostridium tetani. The former produces the seven botulinum neurotoxins (BoNTs) A-G the latter produces tetanus neurotoxin (TeNT). All eight toxins consist of a heavy (H) chain and a light (L) chain that are associated by an interchain S-S bond. The L-chains enter the cytosol of axon terminals. Importantly, BoNT L-chains mainly enter peripheral cholinergic terminals, whereas the TeNT L-chain mainly enters cerebral and spinal cord GABAergic and glycinergic terminals. The L-chains are the active domains of the toxins. They are zinc-endopeptidases and specifically split the three core proteins of exocytosis, i.e. the SNAREs (Fig. 1 inset). Each ofthe eight toxins splits a... 

The mechanism whereby the bacteria produce the disease with its attendant symptoms is often due to the cells ability to produce specific poisons, toxins or aggressins (Chapter 14). Many of these are tissue-destroying enzymes which can damage the cellular structure ofthe body or destroy red blood cells. Others (neurotoxins) are highly specific poisons ofthe central nervous system, for example the toxin produced by Clostridium botulinum is, weight for weight, one ofthe most poisonous substances known. 

Although it had been assumed that only hypoxanthine dehydrogenase is required for the conversion of hypoxanthine (6-hydroxypurine) into uric acid, in Clostridium purinolyti-cum, two enzymes, both of which contain a selenium cofactor, are required. The enzymes differ in the molecular mass of their subunits, in their terminal amino acid sequences, in their kinetic parameters, and in their specific activities for purines (Self and Stadman 2000). Purine hydroxylase converts purine into hypoxanthine and xanthine (2,6-dihy-droxypurine), which is then further hydroxylated to uric acid (2,6,8-trihydroxypurine) by xanthine dehydrogenase (Self 2002). 

All earlier studies [155-158] reported the complexation of berberine with calf thymus DNA and suggested by a mechanism of intercalation. Maiti and coworkers [159-162] demonstrated first the base- and sequence-specificity of berberine from studies with several naturally occurring DNAs (Clostridium perfringenes, cholera bacteriophage 02, calf thymus, Escherichia coli, Micrococcus lysodeikticus) and synthetic DNAs ((poly(dG-dC) poly(dG-dC), poly(dG)-poly(dC), poly(dA-dT) poly(dA-dT), poly(dA)-poly(dT)) using various physicochemical techniques. Several aspects of the interaction were reported ... 

The crystallographic structure of rubredoxin from Clostridium pasteurianum at 2.5 A, a resolution sufficient to reveal the sequence of several of the bulky amino acid side chains, shows the iron coordinated to two pairs of cysteine residues located rather near the termini of the polypeptide chain (Fig. 1). A related rubredoxin, with a three times larger molecular weight, from Pseudomonas oleovorans is believed to bind iron in a similar fashion. This conclusion is based on physical probes, especially electron paramagnetic resonance spectroscopy, all of which indicate that the iron is in each case situated in a highly similar environment however, the proteins display some specificity in catalytic function. 

Diarrhea is a well-known complication of antibiotic therapy. Rates of antibiotic-associated diarrhea (AAD) vary from 5 to 25%. Some antibiotics are more likely to cause diarrhea than others, specifically, those that are broad spectrum and those that target anaerobic flora. This paper reviews the effects of antibiotics on the fecal flora as well as host factors which contribute to AAD. Clinical features and treatment of AAD are also described. Prevention of AAD rests on wise antibiotic policies, the use of probiotics and prevention of acquisition in the hospital setting. Data from clinical trials suggest that poorly absorbed antimicrobials might have a decreased risk of causing AAD and Clostridium difficile-associated disease, as concluded from studies of antibiotics used for preoperative bowel decontamination and poorly absorbed antibiotics used for traveler s diarrhea. Controlled trials would prove this but are not yet available. Probiotics may be a good adjunct to poorly absorbed antibiotics to minimize the risk of diarrhea associated with antibiotics. 

Selenomethionine, CH3SeCH2CH2CHNH2COOH, 50 (Scheme 17) is also incorporated non-specifically in proteins of Clostridium kluyveri126 and in yeast127,128 apparently taking the place of methionine however, no specific biological incorporation of Se-methionine has been found.105... 

Specificity against toxins of type A, B or E Clostridium botulinum... 

Antibodies raised against venom of various spiders Specificity against toxin of Clostridium tetani Specificity against toxin of C. tetani Antibodies against tick-borne encephalitis virus Specificity for causative agent of chicken pox... 

By using the same experimental procedure, the action pattern of pectinesterase produced by Clostridium multifermentans48 was examined. As none of the separation procedures used were suitable for removing the pectinesterase from exopectate lyase,51 a specific lyase was obtained by inactivating the pectinesterase by heating for 30 minutes at 38° and pH 7.0 under these conditions, the activity of the lyase was retained. All the pectinesterase preparations used were contaminated with the lyase, which could not be differentially inac-... 

The detection of flu viruses via a fluorescent sandwich immunoassay was reported by Bucher.(10) However, the method sensitivity was too low for direct detection of the virus. A novel sandwich immunoassay was described by Ogcr((lff7 for the detection of Botulinum Toxin A. Antibodies specific for Clostridium botulinum were covalently attached to the surface of a tapered fiber. After the capture of the antigen, a sandwich was formed with a rhodamine-labeled anti-toxin IgG, and the evanescent wave was measured. The assay was highly specific with detection limits near 5 ppb. 

Classical bacterial exotoxins, such as diphtheria toxin, cholera toxin, clostridial neurotoxins, and the anthrax toxins are enzymes that modify their substrates within the cytosol of mammalian cells. To reach the cytosol, these toxins must first bind to different cell-surface receptors and become subsequently internalized by the cells. To this end, many bacterial exotoxins contain two functionally different domains. The binding (B-) domain binds to a cellular receptor and mediates uptake of the enzymatically active (A-) domain into the cytosol, where the A-domain modifies its specific substrate (see Figure 1). Thus, three important properties characterize the mode of action for any AB-type toxin selectivity, specificity, and potency. Because of their selectivity toward certain cell types and their specificity for cellular substrate molecules, most of the individual exotoxins are associated with a distinct disease. Because of their enzymatic nature, placement of very few A-domain molecules in the cytosol will normally cause a cytopathic effect. Therefore, bacterial AB-type exotoxins which include the potent neurotoxins from Clostridium tetani and C. botulinum are the most toxic substances known today. However, the individual AB-type toxins can greatly vary in terms of subunit composition and enzyme activity (see Table 2). 

Clostridium sticklandii also expresses a proline reductase that can reduc-tively cleave proline to 8-aminovalerate (Seto and Stadtman 1976). PR was first purified by Seto and Stadtman (1976) by following the decomposition of proUne in the presence of dithiothreitol or NADH. They found PR to have a denatured mass of approximately 30kDa (sodium dodecyl sulfate-polyacrylomide gel electrophoresis SDS-PAGE) and a native size of approximately 300 kDa. The addition of selenite to the growth medium of C sticklandii did increase the specific activity of PR in extracts by threefold however, no selenium was detected in the purified enzyme. It should be noted that this purified enzyme had lost the ability to couple reduction of proline to NADH and thus probably was missing one or more components of the complete enzyme complex. 

Within the cellulosome complex, type I dockerin domain is responsible for incorporating its associated glycosyl hydrolase in the bacterial cellulosome via interaction with a reception domain, the cohesin domain. The three-dimensional solution structure of the 69-residue dockerin domain from the thermophilic Clostridium thermocellum (Topt = 55-65 °C) was solved by NMR and was found to consist of two Ca " -binding loop-helix motifs connected by a linker. Each Ca " -binding subdomain is stabilized by a cluster of buried hydrophobic sidechains. Recently, the NMR sequence-specific resonance assignment of type II cohesin module from C. thermocellum has been published. ... 

Further differentiation was obtained using the chromophoric cel-looligodextrins and a rapid and sensitive HPLC analysis of the reaction products (1). Specific degradation patterns were obtained for several enzymes such as those from Clostridium cloned in E. coli (Fig. 2). 

Figure 2. Specificities of Endoglucanases (EGA, EGB, EGC, EGD) from Clostridium thermocellum cloned in E. coli (10). The substrates (MeUmb-Glc , n = 2-5, MeUmbLac) are depicted (symbols A, (3-1,4 galactopyra-nosyl , / -1,4 glucopyranosyl , 4-methylumbelliferyl) and the arrows indicate scission points as determined by HPLC (1).

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