Protoplast formation,L-colony growth,and regeneration ofClostridium beijerinckii NRRL B-592 and B-593 andClostridium acetobutylicum ATCC 10132

Summary Protocols for protoplast formation, L-colony cultivation, and regeneration ofClostridium beijerinckii NRRL B-592, B-593 andC. acetobutylicum ATCC 10132 were developed. Two osmotically reinforced media were formulated. Protoplasts of B-592, B-593, and ATCC 10132 grew as cell wall-deficient forms (L-colonies) when plated on the first medium (BLM) and continued to do so through at least 3 passages on this medium. The second (BRM) permitted the L-colonies to regenerate cell walls after transfer to this medium. TransferredC. beijerinckii B-592 L-colonies reverted to bacillary colonies at a frequency of 25%. Likewise, L-colonies of B-593 andC. acetobutylicum ATCC 10132 could be regenerated at frequencies of 7.0 and 8.6%, respectively. Thus, these procedures are suitable for genetic engineering of these industrial microorganisms using protoplast manipulation techniques.     

Effect of biomass concentration on the specific solvent productivity ofClostridium acetobutylicum in chemostat culture

Summary Using a defined medium in chemostat culture, an inverse relationship between the biomass concentration and the specific butanol productivity has been observed. It is suggested that this is due to the cell population not being homogeneous, and that a change in the nutrient balance leads to a cha in the relative proportions of acidogenic, solventogenic and inert cells (spores).     

Novel alk-1-enyl ether lipids isolated from Clostridium acetobutylicum

Alkenyl ether analogues of phosphatidylglycerol (plasmenylglycerol), bisphosphatidylglycerol (cardiolipin) (plasmenylglycerolphosphatidic acid), monoglycosyldiglyceride and diglycosyldiglyceride were isolated from the polar lipids of Clostridium acetobutylicum and characterized by chemical analyses and degradation. The position of the alkenyl ether bond (at C-1) and of the acyl ester bond (at C-2) as well as the configuration at C-2 of the phospholipids are the same as of the alkenyl ether phospholipids known so far. The alkenyl ether analogue of monoglycosyldiglyceride contains a galactosyl residue, that of diglycosyldiglyceride a glucosyl-galactosyl residue, glucosyl forming the terminal unit.     

Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum

The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specfic activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 M coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60°C. Kinetic constants were measured in the forward reaction. The apparent K _m for pyruvate and coenzyme A were 322 M and 3.7 M, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.Non-standard abbreviations NAD(H) nicotinamide adenine dinucleotide (reduced) - NADP(H) nicotinamide adenine dinucleotide phosphate (reduced) - DTE dithioerythritol - PMS phenazine methosulfate - NBT nitro blue tetrazolium chloride - DMSO dimethyl sulfoxide - DCPIP dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide - TTC triphenyltetrazolium chloride - FAD flavin adenine dinucleotide - FMN flavin mononucleotide     

Effect of carbohydrate source on alpha-amylase and glucoamylase formation byClostridium acetobutylicum SA-1

Summary An examination into the effect of different carbohydrate sources indicated that the production of extracellular alpha-amylase and glucoamylase was under similar biosynthetic control inClostridium acetobutylicum SA-1. Cell-associated starch-hydrolytic enzymes may be more important than extracellular enzymes in the processing of the starch molecule.     

Development of gametophores from isolated protoplasts of the mossAnoectangium thomsvnii Mitt.

Summary Protoplasts of the mossAnoectangium thomsonii Mitt. were isolated from preplasmolyzed protonemal filaments, grown in suspension cultures, after the digestion of the cell wall by the enzymes cellulase and macerozyme or driselase. Driselase was more effective than cellulase and macerozyme. After purification these protoplasts were plated in the form of small agar drops in modified Kofler's medium without hormones and incubated in the dark at 26 ± 2 °C. Cell walls regenerated within three days and cell divisions started seven days after the initiation of the cultures. When the regenerants were transferred to normal protonemal culture medium and illuminated by 3,000 lux continuous light, a multi-cellular protonema developed which formed leafy gametophores on salicylic acid supplemented medium.     

The effect of novobiocin on solvent production byClostridium acetobutylicum

Cells ofClostridium acetobutylicum treated with novobiocin, a DNA gyrase inhibitor, produced higher butyrate levels and lower solvent levels with acetone being the most affected. Seven enzyme activities involved in acid and solvent production were analyzed. Among them, only CoA transferase, required for acetone formation and acid uptake, experienced a significant decrease in activity. As inEscherichia coli andBacillus subtilis, DNA fromC. acetobutylicum became less negatively supercoiled in the early stationary phase (solventogenic stage), as shown by analysis of linking number of a reporter plasmid by agarose gel electrophoresis in the presence of chloroquine.     

Characterization of thermostable Xyn10A enzyme from mesophilic Clostridium acetobutylicum ATCC 824

A thermostable xylanase gene, xyn10A (CAP0053), was cloned from Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the C. acetobutylicum xyn10A gene encoded a 318-amino-acid, single-domain, family 10 xylanase, Xyn10A, with a molecular mass of 34 kDa. Xyn10A exhibited extremely high (92%) amino acid sequence identity with Xyn10B (CAP0116) of this strain and had 42% and 32% identity with the catalytic domains of Rhodothermus marinus xylanase I and Thermoascus aurantiacus xylanase I, respectively. Xyn10A enzyme was purified from recombinant Escherichia coli and was highly active toward oat-spelt and Birchwood xylan and slightly active toward carboxymethyl cellulose, arabinogalactouronic acid, and various p-nitrophenyl monosaccharides. Xyn10A hydrolyzed xylan and xylooligosaccharides larger than xylobiose to produce xylose. This enzyme was optimally active at 60°C and had an optimum pH of 5.0. This is one of a number of related activities encoded on the large plasmid in this strain.     

The internal pH of Clostridium acetobutylicum and its effect on the shift from acid to solvent formation

Clostridium acetobutylicum was unable to keep a constant pH inside the cells when grown on a phosphatelimited synthetic medium which allowed production of organic acids in a first phase and of solvents in a second phase. At external pH values between 5.9 and 4.3, the cells kept a constant pH of 0.9 to 1.3. A similar pH was measured in continuous culture under solventproducing conditions. The pH was abolished by protonovorous uncouplers, such as tetrachlorosalicylanilide (TCS) or carbonyl-p-trifluormethoxyphenylhydrazone (FCCP). n-Butanol at concentration of 150 mM and above led also to a complete abolition of the pH gradient.The internal pH stayed above 5.5 in cultures that shifted from acid to solvent formation. It is concluded that this is a prerequisite for the shift. The possible function of high internal concentrations of butyrate, butyryl phosphate and butyryl coenzyme A in the triggering mechanisms of the shift is discussed.Abbreviations TCS Tetrachlorosalicylanilide - FCCP carbonyl-p-trifluormethyoxyphenylhydrazone     

Plantlet regeneration from mesophyll protoplasts of Digitalis lanata Ehrh.

Summary Protoplasts were isolated from the mesophyll of Digitalis lanata enzymatically and cultured in a liquid regeneration medium (D2a). Protoplast division occurred at a rate of approximately 30%. Mature cell colonies were transferred onto agar medium (D2b)where they developed into cell clusters with a diameter of about 4–5 mm. After transfer onto MS medium, these calli differentiated leaves and shoots which could be rooted on MS medium containing a low hormone concentration.The main part of this work was carried out in the Max-PlanckInstitut für Züchtungsforschung, Cologne (FRG)     

Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase

Summary The conditions for effective isolation of viable protoplasts from Laminaria japonica with an alginase produced by marine bacterium Alteromonas sp. and a commercially available cellulase were investigated. The highest yields of viable protoplasts (7.910.4x10⁶ cells g^–1 FW) were obtained with a hypertonic solution containing 50 % seawater, 25 mM MgCl₂, 5 mM HEPES buffer system, and 0.5 M mannitol. Protoplasts were not obtained from thalli of L. japonica when an abalone alginase (abalone acetone powder; AAP: Sigma) was used instead of the bacterial alginase. The isolated protoplasts were cultured in an PESI medium at 5 °C. Complete cell wall formation was observed within 7 days, and dividing cells were first observed in a 9-day-old culture. Some protoplasts regenerated into sheet-shaped thalli and rhizoid structures were also observed on some thalli after 30 to 40 days in culture. This is the first report of protoplast regeneration into plantlets of L. japonica Areschoug (Laminariales, Phaeophyceae).Abbreviations FW Flesh weight - AAP Abalone acetone powder - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hyrdoxymethyl)aminomethane - PESI Provasoli's enriched seawater with iodine     

Buffering as a means for increasing growth and butanol production byClostridium acetobutylicum

Summary The objective of this work was to optimize butanol formation in the acetone-butanol-ethanol (ABE) fermentation by examining the level of buffering as it affects the dissociation of butyric acid to the less toxic butyrate anion. Experiments were carried out in batch culture using chemically defined (P2) or complex media containing various buffering agents. These included salts of acetate, citrate, phosphate, nitrate, or bicarbonate, representing a range of pK _a values and buffering capacities. Growth in highly buffered medium was found to increase the stationary phase cell density, carbohydrate utilization, and the final butanol concentration. At higher levels of buffering, increased growth and elevated concentrations of butyric acid were required to initiate solventogenesis, suggesting the involvement of a critical threshold level of undissociated butyric acid.     

Analysis of Tn916-induced mutants ofClostridium acetobutylicum altered in solventogenesis and sporulation

Summary The conjugative transposon Tn916 was used for mutagenesis ofClostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be, in the same region of the chromosome. These results predict, that multiple regulatory elements are required to induce solvent production and sporulation.     

Regeneration of fertile plants from protoplasts of different Arabidopsis thaliana genotypes

Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of Arabidopsis thaliana is reported. Protoplasts were isolated from leaves of 21-to 28-day-old Arabidopsis plants grown in a controlled environment. Sustained divisions were achieved when protoplasts were embedded in beads formed by 1.4% sodium alginate in the presence of 50mM CaCl₂ in 0.4 mannitol, which was then exchanged againts modified B5 medium. About 0.4%–0.6% of the protoplasts developed into colonies of which 80%–90% formed shoots and subsequently regenerated to fertile plants. Seeds harvested from more than 200 independently regenerated plants were sown and germination frequencies of more than 95% were obtained. Furthermore, the F₁ plants did not show any evidence of somaclonal variation on visual inspection. This protocol was originally developed for Arabidopsis thaliana Columbia; however it was shown to be applicable also for the genotypes Wassilewskija, Landsberg erecta and Estland though with differing efficiencies.Abbreviations FDA fluorescein diacetate - CM culture medium - SRM shoot regeneration medium - SEM shoot elongation medium - RM rooting medium - PE plating effciency - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - Kin kinetin - 2-iP 2-isopentenyladenine - GA₃ gibberetic acid     

Influence of acetic and butyric acid addition on polysaccharide formation byClostridium acetobutylicum

Summary The production of granulose (an intracellular reserve polygranule), capsule and exopolysaccharide was investigated in a synthetic medium in which the oxido-reduction level was modified by the addition of acetic or butyric acid. After addition of the acids, granulose synthesis increased from 150 to 300 mg glucose equivalents ·1^–1 and capsular synthesis decreased by 25%. Exopolysaccharide production was unchanged under these conditions. A hypothesis that attributes a role to the polymer in the oxido-reduction sequences is discussed.     

Isolation of protoplasts from fern prothallia and their regeneration to gametophytes

Protoplasts were isolated enzymatically from prothallia ofLygodium japonicum. The protoplasts grown in a culture medium containing 0.6 M mannitol and 0.05 M sucrose began to divide within 8 days of culture, and after 30 days 10-cell clusters were present. When the cell-clusters were transferred into fresh media followed by sequential reduction of mannitol concentration, they developed rhizoids and protonemata. The reduction of mannitol concentration to 0.3 M resulted in the regeneration of a common gametophyte within 50 days of culture, and subsequently the regenerated gametophytes produced sporophytic leaves and roots.     

Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisation

Continuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l^–1 h^–1 and a 84% (w/w) glycerol consumption were attained when cultures were grown at pH 6 and D = 0.05 h^–1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.     

Electrotransformation ofClostridium thermosaccharolyticum

Transformation of the thermophileClostridium thermosaccharolyticum ATCC 31960 was achieved using plasmid pCTC1 and electroporation. Evidence supporting transformation was provided by Southern blots, detection of the plasmid in 10 out of 10 erythromycin-resistant clones, retransformation ofE. coli andC. thermosaccharolyticum with plasmid DNA isolated fromC. thermosaccharolyticum, and a proportional relationship between the number of transformants and the amount of DNA added. Transformation efficiencies were very low for plasmid DNA prepared fromE. coli (0.6 transformants mg^–1 DNA), although somewhat higher for plasmid DNA prepared fromC. thermosaccharolyticum (52 transformants mg^–1 DNA). Transformation-dependent erythromycin resistance indicates that an adenosine methylase gene originating fromEnterococcus faecalis, a mesophile, is expressed inC. thermosaccharolyticum. The plasmid pCTC1 appears to be replicated independently of the chromosome, as indicated by visualization of recovered plasmid on gels, and retransformation using recovered plasmid. pCTC1 is maintained inC. thermosaccharolyticum at both 45 and 60°C. Restriction analysis showed little or no rearrangement occurred upon passage through the thermophile.     

Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum

The transposon-containing streptococcal plasmids pAM211, pCF10, and pINY1275 have been transferred at high frequency (10^-2–10^-3 per recipient, selecting for tetracycline resistance) to the Gram-positive anaerobe Clostridium acetobutylicum. Selection in the presence of two antibiotics (tetracycline and erythromycin) with the plasmids pAM 180 and pINY1275 yielded only low numbers of transconjugants (10^-8 per recipient). Matings were done by combining liquid and filter mating procedures under anaerobic conditions. No plasmid DNA could be detected in the transconjugants selected on a minimal medium in the presence of tetracycline. DNA-DNA hybridization experiments with restricted chromosomal DNA using biotinylated pAM120::Tn916 as probe revealed the presence of homologous sequences in the transconjugants but not in Clostridium acetobutylicum wild type. The transconjugants were used as donors in mating experiments with tetracycline-sensitive Bacillus subtilis and Streptococcus lactis subspec. diacetylactis. In both cases tetracycline-resistant strains were found. Transfer frequencies in these experiments were less than 10^-7 per recipient.     

Cloning of Clostridium acetobutylicum genes and their expression in Escherichia coli and Bacillus subtilis

Summary DNA from Clostridium acetobutylicum ABKn8 was partially digested with Sau3A and the fragments obtained were inserted into the unique BamHI site of the cloning vector pHV33. The recombinant plasmids were used to transform Escherichia coli HB101 with selection for ampicillin resistance. A collection of ampicillin-resistant, tetracycline-sensitive clones representative of the Clostridium acetobutylicum genome was made. The clones were shown to carry recombinant plasmids each containing an insert of 2 to 16 kb in size. Several of them complemented the HB101 proA2 or leuB6 auxotrophic mutations. The cloned sequences were shown by Southern blot hybridization to be homologous to the corresponding ABKn8 DNA fragments.