Abstract
Glutamine synthetase is an essential enzyme in ammonium assimilation and glutamine biosynthesis. The Haloferax mediterranei genome has two other glnA-type genes (glnA2 and glnA3) in addition to the glutamine synthetase gene glnA. To determine whether the glnA2 and glnA3 genes can replace glnA in nitrogen metabolism, we generated deletion mutants of glnA. The glnA deletion mutants could not be generated in a medium without glutamine, and thus, glnA is an essential gene in H. mediterranei. The glnA deletion mutant was achieved by adding 40 mM glutamine to the selective medium. This conditional HM26-ΔglnA mutant was characterised with different approaches in the presence of distinct nitrogen sources and nitrogen starvation. Transcriptomic analysis was performed to compare the expression profiles of the strains HM26-ΔglnA and HM26 under different growth conditions. The glnA deletion did not affect the expression of glnA2, glnA3 and nitrogen assimilation genes under nitrogen starvation. Moreover, the results showed that glnA, glnA2 and glnA3 were not expressed under the same conditions. These results indicated that glnA is an essential gene for H. mediterranei and, therefore, glnA2 and glnA3 cannot replace glnA in the conditions analysed.
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Acknowledgements
We thank Jörg Soppa for the useful comments on and assistance with this work. This work was funded by MICINN Grant Number BIO2013-42921P (to MJB), Generalitat Valenciana Grant Number ACIF/2018/200 (to GP) and Universidad de Alicante (VIGROB-016).
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GP and VB designed and characterised the mutants; AV, JE, and MC prepared the array samples and performed the data collection; VHR, MCM and JE analysed the microarray data; GP and VHR wrote the paper; JE, VB, MC and MJB conducted the review and editing; MJB provided funding, project administration, and resources. All authors read and approved the final manuscript.
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Rodríguez-Herrero, V., Payá, G., Bautista, V. et al. Essentiality of the glnA gene in Haloferax mediterranei: gene conversion and transcriptional analysis. Extremophiles 24, 433–446 (2020). https://doi.org/10.1007/s00792-020-01169-x
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DOI: https://doi.org/10.1007/s00792-020-01169-x