Gene transfer to Clostridium cellulolyticum ATCC 35319

Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CceI, is an isoschizomer of MspI (HpaII). Electro-transformation was employed to establish plasmids containing the replication functions of pAMβ1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5′-CCGG-3′ using either BsuFI methylase in vivo or MspI methylase in vitro. Plasmids based on the pAMβ1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 102 transformants per μg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 105 and 107 transformants per μg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.