Cited by 112Bacterial Genetics and BiotechnologyBiofuel production and bioconversionCRISPR and Genetic Engineering

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Gene transfer to Clostridium cellulolyticum ATCC 35319

Jennert, Katrin C. B., Tardif, Chantal, Young, Danielle I., Young, Michael·2000·Microbiology

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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 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 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 microg 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 microg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.

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Authors: Jennert, K. C. B., Tardif, C., Young, D. I., & Young, M.
Affiliations: Aberystwyth University (GB)
Centre National de la Recherche Scientifique (FR)
Corresponding author: Young, Michael
Venue: Microbiology
Publisher: Microbiology Society
Published: 2000-12-01
Volume: 146
Issue: 12
Pages: 3071-3080
ISSN: 1350-0872 (ISSN-L)
1465-2080
DOI: 10.1099/00221287-146-12-3071
OpenAlex: W2170054056
Cited by: 112
References: 72
Citations by year: 2025: 1 · 2024: 3 · 2023: 3 · 2022: 6 · 2021: 10 · 2020: 6 · 2019: 5 · 2018: 4 · 2017: 1 · 2016: 9 · 2015: 10 · 2014: 7 · 2013: 9 · 2012: 6
FWCI: 1.55
Type: journal article
Language: en
Open access: closed
Locations: Microbiology (publishedVersion)
PubMed (publishedVersion)
Funders: Centre National de la Recherche Scientifique
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords: Clostridium cellulolyticum, gene transfer, cellulosome, plasmid conjugation, IncP plasmids, DNA methylation, plasmid replication, electrotransformation, transformation efficiency, cellulose degradation, co-culture
MeSH terms: Cellulose, Clostridium, Conjugation, Genetic, DNA Transposable Elements, DNA, Bacterial, Plasmids, Transformation, Bacterial, Organelles, Electroporation, Deoxyribonuclease HpaII, DNA Methylation

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Gene transfer to Clostridium cellulolyticum ATCC 35319

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