Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

Biotechnology for Biofuels

Lee R Lynd¹  Nicky C Caiazza³  Daniel G Olson¹  Adam M Guss² 

[1]Mascoma Corporation, 67 Etna Rd, Suite 300, Lebanon, NH 03755, USA

[2]Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

[3]Current address: Synthetic Genomics Inc, La Jolla, CA 92037, USA

关键词: Consolidated bioprocessing;    Transformation efficiency;    DNA methylation;    Clostridium thermocellum;   

Others  :  798302
DOI  :  10.1186/1754-6834-5-30

 received in 2012-01-04, accepted in 2012-03-29,  发布年份 2012

【 摘 要 】

Background

Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies.

Results

We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+dcm+E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAMG205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude.

Conclusions

E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

【 授权许可】

   
2012 Guss et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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