1887

Abstract

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 cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for ATCC 35319. Firstly, transfer of Tn has been obtained using an donor. Secondly, IncP-mediated conjugative mobilization of plasmids from 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 with copiotrophic donors. A restriction endonuclease was detected in crude extracts of . This enzyme, named I, is an isoschizomer of I (II). Electro-transformation was employed to establish plasmids containing the replication functions of pAMβ1 (), pIM13 (), pCB102 (), pIP404 () and pWV01 ( subsp. ) in . Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5′-CCGG-3′ using either FI methylase or I methylase . 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. 10 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 10 and 10 transformants per μg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components .

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2000-12-01
2021-10-21
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