Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.

Orf5/SolR: a transcriptional repressor of the sol operon of Clostridium acetobutylicum?

The gene of Orf5 (SolR) of Clostridium acetobutylicum DSM 792 was subcloned and overexpressed in Escherichia coli. The protein was purified with Ni-NTA agarose and used for DNA binding assays. No DNA binding of Orf5 to regions upstream of the sol operon from C. acetobutylicum was observed. Overexpression of Orf5 in C. acetobutylicum led to a change in the organism's pattern of glycosylated exoproteins. The Orf5 protein was localized in the cell membrane fraction and to a small extent in the supernatant medium. Based on these results Orf5 (SolR) appears not to act as a transcriptional repressor in C. acetobutylicum, but instead may be an enzyme involved in glycosylation or deglycosylation.

Differential regulation of two thiolase genes from Clostridium acetobutylicum DSM 792.

Thiolase of Clostridium acetobutylicum is an important enzyme involved in both, acid and solvent fermentation. Two thiolase genes (thlA and thIB) have been cloned and sequenced from Clostridium acetobutylicum DSM 792, showing high homology to each other and to thiolases of PHA-synthesizing bacteria. The thlA gene is identical to the gene already cloned and sequenced from strain ATCC 824 (Stim-Herndon et al., 1995, Gene 154: 81-85). Using primer extension and S1 nuclease analysis a transcriptional start site was identified 102 bp upstream of the thlA start codon. This site was preceded by a region that exhibits high similarity to the sigma70 consensus promoter sequences of Gram-positive and -negative bacteria. Regulation of thlA and thlB was studied at the transcriptional level to elucidate the specific function of each gene. Non-radioactive primer extension analysis using fluorescein-labelled oligonucleotides and Northern blot analysis revealed high levels of thlA transcripts in acid- and solvent-producing cells. During an induced shift of a continuous culture from acid to solvent formation, the transcript level transiently decreased to a minimum, 3 to 7 h after induction. The thlA transcript length is about 1.4 kb, indicating a monocistronic organisation, whereas genetic organization and reverse transcription (RT)-PCR analysis indicated that thlB forms an operon with two other adjacent genes, thlR and thlC. Transcription and regulation of the thlB operon was studied using RTPCR and showed a very low expression in acid- and solvent-producing cells. Heterologously expressed clostridial ThlB showed high thiolase activity in Escherichia coli. The N-terminal part of ThlR possesses a potential helix-turn-helix motif and shows significant homology to regulatory proteins belonging to the TetR/AcrR family of transcriptional regulators. ThlR possibly acts as a transcriptional repressor of thlB operon expression. The data provide strong evidence that ThlA is involved in the metabolism of both acid and solvent formation, whereas the physiological function of ThlB has yet to be elucidated.

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

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

Sequence analysis of the atp operon of Clostridium acetobutylicum DSM 792 encoding the F0F1 ATP synthase.

The atp gene region of Clostridium acetobutylicum DSM 792 has been fully sequenced. It contains the F0F1 ATPase genes in the order atpIBEFHAGDC, whose products share high sequence homology to the respective proteins of a variety of other bacteria. It is the first such sequence available for a mesophilic Clostridium. Significant differences to other reported atp operons are a distal transcription start point 219 bp upstream of the translation start point and a second transcription initiation site (without corresponding promoter sequence) upstream of atpE, indicating posttranscriptional processing for massive expression of this gene product.

Stable Escherichia coli-Clostridium acetobutylicum shuttle vector for secretion of murine tumor necrosis factor alpha.

Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-alpha) from Clostridium acetobutylicum. The shuttle plasmids contained the clostridial endo-beta1, 4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-alpha cDNA. The construction was first tested in Escherichia coli and then introduced in C. acetobutylicum DSM792 by electroporation. Controls confirmed the presence and stability of the recombinant plasmids in this organism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-alpha during growth. Significant levels of biologically active mTNF-alpha were measured in both lysates and supernatants. The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.

Electroporation of, plasmid isolation from and plasmid conservation in Clostridium acetobutylicum DSM 792.

Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 x 10(2) transformants/microgram DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 microF, and 600 omega. The method also allowed the taxonomic group IV strain NI-4082 to be transformed (10(1) transformants/microgram DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be easily preserved as spore suspensions; under all conditions tested plasmids were maintained.

Sporulation and time course expression of sigma-factor homologous genes in Clostridium acetobutylicum.

The gene for the vegetative sigma factor A of Clostridium acetobutylicum was constitutively transcribed during growth and formed an operon together with dnaE. Sporulation-specific sigma factors E, G, and K were sequentially induced shortly before mature endospores could be detected. Maximal transcription in the course of spore formation was found to be in the order sigE-sigG-sigK, thus matching the pattern described for Bacillus subtilis. From primer extension experiments promoter structures could be deduced with high homology to the Bacillus consensus. Upstream of the spoIIGA-sigE operon a gene with significant similarity to ftsZ could be detected.

The kdp system of Clostridium acetobutylicum: cloning, sequencing, and transcriptional regulation in response to potassium concentration.

The complete sequence of the kdp gene region of Clostridium acetobutylicum has been determined. This part of the chromosome comprises two small open reading frames (orfZ and orfY), putatively encoding hydrophobic peptides, and the genes kdpA, kdpB, kdpC, and kdpX, followed by an operon encoding a pair of sensor-effector regulatory proteins (KdpD and KdpE). Except for orfZ, orfY, and kdpX, all genes showed significant homology to the kdp genes of Escherichia coli, encoding a high-affinity potassium transport ATPase and its regulators. The complete genome sequence of Synechocystis sp. strain PCC 6803 and a recently published part of the Mycobacterium tuberculosis genome indicate the existence of a kdp system in these organisms as well, but all three systems comprise neither a second orf upstream of kdpA nor an additional kdpX gene. Expression of the clostridial kdp genes, including the unique kdpX gene, was found to be inducible by low potassium concentrations. A transcription start point could be mapped upstream of orfZ. A promoter upstream of kdpD was active only under noninducing conditions. Lowering the potassium content of the medium led to formation of a common transcript (orfZYkdpABCXDE), with a putative internal RNase E recognition site, which could be responsible for the instability of the common transcript. Except for the two small peptides, all gene products could be detected in in vitro transcription-translation experiments.

Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation.

The enzymes acetoacetate decarboxylase and coenzyme A transferase catalyse acetone production from acetoacetyl-CoA in Clostridium acetobutylicum. The adc gene encoding the former enzyme is organized in a monocistronic operon, while the ctf genes form a common transcription unit with the gene (adhE) encoding a probable polyfunctional aldehyde/alcohol dehydrogenase. This genetic arrangement could reflect physiological requirements at the onset of solventogenesis. In addition to AdhE, two butanol dehydrogenase isozymes and a thiolase are involved in butanol synthesis. RNA analyses showed a sequential order of induction for the different butanol dehydrogenase genes, indicating an in vivo function of BdhI in low level butanol formation. The physiological roles of AdhE and BdhII most likely involve high level butanol formation, with AdhE being responsible for the onset of solventogenesis and BdhII ensuring continued butanol production. Addition of methyl viologen results in artificially induced butanol synthesis which seems to be mediated by a still unknown set of enzymes. Although the signal that triggers the shift to solventogenesis has not yet been elucidated, recent investigations suggest a possible function of DNA supercoiling as a transcriptional sensor of the respective environmental stimuli.

Sigma factor and sporulation genes in Clostridium.

The genus Clostridium, represented by Gram-positive, anaerobic, spore-forming bacteria, is well known for its clinical importance and considerable biotechnological potential. Recently, evidence for a functional role of the transcription factors sigma A, sigma E, sigma G, and sigma K in this genus was provided by cloning and sequencing these genes from C. acetobutylicum. In C. kluyveri, a partially sequenced open reading frame was found to encode the N terminus of the putative sigma factor L with significant similarity to members of the sigma 54 family. The identification of sequences with high similarity to the Bacillus sigma F (C. acetobutylicum), sigma H (several clostridial species), and sigma D (C. thermocellum)-controlled consensus promoters renders the existence of these transcription factors in clostridia very likely. These data are in agreement with information obtained by RNA transcript mapping (sigma A, sigma H), heterologous DNA hybridization (sigma D, sigma H), and immuno characterization of purified proteins (sigma A) from various clostridial species. Thus, the picture emerges that a fundamental similarity exists at the genetic level between the regulation of various cellular responses, in particular sporulation, in the genera Bacillus and Clostridium. The different induction patterns of sporulation in Bacillus spp. (nutrient starvation) and many clostridial species (cessation of growth or exposure to oxygen in the presence of excess nutrients) are most interestingly not reflected in the general regulatory features of this developmental process.

Characterization and expression of the hydrogenase-encoding gene from Clostridium acetobutylicum P262.

The hydrogenase enzyme of Clostridium acetobutylicum plays a pivotal role in controlling electron flow, and hence carbon flow, during the complex biphasic fermentation of carbohydrates to the neutral solvents acetone and butanol. We report here the cloning and molecular characterization of the hydrogenase-encoding gene (hydA) from C. acetobutylicum P262. This gene was isolated by colony hybridization, using the Clostridium pasteurianum hydrogenase-1 gene as a probe. The DNA sequence encoding the hydA gene from C. acetobutylicum was determined, and revealed an ORF (1722 bp) encoding a 574 amino-acid protein. This C. acetobutylicum hydrogenase protein product has 82% similarity and 67% identity with the C. pasteurianum hydrogenase-1 protein. Northern blot analysis of RNA isolated from C. acetobutylicum indicates that the C. acetobutylicum hydrogenase protein product is translated from a monocistronic operon. RNA was isolated from the different morphological and physiological stages of a batch C. acetobutylicum fermentation, and further Northern blot analyses revealed no differences in the expression of the gene during acidogenesis as opposed to solventogenesis. Primer extension experiments confirmed these results and identified the 5' start of the mRNA transcript. These results correlated well with the physiological need for this organism to dispose of excess reducing equivalents.

Sporulation and primary sigma factor homologous genes in Clostridium acetobutylicum.

Using a PCR-based approach, we have cloned various sigma factor homologous genes from Clostridium acetobutylicum DSM 792. The nucleotide sequence of the dnaE-sigA operon has been determined and predicts two genes encoding 69- and 43-kDa proteins. The deduced DnaE amino acid sequence has approximately 30% amino acid identity with protein sequences of other primases. The putative sigA gene product shows high homology to primary sigma factors of various bacteria, most significantly to Bacillus subtilis and Staphylococcus aureus. Northern (RNA) blot analysis revealed that both genes from an operon, which is clearly expressed under conditions that allow for cell division. A promoter sequence with significant homology to the sigma H-dependent Bacillus promoters preceded the determined transcriptional start point, 182 bp upstream of the GUG start codon of dnaE. The homologous genes to Bacillus spp. sporulation sigma factors G, E, and K have been cloned and sequenced. Indirect evidence for the existence of sigma F was obtained by identification of a DNA sequence homologous to the respective Bacillus consensus promoter. Southern hybridization analysis indicated the presence of sigma D and sigma H homologous genes in C. acetobutylicum. A new gene group conserved within the eubacteria, but with yet unspecified functions, is described. The data presented here provide strong evidence that at least some of the complex regulation features of sporulation in B. subtilis are conserved in C. acetobutylicum and possibly Clostridium spp.

Plasmid Transfer into the Homoacetogen Acetobacterium woodii by Electroporation and Conjugation.

Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMbeta1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 x 10 transformants per mug of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene into pAT187. Mating of E. coli containing pKV12 with A. woodii resulted in transfer frequencies of 3 x 10 to 7 x 10 per donor or recipient.

Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis.

A DNA region of Clostridium acetobutylicum contiguous with the adc operon has been cloned and sequenced. Structural genes encoding the acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (ctfB and ctfA) and an alcohol/aldehyde dehydrogenase (adhE) could be identified. These three genes together with a small open reading frame (ORF) of unknown function (upstream of adhE) formed an operon (sol operon), as shown by mRNA analyses. The complete sol operon was transcriptionally induced or derepressed before the onset of solventogenesis, thus confirming earlier results of Northern hybridizations with a ctfB gene probe (U. Gerischer and P. Dürre, J. Bacteriol. 174:426-433, 1992). Upstream of the sol operon, we identified two putative promoters that were located in regions with possible stem-loop structures formed by several inverted repeats. The distal promoter P1 showed only minor transcription initiation in solventogenic C. acetobutylicum cells but was recognized in Escherichia coli, presumably because of its high similarity to the sigma 70 consensus sequence. The adhE-proximal promoter P2 directed the major transcription start point in solventogenic C. acetobutylicum but was not recognized in E. coli. The clostridial AdhE showed high similarity to a novel family (type III) of alcohol dehydrogenases. Two other ORFs (ORF 5 and ORF 6) were found on the cloned DNA region that showed no significant similarity to sequences in various available data bases. mRNA studies revealed that ORF 5 formed a monocistronic operon and showed increased expression before onset of solventogenesis.

Sequence and molecular characterization of a DNA region encoding a small heat shock protein of Clostridium acetobutylicum.

A DNA region of Clostridium acetobutylicum containing a gene (hsp 18) with significant homology to a family of small eukaryotic heat shock proteins was cloned and sequenced. It is the second reported sequence of a low-molecular-weight heat shock protein from gram-positive bacteria and is induced not only by heat shock but also at the onset of solventogenesis, as determined by Northern (RNA) blot analysis, thus confirming the results of an earlier study performed at the protein level (A. Pich, F. Narberhaus, and H. Bahl, Appl. Microbiol. Biotechnol. 33:697-704, 1990). By primer extension analysis, a transcriptional start site was identified 149 bp upstream of hsp18. This site was preceded by a region that exhibits high homology to the consensus promoter sequences of gram-positive bacteria, as well as sigma 70-dependent Escherichia coli. A direct repeat structure was detected in the -35 region. The promoter is located 196 bp from the start of a potential regulatory tRNA(Thr)ACG gene involved in the shift to solventogenesis which is transcribed in the opposite direction. A putative rho-independent transcription termination structure was identified at the 3' end of hsp18.

Possible function of tRNA(Thr)ACG in regulation of solvent formation in Clostridium acetobutylicum.

The mutation of Clostridium acetobutylicum mutant AA2, defective in the formation of acetone and butanol, was shown to be caused by a single insertion of Tn916 close to the structural gene thrA, encoding the tRNA(Thr)ACG. The DNA region containing the thrA gene was cloned and sequenced. Start and end points of the transcript were determined by primer extension and S1-mapping analysis. The results obtained were identical to predictions derived from the DNA sequence by various RNA-analysing computer programs. The rarely used ACG codon seems to be confined to genes expressed at the end of the exponential growth phase or involved in uptake or turnover of minor C or N substrates. Evolutionary aspects of this codon selection and a possible translational regulation mechanism are discussed.