[Show abstract][Hide abstract] ABSTRACT: Background:
Clostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum.
Both systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose (glgA) and amylase (amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1).
The codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Our study also revealed a surprising number of errors in the ATCC 824 genome sequence, while at the same time emphasising the need to re-sequence commonly used laboratory strains.
Full-text · Article · Jan 2016 · Biotechnology for Biofuels
[Show abstract][Hide abstract] ABSTRACT: In recent years the genus Clostridium has assumed greater prominence in terms of both the diseases individual species cause and as a consequence of the renewed importance of certain strains in the production of chemical commodities from renewal biomass, and in particular biofuels. This has precipitated the determination of a plethora of genome sequences, and, more importantly, an acceleration in efforts directed at the development of genetic systems to facilitate the exploitation of the data being generated. As a consequence, new methods have been formulated with which directed mutants can be made to assist reverse genetic studies, most notably insertional systems based on retargeting group II introns, e.g. the ClosTron. In parallel to this, inroads have been made towards the development of efficient, random mutagens for forward genetic approaches, in the form of exemplification of transposome technology and a mariner-based transposon in Clostridium perfringens and Clostridium difficile, respectively. Still to be implemented are rapid and reliable methods for creating in-frame deletions as well as more effective methods for delivering large segments of DNA into the genome. The formulation of such methods has already reached an advanced stage of development in our laboratory. In the meantime, there is still some way to go before clostridial researchers can boast the degree of sophistication available to those researchers studying Bacillus.
[Show abstract][Hide abstract] ABSTRACT: Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.
[Show abstract][Hide abstract] ABSTRACT: Consolidated bioprocessing (CBP) is reliant on the simultaneous enzyme production, saccharification of biomass, and fermentation of released sugars into valuable products such as butanol. Clostridial species that produce butanol are, however, unable to grow on crystalline cellulose. In contrast, those saccharolytic species that produce predominantly ethanol, such as Clostridium thermocellum and Clostridium cellulolyticum, degrade crystalline cellulose with high efficiency due to their possession of a multienzyme complex termed the cellulosome. This has led to studies directed at endowing butanol-producing species with the genetic potential to produce a cellulosome, albeit by localising the necessary transgenes to unstable autonomous plasmids. Here we have explored the potential of our previously described Allele-Coupled Exchange (ACE) technology for creating strains of the butanol producing species Clostridium acetobutylicum in which the genes encoding the various cellulosome components are stably integrated into the genome.
We used BioBrick2 (BB2) standardised parts to assemble a range of synthetic genes encoding C. thermocellum cellulosomal scaffoldin proteins (CipA variants) and glycoside hydrolases (GHs, Cel8A, Cel9B, Cel48S and Cel9K) as well as synthetic cellulosomal operons that direct the synthesis of Cel8A, Cel9B and a truncated form of CipA. All synthetic genes and operons were integrated into the C. acetobutylicum genome using the recently developed ACE technology. Heterologous protein expression levels and mini-cellulosome self-assembly were assayed by western blot and native PAGE analysis.
We demonstrate the successful expression, secretion and self-assembly of cellulosomal subunits by the recombinant C. acetobutylicum strains, providing a platform for the construction of novel cellulosomes.
Full-text · Article · Aug 2013 · Biotechnology for Biofuels
[Show abstract][Hide abstract] ABSTRACT: The bacterium Clostridium acetobutylicum produces acids as an energy-yielding process during exponential growth. An acidic environment, however, is toxic to the cells and two survival mechanisms are in place to prevent them from dying. Firstly, during a solventogenesis phase, the cells
take up these acids and convert them to solvents, thus raising the environmental pH. Secondly, the cells undergo sporulation to form highly resistant spores capable of surviving extreme conditions. One possible regulatory mechanism for these processes is the accessory gene regulatory (agr) quorum-sensing system, which is thought to coordinate cell population density with cell phenotype. We model this system to monitor its putative e.ect upon solventogenesis and the sporulation-initiation network responsible for triggering spore formation. We demonstrate that a high population density should be able to induce both solventogenesis and sporulation, with variations to the parameter set allowing sporulation alone to be triggered; additional distinct signals are capable of restoring the solventogenic response. We compare the agr system of C. acetobutylicum with that of Staphylococcus aureus in order to investigate why the di.erences in feedback between the two systems may have evolved. Our findings indicate that, depending upon the mechanism of interaction between the agr system and the sporulation-initiation network, the clostridial agr circuitry may be in place either to moderate the number of spores that are formed (in order for this number to reflect the urgency of the situation), or simply as an energy-saving strategy.
No preview · Article · Mar 2013 · Mathematical biosciences
[Show abstract][Hide abstract] ABSTRACT: Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.
[Show abstract][Hide abstract] ABSTRACT: The role of the two-component system (TCS) CBO0366/CBO0365 in the cold shock response and growth of the mesophilic Clostridium botulinum ATCC 3502 at 15°C was demonstrated by induced expression of the TCS genes upon cold shock and impaired growth of the TCS
mutants at 15°C.
Full-text · Article · Jun 2012 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Clostridium difficile causes a potentially fatal diarrheal disease through the production of its principal virulence factors, toxin A and toxin
B. The tcdC gene is thought to encode a negative regulator of toxin production. Therefore, increased toxin production, and hence increased
virulence, is often inferred in strains with an aberrant tcdC genotype. This report describes the first allele exchange system for precise genetic manipulation of C. difficile, using the codA gene of Escherichia coli as a heterologous counterselection marker. It was used to systematically restore the Δ117 frameshift mutation and the 18-nucleotide
deletion that occur naturally in the tcdC gene of C. difficile R20291 (PCR ribotype 027). In addition, the naturally intact tcdC gene of C. difficile 630 (PCR ribotype 012) was deleted and then subsequently restored with a silent nucleotide substitution, or “watermark,”
so the resulting strain was distinguishable from the wild type. Intriguingly, there was no association between the tcdC genotype and toxin production in either C. difficile R20291 or C. difficile 630. Therefore, an aberrant tcdC genotype does not provide a broadly applicable rationale for the perceived notion that PCR ribotype 027 strains are “high-level”
toxin producers. This may well explain why several studies have reported that an aberrant tcdC gene does not predict increased toxin production or, indeed, increased virulence.
No preview · Article · Apr 2012 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Analysis of the Gram-positive Clostridium acetobutylicum genome reveals an inexplicable level of redundancy for the genes putatively involved in asparagine (Asn) and Asn-tRNAAsn synthesis. Besides a duplicated set of gatCAB tRNA-dependent amidotransferase genes, there is a triplication of aspartyl-tRNA synthetase genes and a duplication of asparagine
synthetase B genes. This genomic landscape leads to the suspicion of the incoherent simultaneous use of the direct and indirect
pathways of Asn and Asn-tRNAAsn formation. Through a combination of biochemical and genetic approaches, we show that C. acetobutylicum forms Asn and Asn-tRNAAsn by tRNA-dependent amidation. We demonstrate that an entire transamidation pathway composed of aspartyl-tRNA synthetase and
one set of GatCAB genes is organized as an operon under the control of a tRNAAsn-dependent T-box riboswitch. Finally, our results suggest that this exceptional gene redundancy might be interconnected to
control tRNA-dependent Asn synthesis, which in turn might be involved in controlling the metabolic switch from acidogenesis
to solventogenesis in C. acetobutylicum.
[Show abstract][Hide abstract] ABSTRACT: 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
Full-text · Article · Jan 2012 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Class I heat shock genes (HSGs) code for molecular chaperones which play a major role in the bacterial response to sudden
increases of environmental temperature by assisting protein folding. Quantitative reverse transcriptase real-time PCR gene
expression analysis of the food-borne pathogen Clostridium botulinum grown at 37°C showed that the class I HSGs grpE, dnaK, dnaJ, groEL, and groES and their repressor, hrcA, were expressed at constant levels in the exponential and transitional growth phases, whereas strong downregulation of all
six genes was observed during stationary phase. After heat shock from 37 to 45°C, all HSGs were transiently upregulated. A
mutant with insertionally inactivated hrcA expressed higher levels of class I HSGs during exponential growth than the wild type, followed by upregulation of only groES and groES after heat shock. Inactivation of hrcA or of dnaK encoding a major chaperone resulted in lower maximum growth temperatures than for the wild type and reduced growth rates
under optimal conditions compared to the wild type. The dnaK mutant showed growth inhibition under all tested temperature, pH, and NaCl stress conditions. In contrast, the growth of
an hrcA mutant was unaffected by mild temperature or acid stress compared to the wild-type strain, indicating that induced class
I HSGs support growth under moderately nonoptimal conditions. We show that the expression of class I HSGs plays a major role
for survival and growth of C. botulinum under the stressful environmental conditions that may be encountered during food processing or growth in food products, in
the mammalian intestine, or in wounds.
Full-text · Article · Mar 2011 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: The phosphorylated Spo0A transcription factor controls the initiation of endospore formation in Clostridium acetobutylicum, but genes encoding key phosphorelay components, Spo0F and Spo0B, are missing in the genome. We hypothesized that the five orphan histidine kinases of C. acetobutylicum interact directly with Spo0A to control its phosphorylation state. Sequential targeted gene disruption and gene expression profiling provided evidence for two pathways for Spo0A activation, one dependent on a histidine kinase encoded by cac0323, the other on both histidine kinases encoded by cac0903 and cac3319. Purified Cac0903 and Cac3319 kinases autophosphorylated and transferred phosphoryl groups to Spo0A in vitro, confirming their role in Spo0A activation in vivo. A cac0437 mutant hyper-sporulated, suggesting that Cac0437 is a modulator that prevents sporulation and maintains cellular Spo0A∼P homeostasis during growth. Accordingly, Cac0437 has apparently lost the ability to autophosphorylate in vitro; instead it catalyses the ATP-dependent dephosphorylation of Spo0A∼P releasing inorganic phosphate. Direct phosphorylation of Spo0A by histidine kinases and dephosphorylation by kinase-like proteins may be a common feature of the clostridia that may represent the ancestral state before the great oxygen event some 2.4 billion years ago, after which additional phosphorelay proteins were recruited in the evolutionary lineage that led to the bacilli.
Preview · Article · Mar 2011 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: The relative expression of three cold shock protein coding genes (cspA, cspB and cspC) of Clostridium botulinum ATCC 3502 was studied with quantitative RT-PCR analysis following a cold shock shift from 37 °C to 15 °C. A significant increase in the relative expression of all three genes was observed upon the temperature downshift. To validate these findings, single-gene insertional inactivation of cspA, cspB and cspC was undertaken with the ClosTron gene knock-out system. In growth experiments, mutations in cspB or cspC, but not cspA, resulted in a cold-sensitive phenotype. No growth of the cspB mutant was observed at 15 °C over a ten day period, whereas at 20 °C the growth rate was 70% lower than that of wild type strain. The growth rate of cspC mutant was 70% and 80% lower than the growth rate of the wild type strain at 15 °C and 20 °C, respectively. At 37 °C the growth of cspB mutant did not differ from, but the growth rate of cspC mutant was 30% lower than, that of the wild type strain. The cspA mutant grew somewhat faster than the wild type strain at all studied temperatures. Since the inactivation of cspB resulted in the most prominent defect in growth at low temperatures, we suggest that cspB encodes the major cold shock protein of C. botulinum ATCC 3502. Understanding the mechanisms behind cold tolerance of C. botulinum helps to evaluate the safety risks this foodborne pathogen poses in the modern food industry.
Full-text · Article · Mar 2011 · International Journal of Food Microbiology
[Show abstract][Hide abstract] ABSTRACT: The genus Clostridium is a diverse assemblage of Gram positive, anaerobic, endospore-forming bacteria. Whilst certain species have achieved notoriety as important animal and human pathogens (e.g. Clostridium difficile, Clostridium botulinum, Clostridium tetani, and Clostridium perfringens), the vast majority of the genus are entirely benign, and are able to undertake all manner of useful biotransformations. Prominent amongst them are those species able to produce the biofuels, butanol and ethanol from biomass-derived residues, such as Clostridium acetobutylicum, Clostridium beijerinkii, Clostridium thermocellum, and Clostridium phytofermentans. The prominence of the genus in disease and biotechnology has led to the need for more effective means of genetic modification. The historical absence of methods based on conventional strategies for "knock-in" and "knock-out" in Clostridium has led to the adoption of recombination-independent procedures, typified by ClosTron technology. The ClosTron uses a retargeted group II intron and a retro-transposition-activated marker to selectively insert DNA into defined sites within the genome, to bring about gene inactivation and/or cargo DNA delivery. The procedure is extremely efficient, rapid, and requires minimal effort by the operator.
Full-text · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Endospore production is vital for the spread of Clostridium difficile infection. However, in order to cause disease, these spores must germinate and return to vegetative cell growth. Knowledge of germination is therefore important, with potential practical implications for routine cleaning, outbreak management and potentially in the design of new therapeutics. Germination has been well studied in Bacillus, but until recently there had been few studies reported in C. difficile. The role of bile salts as germinants for C. difficile spores has now been described in some detail, which improves our understanding of how C. difficile spores interact with their environment following ingestion by susceptible individuals. Furthermore, with the aid of novel genetic tools, it has now become possible to study the germination of C. difficile spores using both a forward and reverse genetics approach. Significant progress is beginning to be made in the study of this important aspect of C. difficile disease.
No preview · Article · Nov 2010 · Research in Microbiology
[Show abstract][Hide abstract] ABSTRACT: Clostridium difficile infection is the leading cause of healthcare-associated diarrhoea in Europe and North America. During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have indicated that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A or there is prior damage to the gut mucosa. However, a recent study indicated that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent. This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knockout system to inactivate the toxin genes permanently, we found that C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo. Furthermore, by constructing the first ever double-mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue to consider both toxins in the development of diagnostic tests and effective countermeasures against C. difficile.
[Show abstract][Hide abstract] ABSTRACT: Clostridium difficile causes diarrhoeal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis. Endospore production plays a vital role in transmission of infection, and in order to cause disease these spores must then germinate and return to vegetative cell growth. Type BI/NAP1/027 strains of C. difficile have recently become highly represented among clinical isolates and are associated with increased disease severity. It has also been suggested that these 'epidemic' types generally sporulate more prolifically than 'non-epidemic' strains, although the few existing reports are inconclusive and encompass only a small number of isolates. In order to better understand any differences in sporulation rates between epidemic and non-epidemic C. difficile types, we analysed these characteristics using 14 C. difficile clinical isolates of a variety of types. Sporulation rates varied greatly between individual BI/NAP1/027 isolates, but this variation did not appear to be type-associated. Furthermore, a number of BI/NAP1/027 spores appeared to form colonies with a lower frequency than specific non-BI/NAP1/027 strains. The data suggest that (i) careful experimental design is required in order to accurately quantify sporulation; and (ii) current evidence cannot link differences in sporulation rates with the disease severity of the BI/NAP1/027 type.
[Show abstract][Hide abstract] ABSTRACT: The impact of Clostridium difficile-associated disease (CDAD) in healthcare settings throughout the developed world is considerable in terms of mortality, morbidity, and disease management. The incidence of CDAD has risen dramatically since the turn of this century, concomitant with the emergence of so-called hypervirulent strains which are thought to cause a more severe disease, higher relapse rates, and increased mortality. Pre-eminent amongst hypervirulent strains are those belonging to ribotype 027, which were first reported in Canada in 2003 and shortly thereafter in the UK. Since its arrival in Europe, it has spread rapidly and has now been reported in 16 member states and Switzerland. The physiological factors responsible for the rapid emergence of hypervirulent C. difficile strains remain unclear. It is known that they produce a binary toxin (CDT) in addition to toxins A and B, that they are resistant to fluoroquinolones due to mutations in gyrA, and that they are resistant to erythromycin. Representative strains have been suggested to produce more toxin A and B in the 'laboratory flask' (most likely due to a frameshift mutation in the repressor gene tcdC), to be more prolific in terms of spore formation, and also exhibit increased adherence to human intestinal epithelial cells due to altered surface proteins. However, the contribution of these and other as yet unidentified factors to the rapid spread of certain C. difficile variants (e.g., ribotypes 027 and 078) remains unclear at present. The advent of ClosTron technology means that it is now possible to construct genetically stable isogenic mutants of C. difficile and carry out reverse genetic studies to elucidate the role of specific gene loci in causing disease. The identification of virulence factors using this approach should help lead to the rational development of therapeutic countermeasures against CDAD.
Full-text · Article · Aug 2010 · International journal of medical microbiology: IJMM
[Show abstract][Hide abstract] ABSTRACT: Bacillus subtilis cells may opt to forgo normal cell division and instead form spores if subjected to certain environmental stimuli, for example nutrient deficiency or extreme temperature. The resulting spores are extremely resilient and can survive for extensive periods of time, importantly under particularly harsh conditions such as those mentioned above. The sporulation process is highly time and energy consuming and essentially irreversible. The bacteria must therefore ensure that this route is only undertaken under appropriate circumstances. The gene regulation network governing sporulation initiation accordingly incorporates a variety of signals and is of significant complexity. We present a model of this network that includes four of these signals: nutrient levels, DNA damage, the products of the competence genes, and cell population size. Our results can be summarised as follows: (i) the model displays the correct phenotypic behaviour in response to these signals; (ii) a basal level of sda expression may prevent sporulation in the presence of nutrients; (iii) sporulation is more likely to occur in a large population of cells than in a small one; (iv) finally, and of most interest, PhrA can act simultaneously as a quorum-sensing signal and as a timing mechanism, delaying sporulation when the cell has damaged DNA, possibly thereby allowing the cell time to repair its DNA before forming a spore.
No preview · Article · Mar 2010 · Bulletin of Mathematical Biology