A roadmap for gene system development in Clostridium

Abstract

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.

Keywords: Allelic exchange; ClosTron; Counterselection marker; Fluoroorotic acid; Gene transfer; Knock-in; Knock-out; Restriction modification; pyrE.

Fig. 1

Relative stability of the various modular replicons in different species and strains of Clostridium. R = Plasmid loss in media lacking supplementation with antibiotic as a function of time and measured as loss of antibiotic resistance. Each replicon based plasmid is compared to the stability of a cell carrying the antibiotic gene in the chromosome. For comparison, the projected rate of loss of a population carrying a plasmid that cannot replicate (‘Suicide’) is given.

Fig. 2

Comparison of the steps required for complementation using ACE integration [A] or autonomous [B] vectors. Step are: (1) Plasmid construction; (2) Transfer to clostridia (electroporation or conjugation); (3) development of transformant/transconjugant colonies on rich agar containing antibiotic relevant to the resistance gene present on the plasmid backbone; (4) in the case of [A] only, restreaked on minimal media lacking uracil (only double crossover integrants grow) and antibiotic supplementation, and; (5) purification of complemented clonal populations, in the case of [A] on rich media containing antibiotic to select for the plasmid, and in the case of [B] rich media containing antibiotic. ACE integration [A] is characterised by being at appropriate gene dosage, requires no supplementing antibiotic as the complementing gene is entirely stable, this eliminates antibiotic effects on phenotype and, therefore dispenses with the requirement for vector only control. The use of autonomous vectors [B] results in high gene dosage that can affect phenotype, requires antibiotic supplementation to maintain the plasmid which can affect phenotype and necessitates the inclusion of an additional vector only control.

Fig. 3

The three types of ACE integration vectors used to restore the pyrE allele to wildtype in the pyrE mutant host in which a mutation (Gene X) has been made by allelic exchange. Integration cassettes are modular and inserted between the SbfI and AscI sites of the pMTL80000 vectors. Each vector has a long (1200 bp) Right hand homology arm (RHA) and a shorter (300 bp) left homology arm (LHA). The latter is composed of the 3′-end of the pyrE gene, while former comprises the 1200 bp region of the chromosome from immediately downstream of the pyrE gene. In the case of the ACE Correction vector , the 300 bp and 1200 bp regions are in effect a continuous 1500 bp region of homology to the host chromosome in this region. In the case of the Complementation and Overexpression vectors, the two homology arms are separated by a region of DNA comprising a lacZ containing multiple cloning site (MCS) region and a downstream transcriptional terminator (FT). The Expression vector additionally contains a strong promoter (P_fdx) from the C. sporogenes ferredoxin gene immediately before the lacZ′ gene. Using the MCS, the complementation and expression plasmid variants allow delivery of a functional copy of a knocked-out gene (Gene X) under the control of its native promoter, for complementation studies, or under the control of the strong promoter of the C. spororgenes ferredoxin gene (P_fdx), to allow an assessment of the effect of overexpressing the gene. In every case, integration if the ACE plasmid is initially via the longer RHA. Subsequent plasmid excision via the LHA restores the pyrE allele to wildtype, allowing the former pyrE minus host to grow on minimal media lacking uracil.

Fig. 4

Schematic representation of Roadmap stages. For details refer to relevant section (2.1 to 2.9) in the text.