Gene transfer to Clostridium cellulolyticum ATCC 35319

Although much is known about the bacterial cellulosome and its various prot ein components, their contributions to bacterial growth on cellulose and th e process of cellulolysis in vivo cannot currently be assessed. To remedy t his, the authors have developed gene transfer techniques for Clostridium ce llulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained usin g an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobil ization of plasmids from Escherichia coil donors has also been achieved. Th e yield of transconjugants in both cases was low and was probably limited b y the suboptimal growth conditions that must of necessity be employed for t he co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolytic um. This enzyme, named CceI, is an isoschizomer of MspI (HpaII). Electrotra nsformation was employed to establish plasmids containing the replication f unctions of pAM beta1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Cl ostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococc us lactis subsp. cremoris) in C. cellulolyticum. Transformants were only ob tained if the DNA was appropriately methylated on the external C of the seq uence 5'-CCGG-3' using either BsuFI methylase in vivo or MspI methylase in vitro. Plasmids based on the pAM beta1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transforman ts on solid medium led to low apparent transformation efficiencies (approx. 10(2) transformants per mug DNA) which might, in part, reflect the low pla ting efficiency of the organism. Selection of transformants in liquid mediu m led to a higher apparent yield of transformants (between 10(5) and 10(7) transformants per mug DNA). The methods developed here will pave the way fo r functional analysis of the various cellulosome components in vivo.