J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 307 - 13 Orf5/SolR: a transcriptional repressor of the sol operon of Clostridium acetobutylicum? Thormann K, Durre P. The gene of Orf5 (SolR) of Clostridium acetobutylicum DSM 792 was subcloned and overexpressed in Escherichia coli . The protein was purified with Ni-NTA agarose and used for DNA binding assays . No DNA binding of Orf5 to regions upstream of the sol operon from C . acetobutylicum was observed . Overexpression of Orf5 in C . acetobutylicum led to a change in the organism's pattern of glycosylated exoproteins . The Orf5 protein was localized in the cell membrane fraction and to a small extent in the supernatant medium . Based on these results Orf5 (SolR) appears not to act as a transcriptional repressor in C . acetobutylicum, but instead may be an enzyme involved in glycosylation or deglycosylation. J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 298 - 306 Characterization of a maltose transport system in Clostridium acetobutylicum ATCC 824; Tangney M et al.; The utilization of maltose by Clostridium acetobutylicum ATCC 824 was investigated . Glucose was used preferentially to maltose, when both substrates were present in the medium . Maltose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on maltose, but not glucose or sucrose, as the sole carbon source . Extract fractionation and PTS reconstitution experiments revealed that the specificity for maltose is contained entirely within the membrane in this organism . A putative gene system for the maltose PTS was identified (from the C . acetobutylicum ATCC 824 genome sequence), encoding an enzyme II(Mal) and a maltose 6-phosphate hydrolase. J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 292 - 7 ABE production from corn: a recent economic evaluation; Qureshi N et al.; This article details an economic assessment of butanol production from corn using the newly developed hyper-butanol-producing strain of Clostridium beijerinckii BA101 . Butanol is produced in batch reactors and recovered by distillation . For a plant with 153,000 metric tons of acetone, butanol, and ethanol (ABE) production capacity, the production equipment cost and total working capital cost is US$33.47x10(6) and US$110.46x10(6), respectively . Based on a corn price (C(p)) of US$79.23 x ton(-1) (US$2.01 x bushel(-1)), an ABE yield of 0.42 (g ABE/g glucose) butanol price is projected to be US$0.34 x kg(-1) . An improved yield of 0.50 will reduce this price to US$0.29 x kg(-1) . Assumptions, such as by-product credit for gases and complete conversion of corn steep liquor (CSL) to fermentation by-products, have been taken into consideration . An increased price of corn to US$197.10 x ton(-1) would result in a butanol price of US$0.47 x kg(-1) . A grass-rooted plant would result in a butanol price of US$0.73 x kg(-1) (C(p) US$79.23 x ton(-1)) . In a worst case scenario, the price of butanol would increase to US$1.07 x kg(-1) (C(p) 197.10 x ton(-1) for a grass-rooted plant and assuming no credit for gases) . This is based on the assumption that corn price would not increase to more than US$197.10 x ton(-1). J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 287 - 91 Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101; Qureshi N et al.; This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone-butanol-ethanol, ABE) at elevated levels . This organism expresses high levels of amylases when grown on starch . C . beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27-29 g x l(-1) . C . beijerinckii BA101 has been characterized for both substrate and butanol inhibition . Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g x l(-1) . The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors . Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C . beijerinckii BA101, and solvent production as high as 165 g x l(-1) has been achieved . Immobilization of C . beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g x l(-1) x h(-1) . Recent economic studies employing C . beijerinckii BA101 suggested that butanol can be produced at US$0.20-0.25 x lb(-1) by employing batch fermentation and distillative recovery . Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 281 - 6 Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii; Chen JS et al.; Several solvent-producing clostridia, including Clostridium acetobutylicum and C . beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of 15N2 into cellular material . The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron-molybdenum cofactor (FeMoco) of nitrogenase, have now been identified in C . acetobutylicum or C . beijerinckii or both . The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifVomega and nifValpha genes . The corresponding nif genes of these three clostridial species are highly related to each other . However, in the two solvent-producing clostridia, the nifH and nifD genes are interspersed by two glnB-like genes, which are absent in the corresponding region in C . pasteurianum . However, the nifN-B and nifVomega genes of C . pasteurianum are interspersed by the putative modA and modB genes (for molybdate transport), which are absent in the corresponding region in C . acetobutylicum . C . acetobutylicum and C . beijerinckii grew well under nitrogen-fixing conditions, and the acetylene-reducing activity of nitrogenase was measured in the two species . Acetone, butanol, and isopropanol production occurred in nitrogen-fixing cultures, but the peak of nitrogen-fixing activity preceded the active solventogenic phase. J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 271 - 4 Electrotransformation studies in Clostridium cellulolyticum; Tardif C et al.; Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays . Electrotransformation was then performed under optimized conditions (6 to 7.5 kV x cm(-1) field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer) . Transformants were only obtained when 7 or 7.5 kV x cm(-1) pulses were applied . Transformation efficiencies evaluated from the growth curves of transformed cells were between 10(5) and 10(7) transformants per microgram of plasmid DNA for five different replicon-based plasmids . Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain . Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively. Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 660 - 6 Sequence of celQ and properties of celQ, a component of the Clostridium thermocellum cellulosome; Arai T et al.; The nucleotide sequence of the Clostridium thermocellum F1 celQ gene, which codes for the endoglucanase CelQ, consists of 2,130 bp encoding 710 amino acids . The precursor form of CelQ has a molecular weight of 79,809 and is composed of a signal peptide, a family 9 cellulase domain, a family IIIc carbohydrate-binding module (CBM), and a dockerin domain . Truncated derivatives of CelQ were constructed: CelQdeltadoc consisted of the catalytic domain and the CBM; CelQcat consisted of the catalytic domain only . CelQdeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan and low activity toward Avicel, acid-swollen cellulose, lichenan, and xylan . The Vmax and Km values were 235 micromol/min/mg and 3.3 mg/ml, respectively, for CMC . By contrast, CelQcat, which was devoid of the CBM, showed negligible activity toward CMC, i.e., about 1/1,000 of the activity of CelQdeltadoc, supporting the previously proposed idea that family IIIc CBMs participate in the catalytic function of the enzyme . Immunological analysis using an antiserum raised against CelQdeltadoc confirmed that CelQ is a component of the C . thermocellum cellulosome. Presse Med, 2001 Dec 8, 30(37), 1825 - 6 {Clostridium difficile bacteremia}; Duthilly A et al.; BACKGROUND: Extra-digestive manifestations of Clostridium difficile infection are very uncommon . Exceptional cases of C . difficile bacteremia or severe sepsis have been described in intensive care patients, demonstrating the capacity of this agent to generate generalized infection . CASE REPORT: C . difficile bacteremia occurred in a 66-year-old immunodepressed patient treated for acute myeloblastic leukemia . Bacteremia was associated with a abscess of the anal margin . Outcome was favorable after treatment with metronidosole . DISCUSSION: Clostridium difficile is generally selected by prior antibiotic treatment . It is the principal agent of nosocomial diarrhea . In immunodepressed patients, systemic dissemination is a rare but possible development. Chin Med J (Engl), 2000 Sep, 113(9), 858 - 61 Anaerobic bacteria and intrahepatic stones: detections of Clostridium sp . and Bacteroides fragilis; Liu Y et al.; OBJECTIVE: To detect anaerobic bacteria Clostridium sp . and Bacteroides fragilis in intrahepatic stones by molecular genetic method . METHODS: DNA was extracted from 59 stone samples and subjected to polymerase chain reaction (PCR) amplification targeting the 16S rRNA gene of Clostridium sp . and the glutamine synthetase gene of Bacteroides fragilis . Single-strand conformational polymorphism (SSCP) analysis was performed to identify the Clostridium sp . RESULTS: 16S rRNA gene sequences for Clostridium sp . were identified in 49 stones (83%, 49/59) . The two most common groups were detected in 19 (41%) and 17 (37%) of the 46 samples using SSPC analysis, and 25/59 (42%) stones were tested positive for Bacteroides fragilis . CONCLUSIONS: Anaerobes such as Clostridium sp . and Bacteroides fragilis present in intrahepatic stones and may play a role in stone formation . PCR is a useful technique to detect fastidious pathogens, which are difficult to culture . SSCP of PCR products is a rapid method in differentiating bacterial species. Clin Infect Dis, 2002 Feb 1, 34(3), 346 - 53 Epub 2001 Dec 17. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile; Kyne L et al.; A total of 271 patients were prospectively followed up to determine whether patients whose hospital stay is complicated by diarrhea due to Clostridium difficile experience differences in cost and length of stay and survival rates when compared with patients whose stay is not complicated by C . difficile-associated diarrhea . Forty patients (15%) developed nosocomial C . difficile-associated diarrhea . These patients incurred adjusted hospital costs of $3669--that is, 54% (95% confidence interval {CI}, 17%-103%)--higher than patients whose course was not complicated by C . difficile-associated diarrhea . The extra length of stay attributable to C . difficile-associated diarrhea was 3.6 days (95% CI, 1.5-6.2) . C . difficile-associated diarrhea was not associated with excess 3-month or 1-year mortality after adjustment for age, comorbidity, and disease severity . On the basis of the findings of this study, a conservative estimate of the cost of this disease in the United States exceeds $1.1 billion per year. Obstet Gynecol Surv, 2002 Jan, 57(1), 53 - 7 Diagnosis and management of clostridium perfringens sepsis and uterine gas gangrene; Halpin TF et al.; The progression of Clostridium perfringens endomyometritis to gas gangrene is a rare, but greatly feared complication in the obstetrical patient . While endometritis following cesarean delivery is a common complication, recognition of C . perfringens as the pathogen as well as its progression to gas formation in the myometrium is essential to the survival of the patient . We present a patient that we recently cared for, and review the bacteriology, clinical diagnosis, and management. J Clin Microbiol, 2002 Jan, 40(1), 101 - 4 Molecular fingerprinting of Clostridium difficile isolates: pulsed-field gel electrophoresis versus amplified fragment length polymorphism; Klaassen CH et al.; Two molecular fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP), were used to investigate the epidemiological relatedness among Clostridium difficile isolates from suspected outbreaks in three general hospitals . Analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation . Although this degradation could be prevented to a certain extent by the inclusion of thiourea in the electrophoresis buffer, the weak DNA banding patterns obtained in this way were still far from optimal . AFLP data were obtained by using fluorescently labeled PCR primers and analysis on an ABI PRISM automated DNA analysis platform . AFLP analysis yielded high resolution and highly reproducible DNA fingerprinting patterns from which the epidemiological relatedness among the isolates could easily be determined . AFLP results could be readily obtained within 24 h, whereas 3 to 4 days were routinely required to complete the lengthy PFGE protocol . AFLP clearly proved to be a much more fail-safe fingerprinting method for C . difficile isolates, especially for those isolates for which a standard PFGE procedure yielded inconclusive results due to DNA degradation. Appl Environ Microbiol, 2002 Jan, 68(1), 53 - 8 Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering; Guedon E et al.; Cellulolytic clostridia have evolved to catabolize lignocellulosic materials at a seasonal biorhythm, so their biotechnological exploitation requires genetic improvements . As high carbon flux leads to pyruvate accumulation, which is responsible for the cessation of growth of Clostridium cellulolyticum, this accumulation is decreased by heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis . In comparison with that of the wild strain, growth of the recombinant strain at the same specific rate but for 145 h instead of 80 h led to a 150% increase in cellulose consumption and a 180% increase in cell dry weight . The fermentation pattern was shifted significantly: lactate production decreased by 48%, whereas the concentrations of acetate and ethanol increased by 93 and 53%, respectively . This study demonstrates that the fermentation of cellulose, the most abundant and renewable polymer on earth, can be greatly improved by using genetically engineered C . cellulolyticum. J Endod, 2001 Dec, 27(12), 730 - 3 Antibacterial activity of fifth-generation dentin bonding systems; Atac AS et al.; Past concepts that the pulp does not become infected until an actual carious exposure takes place have been challenged . The antibacterial effects of the dentin bonding systems Single Bond, Prime&Bond NT, and Excite were evaluated using the bacteria Streptococcus mutans ATCC 25175, Streptococcus intermedius, Lactobacillus acidophilus, Prevotella oris, Prevotella bivia, Prevotella denticola, Porphyromonas gingivalis, Porphyromonas endodontalis, and Clostridium ramosum with a disk diffusion method . Chlorhexidine (0.2%) was used as a positive control . After incubation zones of inhibited bacterial growth were measured . Prime&Bond NT showed growth inhibition for all bacterial strains . Lactobacillus acidophilus and Streptococcus mutans were remarkably resistant to Single Bond, whereas EX produced no inhibitory effect on Porphyromonas endodontalis, although the adhesive produced the maximum halo inhibition to Streptococcus mutans (15+/-1 mm), showing an antibacterial effect closest to chlorhexidine . The variety of results obtained in this study suggest that antibacterial properties of current dentin adhesives may depend on components that are originally incorporated to promote adhesion. J Food Prot, 2001 Dec, 64(12), 1956 - 60 Occurrence of Clostridium perfringens in the broiler chicken processing plant as determined by recovery in iron milk medium; Craven SE; Over 30 years ago, Clostridium perfringens was reported as a contaminant of the processing plant and processed carcasses of broiler chickens . Poultry processing procedures and methods for detecting C . perfringens have changed since that time . Therefore, a study was conducted to determine the incidence and numbers of C . perfringens in the water of the scald tank, the water of the chill tank, and the rinse water of the processed carcasses from modern broiler chicken processing plants . In trial 1, collected samples were inoculated into iron milk medium (IMM) and incubated at 46 degrees C for 18 h (the traditional method) or at 37 degrees C for 3 h followed by incubation at 46 degrees C for 15 h (an injury recovery method) . Each of three preselected broiler chicken flocks from two integrators were the first processed for that processing shift . The overall incidence of confirmed C . perfringens in samples associated with the three flocks was 40% of postprocessing scald water samples, 13% of preprocessing chill water samples, 13% of postprocessing chill water samples, and 19% of carcass rinses . The incidence of C . perfringens in samples incubated in IMM using the injury recovery procedure was significantly higher than in samples incubated in IMM by the traditional method, but only when all samples associated with the three flocks were pooled . In trial 2, water samples from each tank of a three-tank counterflow scalder, water samples from the prechill and chill tank, and samples of carcass rinses were collected in the middle of a processing shift during multiple visits to a processing plant . Samples were inoculated into IMM with neomycin and polymyxin B sulfate (IMMA) and incubated using the traditional and injury recovery procedures . The incidence of C . perfringens in water samples was 100% from scald tank 1, 100% from scald tank 2, 100% from scald tank 3, 88% from the prechill tank, and 63% from the chill tank . The incidence in carcass rinse samples was 67% . The mean most probably number (MPN) of C . perfringens for contaminated samples decreased from log10 5.07/100 ml of water in scald tank 1 to log10 1.26/100 ml of water in the chill tank . The mean MPN in carcass rinse samples was log10 1.20 C . perfringens per 100 ml . The incidence and mean MPN of C . perfringens in these samples after heat shock at 75 degrees C for 20 min was somewhat less, but high enough to indicate that much of the contamination arises from heat-resistant spores of this organism . In trial 2, there were no differences in incidence and MPN of C . perfringens in samples incubated in IMMA with the traditional method or the injury recovery method. DNA Seq, 2001, 12(2), 115 - 20 ADP-ribosylating binary toxin genes of Clostridium difficile strain CCUG 20309; Chang SY et al.; The cdt genes that encode a binary ADP-ribosylating toxin in Clostridium difficile were first characterized from a toxigenic C . difficile strain CD196 in 1997 . We report here C . difficile strain CCUG 20309 (ATCC 8864), a strain that produces toxin B but not toxin A, also carry a complete set of cdtA and cdtB genes . These genes were sequenced by cycle sequencing method . The 2 ORFs and the intergenic sequences of these 2 strains have a homology of 99.6% . Interestingly, 9 extra bases were found within the cdtA gene of strain CCUG 20309 which do not affect the downstream region of the ORF . Using the same homologous primers, the highly toxigenic reference strain VPI 10463 was found to carry only parts of the 2 ORFs while a nontoxigenic strain ATCC 8884 does not carry any of the cdt genes . Though it remains to be determined whether these genes are expressed, it is significant that strain CCUG 20309 contains the complete set of cdt genes . We speculate that the putative expressed proteins may contribute to pathogenesis, for example, enterotoxicity, of this unique strain of bacteria. Vet Rec, 2001 Nov 17, 149(20), 618 - 20 Clostridium perfringens beta2-toxin in an African elephant (Loxodonta africana) with ulcerative enteritis; Bacciarini LN et al.; A 22-year-old female African elephant (Loxodonta africana) developed diarrhoea of unknown cause which lasted for two days . The animal was euthanased after it remained recumbent and refused to get up . Gross pathological changes were present mainly in the gastrointestinal tract . The intestinal contents were watery and dark brown . Several areas of the mucosa of the small intestine were covered minimally to moderately with fibrin and had a few 0.1 x 10 to 15 cm linear ulcerations . Microscopical lesions consisted of discrete areas of necrosis of the surface and crypt epithelium without overt inflammatory infiltrates . Culture of the small intestinal contents resulted in a moderate growth of Clostridium perfringens . No salmonella were found in the small or large intestine . PCR of the isolate of C . perfringens revealed the presence of the beta2-toxin gene cpb2 and the alpha-toxin gene cpa but no other known toxin genes . The expression of the beta2-toxin gene in vivo was demonstrated by the immunohistochemical localisation of the beta2-toxin to the microscopical lesions in the small intestine. J Med Microbiol, 2001 Dec, 50(12), 1082 - 6 PCR ribotyping of clinically important Clostridium difficile strains from Hungary; Urban E et al.; Isolates of Clostridium difficile from different hospital wards at the University Hospital of Szeged in Hungary were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping) . A total of 15 different ribotypes was detected among the 65 isolates tested . The predominant type, PCR ribotype 087, accounted for 39% of all isolates, in contrast with an international typing study where ribotype 001 was the most common . Two non-toxigenic C . difficile strains were found to exhibit the same pattern, which was distinct from those of all the ribotypes described previously, suggesting that this is a new type. Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2105 - 11 Metabolism of cinnamic acids by some Clostridiales and emendation of the descriptions of Clostridium aerotolerans, Clostridium celerecrescens and Clostridium xylanolyticum; Chamkha M et al.; The ability of Clostridium aerotolerans DSM 5434T, Clostridium celerecrescens DSM 5628T, Clostridium methoxybenzovorans DSM 12182T, Clostridium stercorarium ATCC 35414T, Clostridium subterminale DSM 2636, Clostridium termitidis DSM 5398T, Clostridium thermolacticum DSM 2910T, Clostridium thermopalmarium DSM 5974T and Clostridium xylanolyticum DSM 6555T to metabolize cinnamic acid and various derivatives, with or without glucose supplementation, was examined . Only C aerotolerans DSM 5434T and C . xylanolyticum DSM 6555T, closely related species, transformed cinnamic acid to 3-phenylpropionic acid . Both species also reduced a wide range of cinnamic acid derivatives, including o-, m- and p-coumaric, o-, m- and p-methoxycinnamic, p-methylcinnamic, caffeic, ferulic, isoferulic and 3,4,5-trimethoxycinnamic acids to their corresponding 3-phenylpropionic acid derivatives . C . aerotolerans DSM 5434T, however, also decarboxylated p-coumaric acid into 4-vinylphenol, which was then reduced to 4-ethylphenol . C . celerecrescens was grouped with C . aerotolerans and C . xylanolyticum in subcluster XIVa of the Clostridiales . C . celerecrescens DSM 5628T only metabolized m- and p-methoxycinnamic and p-methylcinnamic acids to their corresponding 3-phenylpropionic acid derivatives, reducing the double bond in the C3 aliphatic side chain . Addition of glucose markedly increased the yield of the biotransformations by these three species . An emendation of the descriptions of C . aerotolerans, C . celerecrescens and C . xylanolyticum is proposed, based on these observations. Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2095 - 103 Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp . nov . and Clostridium saccharobutylicum sp . nov; Keis S et al.; On the basis of 16S rRNA gene sequencing and DNA-DNA reassociation, industrial solvent-producing clostridia have been assigned to four species . In this study, the phenotypic characteristics of Clostridium acetobutylicum, Clostridium beijerinckii, 'Clostridium saccharoperbutylacetonicum', and an unnamed Clostridium sp . represented by the strains NCP 262T and NRRL B643 are compared . In addition, a further 40 strains of solvent-producing clostridia have been classified by biotyping, DNA fingerprinting and 16S rRNA gene sequencing . These included 14 C . beijerinckii strains, two strains currently designated as 'Clostridium kaneboi' and 'Clostridium butanologenum', and 24 production strains used in the commercial acetone-butanol fermentation . All of the C . beijerinckii strains were confirmed to have been classified correctly . The 'C . kaneboi' and 'C . butanologenum' strains require reclassification as C . acetobutylicum and C . beijerinckii, respectively . The commercial production strains were found to belong either to C . beijerinckii or to the unnamed Clostridium sp . For the comparative phenotypic studies of the four species, representative strains were selected from each of the DNA-fingerprint subgroups within each species . These strains were analysed for their ability to utilize different carbohydrates, hydrolyse gelatin or aesculin, and produce indole, and were tested for the presence of catalase and urease . On the basis of these results, several phenotypic traits were found to be useful for differentiating between the four species . The descriptions of C . acetobutylicum and C . beijerinckii have been emended . The names Clostridium saccharoperbutylacetonicum sp . nov . {type strain = N1-4 (HMT) = ATCC 27021T} and Clostridium saccharobutylicum sp . nov . (type strain = DSM 13864T = ATCC BAA-117T) are proposed for the two new species. Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2049 - 54 Isolation of a cinnamic acid-metabolizing Clostridium glycolicum strain from oil mill wastewaters and emendation of the species description; Chamkha M et al.; A strictly anaerobic, gram-positive, motile, sporulated bacterium, designated strain CIN5, was isolated from olive mill wastewaters after enrichment on cinnamic acid . The rod-shaped cells were slightly curved (0.4-1.1 x 2.0-15 microm) and occurred singly or in pairs . Strain CIN5 utilized a limited number of carbohydrates (glucose, fructose, maltose, sorbitol), grew optimally at 37 degrees C and at pH 7.3-7.5 and had a DNA G+C content of 29.1+/-0.3 mol% . Strain CIN5 was very closely related to Clostridium glycolicum DSM 1288T . Both strain CIN5 and the type strain of C . glycolicum transformed cinnamic acid to hydrocinnamic acid and a wide range of other cinnamic acid derivatives, including o-, m- and p-coumaric, o-, m- and p-methoxycinnamic, p-methylcinnamic, caffeic, ferulic and isoferulic acids, to their corresponding 3-phenylpropionic acids by reducing the double bond of the side chain . Glucose supplementation increased the rate of conversion markedly . The emendation of the description of C . glycolicum is proposed to include these new characteristics. Pharmacoepidemiol Drug Saf, 2001 Jun-Jul, 10(4), 303 - 8 Risk factors for the development of Clostridium difficile toxin-associated diarrhoea: a pilot study; Aziz EE et al.; This study was a pilot investigation of risk factors for the development of Clostridium difficile toxin-associated diarrhoea and in particular the differential influence of antimicrobial agents . The study was a retrospective case-control design conducted at Freeman Hospital, Newcastle upon Tyne . Cases were inpatients with stool positive C . difficile toxin diarrhoea and two controls were drawn for each case matched for age (+/- 5 years) and type of admission (emergency or elective) . Using conditional logistic regression analysis, cephalosporins and erythromycin were found to be statistically significantly associated with Clostridium difficile toxin associated-diarrhoea . The results form the basis for designing a larger, prospective study. Trends Microbiol, 2002 Jan, 10(1), 5 - 7 C3stau, a new member of the family of C3-like ADP-ribosyltransferases; Wilde C et al.; C3-like ADP-ribosyltransferases, which are produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus, are exoenzymes lacking a translocation unit . These enzymes specifically inactivate Rho GTPases in host target cells . Recently, a novel C3-like transferase from S . aureus with new properties was identified, raising questions regarding its function . As Rho GTPases are master regulators of several eukaryotic signal processes and S . aureus can invade eukaryotic cells, C3 might play a role as a virulence factor. Nucleic Acids Res, 2002 Jan 1, 30(1), 179 - 82 The tmRNA Website: invasion by an intron; Williams KP; tmRNA (also known as 10Sa RNA or SsrA) plays a central role in an unusual mode of translation, whereby a stalled ribosome switches from a problematic mRNA to a short reading frame within tmRNA during translation of a single polypeptide chain . Research on the mechanism, structure and biology of tmRNA is served by the tmRNA Website, a collection of sequences for tmRNA and the encoded proteolysis-inducing peptide tags, alignments, careful documentation and other information; the URL is Four pseudoknots are usually present in each tmRNA, so the database is rich with information on pseudoknot variability . Since last year it has doubled (227 tmRNA sequences as of September 2001), a sequence alignment for the tmRNA cofactor SmpB has been included, and genomic data for Clostridium botulinum has revealed a group I (subgroup IA3) intron interrupting the tmRNA T-loop. J Bacteriol, 2002 Jan, 184(2), 600 - 4 alpha-Galactosidase Aga27A, an enzymatic component of the Clostridium josui cellulosome; Jindou S et al.; The Clostridium josui aga27A gene encodes the cellulosomal alpha-galactosidase Aga27A, which comprises a catalytic domain of family 27 of glycoside hydrolases and a dockerin domain responsible for cellulosome assembly . The catalytic domain is highly homologous to those of various alpha-galactosidases of family 27 of glycoside hydrolases from eukaryotic organisms, especially plants . The recombinant Aga27A alpha-galactosidase devoid of the dockerin domain preferred highly polymeric galactomannan as a substrate to small saccharides such as melibiose and raffinose. FEMS Microbiol Lett, 2001 Dec 18, 205(2), 305 - 14 Transcriptional regulation of pentose utilisation systems in the Bacillus/Clostridium group of bacteria; Rodionov DA et al.; In Bacillus subtilis, utilisation of xylose, arabinose and ribose is controlled by the transcriptional factors XylR, AraR and RbsR, respectively . Here we apply the comparative approach to the analysis of these regulons in the Bacillus/Clostridium group . Evolutionary variability of operon structures is demonstrated and operator sites for the main transcription factors are predicted . The consensus sequences for the XylR and RbsR binding sites vary in different subgroups of genomes . The functional coupling of gene clusters and the conservation of regulatory sites allow for detection of non-orthologous gene displacement of ribulose kinase in Enterococcus faecium and Clostridium acetobutylicum . Moreover, candidate catabolite responsive elements found upstream of most pentose-utilising genes suggest CcpA-mediated catabolite repression. J Korean Med Sci, 2001 Dec, 16(6), 742 - 4 Pseudomonas aeruginosa as a potential cause of antibiotic-associated diarrhea; Kim SW et al.; Although Pseudomonas aeruginosa is not generally considered as a cause of antibiotic-associated diarrhea, several cases of diarrhea caused by P . aeruginosa have been reported . We experienced seven cases of nosocomial diarrhea presumably caused by P . aeruginosa, which was the predominant organism isolated from stool cultures . Clostridium difficile toxin was also positive in one patient . No other potential or recognized enteropathogens were identified from stools . All patients had underlying diseases and had been receiving antibiotics before the diarrheal onset . All of the seven P . aeruginosa isolates were resistant to previously given antibiotics . Diarrhea stopped three days after withdrawal of probable offending antibiotics without specific treatment in two patients . The other five patients having continuous diarrhea despite withdrawal of probable offending antibiotics, were successfully treated with antipseudomonal agents . The median duration of diarrhea after the initiation of treatment was 6.3 days . These data suggest that P . aeruginosa can be a potential cause of antibiotic-associated diarrhea . Further investigations are warranted to evaluate the possible etiologic role of P . aeruginosa in antibiotic-associated diarrhea. J Biol Chem, 2002 Mar 8, 277(10), 8306 - 11 Epub 2001 Dec 17. The structure of 3-methylaspartase from Clostridium tetanomorphum functions via the common enolase chemical step; Asuncion M et al.; Methylaspartate ammonia-lyase (3-methylaspartase, MAL; EC ) catalyzes the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid . This reaction lies on the main catabolic pathway for glutamate in Clostridium tetanomorphum . MAL requires monovalent and divalent cation cofactors for full catalytic activity . The enzyme has attracted interest because of its potential use as a biocatalyst . The structure of C . tetanomorphum MAL has been solved to 1.9-A resolution by the single-wavelength anomalous diffraction method . A divalent metal ion complex of the protein has also been determined . MAL is a homodimer with each monomer consisting of two domains . One is an alpha/beta-barrel, and the other smaller domain is mainly beta-strands . The smaller domain partially occludes the C terminus of the barrel and forms a large cleft . The structure identifies MAL as belonging to the enolase superfamily of enzymes . The metal ion site is located in a large cleft between the domains . Potential active site residues have been identified based on a combination of their proximity to a metal ion site, molecular modeling, and sequence homology . In common with all members of the enolase superfamily, the carboxylic acid of the substrate is co-ordinated by the metal ions, and a proton adjacent to a carboxylic acid group of the substrate is abstracted by a base . In MAL, it appears that Lys(331) removes the alpha-proton of methylaspartic acid . This motif is the defining mechanistic characteristic of the enolase superfamily of which all have a common fold . The degree of structural conservation is remarkable given only four residues are absolutely conserved. Cell Signal, 2002 Jan, 14(1), 75 - 81 Phospholipase D stimulation is required for sphingosine-1-phosphate activation of actin stress fibre assembly in human airway epithelial cells; Porcelli AM et al.; In human airway epithelial cells, sphingosine-1-phosphate (SPP) and lysophosphatidic acid (LPA) stimulated the production of phosphatidic acid (PA), which was inhibited by the primary alcohol butan-1-ol, but not by the inactive butan-2-ol, clearly indicating phospholipase D (PLD) involvement . Both SPP and LPA stimulated actin stress fibre formation, which was also butan-2-ol-insensitive and inhibited by butan-1-ol . SPP-induced PLD activation and cytoskeletal remodelling were insensitive to brefeldin A and toxin B from Clostridium difficile, which conversely blocked the effect of LPA, suggesting that the monomeric GTPases ADP ribosylation factor (ARF) and Rho are involved in LPA, but not in SPP responses . Pertussis toxin inhibited SPP- but not LPA-induced effects . PLD activation and stress fibre formation by both lysolipids were abolished by the tyrosine kinase inhibitor genistein . Addition of PA to cells caused a massive stress fibre assembly . In conclusion, PLD is one of the signalling components linking SPP-receptor activation to assembly of actin stress fibres. Phytother Res, 2001 Nov, 15(7), 586 - 8 Antimicrobial activity of some Pacific Northwest woods against anaerobic bacteria and yeast; Johnston WH et al.; Extracts of woods commonly used for animal bedding were tested for antimicrobial activity . Essential oils from Alaska cedar (Chamaecyparis nootkatensis), western juniper (Juniperus occidentalis) and old growth Douglas fir (Pseudotsuga menziesii) as well as methanol extracts of wood from these trees plus western red cedar (Thuja plicata) and ponderosa pine (Pinus ponderosa) were tested for antimicrobial activity against anaerobic bacteria and yeast . The test microbes included Fusobacterium necrophorum, Clostridium perfringens, Actinomyces bovis and Candida albicans which are common to foot diseases and other infections in animals . The essential oils and methanol extracts were tested using a standardized broth assay . Only extracts of Alaska cedar and western juniper showed significant antimicrobial activity against each of the microbes tested . The essential oil of Douglas fir did show antimicrobial activity against A . bovis at the concentrations tested . The methanol extracts of the heartwood of Douglas fir and the sapwood of ponderosa pine showed no antimicrobial activity . The major chemical components of western juniper (cedrol and alpha- and beta-cedrene) and Alaska cedar (nootkatin) were also tested . In western juniper, alpha- and beta-cedrene were found to be active components . Nootkatin showed activity only against C . albicans . The inhibitory activity in Alaska cedar oil was high enough to justify further efforts to define the other chemical components responsible for the antimicrobial activity . Int J Mol Med, 2002 Jan, 9(1), 53 - 7 Oral administration of a product derived from Clostridium butyricum in rats; Araki Y et al.; Recent studies have suggested that short chain fatty acids (SCFAs) exert a therapeutic effect on some human and experimental animal diseases . Clostridium butyricum produces high levels of SCFAs in the gut lumen . The aim of the present study was to analyze the product derived from Clostridium butyricum in a culture system, and to develop methods to eliminate the odor derived from SCFAs in the product . Clostridium butyricum was incubated in CS medium for 24 h and subsequently in CS broth for 24 h . The suspension of Clostridium butyricum in the broth was centrifugated and the supernatant was analyzed . The results showed this product contained high levels of SCFAs, especially acetic acid and n-butyric acid . Many food materials were tested in order to eliminate the odor derived from SCFAs in the product . Of the food materials tested, yogurt was shown to most effectively eliminate the odor . Using a yogurt base, we prepared a special food additive . Use of the additive completely eliminated the odor of the product derived from Clostridium butyricum . Finally, we administered the product with the additive to Sprague-Dawley rats for 14 days . The rats grew normally for the duration of the experimental period . It is possible that this novel product with the additive exerts therapeutic effects on some gastointestinal disorders. Dis Colon Rectum, 2001 Dec, 44(12), 1871 - 2 Intracolonic use of vancomycin for treatment of clostridium difficile colitis in a patient with a diverted colon: report of a case; Nathanson DR et al.; Clostridium difficile-associated pseudomembranous colitis (PMC) is a common affliction of postoperative patients . Risk factors include antibiotic therapy, recent surgery, and hospitalization (1,2,3) . We present a case of PMC in a diverted colon and its treatment using vancomycin enemas. J Biol Chem, 2002 Feb 22, 277(8), 6143 - 52 Epub 2001 Dec 10. Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes . Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia; Knapp O et al.; The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail . Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers . The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl . Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner . The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6 . The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific . Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride . The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore . Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane . Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them . In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin . The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed. J Biol Chem, 2002 Feb 15, 277(7), 5074 - 81 Epub 2001 Dec 06. The binary Clostridium botulinum C2 toxin as a protein delivery system: identification of the minimal protein region necessary for interaction of toxin components; Barth H et al.; The binary Clostridium botulinum C2 toxin is composed of the enzyme component C2I and the binding component C2II, which are individual and non-linked proteins . Activated C2IIa mediates cell binding and translocation of C2I into the cytoplasm . C2I ADP-ribosylates G-actin at Arg-177 to depolymerize actin filaments . A fusion toxin containing the N-terminal domain of C2I (residues 1-225) transports C3 ADP-ribosyltransferase from Clostridium limosum into cells (Barth, H., Hofmann, F., Olenik, C., Just, I., and Aktories, K . (1998) Infect . Immun . 66, 1364-1369) . We characterized the adaptor function of C2I and its interaction with C2IIa . The fusion toxin GST-C2I(1-225)-C3 was efficiently transported by C2IIa, indicating that C2IIa translocates proteins into the cytosol even when the C2I(1-225) adaptor was positioned in the middle of a fusion protein . Amino acid residues 1-87 of C2I were sufficient for interaction with C2IIa and for translocation of C2I fusion toxins into HeLa cells . Residues 1-87 were the minimal part of C2I to bind to C2IIa on the cell surface, as detected by fluorescence-activated cytometry . An excess of C2I(1-87) (but not of further truncated C2I fragments) competed with Alexa488-labeled C2I for binding to C2IIa . Also, the fragment C2I(30-431) and the fusion toxin C2I(30-225)-C3 competed with C2I-Alexa488 for binding to C2IIa . C2I(30-225)-C3 did not induce cytotoxic effects on cells when applied together with C2IIa, indicating that amino acid residues 1-29 are involved in translocation of C2I but are not absolutely essential for binding to C2IIa. J Bacteriol, 2002 Jan, 184(1), 76 - 81 Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes; Murashima K et al.; In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex . Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a "designer" cellulosome, or a recombinant cellulosome with a specific function . We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units . The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli . The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis. FEBS Lett, 2001 Dec 7, 509(2), 235 - 8 Phased A-tracts bind to the alpha subunit of RNA polymerase with increased affinity at low temperature; Katayama S et al.; Previously we showed that the expression of a Clostridium perfringens phospholipase C gene (plc) is activated by promoter upstream phased A-tracts in a low temperature-dependent manner . In this paper we characterize the interaction between the alpha subunit of C . perfringens RNA polymerase and the phased A-tracts . Hydroxyl radical footprinting and fluorescence polarization assaying revealed that the alpha subunit binds to the minor grooves of the phased A-tracts through its C-terminal domain with increased affinity at low temperature . The result provides a molecular mechanism underlying the activation of the plc promoter by the phased A-tracts. J Pediatr Gastroenterol Nutr, 2001 Nov, 33(5), 592 - 601 Bacterial translocation in neonatal rats: the relation between intestinal flora, translocated bacteria, and influence of milk; Yajima M et al.; BACKGROUND: A high incidence of bacterial translocation in neonates results not only from immaturity of host-defense functions, but also from the dominant colonization of aerobic bacteria in the intestine . Bacterial colonization develops differently among breast-fed, formula-fed, premature, and full-term infants . The purpose of this study was to examine the incidence of bacterial translocation and to identify the translocated bacterial species, relating these findings to the intestinal microflora and to the type of feeding in neonatal rats . METHODS: Animals were divided into three groups: breast-fed normal pups (MR group), formula-fed pups fed via an intragastric cannula implanted esophageally (AR group), and breast-fed pups after the removal of the cannula (Sham group) . Artificial rearing was achieved using a machine feeding system . Culture and identification of the bacteria in the intestine, mesenteric lymph nodes, liver, portal blood, and lungs were made using a simplified version of Mitsuoka's method . RESULTS: At 14 days of age, the dominant bacteria in the feces of the MR and Sham Groups were Enterobacteriaceae, Lactobacillus, and Enterococcus, but Enterobacteriaceae and Clostridium were significantly more common in the AR group than in the MR group . The dominant bacteria in the mesenteric lymph nodes were Enterobacteriaceae, Lactobacillus, and Staphylococcus . The extent of systemic bacterial translocation decreased earlier in the Sham group than in the AR group . CONCLUSIONS: The frequency with which species of bacteria were cultured from mesenteric lymph nodes and other peripheral sites did not mirror the composition of the intestinal flora . Among the translocated bacteria, Staphylococcus may be especially hard to recognize and difficult for the host-defense systems to destroy . Breast-feeding inhibited systemic bacterial translocation in the suckling period of the rat. Toxicon, 2002 Apr, 40(4), 409 - 18 Isolation and identification of Clostridium perfringens in the venom and fangs of Loxosceles intermedia (brown spider): enhancement of the dermonecrotic lesion in loxoscelism; Monteiro CL et al.; Loxoscelism or the envenoming by the brown spiders (Loxosceles genus spiders), may produce extensive dermonecrosis and hemorrhage at the bite site and, eventually, systemic reactions that may be lethal . Isolation and identification of many different bacteria, among them Clostridium perfringens, of great medical importance due to its involvement in dermonecrotizing and systemic conditions, was carried out from the venomous apparatus (fangs and venom) of spiders obtained directly from nature, through microbiological cultures in aerobic and anaerobic conditions . Working with Loxosceles intermedia venom (alone) and with the venom conjugated with Clostridium perfringens using rabbits as experimental models for dermonecrosis, allowed for the observation that venom and anaerobic bacteria conjugated resulted in a striking increase of the dermonecrotic picture when compared to venom alone, suggesting a role for Clostridium perfringens in the severe dermonecrotic picture of these patients and opening the possibility for the association of antibiotic therapy in treating loxoscelism. Structure (Camb), 2001 Dec, 9(12), 1183 - 90 The structure of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides insights into substrate recognition; Prates JA et al.; BACKGROUND: Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes . In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation . Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications . RESULTS: We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum . The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8% . The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0% . CONCLUSIONS: The C . thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad . Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds . Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate . There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate. Eur J Biochem, 2001 Dec, 268(24), 6473 - 86 The sialate-pyruvate lyase from pig kidney . Elucidation of the primary structure and expression of recombinant enzyme activity; Traving C et al.; The first complete primary structure of a mammalian sialate-pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments . The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template . The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of approximately 35 kDa, which fits well to the value determined for the native enzyme . The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes . The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino-acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group . In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%) . Thirty-one amino acids were found to be absolutely conserved in all lyase sequences . Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction . After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences . After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel. ANZ J Surg, 2001 Nov, 71(11), 647 - 9 Clostridium septicum and malignancy; Chew SS et al.; BACKGROUND: Clostridium septicum is known to be associated with malignancy or immunosuppression . It has a variable clinical presentation and is associated with a high mortality . The aim of the present study was to review the experience at St George Hospital, Sydney, over a 10-year period, with particular reference to the association of this condition with colorectal cancer . METHODS: The records of five patients with blood culture-proven Clostridium septicum infection, among a larger group of 31 patients with clostridial infections, presenting to St George Hospital between 1990 and 2000 were reviewed . RESULTS: Associated malignancy was found in four (80%) of the patients with Clostridium septicum infection . Two infections were related to colorectal cancer, two to haematological malignancies and one to radiation-induced recto-urethral fistula . Those patients who had colorectal cancer presented with septicaemia and vague abdominal symptoms . CONCLUSIONS: Clostridium septicum infections have a strong association with malignancy . When this infection occurs without an obvious underlying aetiology there should be a high index of suspicion about associated malignancy . In the absence of haematological malignancy a colonoscopy is warranted . Early diagnosis and aggressive treatment is essential in order to improve prognosis. Biochem J, 2001 Dec 15, 360(Pt 3), 717 - 26 Influence of electrochemical properties in determining the sensitivity of {4Fe-4S} clusters in proteins to oxidative damage; Tilley GJ et al.; Interconversion between {4Fe-4S} cubane and {3Fe-4S} cuboidal states represents one of the simplest structural changes an iron-sulphur cluster can undertake . This reaction is implicated in oxidative damage and in modulation of the activity and regulation of certain enzymes, and it is therefore important to understand the factors governing cluster stability and the processes that activate cluster conversion . In the present study, protein film voltammetry has been used to induce and monitor the oxidative conversion of {4Fe-4S} into {3Fe-4S} clusters in different variants of Azotobacter vinelandii ferredoxin I (AvFdI; the 8Fe form of the native protein), and DeltaThr(14)/DeltaAsp(15), Thr(14)-->Cys (T14C) and C42D mutants . The electrochemical results have been correlated with the differing oxygen sensitivities of {4Fe-4S} clusters, and comparisons have been drawn with other ferredoxins (Desulfovibrio africanus FdIII, Clostridium pasteurianum Fd, Thauera aromatica Fd and Pyrococcus furiosus Fd) . In contrast with high-potential iron-sulphur proteins (HiPIPs) for which the oxidized species {4Fe-4S}(3+) is inert to degradation and can be isolated, the hypervalent state in these ferredoxins (most obviously the 3+ level) is very labile, and the reduction potential at which this is formed is a key factor in determining the cluster's resistance to oxidative damage. Biochem J, 2001 Dec 15, 360(Pt 3), 651 - 6 Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity; Goyal A et al.; Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase . Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB) . The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue . The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S . The renatured mixture was treated with DTNB and separated on an NAD(+)-agarose column to which only C320S subunits bind tightly . Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry . A fraction highly enriched in the 1:5 hybrid was identified . Homohexamers (C320S with 40 mM glutamate and 1 mM NAD(+) at pH 8.8, or K89L/A163G/S380A with 70 mM norleucine and 1 mM NAD(+) at pH 8.5) showed allosteric activation; succinate activated C320S approx . 50-fold (EC(50)=70 mM, h=2.4), and glutarate gave approx . 30-fold activation (EC(50)=35 mM, h=2.3) . For the triple mutant, corresponding values were 80 mM and 2.2 for succinate, and 75 mM and 1.7 for glutarate, but maximal activation was only about 2-fold . In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer . A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites . On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine . With glutamate at pH 8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer . The "foreign" subunit evidently impedes inter-subunit communication to some extent. Biochemistry, 2001 Dec 18, 40(50), 15327 - 33 Role of the disulfide cleavage induced molten globule state of type a botulinum neurotoxin in its endopeptidase activity; Cai S et al.; Botulinum neurotoxins are produced by anaerobic Clostridium botulinum in an inactive form . The endopeptidase activity of type A botulinum neurotoxin (BoNT/A) is triggered by reduction of its disulfide bond between its heavy chain and light chain . By using circular dichroism spectroscopy, we show that, upon reduction of BoNT/A and under physiological temperature (37 degrees C), the BoNT/A loses most of its native tertiary structure, while retaining most of its secondary structure . This type of structure is characterized as a molten globule type conformation, which was further confirmed for BoNT/A by the characteristic binding of 1-anilinonaphthalene-8-sulfonic acid . Under nonreducing conditions where the interchain disulfide bond is intact, the enzymatically inactive BoNT/A did not show a molten globule type of structure . A temperature profile of the structure and enzyme activity of BoNT/A revealed that, under reducing conditions, there was a strong correlation in the existence of the molten globule structure and optimum endopeptidase activity at about 37 degrees C. Plasmid, 2001 Nov, 46(3), 229 - 32 Induction of pCW3-encoded tetracycline resistance in Clostridium perfringens involves a host-encoded factor; Johanesen PA et al.; The tetracycline resistance determinant Tet P, which is encoded by the conjugative plasmid pCW3 from Clostridium perfringens, is induced by subinhibitory concentrations of tetracycline . In this study we have shown that the inducible phenotype is strain dependent . When pCW3 is present in derivatives of the wild-type strains CW234 and CW362 resistance is inducible . However, transfer to derivatives of strain 13 leads to a constitutive phenotype that is only observed in this strain background . Based on these results it is proposed that induction of the pCW3-encoded tet(P) genes in C . perfringens requires a host-encoded factor that is either absent or nonfunctional in strain 13 derivatives . Mol Cell Probes, 2001 Oct, 15(5), 301 - 6 Electroporation of DNA sequences from the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile into a non-toxigenic strain; Ackermann G et al.; Toxigenic Clostridium difficile is the etiologic agent of C . difficile-associated diarrhoea (CDAD), the most common cause of hospital-acquired infectious diarrhoea . The genes tcdA and tcdB, which encode for the toxin A and B proteins, are part of the pathogenicity locus (PaLoc) of toxigenic C . difficile . Genetic and virulence studies at the molecular level in C . difficile have been hindered by the lack of techniques for DNA manipulation in this species . We describe the electroporation of DNA fragments from a toxigenic isolate into a non-toxigenic strain of C . difficile . Using previously described methods of electroporation into Clostridium spp., the complete toxin B gene and polymerase chain reaction (PCR) fragments of the PaLoc were cloned and electroporated into a non-toxigenic strain of C . difficile . The resulting transformed clones were screened for the introduced gene fragments by PCR, which confirmed their presence . This is the first description of introduction of DNA into C . difficile by electroporation . J Mol Biol, 2001 Dec 7, 314(4), 797 - 806 Recognition of cello-oligosaccharides by a family 17 carbohydrate-binding module: an X-ray crystallographic, thermodynamic and mutagenic study; Notenboom V et al.; The crystal structure of the Clostridium cellulovorans carbohydrate-binding module (CBM) belonging to family 17 has been solved to 1.7 A resolution by multiple anomalous dispersion methods . CBM17 binds to non-crystalline cellulose and soluble beta-1,4-glucans, with a minimal binding requirement of cellotriose and optimal affinity for cellohexaose . The crystal structure of CBM17 complexed with cellotetraose solved at 2.0 A resolution revealed that binding occurs in a cleft on the surface of the molecule involving two tryptophan residues and several charged amino acids . Thermodynamic binding studies and alanine scanning mutagenesis in combination with the cellotetraose complex structure allowed the mapping of the CBM17 binding cleft . In contrast to the binding groove characteristic of family 4 CBMs, family 17 CBMs appear to have a very shallow binding cleft that may be more accessible to cellulose chains in non-crystalline cellulose than the deeper binding clefts of family 4 CBMs . The structural differences in these two modules may reflect non-overlapping binding niches on cellulose surfaces . Infect Control Hosp Epidemiol, 2001 Sep, 22(9), 572 - 5 Quinolone use as a risk factor for nosocomial Clostridium difficile-associated diarrhea; Yip C et al.; OBJECTIVE: To determine modifiable risk factors for nosocomial Clostridium difficile-associated diarrhea (CDAD) . DESIGN: Case-control study . SETTING: 300-bed tertiary-care hospital . PARTICIPANTS: Hospital inpatients present during the 3-month study period . METHODS: Case-patients identified with nosocomial CDAD over the study period were compared to two sets of control patients: inpatients matched by age, gender, and date of admission; and inpatients matched by duration of hospital stay . Variables including demographic data, comorbid illnesses, antibiotic exposure, and use of gastrointestinal medications were assessed for case- and control-patients . Conditional logistic regression was performed to identify risk factors for nosocomial CDAD . RESULTS: 27 case-patients were identified and were compared to the two sets of controls (1:1 match for each comparison set) . For the first set of controls, use of ciprofloxacin (odds ratio {OR}, 5.5; 95% confidence interval {CI 95}, 1.2-24.8; P=.03) was the only variable that remained significant in the multivariable model . For the second set of controls, prior exposure to cephalosporins (OR, 6.7; CI 95, 1.3-33.7; P=.02) and to ciprofloxacin (OR, 9.5; CI 95, 1.01-88.4; P=.05) were kept in the final model . CONCLUSIONS: Along with cephalosporins, prior quinolone use predisposed hospitalized patients to nosocomial CDAD . Quinolones should be used judiciously in acute-care hospitals, particularly in those where CDAD is endemic. Biotechniques . 2001 Nov;31(5):1064, 1066, 1068. New E . coli cloning vector using a cellulase gene (celA) as a screening marker; Jang SJ et al.; The extracellular endoglucanase A gene of Clostridium thermocellum (celA) was used as a screening marker for E . coli cloning vector A 1.4-kb EcoRI fragment containing celA from pTvec/celA was isolated and cloned into a pUC18 deleting beta-galactosidase gene fragment . The constructed vectors, pCEL1, pCEL10, pCEL11, and pCEL20, have different multiple cloning sites within celA . If the cellulase, CelA, is inactivated by insertion of a foreign DNA fragment into multiple cloning sites, the recombinant transformants show no clear halos on an agar plate containing cellulose . This process overcomes the ambiguity of color screening in the X-gal/beta-galactosidase system, and over 90% of the recombinant transformants with no halos have foreign DNA inserts . Several E . coli strains were transformed successfully with pCEL series vectors regardless of mutation for alpha-complementation . Because E . coli strains do not have a cellulase gene, a vector using a cellulase gene screening marker can be used in any E . coli strain without limit . The new cloning system is very efficient, convenient, and cost effective. J Biol Chem, 2002 Feb 8, 277(6), 4247 - 54 Epub 2001 Nov 29. Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A; Chen ML et al.; Clostridium difficile toxin A increases paracellular permeability in colonic epithelial T84 cells by mechanisms involving RhoA glucosylation and actin depolymerization . However, we previously observed that toxin A-mediated decline in transepithelial electrical resistance preceded changes in cell morphology and tight junction ultrastructure (Hecht, G., Pothoulakis, C., LaMont, J . T., and Madara, J . L . (1988) J . Clin . Invest . 82, 1516-1524) . Recent studies also showed that C . difficile toxins induce early cellular responses, including activation of mitogen-activated protein kinases, generation of reactive oxygen metabolites, and calcium influx . The aim of this study was to investigate whether toxin A-induced early cellular responses contribute to the permeability changes . We found that toxin A stimulated the activities of membrane and cytosolic protein kinase Calpha (PKCalpha) and cytosolic PKCbeta . A specific PKCalpha/beta antagonist (myristoylated PKCalpha/beta peptide) blocked toxin A-mediated RhoA glucosylation . Furthermore, decreased transepithelial electrical resistance and increased translocation of ZO-1 from tight junction occurred within 2-3 h of toxin A exposure and were also inhibited by PKCalpha/beta antagonist . During this time period, toxin exposure did not induce translocation of ZO-2, dephosphorylation or translocation of occludin, or cell rounding . Our data indicate that PKC signaling regulates toxin A-mediated paracellular permeability changes and ZO-1 translocation. Biochem Pharmacol, 2001 Dec 1, 62(11), 1459 - 68 Positive modulation by Ras of interleukin-1beta-mediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells; Tannous M et al.; In the present study, we have shown that exposure of insulin-secreting clonal beta (HIT-T15) cells to interleukin-1beta (IL-1beta) results in a time- and concentration-dependent increase in nitric oxide (NO) release . These effects by IL-1beta on NO release were mediated by induction of inducible nitric oxide synthase (iNOS) from the cells . Preincubation of HIT cells with Clostridium sordellii lethal toxin-82, which irreversibly glucosylates and inactivates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1beta-induced NO release . Pre-exposure of HIT cells to C . sordellii lethal toxin-9048, which monoglucosylates and inhibits Ras, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1beta-mediated NO release . These data indicate that activation of Ras and/or Rac may be necessary for IL-1beta-mediated NO release . Preincubation of HIT cells with C . difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no demonstrable effects on IL-mediated NO release, ruling out the possibility that Rac may be involved in this signaling step . Further, two structurally dissimilar inhibitors of Ras function, namely manumycin A and damnacanthal, inhibited, in a concentration-dependent manner, the IL-1beta-mediated NO release from these cells . Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1beta-mediated NO release and, presumably, the subsequent dysfunction of the pancreatic beta cell . Our data also provide a basis for future investigations to understand the mechanism of cytokine-induced beta cell death leading to the onset of insulin-dependent diabetes mellitus. Scand J Infect Dis, 2001, 33(10), 731 - 3 The role of Clostridium difficile in childhood nosocomial diarrhea; Oguz F et al.; The role of Clostridium difficile was investigated in 100 children with nosocomial diarrhea . An etiologic agent was identified in 69 cases, 8 of whom had dual infection . C . difficile-associated diarrhea (Cdad) was defined in 16 children (16%) . The mean age of the patients with Cdad was 5.4 y (range 2 months to 13 y) and the male:female ratio was 1.2 . All cases with Cdad were on antibiotic therapy . Cdad occurred more frequently in the cases given combined antibiotic treatment than in those given single antibiotic treatment (p < 0.05) . One case with neutropenic sepsis died . C . difficile was also investigated in the stool samples of 50 hospitalized children treated with antibiotics who did not develop diarrhea . C . difficile toxins A and B were found in 5 children aged < 2 y in the control group . This study shows that C . difficile is an important cause of nosocomial diarrhea in our hospital population. J Biomol NMR, 2001 Oct, 21(2), 107 - 16 Simultaneous measurement of intra- and intermolecular NOEs in differentially labeled protein-ligand complexes; Eichmuller C et al.; A new NOE strategy is presented that allows the simultaneous observation of intermolecular and intramolecular NOEs between an unlabeled ligand and a 13C,15N-labeled protein . The method uses an adiabatic 13C inversion pulse optimized to an empirically observed relationship between 1 J(CH) and carbon chemical shift to selectively invert the protein protons (attached to 13C) . Two NOESY data sets are recorded where the intermolecular and intramolecular NOESY cross peaks have either equal or opposite signs, respectively . Addition and subtraction yield two NOESY spectra which contain either NOEs within the labeled protein (or unlabeled ligand) or along the binding interface . The method is demonstrated with an application to the B12-binding subunit of Glutamate Mutase from Clostridium tetanomorphum complexed with the B12-nucleotide loop moiety of the natural cofactor adenosylcobalamin (Coenzyme B12). Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15155 - 60 Epub 2001 Nov 27. Combination bacteriolytic therapy for the treatment of experimental tumors; Dang LH et al.; Current chemotherapeutic approaches for cancer are in part limited by the inability of drugs to destroy neoplastic cells within poorly vascularized compartments of tumors . We have here systematically assessed anaerobic bacteria for their capacity to grow expansively within avascular compartments of transplanted tumors . Among 26 different strains tested, one (Clostridium novyi) appeared particularly promising . We created a strain of C . novyi devoid of its lethal toxin (C . novyi-NT) and showed that intravenously injected C . novyi-NT spores germinated within the avascular regions of tumors in mice and destroyed surrounding viable tumor cells . When C . novyi-NT spores were administered together with conventional chemotherapeutic drugs, extensive hemorrhagic necrosis of tumors often developed within 24 h, resulting in significant and prolonged antitumor effects . This strategy, called combination bacteriolytic therapy (COBALT), has the potential to add a new dimension to the treatment of cancer. J Biol Chem, 2002 Mar 8, 277(10), 8366 - 71 Epub 2001 Nov 27. Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library; Hu Y et al.; The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method . An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence . This immobilized peptide library was incubated with C . histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase . The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase . Edman sequencing was used to assign structure to subsites of active substrate mixtures . Collectively, the substrate preference for subsites (P(3)-P(4)') of C . histolyticum beta-collagenase was determined . The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position . The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser . The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C . histolyticum beta-collagenase . S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity . The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C . histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest. Biochemistry, 2001 Dec 4, 40(48), 14384 - 91 Reduced temperature dependence of collective conformational opening in a hyperthermophile rubredoxin; Hernandez G et al.; Spatially localized differences in the conformational dynamics of the rubredoxins from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements . As shown previously for the hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX(2) hydrogen exchange kinetics with conformational opening rates of >1 s(-)(1) . Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded beta-sheet, while for the rest of the structure, the exchange rates at 23 degrees C are similar for both proteins . For the multiple-turn region comprising residues 14-32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening . In contrast to the common expectation of increased rigidity in the hyperthermophile proteins, below room temperature Pf rubredoxin exhibits a larger apparent flexibility in this multiple-turn region . The smaller enthalpy for the conformational opening process of this region in Pf rubredoxin reflects the much weaker temperature dependence of the underlying conformational equilibrium in the hyperthermophile protein. Appl Environ Microbiol, 2001 Dec, 67(12), 5656 - 67 Percent G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens; Apajalahti JH et al.; Broiler chickens from eight commercial farms in Southern Finland were analyzed for the structure of their gastrointestinal microbial community by a nonselective DNA-based method, percent G+C-based profiling . The bacteriological impact of the feed source and in-farm whole-wheat amendment of the diet was assessed by percent G+C profiling . Also, a phylogenetic 16S rRNA gene (rDNA)-based study was carried out to aid in interpretation of the percent G+C profiles . This survey showed that most of the 16S rDNA sequences found could not be assigned to any previously known bacterial genus or they represented an unknown species of one of the taxonomically heterogeneous genera, such as Ruminococcus or Clostridium . The data from bacterial community profiling were analyzed by t-test, multiple linear regression, and principal-component statistical approaches . The percent G+C profiling method with appropriate statistical analyses detected microbial community differences smaller than 10% within each 5% increment of the percent G+C profiles . Diet turned out to be the strongest determinant of the cecal bacterial community structure . Both the source of feed and local feed amendment changed the bacteriological profile significantly, whereas profiles of individual farms with identical feed regimens hardly differed from each other . This suggests that the management of typical Finnish farms is relatively uniform or that hygiene on the farm, in fact, has little impact on the structure of the cecal bacterial community . Therefore, feed compounders should have a significant role in the modulation of gut microflora and consequently in prevention of gastrointestinal disorders in farm animals. J Appl Microbiol, 2001 Nov, 91(5), 814 - 21 Preliminary microbiological investigation of the preparation of two traditional Maori foods (Kina and Tiroi); Hudson JA et al.; AIMS: Little information exists regarding the microbiology of two traditional Maori food preparation processes which may involve fermentations . Preliminary microbiological and chemical analyses were carried out on these two foods in order to identify the fermentations involved (if any) . METHODS AND RESULTS: Testing was carried out on freshly-prepared foods and on those that had been processed and stored . Kina (sea urchins, Evechinus chloroticus) are harvested and then stored either under fresh water or buried underground . The most frequently-occurring process appeared to be an alkaline fermentation . Large numbers of Clostridium perfringens were detected in one set of samples prepared outside of the traditional season, but this was the only pathogen detected . In Kina stored in buried plastic bottles during the traditionally-accepted time of the year, bacterial numbers decreased . Tiroi is prepared from mussels and Puha (sow thistle, Sonchos asper) that have been cooked to some degree, combined and stored . Of three methods used to prepare and store Tiroi, the results for one indicated the possible involvement of a lactic acid fermentation, but the other two methods were effectively only cooking and bottling processes . CONCLUSIONS: In the case of Kina, the use of an alkaline fermentation to prepare a seafood for consumption is unusual . One method of Tiroi production is a lactic acid fermentation . SIGNIFICANCE AND IMPACT OF THE STUDY: If these foods are produced as described and are not either eaten immediately or cooked before consumption, then growth, and intoxication by, Clostridium botulinum might occur. J Anim Sci, 2001 Oct, 79(10), 2558 - 64 Clostridial antibody response from injection-site lesions in beef cattle, long-term response to single or multiple doses, and response in newborn beef calves; Troxel TR et al.; Experiments were conducted to compare clostridial antibody response of beef heifers that do and do not develop injection-site lesions, evaluate long-term antibody response of a single- and multiple-dose toxoid, and evaluate the ability of a clostridial toxoid to elicit an active antibody response in newborn calves . In Exp . 1, 37 weaned heifers were vaccinated (d 0) with a clostridial vaccine (Alpha-7, 2 mL, s.c.) . Serum samples were collected on d 0, 28, 56, 84, and 112 to determine clostridial antibody titers . On d 28, heifers were visually inspected and palpated for injection-site lesions . The percentage of heifers that developed lesions was 64.9% . Lesioned heifers had elevated antibody titers for Clostridium chauvoei (CC) on d 28 (P < 0.08) and 84 (P < 0.07) compared with non-lesioned heifers . Clostridium sordellii (CS) and perfringens type D (CPD) antibody titers were greater in lesioned heifers than in non-lesioned heifers on d 28 and 56 . In Exp . 2, long-term antibody response of Alpha-7 (A7) and Ultrabac 7 (UB7) was investigated in stocker heifers . The A7 heifers (n = 15) received one 2-mL vaccination (d 0), and the UB7 heifers (n = 15) received a 5-mL vaccination on d 0 and 28 . Blood samples were collected on d 0, 28, 56, 84, 112, 140, and 180 . Clostridium chauvoei, CPD, and Cl . novyi (CN) antibody titers from the A7 heifers were greater than those from the UB7 heifers on d 28 . Due to the second UB7 injection, CC, CS, CN, and Cl . perfringens type C (CPC) antibody titers were greater in UB7 heifers than in A7 heifers on d 56 . By d 112, titers were not different, and by d 140 all antibody titers were below detectable levels . In Exp . 3, 58 pregnant, mature, crossbred cows were vaccinated with A7 before calving . At birth, calves were carefully observed to ensure consumption of colostrum . Calves were blocked according to parturition date, and calves in each block were randomly allocated to receive A7 (s.c . at 3 +/- 3 d of age) or remain unvaccinated controls . Calves were bled at the time of vaccination (d 0) and on d 28, 56, 84, and 112 . Antibody titers for CC, CPC, and CPD were elevated on d 0 and decreased throughout the experimental period (P < 0.01), but no titer differences (P > 0.10) were detected between treatment groups on any of the days sampled . These data indicated that antibody titers against clostridial diseases are enhanced when injection-site lesions develop . One injection of Alpha-7 seemed to provide the same length of protection as two injections of Ultrabac 7. Equine Vet J, 2001 Nov, 33(6), 547 - 53 Systemic antibodies to Clostridium botulinum type C: do they protect horses from grass sickness (dysautonomia)? Hunter LC, Poxton IR. The aetiology of equine grass sickness (EGS) is still unknown . There is increasing evidence that toxicoinfection with Clostridium botulinum type C is involved . Epidemiological evidence shows that resistance to EGS can occur in older horses and those that have been on a particular pasture for longer or have been in prior contact with the disease . This resistance may be in the form of an immune response to the aetiological agent . Levels of systemic antibodies to the surface antigens of C . botulinum type C (using the closely related and safe C . novyi type A as a phenotypic marker) and to the botulinum type C neurotoxin (BoNT/C) were investigated in horses with and without EGS . Horses with grass sickness were found to have significantly lower levels of systemic IgG to both surface antigens and BoNT/C . Horses with low levels of systemic immunity to these antigens may be more susceptible to developing EGS . There were no significant differences in antibody levels between the different categories of EGS, suggesting systemic immunity to C . botulinum type C does not play a significant role in influencing the severity of the disease . However, horses that had been in contact with EGS or that were grazing land where it had occurred frequently in the past had significantly higher antibody levels to these antigens . These horses may have been exposed to subclinical doses of C . botulinum type C and BoNT/C, resulting in the production of a protective immune response against the putative aetiological agent . This finding is of potential significance for the prospect of prevention of EGS by vaccination against C . botulinum type C. FEBS Lett, 2001 Nov 16, 508(2), 253 - 8 Protein-binding partners of the tobacco syntaxin NtSyr1; Kargul J et al.; Syntaxins and other SNARE (soluble NSF-attachment protein receptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion . Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid . However, partner proteins contributing to its function(s) remain unknown . We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by alphaAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue . Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa . Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo. J Bacteriol, 2001 Dec, 183(24), 7110 - 9 Transcriptional analysis of the tet(P) operon from Clostridium perfringens; Johanesen PA et al.; The Clostridium perfringens tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes, tetA(P) and tetB(P), which mediate resistance by different mechanisms . Detailed transcriptional analysis has shown that the inducible tetA(P) and tetB(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the tetA(P) start codon . Deletion of P3 or alteration of the spacing between the -35 and -10 regions significantly reduced the level of transcription in a reporter construct . Induction was shown to be mediated at the level of transcription . Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of the tetA(P) gene . Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct . It is postulated that the T1 terminator is an intrinsic control element of the tet(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline. J Bacteriol, 2001 Dec, 183(24), 7037 - 43 Characterization of xylanolytic enzymes in Clostridium cellulovorans: expression of xylanase activity dependent on growth substrates; Kosugi A et al.; Xylanase activity of Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium, was characterized . Most of the activity was secreted into the growth medium when the bacterium was grown on xylan . Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions . Each of these fractions contained at least two major and three minor xylanase activities . In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis . The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000 . High alpha-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions . Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose- and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan . These results strongly indicated that C . cellulovorans is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate. J Hosp Infect, 2001 Nov, 49(3), 204 - 9 Prospective evaluation of environmental contamination by Clostridium difficile in isolation side rooms; Verity P et al.; We determined prospectively the frequency, persistence and molecular epidemiology of Clostridium difficile environmental contamination after detergent-based cleaning in side rooms used to isolate patients with C . difficile diarrhoea . Approximately one-quarter of all environmental sites in side rooms sampled over four-week periods were contaminated with C . difficile . The overall side room prevalence of environmental C . difficile declined from 35% initially, to 24% in week 2, 18% in week 3, and 16% in week 4 . The bed frame was the most common site from which C . difficile was recovered, although the floor was the most contaminated site in terms of total numbers of colonies . C . difficile was recovered significantly more frequently from swabs plated directly on to C . difficile selective media containing lysozyme than from enrichment broth (P< 0.001), emphasizing the benefit of lysozyme supplementation . The great majority of C . difficile isolates (87% of all isolates, 84% of patient isolates) was indistinguishable from the UK epidemic strain (PCR ribotype 1) . It thus could not be determined whether environmental contamination was a cause or a consequence of diarrhoea . Our findings highlight the need for improved approaches to hospital environmental hygiene, and call into question current UK guidelines that recommend detergent-based cleaning to remove environmental C . difficile . In particular, improved cleaning of frequently touched sites in the immediate bed space area is required . Biochem Biophys Res Commun, 2001 Nov 30, 289(2), 623 - 9 High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein; Munro P et al.; Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis . Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT . We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP . Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC . In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT . Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin. J Biol Chem, 2002 Jan 25, 277(4), 2650 - 6 Epub 2001 Nov 16. In vitro reconstitution of the Clostridium botulinum type D progenitor toxin; Kouguchi H et al.; Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing . The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33) . The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents . We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17 . The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice . M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents . We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17. Microb Pathog, 2001 Nov, 31(5), 255 - 60 Analysis of expression of GroEL (Hsp60) of Clostridium difficile in response to stress; Hennequin C et al.; Our laboratory has previously shown that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses and that GroEL, a heat shock protein, serves an adhesive function in this bacterium . In this communication, RT-PCR, SDS-PAGE and immunoblotting were used to study the stress response in C . difficile following heat, acid or osmotic shock, iron deprivation or presence of a subinhibitory concentration of ampicillin in the culture medium . All these stresses increased transcription of groEL and production of GroEL to various degrees . Furthermore, the protein was found in membrane fractions and in the extracellular space after heat stress . FEBS Lett, 2001 Nov 9, 508(1), 95 - 8 Sialic acids in gastropods; Burgmayr S et al.; The occurrence of N-acetylneuraminic acid and N-glycolylneuraminic acid residues in preparations of the slug Arion lusitanicus (Gastropoda) was determined by sodium dodecyl sulphate electrophoresis of the proteins followed by lectin blots stained with the sialic acid specific lectin from Maackia amurensis, by the sensitivity of this binding to sialidase from Clostridium perfringens, by specific fluorescent labelling of sialic acids with 1,2-diamino-4,5-methylenedioxybenzene, by the determination of the sensitivity to sialate-pyruvate-lyase, by co-migration with standards on high performance anion exchange chromatography with pulsed amperometric detection and by identification of the typical masses in the fragmentation patterns of the trimethylsilyl derivatives after gas chromatography . It is the first time sialic acids are identified in gastropods. Infect Immun, 2001 Dec, 69(12), 7937 - 40 Role of FliC and FliD flagellar proteins of Clostridium difficile in adherence and gut colonization; Tasteyre A et al.; In vitro and in vivo adhesive properties of flagella and recombinant flagellin FliC and flagellar cap FliD proteins of Clostridium difficile were analyzed . FliC, FliD, and crude flagella adhered in vitro to axenic mouse cecal mucus . Radiolabeled cultured cells bound to a high degree to FliD and weakly to flagella deposited on a membrane . The tissue association in the mouse cecum of a nonflagellated strain was 10-fold lower than that of a flagellated strain belonging to the same serogroup, confirming the role of flagella in adherence. Infect Immun, 2001 Dec, 69(12), 7904 - 10 Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene; Awad MM et al.; To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of Clostridium perfringens . The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene (plc), the perfringolysin O gene (pfoA), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model . Synergistic effects were clearly observed in these experiments . Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted . These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons . Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis . These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed . Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain . The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels . The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene. Am J Physiol Renal Physiol, 2001 Dec, 281(6), F1092 - 101 Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells; Tamma G et al.; First published August 8, 2001; 10.1152/ajprenal.00091.2001.-We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29) . The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton . In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C . limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin . Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient . Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin . Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation . Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton. Vox Sang, 2001 Oct, 81(3), 154 - 60 Evaluation of the BacT/Alert automated blood culture system for detecting bacteria and measuring their growth kinetics in leucodepleted and non-leucodepleted platelet concentrates; McDonald CP et al.; BACKGROUND AND OBJECTIVE: To evaluate the BacT/Alert automated blood culture system for the detection of bacteria in platelet concentrates, and to determine bacterial growth kinetics in leucodepleted and non-leucodepleted units . MATERIALS AND METHODS: Apheresis (Cobe Leucocyte Reduction System {LRS}) and pooled buffy coat-derived (Optipress) platelet concentrates (PCs) were tested . Six organisms were used for spiking the PCs: Clostridium perfringens, Bacillus cereus, Group B Streptococcus, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli . Units were inoculated to give a final concentration of approximately equal to 1 and 50 colony-forming units (CFU)/ml . On days 0, 2 and 5, BacT/Alert standard aerobic and anaerobic bottles were inoculated with a 5-ml fill volume and bacteria were enumerated . RESULTS: The BacT/Alert Automated blood culture system gave rapid determination times of spiked units, with all positives detected within 48 h and 98.1% detected within 24 h . In general, as the inoculum concentration increased, the detection time decreased . Rapid growth was obtained with all organisms tested except for B . cereus, which failed to grow on four occasions . Bacterial numbers on day 2 ranged from 10(5) to 10(11) CFU/ml and on day 5 ranged from 10(4) to 10(12) CFU/ml . Growth was not significantly greater in leucodepleted units . CONCLUSIONS: The study confirmed that PCs are an excellent growth medium for bacteria . Rapid and substantial growth was obtained with all organisms under test . Leucodepletion does not appear to enhance bacterial proliferation . The BacT/Alert automated blood culture system could rapidly detect contamination of units . Bacterial screening using an automated blood culture system is therefore a potential option. Folia Microbiol (Praha), 2001, 46(3), 197 - 204 Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone-1-butanol-ethanol solvent by Clostridium acetobutylicum; Madihah MS et al.; A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch by Clostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent, i.e . 1.5-2 times more than with pure maltose or glucose used as carbon sources . Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch . The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation . The pH optima for alpha-amylase and glucoamylase were found to be at 5.3 and 4.0-4.4, respectively . alpha-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0-8.0 after a 1-d incubation at 37 degrees C . Since C . acetobutylicum alpha-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch . However, the C . acetobutylicum glucoamylase was only stable at pH 4-5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37 degrees C. Eur J Clin Microbiol Infect Dis, 2000 Jan, 19(1), 9 - 15 Factors associated with nosocomial diarrhea and Clostridium difficile-associated disease on the adult wards of an urban tertiary care hospital; Schwaber MJ et al.; A prospective survey of the adult inpatient population of an urban tertiary care hospital was conducted to determine factors associated with the development of nosocomial diarrhea and the acquisition of Clostridium difficile-associated disease . During the 3-month survey, 98 patients with nosocomial diarrhea were enrolled, and 38 controls were recruited . The controls were patients without diarrhea lying in beds adjacent to the affected patients . Factors significantly associated with nosocomial diarrhea were the administration of a special diet (P=0.02) and receipt of a greater number of different antibiotics (P=0.02) . Among the 98 patients with diarrhea, Clostridium difficile toxin B was identified in the stool of 13 . Factors found to be associated with the presence of toxin B as compared to other causes of nosocomial diarrhea were a greater number of individual antibiotics used during hospitalization (P=0.02) and receipt of a cephalosporin (P=0.03) or, more specifically, a third-generation cephalosporin (P=0.02) . Among patients with nosocomial diarrhea, those who had toxin in their stool had a significantly higher total antibiotic burden (expressed as antibiotic days) than those with diarrhea due to other causes (P=0.01). Am J Physiol Cell Physiol, 2001 Dec, 281(6), C2010 - 9 Enhancement of survival by LPA via Erk1/Erk2 and PI 3-kinase/Akt pathways in a murine hepatocyte cell line; Sautin YY et al.; First published September 5, 2001; 10.1152/ajpcell.00077.2001.-Protective mechanisms for lysophosphatidic acid (LPA) against cell death caused by Clostridium difficile toxin, or tumor necrosis factor-alpha (TNF-alpha) plus D-galactosamine, were investigated in a murine hepatocyte cell line AML12 expressing Edg2 LPA receptor . In these models of hepatocellular injury, LPA prevented hepatocyte damage, suppressed apoptosis, and enhanced cell survival in a dose-dependent fashion . The protective effects of LPA were abolished by wortmannin and LY-294002, specific inhibitors of phosphatidylinositol 3-phosphate kinase (PI 3-kinase), and by PD-98059 and U-0126, inhibitors of MEK1/MEK2 . In nontreated hepatocytes, LPA elicited a gradual and sustained increase in phosphorylation of Erk1/Erk2 over 180 min of stimulation and downstream phosphorylation of p90RSK and transcription factor Elk-1 . In C . difficile toxin-treated cells, LPA-induced phosphorylation of Erk1/Erk2 was rapid but transient, while p90RSK and Elk-1 phosphorylation did not change significantly . LPA stimulated phosphorylation of Akt in a time-dependent manner in both intact and toxin-treated AML12 hepatocytes . Wortmannin and LY-294002 abolished phosphorylation of Akt, further supporting activation of PI 3-kinase/Akt as a signaling pathway, which mediates hepatocyte protection by LPA . Taken together, these results demonstrate that LPA prevents cell apoptosis induced by C . difficile toxin and TNF-alpha/D-galactosamine in the AML12 murine hepatocyte cell line . Cell protection by LPA involves activation of the mitogen-activated protein kinase Erk1/Erk2 cascade and PI 3-kinase-dependent phosphorylation of Akt. Clin Exp Dermatol, 2001 Oct, 26(7), 619 - 30 Botulinum toxin A in the therapy of mimic facial lines; Becker-Wegerich P et al.; In aesthetic medicine, many different methods of skin rejuvenation are available . At the end of the 1980s, the neurotoxin Botulinum toxin A (BT-A) led to a revolution in aesthetic-corrective dermatology for the treatment of mimic facial wrinkles . The toxin is produced by Clostridium botulinum and causes a reversible, selective muscle relaxation that leads to a temporary flattening of the mechanical part of wrinkling without the stigmata of invasive surgery . After two decades of experience in different medical disciplines, there has been remarkable clinical development and progress in research, the identification of new botulinum toxin serotypes, and also innovation in indications and combined modalities . These lead to new and interesting questions . BT-A offers the experienced, critical dermatologist a time-saving, effective, cosmetically satisfactory, non-invasive treatment for mimic facial wrinkles and neck and decollete lines, with only minor side effects . Dermatologists should have a profound anatomical knowledge and should be able to perform all injection techniques to meet the needs of ever more demanding patients and to ensure optimization of patient satisfaction . The following review summarizes the historical development and the mechanism of action of both frequently and rarely used injection techniques with BT-A for the treatment of wrinkles and lines of the upper face, neck and decollete, and gives an update of different experiences encountered. Biochemistry, 2001 Nov 13, 40(45), 13548 - 55 Alanine-scanning of the 50's loop in the Clostridium beijerinckii flavodoxin: evaluation of additivity and the importance of interactions provided by the main chain in the modulation of the oxidation-reduction potentials; Kasim M et al.; The four-residue reverse turn -Met56-Gly-Asp-Glu59- in the Clostridium beijerinckii flavodoxin provides the majority of the critical interactions with the isoalloxazine ring of the flavin mononucleotide (FMN) cofactor that contribute to the binding and the differential stabilization of its three redox states . Direct side chain contacts include the sulfur-ring interaction of Met56, which primarily influences the oxidized and hydroquinone states, and the hydrogen bond by Glu59 with the N3H, which directly (and indirectly through its "anchoring" function) influences all three states to various extents . Involving a novel redox-dependent conformational change, the hydrogen bond formed between the carbonyl group of Gly57 and the N5H of the reduced cofactor strongly influences the stability of the semiquinone state . In this study, the sequential elimination of all side chain interactions in various combinations through a systematic alanine-scanning mutagenesis approach was conducted to more completely understand the functional inter-relationships as well as any synergistic interactions that might occur within the loop . In general, additive effects for each side chain on the midpoint potentials for both couples were ob