Superiority of the PCR-based Approach for Cloning the Acetate Kinase Gene of Clostridium Thermocellum

Abstract

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.

Department(s)

Biological Sciences

Sponsor(s)

J. William Fulbright Scholarship

Comments

G Ozcengiz gratefully acknowledges a J William Fulbright Scholarship.

Keywords and Phrases

Acetate Kinase; Acetic Acid; Acetyl Coenzyme a Synthetase; DNA Fragment; Phosphate Acetyltransferase; Bacterial Gene; Bacterium Mutant; Clostridium thermocellum; Controlled Study; DNA Probe; Escherichia coli; Gene Isolation; Genetic Complementation; Methanosarcina; Molecular Cloning; Nonhuman; Polymerase Chain Reaction; Restriction Mapping; Sequence Homology; Technique; Bacteria (Microorganisms); Methanosarcina thermophila; Prokaryota; Gene Cloning; PCR; Phosphotransacetylase; Thermophilic Bacteria

International Standard Serial Number (ISSN)

1367-5435;1476-5535

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 1998 Springer Verlag, All rights reserved.

Publication Date

01 Sep 1998

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