Gene replacement of adenylate kinase in the gram-positive thermophile Geobacillus stearothermophilus disrupts adenine nucleotide homeostasis and reduces cell viability

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 stearothermophilus strain 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 mesophile Escherichia coli. To study the role of AK in the maintenance of adenylate energy charge (EC) and cell viability of G. 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 the adk gene from the mesophile Bacillus 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 degrees C lower than for wild-type cells (56 vs. 75 degrees C). This temperature-sensitive phenotype was secondary to heat inactivation of B. subtilis AK, as evidenced by enzyme activity assays and EC measurements. At higher temperatures (65 degrees 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.