학술논문
Gene replacement of adenylate kinase in the gram-positive thermophileGeobacillus stearothermophilusdisrupts adenine nucleotide homeostasis and reduces cell viability.
Document Type
Article
Author
Source
Subject
*THERMOPHILIC bacteria
*BACTERIA
*THERMOPHILIC microorganisms
*ADENINE nucleotides
*PURINE nucleotides
*BACILLUS subtilis
*BACILLUS (Bacteria)
*PHYSIOLOGICAL control systems
*NUCLEIC acids
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Language
ISSN
1431-0651
Abstract
Thermophilic bacteria are of great value for industry and research communities. Unfortunately, the cellular processes and mechanisms of these organisms remain largely understudied. In the present study, we investigate how the inactivation of adenylate kinase (AK) affects the adenine nucleotide homeostasis of a gram-positive moderate thermophile,Geobacillus stearothermophilusstrain NUB3621-R. AK plays a major role in the adenine nucleotide homeostasis of living cells and has been shown to be essential for the gram-negative mesophileEscherichia coli. To study the role of AK in the maintenance of adenylate energy charge (EC) and cell viability ofG. stearothermophilus, we generated a recombinant strain of this organism in which its endogenous gene coding for the essential protein adenylate kinase (AK) has been replaced with theadkgene from the mesophileBacillus subtilis. PCR, DNA sequencing and Southern analysis were performed to confirm proper gene replacement and preservation of neighboring genes. The highest growing temperature for recombinant cells was almost 20°C lower than for wild-type cells (56 vs. 75°C). This temperature-sensitive phenotype was secondary to heat inactivation ofB. subtilisAK, as evidenced by enzyme activity assays and EC measurements. At higher temperatures (65°C), recombinant cells also had lower EC values (0.09) compared to wild-type cells (0.45), which reflects a disruption of adenine nucleotide homeostasis following AK inactivation. [ABSTRACT FROM AUTHOR]