KdpE of Clostridium acetobutylicum is a highly specific response regulator controlling only the expression of the kdp operon

KdpE from Clostridium acetobutylicum was enriched in form of its Strep-tag-derivative to allow easy immunodetection. It could be artificially phosphorylated by acetyl phosphate or carbamyl phosphate. Only phosphorylated clostridial KdpE was able to bind to a region upstream of the clostridial kdp structural genes. The minimal sequence requirements for binding were determined and found to share significant similarity with the Escherichia coli KdpE binding motif. However, the clostridial protein proved to be much more specific and did not bind in unphosphorylated form or to other similar sequences either from C. acetobutylicum or E. coli. In contrast, the enterobacterial protein recognized the clostridial binding motif. An HPt domain has been detected in KdpD from C. acetobutylicum, the cognate sensor kinase of KdpE. The data reported indicate that in E. coli, KdpE might represent a regulatory checkpoint for different phosphorelay signalling pathways, whereas in C. acetobutylicum KdpD might serve this function.     

Heterologous expression of clostridial hydrogenase in the Cyanobacterium synechococcus PCC7942

The Clostridium pasteurianum hydrogenase I has been expressed in the cyanobacterium Synechococcus PCC7942. The Shine-Dalgarno sequence of the structural gene encoding hydrogenase I from C. pasteurianum was changed to that of the cat (chloramphenicol acetyltransferase) gene. The hydrogenase gene was cloned downstream of a strong promoter, isolated from Synechococcus PCC7942, with the cat gene as a reporter gene. Expression of clostridial hydrogenase was confirmed by Western and Northern blot analyses in Synechococcus and Escherichia coli, whereas in vivo/in vitro measurements and activity staining of soluble proteins separated on non-denaturing polyacrylamide gels revealed functional expression of hydrogenase only in cyanobacterial cells. The changed Shine-Dalgarno sequence appeared to be essential for the functional expression of clostridial hydrogenase in Synechococcus, but had no influence on the expression and activity of clostridial hydrogenase expressed in E. coli.     

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.     

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.     

Detection of Clostridial Hemolysin Formed In Vivo

Experimental clostridial myonecrosis closely resembling gas gangrene was produced in goats by the intramuscular injection of washed clostridia suspended in adrenalin. The preparation provides a model for the study of clostridial myonecrosis that is relatively isolated from preceding trauma. A new test for the demonstration of clostridial exotoxins, the hemolysin indicator plate test, was described. This test gave reliable results within 6 hr, and only minute quantities of test material were required. Application of this test to would exudates of goats with experimental clostridial myonecrosis demonstrated the presence of the gamma toxin of Clostridium novyi, alpha toxin of C. septicum, and the alpha toxin of C. perfringens in exudates from animals injected with washed Clostridium bacilli suspended in adrenalin. Exudates from goats injected with C. perfringens were lethal for mice and produced necrosis of guinea pig skin typical of C. perfringens. The convincing demonstration of clostridial exotoxins formed in vivo in this experimental model could be the basis for definition of clostridial exotoxin actions which have evaded study in the past.     

The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli.

The gene encoding the NAD(+)-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M(r) 49,295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site-directed mutagenesis.     

Characterization of a psychrotrophic Clostridium causing spoilage in vacuum-packed cooked pork: description of Clostridium algidicarnis sp. nov.

A Clostridium species causing spoilage of vacuum-packed refrigerated pork was isolated and characterized. The unknown organism differed phenotypically from other clostridial species usually associated with spoilage. Phylogenetic analyses based on 16S rRNA gene sequencing demonstrated that the psychrotroph represents a distinct line of descent within the genus Clostridium. It is proposed that the organism be classified as a new species of the genus Clostridium, Clostridium algidicarnis .     

High-multiplicity spin states of 2[4Fe-4Se]+ clostridial ferredoxins

The electron paramagnetic resonance (EPR) spectra of the reduced selenium-substituted 2-[4Fe-4Se]+ ferredoxins from three bacteria of the Clostridium genus display low-field signals at g = 5.17, g = 10.11, and g = 12.76. The positions, shapes, and temperature dependencies of these signals have allowed their assignments to the three excited states of an S = 7/2 spin multiplet, the fundamental state of which is observed as unusual features in low-temperature (T less than or equal to 20 K) M?ssbauer spectra. The S = 7/2 spin state is present in 2[4Fe-4Se]+ clostridial ferredoxins together with the classical S = 1/2 state and with a S = 3/2 state, the fundamental doublet of which is observed as a broad signal in the g = 3-4 region. The relative intensities of the EPR signals corresponding to these spin states depend on the species of Clostridium that the ferredoxin is extracted from. In contrast with clostridial ferredoxins, the reduced selenium-substituted ferredoxin from Bacillus stearothermophilus, which differs significantly from the clostridial proteins by its primary structure and by its containing only one tetranuclear cluster, displays only the S = 1/2 state. Thus, the high-multiplicity spin states arise from a specific interaction between the clostridial ferredoxin polypeptide chain and the reduced [4Fe-4Se]+ clusters.     

Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824.

In Clostridium acetobutylicum, conversion of butyraldehyde to butanol is enzymatically achieved by butanol dehydrogenase (BDH). A C. acetobutylicum gene that encodes this protein was identified by using an oligonucleotide designed on the basis of the N-terminal amino acid sequence of purified C. acetobutylicum NADH-dependent BDH. Enzyme assays of cell extracts of Escherichia coli harboring the clostridial gene demonstrated 15-fold-higher NADH-dependent BDH activity than untransformed E. coli, as well as an additional NADPH-dependent BDH activity. Kinetic, sequence, and isoelectric focusing analyses suggest that the cloned clostridial DNA contains two or more distinct C. acetobutylicum enzymes with BDH activity.     

Sequence of the lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC824: comparison with other lytic enzymes

The lyc gene, encoding an autolytic lysozyme from Clostridium acetobutylicum ATCC824, has been cloned. The nucleotide sequence of the lyc gene has been determined and found to encode a protein of 324 amino acids (aa) with a deduced Mr of 34,939. The lyc gene is preceded by two open reading frames with unknown functions, suggesting that this gene is part of an operon. Comparison between the deduced aa sequence of the lyc gene and the directly determined N-terminal sequence of the extracellular clostridial lysozyme suggests that the enzyme is synthesized without a cleavable signal peptide. Moreover, the comparative analyses between the clostridial lysozyme and other known cell-wall lytic enzymes revealed a significant similarity with the N-terminal portion of the lysozymes of Streptomyces globisporus, the fungus Chalaropsis, the Lactobacillus bulgaricus bacteriophage mv1, and the Streptococcus pneumoniae bacteriophages of the Cp family (CPL lysozymes). In addition, the analyses showed that the C-terminal half of the clostridial lysozyme was homologous to the N-terminal domain of the muramoyl-pentapeptide-carboxypeptidase of Streptomyces albus, suggesting a role in substrate binding. The existence of five putative repeated motifs in the C-terminal region of the autolytic lysozyme suggests that this region could play a role in the recognition of the polymeric substrate.     

Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.     

Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases

The product of yjeK in Escherichia coli is a homologue of lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4, and both enzymes catalyze the isomerization of (S)- but not (R)-alpha-lysine by radical mechanisms. The turnover number for LAM from E. coli is 5.0 min(-1), 0.1% of the value for clostridial LAM. The reaction of E. coli LAM with (S)-alpha-[3,3,4,4,5,5,6,6-(2)H8]lysine proceeds with a kinetic isotope effect (kH/kD) of 1.4, suggesting that hydrogen transfer is not rate-limiting. The product of the E. coli enzyme is (R)-beta-lysine, the enantiomer of the clostridial product. Beta-lysine-related radicals are observed in the reactions of both enzymes by electron paramagnetic resonance (EPR). The radical in the reaction of clostridial LAM has the (S)-configuration, whereas that in the reaction of E. coli LAM has the (R)-configuration. Moreover, the conformations of the beta-lysine-related radicals at the active sites of E. coli and clostridial LAM are different. The nuclear hyperfine splitting between the C3 hydrogen and the unpaired electron at C2 shows the dihedral angle to be 6 degrees, unlike the value of 77 degrees reported for the analogous radical bound to the clostridial enzyme. Reaction of (S)-4-thialysine produces a substrate-related radical in the steady state of E. coli LAM, as in the action of the clostridial enzyme. While (S)-beta-lysine is not a substrate for E. coli LAM, it undergoes hydrogen abstraction to form an (S)-beta-lysine-related radical with the same stereochemistry of hydrogen transfer from C2 of (S)-beta-lysine to the 5'-deoxyadenosyl radical as in the action of the clostridial enzyme. The resulting beta-lysyl radical has a conformation different from that at the active site of clostridial LAM. All evidence indicates that the opposite stereochemistry displayed by E. coli LAM is determined by the conformation of the lysine side chain in the active site. Stereochemical models for the actions of LAM from C. subterminale and E. coli are presented.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme's unusual pH dependence

Glutamate dehydrogenase from Clostridium symbiosum displays unusual kinetic behaviour at high pH when compared with other members of this enzyme family. Structural and sequence comparisons with GDHs from other organisms have indicated that the Asp residue at position 114 in the clostridial enzyme may account for these differences. By replacing this residue by Asn, a mutant protein has been created with altered functional properties at high pH. This mutant protein can be efficiently overexpressed in Escherichia coli, and several criteria, including mobility in non-denaturing electrophoresis, circular dichroism (CD) spectra and initial crystallisation studies, suggest a folding and an assembly comparable to those of the wild-type protein. The D114N mutant enzyme shows a higher optimum pH for activity than the wild-type enzyme, and both CD data and activity measurements show that the distinctive time-dependent reversible conformational inactivation seen at high pH in the wild-type enzyme is abolished in the mutant.     

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.     

Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases

Large clostridial cytotoxins catalyze the glucosylation of Rho/Ras GTPases using UDP-glucose as a cosubstrate. By site-directed mutagenesis of Clostridium sordellii lethal toxin and Clostridium difficile toxin B fragments, we identified tryptophan 102, which is located in a conserved region within the catalytic domain of all clostridial cytotoxins, to be crucial for UDP-glucose binding. Exchange of Trp-102 with alanine decreased the glucosyltransferase activity by about 1,000-fold and blocked cytotoxic activity after microinjection. Replacement of Trp-102 by tyrosine caused a 100-fold reduction in enzyme activity, indicating a partial compensation of the tryptophan function by tyrosine. Decrease in glucosyltransferase and glycohydrolase activity was caused predominantly by an increase in the K(m) for UDP-glucose of these mutants. The data indicate that the conserved tryptophan residue is implicated in the binding of the cosubstrate UDP-glucose by large clostridial cytotoxins. Data bank searches revealed different groups of proteins sharing the recently identified DXD motif (Busch, C., Hofmann, F., Selzer, J., Munro, J., Jeckel, D., and Aktories, K. (1998) J. Biol. Chem. 273, 19566-19572) and a conserved region defined by a tryptophan residue equivalent to Trp-102 of C. sordellii lethal toxin. From our findings, we propose a novel family of glycosyltransferases which includes both prokaryotic and eukaryotic proteins.     

Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.     

Characterization of a Human Serum Inhibitor of Clostridium histolyticum Proteinase(s)

Normal animal sera inhibit at least one Clostridium histolyticum proteinase. An assay procedure based on immune hemolysis was developed for the estimation of this inhibition. This inhibitory activity occurs in various levels in the sera of different animal species. The highest titers have been obtained with rat sera. The inhibitory activity from human serum was isolated and purified 16- to 27-fold by Sephadex G-200 gel filtration and diethylaminoethyl cellulose or hydroxylapatite chromatography. The properties of the human serum inhibitor of the clostridial proteinase were compared with a trypsin inhibiting factor found in the partially purified preparations. Identical behavior of the two inhibitory factors was observed when measured by heat inactivation, beta-mercaptoethanol sensitivity, pH stability, and sucrose gradient centrifugation. The inhibitory factor has an approximate sedimentation coefficient (S(20,w)) of 17. Goat anti-alpha-2-macroglobulin specifically precipitated the clostridial proteinase inhibitor from a partially purified preparation.     

Characterisation of a transposon-induced pleiotropic mutant of Clostridium acetobutylicum P262

Transposon-induced metronidazole resistance was used as a selection system for the isolation of Clostridium acetobutylicum P262 mutants with altered electron transport pathways. The metronidazole resistant transconjugant of interest, mutant 3R, displayed resistance to DNA damaging agents, UV and bleomycin, and harboured a single transposon insertion within a structural gene, designated sum(susceptibility to metronidazole). The sum gene encoded a 334 amino-acid protein, with 36% identity and 57-58% similarity at the amino acid level to two archaebacterial protein sequences which appear to represent a class of uncharacterised reductase enzymes. Physiological studies of mutant 3R revealed a number of pleiotropic characteristics which included enhanced autolysin activity, increased motility, impaired clostridial cell formation, and resistance to the toxic tripeptide analogue, bialaphos. The introduction of the sum gene in multiple copies on a plasmid vector into the related strain Clostridium beijerinckii NCIMB 8052, resulted in inhibition of cell division, motility and autolysin activity. The sum gene appears to be a member of a new subgroup of activases with reducing activity, which may control a regulon affecting different stationary phase processes such as clostridial differentiation and sporulation in C. acetobutylicum P262. The metronidazole resistant phenotype of the sum mutant can be attributed to an increased capacity for DNA repair.