Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

Abstract

Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. These results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.

Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

“…While it is not an inherent feature of rolling-circle plasmids to require recombination, there is a Xer-like recombinase encoded on pBAS2 [9, 19]. The presence of this recombinase suggests that it may be required for replication as well as RecA.…”

“…The other is 5′-AGTCTTCTGGAAAACAATCACAATGAA GATA-3′ at bases 482–512 on the reverse strand. The latter sequence is located less than 100 bp from the previously identified predicted ArgR-binding site [19], a site involved in XerCD-mediated recombination in other plasmids [46]. Motif discovery software could not recognize dif , cer , or psi sites associated with other XerCD-associated plasmids [19].…”

“…The latter sequence is located less than 100 bp from the previously identified predicted ArgR-binding site [19], a site involved in XerCD-mediated recombination in other plasmids [46]. Motif discovery software could not recognize dif , cer , or psi sites associated with other XerCD-associated plasmids [19]. This dif SL -like sequence could allow for intramolecular recombination and dimer resolution by the plasmid-encoded Xer-like recombinase.…”

“…These vectors have proven useful to extend genetic methods developed for C. bescii to another member of this genus, Caldicellulosiruptor hydrothermalis , a naïve host for the study of cellulolytic enzymes [20] and surprisingly, they have also proven to be useful for the more distantly related Clostridium ther-mocellum. Vectors derived from C. bescii plasmid pBAS2 transform C. thermocellum DSM 1313 at 60 °C, the optimal growth temperature of this bacterium, with a transformation efficiency greater than 3000 colony forming units per µg of transformed DNA and a copy number ~ 10 per bacterial chromosome [19]. pBAS2 has recently been used to express a thermostable butanol dehydrogenase gene in C. thermocellum that provides resistance to 5-hydroxym-ethylfurfural, a plant biomass-derived microbial inhibitor [25].…”

“…To avoid this integration and to preserve the integrity of the background strain, we have relied on the use of only heterologous sequences on the plasmid [19]. Several promoters from Caldicellulosiruptor bescii have been shown to function in C. thermocellum [19, 25], but many of the tools developed for C. thermocellum are encoded by endogenous (i.e. homologous) sequences [38].…”