Journal of Industrial Microbiology and Biotechnology

, Volume 21, Issue 3, pp 145-149

Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum

  • G OzcengizAffiliated withBiology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • , J-H KimAffiliated withThayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA
  • , W R LinAffiliated withBiology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • , E OzcengizAffiliated withBiology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • , D WestenbergAffiliated withDepartment of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA
  • , L R LyndAffiliated withThayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA
  • , A L DemainAffiliated withBiology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Rent the article at a discount

Rent now

* Final gross prices may vary according to local VAT.

Get Access

Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR-based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then successfully used as a probe for the isolation of the corresponding gene and restriction analysis of that region.

Keywords: Clostridium thermocellum; acetate kinase; phosphotransacetylase; thermophilic bacteria; PCR; gene cloning