Host-plasmid interactions in recombinant strains of Clostridium acetobutylicum ATCC 824

Abstract Plasmid-containing strains of Clostridium acetobutylicum produced higher levels of solvents and lower levels of acids than wild-type cells in controlled pH 4.5 batch fermentations. This effect was observed regardless of whether or not the plasmids contained C. acetobutylicum genes. The effect was less prevalent in higher pH fermentations and apparently independent of the actual DNA sequences contained on these plasmids. The plasmid-containing strains were found to have lower growth-rates and higher solventogenic enzyme activities than wild-type cells. However, similar activity levels were found for both butyrate-pathway enzymes.     

Comparative study of the symbiotic plasmid DNA in free living bacteria and bacteroids of Rhizobium leguminosarum

Plasmids pIM13, pT127 and pBC16 delta 1, introduced by transformation into Clostridium acetobutylicum N1-4081, were shown to replicate in, and to confer antibiotic resistance upon this new host. Recombinant plasmids were constructed by inserting erythromycin-resistant plasmid pIM13 into the unique ClaI site of pBR322 or by ligating a tetracycline-resistant determinant of plasmid pT127 to HindIII-linearized pIM13. The hybrid plasmids replicated and expressed erythromycin resistance in C. acetobutylicum strain N1-4081 and in Escherichia coli or Bacillus subtilis, indicating that they might be useful as shuttle vectors for transferring genes between these strains. The efficiency and stability of different replicons in C. acetobutylicum were compared.     

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum.     

Genetic manipulation of acid and solvent formation in clostridium acetobutylicum ATCC 824

The genes coding for enzymes involved in butanol or butyrate formation were subcloned into a novel Escherichia coli-Clostridium acetobutylicum shuttle vector constructed from pIMP1 and a chloramphenicol acetyl transferase gene. The resulting replicative plasmids, referred to as pTHAAD (aldehyde/alcohol dehydrogenase) and pTHBUT (butyrate operon), were used to complement C. acetobutylicum mutant strains, in which genes encoding aldehyde/alcohol dehydrogenase (aad) or butyrate kinase (buk) had been inactivated by recombination with Emr constructs. Complementation of strain PJC4BK (buk mutant) with pTHBUT restored butyrate kinase activity and butyrate production during exponential growth. Complementation of strain PJC4AAD (aad mutant) with pTHAAD restored NAD(H)-dependent butanol dehydrogenase activity, NAD(H)-dependent butyraldehyde dehydrogenase activity and butanol production during solventogenic growth. The development of an alternative selectable marker makes it is possible to overexpress genes, via replicative plasmids, in mutant strains that lack specific enzyme activities, thereby expanding the number of possible genetic manipulations that can be performed in C. acetobutylicum. Copyright 1998 John Wiley & Sons, Inc.     

Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Abstract A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb Cla I/ Sph I chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum , SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.     

In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824.

The restriction endonuclease Cac824I has been shown to be a major barrier to electrotransformation of Clostridium acetobutylicum ATCC 824 (L. D. Mermelstein, N. E. Welker, G. N. Bennett, and E. T. Papoutsakis, Bio/Technology 10:190-195, 1992). Methylation by the phi 3T I methyltransferase encoded by Bacillus subtilis phage phi 3T was shown to protect plasmid DNA from restriction by Cac824I. Expression in Escherichia coli of the phi 3tI gene (which encodes the phi 3T I methyltransferase) from pAN1, which replicates via the p15A origin of replication, was sufficient to completely methylate coresident E. coli-C. acetobutylicum shuttle vectors with ColE1 origins of replication. Three shuttle vectors (pIMP1, pSYL2, and pSYL7) methylated in this manner were used to efficiently electrotransform strain ATCC 824. These vectors could not be introduced into strain ATCC 824 when unmethylated because the E. coli portions of the plasmids contain a large number of Cac824I sites. This method obviates the need to use B. subtilis-C. acetobutylicum shuttle vectors with few Cac824I sites to introduce DNA into C. acetobutylicum ATCC 824.     

Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli.

A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pP58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.     

Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli

A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pP58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.     

Prepared and screened a modified TNF-alpha molecule as TNF-alpha autovaccine to treat LPS induced endotoxic shock and TNF-alpha induced cachexia in mouse

Overexpression of TNF-alpha in the body is critically involved in many diseases. A strategy to construct TNF-alpha autovaccine by introducing a T cell helper epitope to the protein has been developed and may be an alternative because it is cheaper and highly efficient. However, the induction of high level anti-TNF-alpha neutralizing autoantibodies by TNF-alpha autovaccine is depend on a proper T cell help epitope. In order to evaluate the effect of different T helper cell epitopes on the immunogenicity of mouse TNF-alpha (mTNF-alpha), three T helper cell epitopes, TT (QYIKANSKFIGITEL), HEL (NTDGSTDYGILQINSR), and PADRE (AKFVAAWTLKA), were chosen for this study. The sequence (amino acids 126-140) of mTNF-alpha was replaced with those of the T cell help epitopes, respectively. The three fusion proteins (mTNF-TT, mTNF-HEL, mTNF-PADRE) were expressed in Escherichia coli and purified with a simple strategy. The abilities of the proteins elicited TNF-alpha autoantibodies in BALB/c mice were investigated. The results showed that mTNF-PADRE is the most effective among the three modified TNF-alpha molecules. In the absence of adjuvant, the therapeutic effect of TNF-PADRE on LPS induced endotoxic shock mice and mTNF-alpha induced cachexia mice was observed. This study suggests that mTNF-PADRE may be a better candidate of mTNF-alpha autovaccine.     

Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress

The toxicity of n-butanol in microbial fermentations limits its formation. The stress response of Clostridium acetobutylicum involves various stress proteins and therefore, over-expression of genes encoding stress proteins constitutes an option to improve solvent tolerance. Over-expression of groESL, grpE and htpG, significantly improved butanol tolerance of C. acetobutylicum. Whereas the wild type and vector control strain did not survive 2?% (v/v) butanol for 2?h, the recombinant strains showed 45?% (groESL), 25?% (grpE) and 56?% (htpG), respectively, of the initial c.f.u. after 2?h of butanol exposure. As previously, over-expression of groESL led to higher butanol production rates, but the novel strains over-expressing grpE or htpG produced only 51 and 68?%, respectively, of the wild type butanol concentrations after 72?h clearly differentiating butanol tolerance and production. Not only butanol tolerance but also the adaptation to butanol in successive stress experiments was significantly facilitated by increased levels of GroESL, GrpE and HtpG. Re-transformation and sequence analyses of the plasmids confirmed that not the plasmids, but the host cells evolved to a more robust phenotype.     

Inhibition of nuclear factor-kappaB activation is essential for membrane-associated TNF-alpha-induced apoptosis in HL-60 cells

The killing of tumour cells that are resistant to soluble TNF-alpha (sTNF-alpha) by the membrane-bound form of TNF-alpha (mTNF-alpha) suggests that different intracellular signalling pathways are involved. We found that mTNF-alpha induced apoptosis in HL-60 cells and failed to cause degradation of inhibitor of kappa B alpha (IkappaB-alpha) and translocation and activation of nuclear factor kappa B (NF-kappaB), whereas sTNF-alpha failed to induce apoptosis, but lowered cytoplasmic inhibitor of kappa B alpha, induced translocation of NF-kappaB to the nucleus and experimentally increased activity of the regulated luciferase. Furthermore, mTNF-alpha upregulated the expression of TNF receptor associated factor (TRAF) 1 and failed to induce TRAF1 and TRAF2 membrane translocation, but led to cytoplasmic colocalization. In contrast, sTNF-alpha stimulated the expression of TRAF1 and TRAF2, recruiting both molecules onto the cell membrane poststimulation. These results suggest that the increased susceptibility of HL-60 cells to mTNF-alpha may be due to the failure of TRAF2 membrane translocation caused by the upregulation of TRAF1 expression and formation of a TRAF1/TRAF2 complex in the cytoplasm, thereby inhibiting NF-kappaB activation and inducing apoptosis.     

Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker

Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms.     

Microbial conversion of glycerol to 1,3-propanediol: physiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1(pSPD5)

Clostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3-propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same "global behavior" was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH2 flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD+ reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.     

Transfer of Tn1545 and Tn916 to Clostridium acetobutylicum

Tn1545, a conjugative transposon originally discovered in Streptococcus pneumoniae, has been transferred from Enterococcus faecalis and Bacillus subtilis to Clostridium acetobutylicum NCIB 8052. Transfer between different strains of C. acetobutylicum has also been observed. Insertion of Tn1545 into the C. acetobutylicum chromosome occurred at multiple sites, as shown by Southern hybridization. Although ermAM (erythromycin-resistance) was the most satisfactory marker for primary selection of transconjugants, all three Tn1545-encoded antibiotic resistance genes (aphA-3, ermAM, and tetM) were apparently expressed in C. acetobutylicum. Our results indicate that Tn1545 is potentially useful for undertaking mutagenesis and mutational cloning in this industrially important organism. Transfer of another conjugative transposon, Tn916, from E. faecalis to C. acetobutylicum NCIB 8052 was also apparently detected. Circumstantial evidence suggests that there may be a hot spot for Tn916 insertion in the C. acetobutylicum NCIB 8052 chromosome.     

Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity.

Clostridium acetobutylicum mutants BA 101 (hyperamylolytic) and BA 105 (catabolite depressed) were isolated by using N-methyl-N'-nitro-N-nitrosoguanidine together with selective enrichment on the glucose analog 2-deoxyglucose. Amylolytic enzyme production by C. acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown in starch and glucose, respectively. C. acetobutylicum BA 105 produced 6.5-fold more amylolytic activity on glucose relative to that of the wild-type strain. The addition of glucose at time zero to starch-based P2 medium reduced the total amylolytic activities of C. acetobutylicum BA 101 and BA 105 by 82 and 25%, respectively, as compared with the activities of the same strains grown on starch alone. Localization studies demonstrated that the amylolytic activities of C. acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested.     

The issue of secretion in heterologous expression of Clostridium cellulolyticum cellulase-encoding genes in Clostridium acetobutylicum ATCC 824

The genes encoding the cellulases Cel5A, Cel8C, Cel9E, Cel48F, Cel9G, and Cel9M from Clostridium cellulolyticum were cloned in the C. acetobutylicum expression vector pSOS952 under the control of a Gram-positive constitutive promoter. The DNA encoding the native leader peptide of the heterologous cellulases was maintained. The transformation of the solventogenic bacterium with the corresponding vectors generated clones in the cases of Cel5A, Cel8C, and Cel9M. Analyses of the recombinant strains indicated that the three cellulases are secreted in an active form to the medium. A large fraction of the secreted cellulases, however, lost the C-terminal dockerin module. In contrast, with the plasmids pSOS952-cel9E, pSOS952-cel48F, and pSOS952-cel9G no colonies were obtained, suggesting that the expression of these genes has an inhibitory effect on growth. The deletion of the DNA encoding the leader peptide of Cel48F in pSOS952-cel48F, however, generated strains of C. acetobutylicum in which mature Cel48F accumulates in the cytoplasm. Thus, the growth inhibition observed when the wild-type cel48F gene is expressed seems related to the secretion of the cellulase. The weakening of the promoter, the coexpression of miniscaffoldin-encoding genes, or the replacement of the native signal sequence of Cel48F by that of secreted heterologous or endogenous proteins failed to generate strains secreting Cel48F. Taken together, our data suggest that a specific chaperone(s) involved in the secretion of the key family 48 cellulase, and probably Cel9G and Cel9E, is missing or insufficiently synthesized in C. acetobutylicum.