Differential levels of ferredoxin and rubredoxin in Clostridium acetobutylicum

Ferredoxin and rubredoxin levels have been determined in DEAE-cellulose treated extracts of Clostridium acetobutylicum using specific enzymatic assays. In contrast to ferredoxin, the content of rubredoxin is affected by various culture conditions; it fluctuates in the proportions of 1 to 3 according to the growth phase, 1 to 8 according to the medium composition, and 1 to 40 according to the pH. Highest rubredoxin level is obtained at the end of the acid phase when the cells grow in chemically defined medium, the pH of which is not controlled. Such variations suggest that rubredoxin as well as rubredoxin-reductase take part in an electron transport chain system inducible by the culture conditions.     

Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high-molecular-weight rubredoxin

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.     

Rapid species identification and partial strain differentiation of Clostridium butyricum by PCR using 16S-23S rDNA intergenic spacer regions

Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain-specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S-23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.     

Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin.

A synthetic gene based on the published amino acid sequence for Clostridium pasteurianum rubredoxin was constructed, cloned in Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promoter system in E. coli HMS273. UV/visible spectroscopy and metal analyses indicated that the as-isolated synthetic gene product is a mixture of holo-(i.e. iron-containing) rubredoxin and zinc-substituted rubredoxin, with the latter amounting to approximately 70% of the total rubredoxin. The UV/visible absorption and resonance Raman spectra of the cloned holorubredoxin are characteristic of the native rubredoxin-type iron site. N-terminal amino acid sequencing suggests that the gene product consists of at least three polypeptide species with the initial sequences (approximate relative abundances): Met-Met-Lys-... (63%), blocked (30%) and Met-Lys-... (7%). The blocked portion presumably consists of a mixture of nMet-Met-Lys-... and nMet-Lys-..., where nMet represents an amino-blocked methionine residue.     

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

AIMS: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.     

Identification of the gene encoding NADH-rubredoxin oxidoreductase in Clostridium acetobutylicum.

NADH-rubredoxin oxidoreductase (NROR), a flavoprotein from the obligately anaerobe Clostridium acetobutylicum is encoded by an ORF (nror) of 1140 nucleotides. Whereas primary structure analysis reveals that NROR has amino acid sequence patterns homologous with those involved in FAD and NAD-binding, the enzyme is distantly related to other flavoproteins in the databank. NROR is highly active for reducing clostridial rubredoxin (Rd) especially against C. acetobutylicum Rd with an efficiency (k(cat)/K(m)) of 400,000 mM(-1)s(-1). These results suggest that Rd from C. acetobutylicum, C. pasteurianum, C. butyricum, and C. cellulolyticum can be interchanged with each other. Since C. acetobutylicum is the sole Clostridium strain that possesses such an enzyme, possible functions are discussed with regard to Desulfovibrio gigas and Pyrococcus furiosus, the only two other anaerobic systems for which a similar activity was reported, but no gene isolated.     

Identification of novel linear megaplasmids carrying a ß-lactamase gene in neurotoxigenic Clostridium butyricum type E strains

Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P) and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ?-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ?-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human pathogen.     

Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1,3-propanediol from glycerol

Clostridium butyricum is to our knowledge the best natural 1,3-propanediol producer from glycerol and the only microorganism identified so far to use a coenzyme B12-independent glycerol dehydratase. However, to develop an economical process of 1,3-propanediol production, it would be necessary to improve the strain by a metabolic engineering approach. Unfortunately, no genetic tools are currently available for C. butyricum and all our efforts to develop them have been so far unsuccessful. To obtain a better "vitamin B12-free" biological process, we developed a metabolic engineering strategy with Clostridium acetobutylicum. The 1,3-propanediol pathway from C. butyricum was introduced on a plasmid in several mutants of C. acetobutylicum altered in product formation. The DG1(pSPD5) recombinant strain was the most efficient strain and was further characterized from a physiological and biotechnological point of view. Chemostat cultures of this strain grown on glucose alone produced only acids (acetate, butyrate and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized in chemostat culture, 1,3-propanediol became the major product, the specific rate of acid formation decreased and a very low level of hydrogen was observed. In a fed-batch culture, the DG1(pSPD5) strain was able to produce 1,3-propanediol at a higher concentration (1104 mM) and productivity than the natural producer C. butyricum VPI 3266. Furthermore, this strain was also successfully used for very long term continuous production of 1,3-propanediol at high volumetric productivity (3 g L-1 h-1) and titer (788 mM).     

Role of acetate and butyrate in the induction of NADH: rubredoxin oxidoreductase in Clostridium acetobutylicum

Study of the biosynthesis of NADH: rubredoxin oxidoreductase in resting cells of Clostridium acetobutylicum shows that this enzyme is synthesized at a maximal rate in the presence of acetic acid at a concentration of 3 g . l-1 and at pH 4.8. Protons do not play any role in this biosynthesis since no induction is observed in a medium without acetate for the same values of pH. Butyric acid at a concentration of 0.5 g . l-1 gives 50% induction and formic acid, isobutyric acid and propionic acid have no inductive action on NADH: rubredoxin oxidoreductase. These results are confirmed by studies using a dialysis bag. Only a culture against acetic acid at an initial concentration of 2 g . l-1 gives maximal biosynthesis of the enzyme, whereas a culture in which all products of metabolism are eliminated gives an activity which is 80% lower.     

酪酸梭菌与婴儿型双歧杆菌对产气荚膜梭菌试管内生物拮抗作用的研究

用酪酸权菌（Clostridium butyricum）和婴儿型双歧杆菌（Bifidobacterium Infantis）对产气荚膜梭菌（Clostriduim perfringens）进行试管内的生物拮抗试验。将酪酸梭菌、婴儿型双歧杆菌及酪酸梭菌和婴儿型双歧杆菌分别对产气荚膜酸菌以一定的比例等量混合接种于GAM液体培养基中进行厌氧培养。实验证明酪酸梭菌和婴儿型双歧能明显抑制产气荚膜梭菌的生长,并且比各自单独培养时显示了较强的生物拮抗作用。     

Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H(+) ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.     

Regulation of the biosynthesis of NADH-Rubredoxin oxidoreductase inClostridium acetobutylicum

The effects of the pH of the culture medium on the biosynthesis of NADH-rubredoxin oxidoreductase byClostridium acetobutylicum, strain ATCC 824, have been studied. Between pH 6.8 and pH 4.2, the rate of enzyme formation fluctuated in the proportions of 1–50 respectively. The induction of the rubredoxin reductase synthesis, normally observed at pH 4.3, was stopped immediately after the addition of rifampicin. It is suggested that NADH-rubredoxin reductase could play a role in some deacidification mechanism in relation to proton transport.     

Sequences of the botulinal neurotoxin E derived from Clostridium botulinum type E (strain Beluga) and Clostridium butyricum (strains ATCC 43181 and ATCC 43755).

Recently, it has been shown that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755), isolated from cases of infant botulism, produce a botulinal neurotoxin type E (BoNT/E). Here we have determined the nucleotide sequences of the BoNT/E genes of these two C. butyricum strains and from C. botulinum E strain Beluga. We show that the sequences of the BoNT/E genes from the two C. butyricum strains are identical and differ in only 64 positions resulting in 39 amino acid changes (97% identity at the amino acid level) from that derived from C. botulinum. Our data suggest a transfer of the BoNT/E gene from C. botulinum to the originally nontoxigenic C. butyricum strains.     

酒窖底泥中丁酸梭菌的分离及其特性研究

通过厌氧培养法从酒窖底泥中分离得到14个菌株,生理生化实验和16SrDNA序列分析鉴定出2株丁酸梭菌。选取丁酸梭菌菌株B1研究了其生长特性和安全性,体外研究表明其具有较强的耐酸、耐胆汁和耐抗生素能力,并能显著抑制常见肠道致病菌的生长,具有作为饲用微生态制剂应用的潜力。     

Plasmid localization of a type E botulinal neurotoxin gene homologue in toxigenic Clostridium butyricum strains, and absence of this gene in non-toxigenic C. butyricum strains

It has been shown recently that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755) contain a botulinal neurotoxin type E (BoNT/E) gene closely related to that of C. botulinum type E. In this study, we show that this gene is located on a large plasmid in the two toxigenic C. butyricum strains and is absent in 18 non-toxigenic C. butyricum and C. beijerinckii strains. Interestingly, the 230 bp upstream and the 1260 bp downstream of the neurotoxin coding sequence are not present in either the non-toxigenic C. butyricum or C. beijerinckii strains. Our data suggest a BoNT/E gene transfer from C. botulinum E to originally non-toxigenic C. butyricum strains.