Gene transfer to Clostridium cellulolyticum ATCC 35319

Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Penglais SY23 3DD, UK 1 Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France 2 Université de Provence, Marseille, France 3 Author for correspon...

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Published in:Microbiology (Society for General Microbiology) 2000-12, Vol.146 (12), p.3071-3080
Main Authors: Jennert, Katrin C. B, Tardif, Chantal, Young, Danielle I, Young, Michael
Format: Article
Language:English
Subjects:
Bacteriology
Biological and medical sciences
Biotechnology
Cellulose - metabolism
cellulosomes
Clostridium - genetics
Clostridium - growth & development
Clostridium - metabolism
Clostridium cellulolyticum
Conjugation, Genetic
Deoxyribonuclease HpaII - metabolism
DNA Methylation
DNA Transposable Elements
DNA, Bacterial - genetics
Electroporation - methods
Enterococcus faecalis
Fundamental and applied biological sciences. Psychology
Gene transfer
Genetic engineering
Genetic technics
Genetics
Methods. Procedures. Technologies
Microbiology
Organelles - enzymology
Organelles - genetics
Plasmids - genetics
Synthetic digonucleotides and genes. Sequencing
Transformation, Bacterial - genetics
transposon Tn1545
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Summary:Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Penglais SY23 3DD, UK 1 Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France 2 Université de Provence, Marseille, France 3 Author for correspondence: Michael Young. Tel: +44 1970 622348. Fax: +44 1970 622307. e-mail: miy{at}aber.ac.uk 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 Tn 1545 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 Cce I, is an isoschizomer of Msp I ( Hpa II). Electro-transformation was employed to establish plasmids containing the replication functions of pAMß1 ( 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 Bsu FI methylase in vivo or Msp I methylase in vitro . Plasmids based on the pAMß1 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 2 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 5 and 10 7 transformants per µg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo . Keywords: conjugation, electro-transformation, methylation, cellulosome a Present address: Department of Biological Sciences, The Open University, Walton H
ISSN:1350-0872
1465-2080
DOI:10.1099/00221287-146-12-3071