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

Date

1998-09-01

Author

Özcengiz, Gülay
Kim, JH
Lin, WR
Ozcengiz, E
Westenberg, D
Lynd, LR
Demain, AL

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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.

Subject Keywords

Clostridium thermocellum, Acetate kinase, Phosphotransacetylase, Thermophilic bacteria, PCR, Gene cloning

URI

https://hdl.handle.net/11511/40808

Collections

Department of Biology, Article