Acetone-butanol fermentation revisited.

Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.

Cellulase, Clostridia, and Ethanol

A modular system for Clostridium shuttle plasmids.

Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes.

/pdf/effect-of-site-specific-modification-on-restriction-2my7dk9v8t.pdf

Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

The effects of challenges with low (0.25%, vol/vol) and high (0.75%) concentrations of butanol on the growth, glucose metabolism, product formation, and transcriptional program of the solvent-tolerant Clostridium acetobutylicum strain 824(pGROE1) and the plasmid control strain 824(pSOS95del) were used to study solvent tolerance and stress response. Strain 824(pGROE1) was generated by groESL overexpression. The growth of 824(pGROE1) was less inhibited than that of 824(pSOS95del), and 824(pGROE1) was able to metabolize glucose over the entire course of the culture (60 h postchallenge) while glucose metabolism in 824(pSOS95del) lasted 24 h. A comparison of their respective DNA array-based transcriptional profiles identified genes with similar expression patterns (these genes are likely to be part of a general butanol stress response) and genes with opposite expression patterns (these genes are likely to be associated with increased tolerance to butanol). Both strains exhibited a butanol dose-dependent increase in expression of all major stress protein genes, including groES, dnaKJ, hsp18, and hsp90; all major solvent formation genes, including aad, ctfA and -B, adc, and bdhA and -B (an unexpected and counterintuitive finding); the butyrate formation genes (ptb and buk); the butyryl coenzyme A biosynthesis operon genes; fructose bisphosphate aldolase; and a gene with homology to Bacillus subtilis kinA. A dose-dependent decrease in expression was observed for the genes of the major fatty acid synthesis operon (also an unexpected and counterintuitive finding), several glycolytic genes, and a few sporulation genes. Genes with opposite expression kinetics included rlpA, artP, and a gene encoding a hemin permease. Taken together, these data suggest that stress, even when it derives from the solvent product itself, triggers the induction of the solvent formation genes.

Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum.

A small collection of clostridia was surveyed for type II restriction endonucleases. Enzymes were detected in two organisms. Clostridium pasteurianum ATCC 6013 contains an isoschizomer of ThaI (FnuDII) [5'-CGCG-3'] and preliminary evidence suggests that cleavage generates bluntended fragments. Clostridium thermohydrosulfuricum DSM 568 contains an isoschizomer of MboI (Sau3A) [5'-GATC-3'] that is inactive on dam methylated substrates. The DNA of this latter organism shows dam methylation.

/pdf/restriction-endonucleases-in-clostridium-pasteurianum-atcc-2gv9zzo5ks.pdf

Restriction endonucleases in Clostridium pasteurianum ATCC 6013 and C. thermohydrosulfuricum DSM 568.

Summary: Clostridium butyricum NCIB 7423 carries two cryptic plasmids, pCB101 (6·05 kbp) and pCB102 (7·8 kbp). Sites for the restriction enzymes EcoRI, EcoRV, HindIII, ClaI and PstI have been found in one or both of these plasmids and their relative positions determined. Restriction fragments from both plasmids have been inserted into a vector plasmid (pJAB1) that is able to replicate in Escherichia coli but not in Bacillus subtilis and the recombinant plasmids have been established in E. coli. A 3·3 kbp Sau3A fragment of pCB101 conferred upon the vector the ability to transform both Rec+and Rec-strains of B. subtilis. Plasmid pRB1, a representative chimaera carrying only the 3·3 kbp Sau3A fragment of pCB101, was successfully transferred from B. subtilis back to E. coli. Plasmid pRB1 was readily lost from B. subtilis in the absence of selection. This evidence, together with the results of hybridization experiments, suggests that pRB1 is present as a weakly replicating autonomous element in B. subtilis. A recombinant plasmid carrying a 2·0 kbp Sau3A fragment of pCB102 underwent integration into the B. subtilis chromosome.

/pdf/identification-of-restriction-fragments-from-two-cryptic-1pofgud7g6.pdf

Identification of restriction fragments from two cryptic Clostridium butyricum plasmids that promote the establishment of a replication-defective plasmid in Bacillus subtilis.

The genus Clostridium contains some of the most toxic bacteria known to man (e.g., Clostridium tetani, Clostridium botulinum) as well as solvent-producing organisms of biotechnological interest (e.g., Clostridium acetobutylicum). Developments within the areas of plasmid vector construction and plasmid delivery methodology now provide the opportunity for applying recombinant DNA technology to this important group of bacteria.

Host: vector systems for gene cloning in Clostridium

Genetic exchange and prospects for cloning in clostridia1

Conjugal gene transfer in clostridium acetobutylicum

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