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Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Sequenced genomes provide completely new insights into the diversity of clostridial hydrogenases. Building on previous reports, we found that [FeFe] hydrogenases are not a homogeneous group of enzymes, but exist in multiple forms with different modular structures and are especially abundant in members of the genus Clostridium. This unusual diversity seems to support the central role of hydrogenases in cell metabolism. In particular, the presence of multiple putative operons encoding multisubunit [FeFe] hydrogenases highlights the fact that hydrogen metabolism is very complex in this genus. In contrast with [FeFe] hydrogenases, their [NiFe] hydrogenase counterparts, widely represented in other bacteria and archaea, are found in only a few clostridial species. Surprisingly, a heteromultimeric Ech hydrogenase, known to be an energy-converting [NiFe] hydrogenase and previously described only in methanogenic archaea and some sulfur-reducing bacteria, was found to be encoded by the genomes of four cellulolytic strains: Clostridum cellulolyticum, Clostridum papyrosolvens, Clostridum thermoceflum and Clostridum phytofermentans. DOI 10.1099/mic.0.032771-0

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyI-L-arginine p-nitroanilide (Bz-L-arginine pNA), as demonstrated by assaying culture supematants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-DL-arginine pNA rather than Bz-DL-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis. DOI 10.1099/mic.0.031609-0

Clostridium perfringens, a Gram-positive anaerobe, is a human pathogen that causes gas gangrene in muscle tissues. Its ability to grow and survive in the host is believed to be due to the production of numerous enzymes that enable the organism to obtain essential nutrients from the host. In this study, CPE0201, a putative acid phosphatase gene deduced by genome analysis, was shown to encode a non-specific acid phosphatase in C. perfringens. Multiple alignments of the amino acid sequence showed that CPE0201 shares two signature motifs that belong to a class C acid phosphatase family. Expression of CPE0201 was shown to be positively regulated by the global VirR/VirS-VR-RNA regulatory cascade at the transcriptional level. To determine the acid phosphatase activity of the CPE0201-encoded protein, cloning, expression, purification and several biochemical characterizations were carried out. The optimum pH for activity of the CPE0201 enzyme was 4.8, and its [V.sub.max] and [K.sub.m] were 3.08 nmol [ml.sup.-1] [min.sup.-1] and 2.84 mM, respectively, with p-nitrophenyl phosphate (PNPP) as substrate. A CPE0201 mutant did not grow in a minimal medium containing PNPP, while it showed normal growth when [Na.sub.2]HP[O.sub.4] was added to the medium. The enzyme appears to be associated with the surface of the cell, where it may function to acquire inorganic phosphate from organic phosphomonoesters in acidic conditions, which could play an important role in the survival and growth of C. perfringens in the host tissue. DOI 10.1099/mic.0.030395-0

Loss of the apical V-ATPase a-subunit VHA-6 prevents acidification of the intestinal lumen during a rhythmic behavior in C. elegans. Am J Physiol Cell Physiol 297: C1071-C1081, 2009. First published September 9, 2009; doi:10.1152/ajpcell.00284.2009.--In Caenorhabditis elegans, oscillations of intestinal pH contribute to the rhythmic defecation behavior, but the acid-base transport mechanisms that facilitate proton movement are not well understood. Here, we demonstrate that VHA-6, an intestine-specific a-subunit of the [H.sup.+]-[K.sup.+]-ATPase complex (V-ATPase), resides in the apical membrane of the intestinal epithelial cells and is required for luminal acidification. Disruption of the vha-6 gene led to early developmental arrest; the arrest phenotype could be complemented by expression of a fluorescently labeled vha-6 transgene. To study the contribution of vha-6 to pH homeostasis in larval worms, we used a partial reduction of function through postembryonic single-generation RNA interference. We demonstrate that the inability to fully acidify the intestinal lumen coincides with a defect in pH recovery of the intestinal epithelial cells, suggesting that VHA-6 is essential for proton pumping following defecation. Moreover, intestinal dipeptide accumulation and fat storage are compromised by the loss of VHA-6, suggesting that luminal acidification promotes nutrient uptake in worms, as well as in mammals. Since acidified intracellular vesicles and autofluorescent storage granules are indistinguishable between the vha-6 mutant and controls, it is likely that the nutrient-restricted phenotype is due to a loss of plasma membrane V-ATPase activity specifically. These data establish a simple genetic model for proton pump-driven acidification. Since defecation occurs at 45-s intervals in worms, this model represents an opportunity to study acute regulation of V-ATPase activity on a short time scale and may be useful in the study of alternative treatments for acid-peptic disorders. biorhythm; nematode; proton doi: 10.1152/ajpcell.00284.2009.

The genome of the Clostridium perfringens food poisoning isolate SM101 encodes a subtilisin-like protease, CspB, upstream of the sleC gene encoding the enzyme essential for degradation of the peptidoglycan cortex during spore germination. SleC is an inactive pro-SleC in dormant spores that is converted to active SleC during spore germination and Csp proteases convert pro-SleC to the active enzyme in vitro. In this work, the germination and viability of spores of a cspB deletion mutant of strain SM101, as well as cspB expression, were studied. The cspB gene was expressed only during sporulation, and only in the mother cell compartment, cspB spores were unable to germinate significantly with either a rich nutrient medium, KCl, or a 1 : 1 chelate of [Ca.sup.2+] and dipicolinic acid (DPA); the viability of these spores was ~[10.sup.4]-fold lower than that of wild-type spores, although cspB and wild-type spores had similar viability on plates containing lysozyme, and cspB spores could not process inactive pro-SleC into active SleC during spore germination. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis, and germination and viability defects in these spores were complemented by an ectopic cspB. These results indicate that Csp proteases are essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. However, Csp proteases likely play another role in spore germination, since cspB spores did not release DPA upon exposure to germinants, while sleC spores have been shown previously to release DPA, albeit slowly, upon exposure to germinants. DOI 10.1099/mic.0.030965-0

In the strict anaerobe Clostridium acetobutylicum, a PerR-homologous protein has recently been identified as being a key repressor of a reductive machinery for the scavenging of reactive oxygen species and molecular [O.sub.2]. In the absence of PerR, the full derepression of its regulon resulted in increased resistance to oxidative stress and nearly full tolerance of an aerobic environment. In the present study, the complementation of a Bacillus subtilis PerR mutant confirmed that the homologous protein from C. acetobutylicum acts as a functional peroxide sensor in vivo. Furthermore, we used a transcriptomic approach to analyze gene expression in the aerotolerant PerR mutant strain and compared it to the [O.sub.2] stimulon of wild-type C. acetobutylicum. The genes encoding the components of the alternative detoxification system were PerR regulated. Only few other targets of direct PerR regulation were identified, including two highly expressed genes encoding enzymes that are putatively involved in the central energy metabolism. All of them were highly induced when wild-type cells were exposed to sublethal levels of [O.sub.2]. Under these conditions, C. acetobutylicum also activated the repair and biogenesis of DNA and Fe-S clusters as well as the transcription of a gene encoding an unknown CO dehydrogenase-like enzyme. Surprisingly few genes were downregulated when exposed to [O.sub.2], including those involved in butyrate formation. In summary, these results show that the defense of this strict anaerobe against oxidative stress is robust and by far not limited to the removal of [O.sub.2] and its reactive derivatives. doi: 10.1128/JB.00351-09

The multi-modular non-cellulosomal endo-1,3(4)-[beta]-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. [Ca.sup.2+] ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of [Ca.sup.2+]. The highest affinity to CBMX was shown for xylan (binding constant K=3.1 x [10.sup.4] [M.sup.-1]), yeast cell wall glucan (K = 1.4 x [10.sup.5] [M.sup.-1]) and chitin (K=3.3.[10.sup.5] [M.sup.-1] in the presence of [Ca.sup.2+]). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.

The genome of the pathogen Clostridium perfringens encodes two proteins, GerO and GerQ, homologous to monovalent cation transporters suggested to have roles in the germination of spores of some Bacillus species. GerO and GerQ were able to transport monovalent cations ([K.sup.+] and/or [Na.sup.+]) in Escherichia coli, and gerO and gerQ were expressed only in the mother cell compartment during C. perfringens sporulation. C. perfringens spores lacking GerO were defective in germination with a rich medium, KCl, L-asparagine, and a 1:1 chelate of [Ca.sup.2+] and dipicolinic acid (DPA), but not with dodecylamine, and the defect was prior to DPA release in germination. All defects in gerO spores were complemented by ectopic expression of wild-type gerO. Loss of GerQ had much smaller effects on spore germination, and these effects were most evident in spores also lacking GerO. A modeled structure of GerO was similar to that of the E. coli [Na.sup.+]/[H.sup.+] antiporter NhaA, and GerO, but not GerQ contained two adjacent Asp residues thought to be important in the function of this group of cation transporters. Replacement of these adjacent Asp residues in GerO with Asn reduced the protein's ability to complement the germination defect in gerO spores but not the ability to restore cation transport to E. coli cells defective in [K.sup.+] uptake. Together, these data suggest that monovalent cation transporters play some role in C. perfringens spore germination. However, it is not clear whether this role is directly in germination or perhaps in spore formation.

An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upregulated in response to oxidative stress in the strictly anaerobic bacterium Clostridium acetobutylicum. In the present study both in vivo and in vitro results demonstrate an [H.sub.2][O.sub.2] and [O.sub.2] detoxification pathway in C. acetobutylicum involving revRbr, rubredoxin (Rd) and NADH : rubredoxin oxidoreductase (NROR). RevRbr exhibited both NADH peroxidase (NADH : [H.sub.2][O.sub.2] oxidoreductase) and NADH oxidase (NADH : [O.sub.2] oxidoreductase) activities in in vitro assays using NROR as the electron-transfer intermediary from NADH to revRbr. Rd increased the NADH consumption rate by serving as an intermediary electron-transfer shuttle between NROR and revRbr. While [H.sub.2][O.sub.2] was found to be the preferred substrate for revRbr, its relative oxidase activity was found to be significantly higher than that reported for other Rbrs. A revRbr-overexpressing strain of C. acetobutylicum showed significantly increased tolerance to [H.sub.2][O.sub.2] and [O.sub.2] exposure. RevRbr thus appears to protect C. acetobutylicum against oxidative stress by functioning as the terminal component of an NADH peroxidase and NADH oxidase.

Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble [beta]-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.

The crystal structure studies of a second conformational state of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) are reported. The studies confirm the swivel motion of the His domain adjacent to the phosphoenolpyruvate (PEP)-binding domain.

The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity.

Antisense RNA is used to decrease the levels of [alpha]/[beta]-type small, acid-soluble spore proteins (SASP) in Clostridium perfringens spores, which in turn have reduced the resistance to both moist heat and ultraviolet (UV) radiation but not to dry heat. The results have highlighted the [alpha]/[beta]-type SASP's role in the resistance of Clostridium perfringens spores.

An unknown cytotoxin was identified in the culture supernatant of Clostridium perfringens type C. The cytotoxin, named TpeL, which was purified using mAb-based affinity chromatography, had a lethal activity of 62 minimum lethal dose (MLD) [mg.sup.-1] in mice and a cytotoxic activity of 6.2 x [10.sup.5] cytotoxic units (CU) [mg.sup.-1] in Vero cells. The nucleotide sequence of TpeL was determined. The entire ORF had a length of 4953 bases, and the same nucleotide sequence was not recorded in the GenBank/EMBL/DDBJ databases. The molecular mass calculated from the deduced amino acid sequence was 191 kDa, and a signal peptide region was not found within the ORF. The deduced amino acid sequence exhibited 30-39% homology to Clostridium difficile toxins A (TcdA) and B (TcdB), Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi alpha-toxin (TcnA). The amino acid sequence of TpeL is shorter than these toxins, and the homologous region was located at the N-terminal site. Eighteen strains of C. perfringens types A, B and C were surveyed for the presence of the tpeL gene by PCR. The tpeL gene was detected in all type B (one strain) and C strains (five strains), but not in any type A strains (12 strains). TpeL was detected in culture filtrates of the five type C strains by dot-blot analysis, but not in the type B strain. It was concluded that TpeL is a novel toxin similar to the known large clostridial cytotoxins. Furthermore, the data indicated that TpeL is produced by many C. perfringens type C strains.

The uptake and metabolism of lactose, the major sugar in industrial whey waste, by Clostridium acetobutylicum ATCC 824 is investigated. Lactose is taken up via a phosphoenolpyruvate-dependent phosphotransferase system and a pathway is proposed for the uptake and metabolism of lactose by an industrially important organism.

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Ce148F, Ce18C, Ce19G, Ce19E, Man5K, Ce19M, and Ce15A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Ce15N, Ce19J, and Ce1440 were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.

A DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant of C. perfringens strain CPBC16ML had the expected molecular weight detected in reference strains of C. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid of C. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in the cpa ORF of C. perfringens.

Clostridium paradoxum is an anaerobic thermoalkaliphilic bacterium that grows rapidly at pH 9.8 and 56[degrees]C. Under these conditions, growth is sensitive to the F-type ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), suggesting an important role for this enzyme in the physiology of C. paradoxum. The ATP synthase was characterized at the biochemical and molecular levels. The purified enzyme (30-fold purification) displayed the typical subunit pattern for an [F.sub.1][F.sub.o]-ATP synthase but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The purified ATPase was stimulated by sodium ions, and sodium provided protection against inhibition by DCCD that was pH dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed chemical gradient of sodium ions in the presence of a transmembrane electrical potential that was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., [Q.sup.28], [E.sup.61], and [S.sup.62]). On the basis of these properties, the [F.sub.1][F.sub.o]-ATP synthase of C. paradoxum is a sodium-translocating ATPase that is used to generate an electrochemical gradient of [Na.sup.+] that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the [epsilon] subunit that has been shown to be essential for coupled ATP synthesis.

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.

Clostridium perfringens is an important human and animal pathogen that causes a number of diseases that vary in their etiology and severity. Differences between strains regarding toxin gene composition and toxin production partly explain why some strains cause radically different diseases than others. However, they do not provide a complete explanation. The purpose of this study was to determine if there is a phylogenetic component that explains the variance in C. perfringens strain virulence by assessing patterns of genetic polymorphism in genes (colA gyrA, plc, pfoS, and rplL) that form part of the core genome in 248 type A strains. We found that purifying selection plays a central role in shaping the patterns of nucleotide substitution and polymorphism in both housekeeping and virulence genes. In contrast, recombination was found to be a significant factor only for the virulence genes plc and colA and the housekeeping gene gyrA. Finally, we found that the strains grouped into five distinct evolutionary lineages that show evidence of host adaptation and the early stages of speciation. The discovery of these previously unknown lineages and their association with distinct disease presentations carries important implications for human and veterinary clostridial disease epidemiology and provides important insights into the pathways through which virulence has evolved in C. perfringens.

The rubredoxin from Clostridium pasteurianum (CpRd) provides an excellent system for investigating how the protein sequence modulates the reduction potential of the active site in an iron-sulfur protein, [sup.15]N NMR spectroscopy has allowed us to determine with unprecedented accuracy the strengths of all six key hydrogen bonds between protein backbone amides and the sulfur atoms of the four cysteine residues that ligate the iron in the oxidized ([Fe.sup.III]) and reduced ([Fe.sup.III]) forms of wild-type CpRd and nine mutants (V44G, V44A, V44I, V44L, V8G, V8A, V8I, V8L, and V8G/V44G). The length (or strength) of each hydrogen bond was inferred from the magnitude of electron spin delocalized across the hydrogen bond from the iron atom onto the nitrogen. The aggregate lengths of these six hydrogen bonds are shorter in both oxidation states in variants with higher reduction potential than in those with lower reduction potential. Differences in aggregate hydrogen bonding upon reduction correlate linearly with the published reduction potentials for the 10 CpRd variants, which span 126 mV. Sequence effects on the reduction potential can be explained fully by their influence on hydrogen-bond strengths. iron-sulfur protein | reduction potential tuning | [sup.15]N NMR | paramagnetic NMR

The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by Clostridium beijerinckii provide evidence for the operation of two uptake systems, a phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system. The potential presence of such a transport system for glucose in both Clostridium beijerinckii BA101 and the parent strain NCIMB 8052 was investigated.

The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, [Y.sup.True.sub.x/ATP] = 16.44 g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for [G.sup.P-T.sub.ATP] (ATP from phosphorolytic [beta]-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed [beta]-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-1)/n (n = mean cellodextrin DP assimilated). A mean [G.sup.P-T.sub.ATP] value of 0.52 [+ or -] 0.06 was calculated for cellulose-grown chemostat cultures, corresponding to n = 4.20 [+ or -] 0.46. Determination of intracellular [beta]-glucan radioactivity resulting from [14.sup.C]-labeled substrates showed that uptake is different for cellulose and cellobiose (G2). For [14.sup.C]-cellobiose, radioactivity was greatest for G2; substantially smaller but measurable for G1, G3, and G4; undetectable for G5 and G6; and n was [approximately equal to] 2. For [14.sup.C]-cellulose, radioactivity was greatest for G5; lower but substantial for G6, G2, and G1; very low for G3 and G4; and n was [approximately equal to] 4. These results indicate that: (i) C. thermocellum hydrolyzes cellulose by a different mode of action from the classical mechanism involving solubilization by cellobiohydrolase; (ii) bioenergetic benefits specific to growth on cellulose are realized, resulting from the efficiency of oligosaccharide uptake combined with intracellular phosphorolytic cleavage of [beta]-glucosidic bonds; and (iii) these benefits exceed the bioenergetic cost of cellulase synthesis, supporting the feasibility of anaerobic biotechnological processing of cellulosic biomass without added saccharolytic enzymes. cellulose hydrolysis | cellulase | cellulosome | anaerobic | thermophilic | ABC transport

Clostridium cellulovorans produces a cellulase enzyme complex called the cellulosome. When cells were grown on different carbon substrates such as Avicel, pectin, xylan, or a mixture of all three, the subunit composition of the cellulosomal subpopulations and their enzymic activities varied significantly. Fractionation of the cellulosomes (7-11 fractions) indicated that the cellulosome population was heterogeneous, although the composition of the scaffolding protein CbpA, endoglucanase EngE and cellobiohydrolase ExgS was relatively constant. One of the cellulosomal fractions with the greatest endoglucanase activity also showed the highest or second highest cellulase activity under all growth conditions tested. The cellulosomal fractions produced from cells grown on a mixture of carbon substrates showed the greatest cellulase activity and contained CbpA, EngE/EngK, ExgS/EngH and EngL. High xylanase activity in cellulose, pectin and mixed carbon-grown cells was detected with a specific cellulosomal fraction which had relatively larger amounts of XynB, XynA and unknown proteins (35-45 kDa). These results in toto indicate that the assembly of cellulosomes occurs in a non-random fashion.

The expression of scaffoldin-anchoring genes and one of the major processive endoglucanases (CelS) from the cellulosome of Clostridium thermocellum has been shown to be dependent on the growth rate. For the present work, we studied the gene regulation of selected cellulosomal endoglucanases and a major xylanase in order to examine the previously observed substrate-linked alterations in cellulosome composition. For this purpose, the transcript levels of genes encoding endoglucanases CelB, CelG, and CelD and the family 10 xylanase XynC were determined in batch cultures, grown on either cellobiose or cellulose, and in carbon-limited continuous cultures at different dilution rates. Under all conditions tested, the transcript levels of celB and celG were at least 10-fold higher than that of celD. Like the major processive endoglucanase CelS, the transcript levels of these endoglucanase genes were also dependent on the growth rate. Thus, at a rate of 0.04 [h.sup.-1], the levels of celB, celG, and celD were threefold higher than those obtained in cultures grown at maximal rates (0.35 [h.sup.-1]) on cellobiose. In contrast, no clear correlation was observed between the transcript level of xynC and the growth rate--the levels remained relatively high, fluctuating between 30 and 50 transcripts per cell. The results suggest that the regulation of C. thermocellum endoglucanases is similar to that of the processive endoglucanase celS but differs from that of a major cellulosomal xylanase in that expression of the latter enzyme is independent of the growth rate.

The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane [DELTA]pH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The [DELTA]pH was measured by the accumulation of [[14.sup]C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the [DELTA]pH component of the protonmotive force) and supplemented with glucose and [Mg.SUP.2] +. The [DELTA]pH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a [DELTA]pH of 1.4 when butanol was added to a concentration of 1.5% (w/v), while the wild-type [DELTA]pH was reduced to 0.1. The [DELTA]pH of both strains was completely dissipated with 1.75% butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the [DELTA]pH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0.8%) prevented this [DELTA]pH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in [DELTA]pH occurred more slowly than in the mutant, and sparing of the [DELTA]pH by 0.8% butanol was less pronounced. The authors interpret these data to mean that the [DELTA]pH is dissipated in the absence of [Mg.sup.2+] by a [Na.sup.+] - or [K.sup.+] -linked process, possibly by a [Na.sup.+]/[H.sup.+] or a [K.sup.+]/[H.sup.+] antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher [Na.sup.+]/[H.sup.+] antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.

Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves [TcdB.sub.10463] between [Leu.sub.543] and [Gly.sub.544] and the naturally occurring variant [TcdB.sub.8864] between [Leu.sub.544] and [Gly.sub.545]. The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 1 21 Da ([TcdB.sub.10463]) and 62 761 Da ([TcdB.sub.8864])] harbour the cytotoxic and glucosyltransferase activities of the toxins. When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time. Twenty minutes after the start of internalization of TcdB, the larger cleavage products [206 609 Da ([TcdB.sub.10463]) and 206 245 Da ([TcdB.sub.8864])] are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane. This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes. Present address: Natural and Medical Sciences Institute at the University of Tubingen, Department of Biochemistry, Reutlingen, Germany. Abbreviations: FBS, fetal bovine serum; LCT, large clostridial toxin; MALDI-TOF, matrix-assisted laser desorption ionization time of flight. The online version of this paper contains five supplementary figures (Figs S1-S5).

The nucleotide sequence of the Clostridium cellulovorans xynB gene, which encodes the XynB xylanase, consists of 1,821 bp and encodes a protein of 607 amino acids with a molecular weight of 65,976. XynB contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 147-amino-acid sequence that is homologous to the family 4-9 (subfamily 9 in family 4) carbohydrate-binding domain. Downstream of this domain is a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The XynB sequence from mass spectrometry and N-terminal amino acid sequence analyses agreed with that deduced from the nucleotide sequence. XynB was highly active toward xylan, but not active toward carboxymethyl cellulose. The enzyme was optimally active at 40[degrees]C and pH 5.0. Northern hybridizations revealed that xynB is transcribed as a monocistronic 1.9-kb mRNA. RNA ligase-mediated rapid amplification of 5' cDNA ends by PCR (RLM-5'RACE PCR) analysis of C. cellulovorans RNA identified a single transcriptional start site of xynB located 47 bp upstream from the first nucleotide of the translation initiation codon. Alignment of the xynB promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the [[sigma].sup.A] consensus promoter sequences of gram-positive bacteria. Expression of xynB mRNA increased from early to middle exponential phase and decreased during the early stationary phase when the cells were grown on cellobiose. No alternative promoter was observed by RLM-5'RACE PCR and reverse transcriptase PCR analyses during expression. The analysis of the products from xylan hydrolysis by thin-layer chromatography indicated its endoxylanase activity. The results suggest that XynB is a consistent and major cellulosomal enzyme during growth on cellulose or xylan.

Clostridial populations and their phylogenetic structures in a strand of the grass Miscanthus sinensis in Japan assessed by a 16s rRNA gene-targeted terminal restriction fragment length polymorphism (TRFLP) analysis combined with most-probable number (MPN) counts is discussed. Group II clostridia were found to exist exclusively in M. Sinensis, when phylogenetic populations from M. Sinensis and the soil around the plants are compared.

The suitability of a real-time PCR (5' nuclease PCR assay) to detect the quantitative C-bioteae and some clostridium groups in fecal specimens of autistic and control children is discussed. The assay is a reliable and rapid method, and has great potential for quantitation of other bacteria in the intestinal tract.

The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans. Two forms of the protein were purified from E. coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in C. cellulolyticum by using expression vectors. The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.

Cellulosomes and noncellulosomal (hemi)cellulolytic enzymes are produced by Clostridium cellulovorans to degrade plant cell walls. To understand their synergistic relationship, changes in mRNA and protein expression in cellulosomes and noncellulosomal (hemi)cellulolytic enzymes (hereafter called noncellulosomal enzymes) of cultures grown on cellobiose, cellulose, pectin, xylan, and corn fiber or mixtures thereof were examined. Cellulase expression, favored particularly by the presence of Avicel, was found with all substrates. Comparison of cellulosome and noncellulosomal enzymes showed that expression profiles were strongly affected by the carbon source. High xylanase or pectate lyase expression was observed when C. cellulovorans was grown on xylan or pectin, respectively. Mixed carbon substrates (cellulose-pectin-xylan mixture or corn fiber) induced a wider variety of enzymes than a single carbon source, such as cellobiose, pectin, or xylan. Cellulosomal proteome profiles were more affected by the carbon source than the noncellulosomal enzymes. Transcription and protein analyses revealed that cellulosomes and noncellulosomal enzymes were expressed simultaneously on mixed carbon sources, but their degree of inducibility varied when the substrate was either cellulose or cellobiose. Cellulosomes and noncellulosomal enzymes had synergistic activity on various carbon substrates. These results indicated that expression of plant cell wall-degrading enzymes is highly influenced by the available carbon source and that synergy between cellulosomes and noncellulosomal enzymes contribute to plant cell wall degradation.

Four extracellular enzymes of the thermophilic bacterium Clostridium stercorarium are involved in the depolymerization of de-esterified arabinoxylan: Xyn 11A, Xyn10C, Bxl3B, and Arf51 B. They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA, xynB, xynC), [beta]-xyloside (bxlA, bxlB, bglZ) and [alpha]-arabinofuranoside (arfA, arfB). The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant of C. stercorarium. Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced. Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated. The other glycosidases either did not attack or only marginally attacked these oligosaccharides. Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan. This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes produced in vivo. The active enzyme system was reconstituted in vitro from recombinant enzymes.

We have studied the basis for intrinsic resistance to low levels of vancomycin in Clostridium innocuum NCIB 10674 (MIC = 8 [micro]g/ml). Analysis by high-pressure liquid chromatography (HPLC) and mass spectrometry of peptidoglycan nucleotide precursors pools revealed the presence of two types of UDP-MurNac-pentapeptide precursors constitutively produced, an UDP-MurNAc-pentapeptide with a serine at the C terminus which represented 93% of the pool and an UDP-MurNAc-pentapeptide with an alanine at the C terminus which represented the rest of the pool. C. innocuum cell wall muropeptides containing pentapeptide[Ser], either dialanine substituted on the epsilon amino group of lysine or not, were identified and represented about 10% of the monomers while only 1% of pentapeptide[D-Ala] monomers were found. The sequence of a 2,465-bp chromosomal fragment from C. innocuum was determined and revealed the presence of [ddl.sub.c.innocuum] and C. innocuum racemase genes putatively encoding homologues of D-Ala:D-X ligases and amino acid racemases, respectively. Analysis of the pool of precursors of Enterococcus faecalis JH2-2, containing cloned [ddl.sub.c.innocuum] and C. innocuum racemase genes showed in addition to the UDP-MurNAc-pentapeptide[n-Ala], the presence of an UDP-MurNAc-pentapeptide [n-Ser] precursor. However, the expression of low-level resistance to vancomycin was observed only when both genes were cloned in E. faecalis JH2-2 together with the vanX[Y.sub.c] gene from Enterococcus gallinarum BM4174 which encodes a D,D-peptidase which eliminates preferentially the high affinity vancomycin UDP-MurNAc-pentapeptide [D-Ala] precursors produced by the host. We conclude that resistance to vancomycin in C. innocuum NCIB 10674 was related to the presence of the two chromosomal [ddl.sub.c. innocuum] and C. innocuum racemase genes allowing the synthesis of a peptidoglycan precursor terminating in serine with low affinity for vancomycin.

The transcriptional regulation of toxin production in the gram-positive anaerobe Clostridium perfringens involves a two-component signal transduction system that comprises the VirS sensor histidine kinase and its cognate response regulator, VirR. Previous studies showed that VirR binds independently to a pair of imperfect direct repeats, now designated VirR box 1 and VirR box 2, located immediately upstream of the promoter of the pfoA gene, which encodes the cholesterol-dependent cytolysin, perfringolysin O. For this study, we introduced mutated VirR boxes into a C. perfringens pfoA mutant and found that both VirR boxes are essential for transcriptional activation. Furthermore, the spacing between the VirR boxes and the distance between the VirR boxes and the -35 region are shown to be critical for perfringolysin O production. Other VirR boxes that were previously identified from the strain 13 genome sequence were also analyzed, with perfringolysin O production used as a reporter system. The results showed that placement of the different VirR boxes at the same position upstream of the pfoA promoter yields different levels of perfringolysin O activity. In all of these constructs, VirR was still capable of binding to the target DNA, indicating that DNA binding alone is not sufficient for transcriptional activation. Finally, we show that the C. perfringens RNA polymerase binds more efficiently to the pfoA promoter in the presence of VirR, indicating that interactions must occur between these proteins. We propose that these interactions are required for VirR-mediated transcriptional activation.

Clostridium perfringens, a strictly anaerobic bacterium, is able to survive when exposed to oxygen for short periods of time and exhibits a complex adaptive response to reactive oxygen species, both under aerobic and anaerobic conditions. However, this adaptive response is not completely understood. C. perfringens possesses specialized genes that might be involved in this adaptive process, such as those encoding superoxide dismutase (SOD), superoxide reductase and alkyl hydroperoxide reductase, but their contribution to the oxidative stress response and their control mechanisms are unknown. By a combination of functional complementation of Escherichia coil strains impaired in either SOD, alkyl hydroperoxide reductase (AhpC) or catalase activity (Cat), transcription analysis and characterization of mutants impaired in regulatory genes, it was concluded that: (i) the product of the sod gene is certainly essential to scavenge superoxide radicals, (ii) the ahpC gene, which is fully induced in all oxidative stress conditions, is probably involved in the scavenging of all intracellular peroxides, (iii) the three rubrerythrin (rbr) genes of C. perfringens do not encode proteins with in vivo [H.sub.2][O.sub.2] reductase activity, and (iv) the two rubredoxin (rub) genes do not contribute to the hypothetical superoxide reductase activity, but are likely to belong to an electron transfer chain involved in energy metabolism.

Clostridium populations that are responsible for cellulose degradation in a methanogenic landfill leachate bioreactor are identified, detected and their spatial resolution is determined based on their proximity to the cellulose surface, identity and relative abundance. The presence, abundance and spatial arrangements of different microorganisms in the bioreactor were confirmed by Fluorescence in situ hybridization (FISH).

Clostridium thermocellum is an anaerobic endospore forming bacteria capable of fermenting cellulose and other beta-glucan substrates to ethanol and acetic acid. Electrotransformation of many strains of clostidium thermocellum was achieved by using plasmid pIkm1based on resistance to erythromycin and lincomycin.

Sperm--egg interaction was investigated in mouse eggs freed from the zona pellucida and injected with Clostridium difficile toxin B, the inhibitor of Rho family small G proteins. Toxin B reduced in a dose-dependent manner the percentage of eggs associated with sperm fusion on the surface or sperm nucleus decondensation in the ooplasm, examined by injection of a DNA-staining dye into the egg and transfer of the dye to the fused sperm head after recording intracellular [Ca.sup.2+] responses for 100 min postinsemination. The mean number of decondensed sperm nuclei per egg was remarkably decreased by ~1 [micro]g/ml toxin B in the ooplasm. This was because spermatozoa were arrested at the fusion state without developing to sperm incorporation and tended to lose cytoplasmic continuity to the egg. The fusion-arrested spermatozoa caused transient small [Ca.sup.2+] oscillations in most of eggs, while an injected spermatozoon produced repetitive large [Ca.sup.2+] spikes unaffected by toxin B. A decrease in the rate of fused spermatozoa and decondensed sperm nuclei was also caused by 20-40 [micro]M cytochalasin D, the inhibitor of actin polymerization. Immunostaining of Rho proteins showed that Rac1 and RhoB are present in the cortical ooplasm, but Cdc42 is absent. Actin filaments in the cortex appeared to be reduced in toxin B-injected eggs. This study suggests that Rho protein(s) regulating actin-based cytoskeletal reorganization is involved in the process leading to sperm incorporation. Keywords: Mouse egg; Fertilization; Clostridium difficile toxin B; Rho family G protein; Actin filament; Gamete fusion; Sperm incorporation; Calcium oscillation

Acetogenic bacteria such as Moorella thermoacetica (f. Clostridium thermoaceticum) can grow autotrophically on the greenhouse gas C[O.sub.2]. These organisms use the Wood-Ljungdahl carbon fixation pathway to convert C[O.sub.2] to acetyl-CoA [...], A metalocofactor containing iron, sulfur, copper, and nickel has been discovered in the enzyme carbon monoxide dehydrogenase/acetyl-CoA (coenzyme A) synthase from Moorella thermoacetica (f. Clostridium thermoaceticum). Our structure at 2.2 angstrom resolution reveals that the cofactor responsible for the assembly of acetyl-CoA contains a [[Fe.sub.4][S.sub.4]] cubane bridged to a copper-nickel binuclear site. The presence of these three metals together in one cluster was unanticipated and suggests a newly discovered role for copper in biology. The different active sites of this bifunctional enzyme comp[ex are connected via a channel, 138 angstroms long, that provides a conduit for carbon monoxide generated at the C-cluster on one subunit to be incorporated into acetyl-CoA at the A-duster on the other subunit.

Byline: I. V. Goldenkova (1), K. A. Musiychuk (1), E. S. Piruzian (1) Keywords: reporter gene; licB; lichenase; thermostable enzyme; prokaryotic and eukaryotic transformed cells Abstract: A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells. The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter. The gene product was active and did not undergo modification in heterologous hosts. Simple and sensitive methods were used to detect and to quantitate the lichenase activity. Inducible licBM2 expression was demonstrated with E. coli and yeast cells, allowing the system to be employed in dynamic studies. Author Affiliation: (1) Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991, Russia Article History: Registration Date: 09/10/2004

Clostridium cellulovorans produces and extracellular enzyme complex called a cellulosome that can produce protoplasts from plants more effectively than commercially available enzymes. Protoplasts are used for cell fusion and transformation in plant biotechnology. The cellulosome from C. cellulovorans represents a highly purified enzyme for industrial use.

The aldehyde/alcohol dehydrogenase (AADH) coding gene adhE2 of Clostridium acetobutylicum ATC 824 is the second gene identified in this bacterium besides adhE. The adhE2 is localized to the pSOL1 megaplasmid, is preferentially expressed in alcohologenic cultures, and is a NADH-dependent bifunctional AADH.

Byline: N. G. Belogurova (1), E. E. Davydova (1), M. V. Sobennikova (1), S. D. Varfolomeev (1), E. P. Delver (2), A. A. Belogurov (2) Keywords: DNA; replication; single-stranded plasmids; plasmid replication proteins; thermophilic bacteria Abstract: Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif without appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for stable maintenance of the AT-rich plasmid in thermophilic bacterial cells. Author Affiliation: (1) Chemical Faculty, Moscow State University, Moscow, 117899, Russia (2) Institute of Experimental Cardiology, Cardiology Research Center, Ministry of Health of the Russian Federation, Moscow, 121552, Russia Article History: Registration Date: 09/10/2004

Researchers have genetically engineered Clostridium beijeinckii with glycoside hydrolase genes from the rumen fungus Neocallimastix patriciarum that allow C. beijerinckii to grow on lichenan. Clostridium spp. can produce butanol and ethanol from carbohydrates and biomass is the most widely available substrate for this conversion.

Clostridium beijerinckii BA101 appears to have a defective phosphoenolpyruvate-sugar phosphotransferase system even though it metabolizes glucose more efficiently than C. beijerinckii NCIMB 8052 and produces more butanol. It may have an alternative glucose transport system and enhanced glucokinase activity.