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

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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 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°C lower than for wild-type cells (56 vs. 75°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°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.

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Abbreviations

adk :

Adenylate kinase gene

AK:

Adenylate kinase protein

Ap5A:

P1, P5 -Di(adenosine-5′)pentaphosphate

cat :

Chloramphenicol acetyl-transferase gene

EC:

Energy charge

infA :

Initiation factor IF-I gene

secY:

Pre-protein translocase secY subunit gene

map :

Methionine aminopeptidase gene

RBS:

Ribosomal binding site

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Acknowledgements

This work was in part funded by a grant from the National Science Foundation (NSF/MCB - 0212417). R.C. is the recipient of a training fellowship from the W.M. Keck Foundation to the Gulf Coast Consortia through the Keck Center for Computational and Structural Biology. The authors would like to thank the MacKenzie Lab (Rice University, Houston, TX) for equipment, helpful discussions and suggestions. The authors are indebted to Dr. N. Welker (Northwestern University, Evanston, IL, USA) for help with G. stearothermophilus NUB3621-R transformation.

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Authors and Affiliations

  1. Biochemistry and Cell Biology Department, Rice University, 6100 Main st. MS 140, Houston, TX, 77251-1892, USA

    Rafael Couñago & Yousif Shamoo

  2. W.M. Keck Center for Computational and Structural Biology, Houston, TX, 77005, USA

    Rafael Couñago

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  1. Rafael Couñago
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  2. Yousif Shamoo
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Correspondence to Yousif Shamoo.

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Communicated by G. Antranikian

The authors would like to dedicate this paper to the memory of Dr. Neil Welker

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Couñago, R., Shamoo, Y. Gene replacement of adenylate kinase in the gram-positive thermophile Geobacillus stearothermophilus disrupts adenine nucleotide homeostasis and reduces cell viability. Extremophiles 9, 135–144 (2005). https://doi.org/10.1007/s00792-004-0428-x

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  • DOI: https://doi.org/10.1007/s00792-004-0428-x

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