Search

Acetogenic bacteria produce acetate following the fixation of CO

The production of TcdA, TcdB and CDT in Clostridium difficile PCR ribotype 027, is regulated by the two-component system response regulator CdtR. Despite this, little is known about the signal transduction pathway leading to the activation of CdtR. In this study, we generated R20291ΔPalocΔcdtR model strains expressing CdtR phospho-variants in which our predicted phospho-accepting Asp, Asp61 was mutated for Ala or Glu. The constructs were assessed for their ability to restore CDT production. Dephospho-CdtR-Asp61Ala was completely non-functional and mirrored the cdtR-deletion mutant, whilst phospho-CdtR-Asp61Glu was functional, possessing 38–52% of wild-type activity. Taken together, these data suggest that CdtR is activated by phosphorylation of Asp61. The same principles were applied to assess the function of PCR ribotype 078-derived CdtR, which was shown to be non-functional owing to polymorphisms present within its coding gene. Conversely, polymorphisms present within its promoter region, provide significantly enhanced promoter activity compared with its PCR ribotype 027 counterpart. To ensure our data were representative for each ribotype, we determined that the cdtR nucleotide sequence was conserved in a small library of eight PCR ribotype 027 clinical isolates and nineteen PCR ribotype 078 isolates from clinical and animal origin., Highlights • R20291ΔPaLocΔcdtR model strains were applied to study the toxin regulator CdtR. • (de)phosphomimetic substitutions revealed that CdtR is activated by phosphorylation of Asp61. • Ribotype 078 CdtR was shown to be non-functional. • PcdtR derived from ribotype 078 has much stronger activity than its ribotype 027 counterpart. • cdtR nucleotide sequence is conserved within eight ribotype 027 and nineteen ribotype 078 strains.

This study reports engineering of a hypertransformable variant of C. pasteurianum for bioconversion of glycerol into hydrogen (H2). A functional glycerol-triggered hydrogen pathway was engineered based on two approaches: (1) increasing product yield by overexpression of immediate enzyme catalyzing H2 production, (2) increasing substrate uptake by overexpression of enzymes involved in glycerol utilization. The first strategy aimed at overexpression of hydA gene encoding hydrogenase, and the second one, through combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase. These genetic manipulations resulted in two recombinant strains (hydA ++ /dhaD1K ++) capable of producing 97% H2 (v/v), with yields of 1.1 mol H2/mol glycerol in hydA overexpressed strain, and 0.93 mol H2/mol glycerol in dhaD1K overexpressed strain, which was 1.5 fold higher than wild type. Among two strains, dhaD1K ++ consumed more glycerol than hydA ++ which proves that overexpression of glycerol enzymes has enhanced glycerol intake rate.

Parageobacillus thermoglucosidasius is a promising chassis for producing chemicals and fuels. Here we designed, built and tested the pMTL60000 modular plasmids containing standardised Gram-positive and Gram-negative replicons, selectable markers and application-specific modules. The pMTL60000 modular plasmids were characterised with regard to transformation efficiency, segregational stability, copy number and compatibility.

The future sustainable production of chemicals and fuels from non-petrochemical sources, while at the same time reducing greenhouse gas (GHG) emissions, represent two of society's greatest challenges. Microbial chassis able to grow on waste carbon monoxide (CO) and carbon dioxide (CO2) can provide solutions to both. Ranging from the anaerobic acetogens, through the aerobic chemoautotrophs to the photoautotrophic cyanobacteria, they are able to convert C1 gases into a range of chemicals and fuels which may be enhanced and extended through appropriate metabolic engineering. The necessary improvements will be facilitated by the increasingly sophisticated gene tools that are beginning to emerge as part of the Synthetic Biology revolution. These tools, in combination with more accurate metabolic and genome scale models, will enable C1 chassis to deliver their full potential.

Highlights • We investigate a kinetic model for the mevalonate pathway which includes inhibition effects and a sink of acetyl-CoA. • Of the enzymes in the pathway, upregulating HMG-CoA reductase has the most significant positive effect on improving pathway efficiency. • Upregulating pyruvate dehydrogenase complex and HMG-CoA synthase can also help, but only in conjunction with the upregulation of HMG-CoA reductase. • We confirm our theoretical predictions by introducing the mevalonate pathway into Cupriavidus necator., The mevalonate pathway is normally found in eukaryotes, and allows for the production of isoprenoids, a useful class of organic compounds. This pathway has been successfully introduced to Escherichia coli, enabling a biosynthetic production route for many isoprenoids. In this paper, we develop and solve a mathematical model for the concentration of metabolites in the mevalonate pathway over time, accounting for the loss of acetyl-CoA to other metabolic pathways. Additionally, we successfully test our theoretical predictions experimentally by introducing part of the pathway into Cupriavidus necator. In our model, we exploit the natural separation of time scales as well as of metabolite concentrations to make significant asymptotic progress in understanding the system. We confirm that our asymptotic results agree well with numerical simulations, the former enabling us to predict the most important reactions to increase isopentenyl diphosphate production whilst minimizing the levels of HMG-CoA, which inhibits cell growth. Thus, our mathematical model allows us to recommend the upregulation of certain combinations of enzymes to improve production through the mevalonate pathway.

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of ‘pseudo-suicide’ vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius., Highlights • A simple roadmap for the development and deployment of gene systems in clostridia. • Allelic exchange using pyrE alleles and pseudo-suicide vectors. • Knock-out and knock-in using allele-coupled exchange (ACE). • Complementation studies through genome insertion. • Genome insertion of application specific modules.

Effective microbial metabolic engineering is reliant on efficient gene transfer. Here we present a simple screening strategy that may be deployed to isolate rare, hypertransformable variants. The procedure was used to increase the frequency of transformation of the solvent producing organism Clostridium pasteurianum by three to four orders of magnitude., Highlights • A procedure to isolate C. pasteurianum mutants capable of high efficient DNA transfer. • Increase DNA transfer efficiency by three to four orders of magnitude. • Structural maintenance of chromosome proteins of C. pasteurianum are involved in DNA transfer.

Clostridium difficile is widely publicised as a problem in the health-care system. Disruption of the normal gut microbiota by antibiotic therapy allows C. difficile to colonise the colon. On colonisation, C. difficile produces two toxins that lead to disease, with symptoms ranging from mild-to-severe diarrhoea, to fulminant and often fatal pseudomembranous colitis (PMC). How C. difficile establishes initial colonisation of the host is an area of active investigation. Recently there has been increased research into the role of C. difficile flagella in colonisation and adherence. Novel research has also elucidated a more complex role of flagella in C. difficile virulence pertaining to the regulation of toxin gene expression. This review focuses on new insights into the specific role of C. difficile flagella in colonisation and toxin gene expression.

Despite substantial investment in prevention, treatment and aftercare, cancer remains a leading cause of death worldwide. More effective and accessible therapies are required. A potential solution is the use of endospore forming Clostridium species, either on their own, or as a tumour delivery vehicle for anti-cancer drugs. This is because intravenously injected spores of these obligate anaerobes can exclusively germinate in the hypoxic/necrotic regions present in solid tumours and nowhere else in the body. Research aimed at exploiting this unique phenomenon in anti-tumour strategies has been ongoing since the early part of the 20th century. Only in the last decade, however, has there been significant progress in the development and refinement of strategies based on spore-mediated tumour colonisation using a range of clostridial species. Much of this progress has been due to advances in genomics and our ability to modify strains using more sophisticated gene tools.

Declining fossil fuel reserves, coupled with environmental concerns over their continued extraction and exploitation have led to strenuous efforts to identify renewable routes to energy and fuels. One attractive option is to convert glycerol, a by-product of the biodiesel industry, into n-butanol, an industrially important chemical and potential liquid transportation fuel, using Clostridium pasteurianum. Under certain growth conditions this Clostridium species has been shown to predominantly produce n-butanol, together with ethanol and 1,3-propanediol, when grown on glycerol. Further increases in the yields of n-butanol produced by C. pasteurianum could be accomplished through rational metabolic engineering of the strain. Accordingly, in the current report we have developed and exemplified a robust tool kit for the metabolic engineering of C. pasteurianum and used the system to make the first reported in-frame deletion mutants of pivotal genes involved in solvent production, namely hydA (hydrogenase), rex (Redox response regulator) and dhaBCE (glycerol dehydratase). We were, for the first time in C. pasteurianum, able to eliminate 1,3-propanediol synthesis and demonstrate its production was essential for growth on glycerol as a carbon source. Inactivation of both rex and hydA resulted in increased n-butanol titres, representing the first steps towards improving the utilisation of C. pasteurianum as a chassis for the industrial production of this important chemical., Highlights • A comprehensive molecular tool set for Clostridium pasteurianum. • Abolishment of by-product (1,3-propanediol) formation by dhaBCE KO. • First reported deletion of main hydrogenase HydA in Clostridium spp. • Inactivation of rex and hydA leads to increased n-butanol titres.

The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone-butanol-ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to ethanol and butanol formation under the standard batch culture conditions employed in this study. All phenotypic changes observed could be reversed by genetic complementation, with exception of those seen for the ptb mutant. This mutant produced around 100 mM ethanol, no acetone and very little (7 mM) butanol. The genome of the ptb mutant was therefore re-sequenced, together with its parent strain (ATCC 824 wild type), and shown to possess a frameshift mutation in the thl gene, which perfectly explained the observed phenotype. This finding reinforces the need for mutant complementation and Southern Blot analysis (to confirm single ClosTron insertions), which should be obligatory in all further ClosTron applications.

Clostridium difficile is a leading cause of healthcare-associated diarrhoea. In recent years, certain C. difficile types have become highly represented among clinical isolates and are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Endospores, produced during sporulation, play a pivotal role in infection and disease transmission and it has been suggested in the literature that these so-called ‘hypervirulent’ C. difficile types are more prolific in terms of sporulation in vitro. However, work in our laboratory has provided evidence to the contrary suggesting that although there is significant strain-to-strain variation in C. difficile sporulation characteristics this variation does not appear to be type-associated. On analysis of the literature, it is apparent that the methods used to quantify sporulation in previous studies have varied greatly and sample sizes have remained small. The conflicting data in the literature may, therefore, not necessarily be generally representative of C. difficile sporulation. Instead, these inconsistencies may reflect differences in the experimental design of each study. In this review, the need for further investigations of C. difficile sporulation rates is highlighted. Specifically, the advantages and disadvantages of the different experimental approaches previously used are discussed and a standard set of principles for measuring C. difficile sporulation in the future is proposed.

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.

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.

The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully.

Despite their medical and industrial importance, our basic understanding of the biology of the genus Clostridium is rudimentary in comparison to their aerobic counterparts in the genus Bacillus. A major contributing factor has been the comparative lack of sophistication in the gene tools available to the clostridial molecular biologist, which are immature, and in clear need of development. The transfer and maintenance of recombinant, replicative plasmids into various species of Clostridium has been reported, and several elements suitable as shuttle plasmid components are known. However, these components have to-date only been available in disparate plasmid contexts, and their use has not been broadly explored. Here we describe the specification, design and construction of a standardized modular system for Clostridium-Escherichia coli shuttle plasmids. Existing replicons and selectable markers were incorporated, along with a novel clostridial replicon. The properties of these components were compared, and the data allow researchers to identify combinations of components potentially suitable for particular hosts and applications. The system has been extensively tested in our laboratory, where it is utilized in all ongoing recombinant work. We propose that adoption of this modular system as a standard would be of substantial benefit to the Clostridium research community, whom we invite to use and contribute to the system.

Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of Clostridium acetobutylicum and 5 of Clostridium difficile, exceeding the number of published mutants ever generated in these species. Genes have also been inactivated for the first time in Clostridium botulinum and Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.

Putative TB vaccine candidates were selected from lists of genes induced in response to in vivo-like stimuli, such as low oxygen and carbon starvation or growth in macrophages, and tested as plasmid DNA vaccines for their ability to protect against Mycobacterium tuberculosis challenge in a guinea pig aerosol infection model. This vaccination method was chosen as it induces the Th1 cell-mediated immune response required against intracellular pathogens such as M. tuberculosis. Protection was assessed in the guinea pig model in terms of mycobacteria present in the lungs at 30 days post-challenge. Protection achieved by the novel candidates was compared to BCG (positive control) and saline (negative control). Four vaccines encoding for proteins such as PE and PPE proteins, a zinc metalloprotease and an acyltransferase, gave a level of protection that was statistically better than saline in the lungs. These findings have enabled us to focus on a sub-set of vaccine candidates for further evaluation using additional vaccination strategies.

A synthetic gene encoding the Hc (binding) domain of Clostridium botulinum neurotoxin F (FHc) was expressed in Escherichia coli fused to maltose binding protein (MBP). The purified MBP–FHc and FHc isolated after removal of MBP were evaluated in mice for their ability to protect against toxin challenge. Balb/c mice developed a protective immune response following administration of either protein via the intraperitoneal or intramuscular routes. A comparison of antibody titres and protection following single and multiple vaccinations and the effects of dosage are shown. The long term protection afforded by the vaccines was also investigated. Ten months following vaccination mice were still protected when challenged with 10 4 MLD 50 doses of botulinum toxin F.

Background: There is considerable interest in the specific targeting of therapeutic agents to cancer cells. Of particular promise is a technique known as Antibody-Directed Enzyme Prodrug Therapy (ADEPT). In this approach an enzyme is targeted to the tumour by its conjugation to a tumour specific-antibody tumour. After allowing sufficient time for the conjugate to localise at the tumour and clear from the circulatory system, a relatively non-toxic prodrug is administered. This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localised at the tumour site. Objectives: To construct gene fusions between the pseudomonad carboxypeptidase G2 (CPG2) gene and DNA encoding MFE-23 (an anti-carcinoembryonic antigen (CEA) single-chain Fv (scFv) molecule), derived from a phage display library. To overexpress the resultant gene fusions in Escherichia coli, and assess the in vitro and in vivo properties of the purified fusion proteins. Study design: To introduce unique cloning restriction sites into the 5′-end of the CPG2 gene by site-directed mutagenesis to facilitate fusion to the 3′-end of the gene encoding MFE-23 (constructs with or without a flexible (Gly4Ser)3 linker-encoding sequence were designed). To overexpress the resultant gene fusions under transcriptional control of the lac promoter and to direct the fusion proteins produced to the periplasmic space of E. coli through translational coupling to the pelB signal peptide. Results: Biologically active recombinant CPG2::MFE-23 scFv fusion proteins were produced in E. coli and shown to possess enzyme and anti-EA activity. Affinity chromatography followed by size exclusion gel filtration yielded approximately 0.7 – 1.4 mg/l from shake flask culture. The fusion protein in which the enzyme and antibody moieties were joined by a linker peptide was shown to be effectively localised in nude mice bearing human colon tumour xenografts, giving favourable tumour to blood ratios. Conclusion: MFE-23 scFv serves as an ideal candidate for the antibody arm of a bacterially expressed fusion protein with CPG2. The biological properties of this recombinant protein suggest that it may be employed for tumour specific prodrug activation. However, further assessment of its stability and pharmokinetics is required if genetic fusion is to be considered as an alternative to chemical conjugation.

Summary Botulinum neurotoxin type F can be produced by proteolytic or non-proreolytic strains of C. botulinum , or from C. baratii . In recent years, the sequences of all toxin types except that of proteolytic type F toxin have been determined. We have cloned the neurotoxin gene from proteolytic C. botulinum (type F Langeland) as a series of overlapping PCR fragments, and determined the nucleotide sequence. The translated sequence demonstrates a toxin of 1278 amino acid residues, which has highest similarity with non-proteolytic type F toxin. Phylogenetic trees based on toxin sequences show that the type F sequences fall into a naturally distinct group, although the strains producing these toxins are not phylogenetically closely related.

A chimeric yeast promoter (pPGK::REP2), capable of directing high-level gene expression in both Saccharomyces cerevisiae and Escherichia coli, has been constructed. It was derived by fusing the promoter of the yeast PGK gene (encoding phosphoglycerate kinase) to a region residing immediately 5' to the yeast 2 mu plasmid REP2 gene (encoding a trans-acting plasmid maintenance protein). In S. cerevisiae, transcripts initiated within the REP2-derived moiety of the promoter, but the transcription start point was dictated by the PGK determinator sequence. Promoter function in E. coli was due to the presence of consensus prokaryotic -35 and -10 motifs in the REP2 moiety. To facilitate expression studies, the promoter was incorporated into a versatile series of S. cerevisiae/E. coli shuttle vectors which provided a choice of selectable marker and copy number in S. cerevisiae. To maximise translational efficiency, a novel cloning strategy was devised which allows the juxtaposition of genes to the promoter such that the heterologous AUG replaces that of the REP2 AUG, without any alteration in the surrounding nucleotide (nt) context. This strategy was used to place both the Tn903 neo gene and the Rhodosporidium toruloides phenylalanine ammonia lyase (PAL)-encoding gene under the transcriptional control of pPGK::REP2. In the former case, cells became resistant to extremely high levels of Geneticin (> 3 mg/ml in the case of S. cerevisiae). In the case of the latter, PAL was shown to accumulate to approx. 9 and 10% of total soluble protein in S. cerevisiae and E. coli, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

An 8.1-kb fragment of chromosomal DNA from Clostridium acetobutylicum NCIMB 8052 (formerly NCIB 8052) has been cloned into plasmid pAT153 and shown to allow the growth of Escherichia coli LJ32 (F + ato C2 c ato D32 fadR ) on butyrate as the sole source of carbon and energy. Deletion analysis delineated a 3.9-kb subfragment capable of complementation. The nucleotide sequence of this fragment was determined and it was shown to encode three complete, and two incomplete open reading frames (ORFs). Based on enzymic studies of recombinant clones, two of these ORFs were shown to encode phosphotransbutyrylase and butyrate kinase. The above enzymes are involved in the acidogenic phase of fermentation in C. acetobutylicum . The fragment also carries an incomplete ORF encoding a polypeptide exhibiting substantial similarity to dihydropteroate synthase.

The complete nucleotide (nt) sequence of a 5.1-κb Eco RI ONA restriction fragment carrying the replication region of the Streptococcus faecalis plasmid pAMβ1 has been determined. Ofthe seven major open reading frames (ORF A-G) identified within this fragment, two (C and E) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that synthesis of the polypeptide (M, 57380) encoded by the largest ORF (E) was essential for replication. Deletion analysis indicated that the minimum unit of DNA required for replication resided on a 2.59-κb AccI- HpaI subfragment. ORF C resided outside of this fragment and encompassed an extensive region of directly repeated nt sequence. The encoded polypeptide (Mr, 30471) was therefore composed oflarge tracts of reiterated amino acid sequence (11 × VDP and 35 × TEP tripeptides) which probably caused the observed anomalous electrophoretic mobility of the synthesised protein (equivalent to 61 κDa). Deletion of a 416-bp segment of DNA between unique KpnI and StyI sites caused an increase in copy number, which correlated with the in vitro production of higher levels of 0 RF E polypeptide. Although homology was detected between the sequenced DNA, and the replicon of a closely related streptococcal plasmid (pSM19035),none was evident to any other characterised Gram+ plasmid.

Background: Clostridium sporogenes is part of a highly diverse group of Gram positive, spore forming, anaerobic bacteria. C. sporogenes can be used as a delivery vehicle for chemotherapeutics in cancer treatment due to the inactive spore form of C. sporogenes only germinating in the microenvironment of the hypoxic tumour. The directed enzyme pro-drug therapy (DEPT) strategy has been tested with several different delivery vectors, and Clostridial DEPT (CDEPT) has previously been developed with one pro-drug converting enzyme (nitroreductase). In this treatment the non-toxic pro-drugs are administered to the patient, then a pro-drug converting enzyme is delivered through genetically altered C. sporogenes. This enzyme breaks the pro-drug down into a toxic component. The aim of this project is to develop the system further with an alternative pro-drug converting enzyme which digests its potential substrate into a 100 fold more toxic subcomponent. Materials and Methods: To efficiently implement the use of this enzyme in the CDEPT strategy, the delivery system needed to be optimised to allow for its effective export and binding to the cell wall of the bacteria to maintain site specificity. To do this, the use of a panel of different signal peptides was investigated. Concurrently to that different sortase signal motifs were identified in Clostridium species through psiBLAST, and their use to anchor the protein to the cell wall was investigated. Successful attachment and protein export was judged by assaying the presence of enzyme activity in the different fractions of bacterial cultures. Conclusion: Results showed an enhanced export of the enzyme into the culture supernatant, and significant cell wall anchoring. This data gives a good basis from which development of an optimized CDEPT system should be achievable, which would result in a highly site specific, hypoxic tumour therapy system.

The complete nucleotide sequence of the Pseudomonas chromosomal gene coding for the enzyme carboxypeptidase G2 (CPG2) has been determined. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that of randomly derived peptide fragments and by N-terminal sequencing of the purified protein. The gene has been shown to code for a 22 amino acid signal peptide at its N-terminus which closely resembles the signal peptides of other secreted proteins. An alternative 36 amino acid signal peptide which may function in Pseudomonas has also been identified. The codon utilisation of the gene is influenced by the high G + C (67.2%) content of the DNA and exhibits a 92.8 % preference for codons ending in G or C. This unusual codon preference may contribute to the generally observed weak expression of Pseudomonas genes in Escherichia coli . A region of DNA upstream of the structural gene has also been sequenced and a ribosome binding site and two putative promoter sequences identified.

Nucleotide sequence analysis of the gene (spa) for staphylococcal protein A (SPA) from Staphylococcus aureus strain Cowan l (NCTC8530) shows that the sequence differs from previously reported SPA nucleotide sequences, especially in the number of repeat units in the cell-wall-binding region of the gene. Dot matrix comparison with streptococcal protein G and the macrophage receptor for the constant fragment (Fc) of immunoglobulins shows a limited but significant homology. The homology to the latter probably identifies the Fc-binding region in the immunoglobulin-binding domains of SPA. Enhanced production of SPA in Escherichia coli was achieved using the lac promoter immediately upstream from the spa gene.

The structural gene for l -lactate dehydrogenase (LDH; EC 1.1.1.27) from Bacillus stearothermophilus NCA1503 has been cloned in Escherichia coli and its complete nucleotide sequence determined. The predicted amino acid (aa) sequence of the LDH enzyme agrees with the previously determined aa sequence except to three positions: aa 125 and 126, Ser-Glu, are inverted whilst His at position 130 has been replaced by Ser m our sequence. The lct gene consists of an open reading frame (ORF) commencing from the ATG start codon of 951 bp followed by a TGA stop codon. Upstream from the start codon is a strong (ΔG = −14.4kcal) Shine-Dalgamo (SD) sequence, a feature typical of Gram-positive ribosome binding sites. Putative RNA polymerase recognition signals (−35 and −10 regions) have been identified upstream from the lct structural gene but there are no structures resembling Rho-independent transcription termination signals downstream from the TGA stop codon. Two further ORFs, preceded by S D sequences, are present downstream from the lct gene. Thus the lct gene may constitute the first gene of an operon. Subclones of the lct gene have been constructed in the expression plasmid pKK223-3 and the LDH enzyme produced in soluble form at levels of up to 36% of the E. coli soluble cell protein.