Acid Experimental Evolution of the Haloarchaeon Halobacterium sp. NRC-1 Selects Mutations Affecting Arginine Transport and Catabolism

Abstract

Halobacterium sp. NRC-1 (NRC-1) is an extremely halophilic archaeon that is adapted to multiple stressors such as UV, ionizing radiation and arsenic exposure; it is considered a model organism for the feasibility of microbial life in iron-rich brine on Mars. We conducted experimental evolution of NRC-1 under acid and iron stress. NRC-1 was serially cultured in CM + medium modified by four conditions: optimal pH (pH 7.5), acid stress (pH 6.3), iron amendment (600 µM ferrous sulfate, pH 7.5), and acid plus iron (pH 6.3, with 600 µM ferrous sulfate). For each condition, four independent lineages of evolving populations were propagated. After 500 generations, 16 clones were isolated for phenotypic characterization and genomic sequencing. Genome sequences of all 16 clones revealed 378 mutations, of which 90% were haloarchaeal insertion sequences (ISH) and ISH-mediated large deletions. This proportion of ISH events in NRC-1 was five-fold greater than that reported for comparable evolution of Escherichia coli. One acid-evolved clone had increased fitness compared to the ancestral strain when cultured at low pH. Seven of eight acid-evolved clones had a mutation within or upstream of arcD, which encodes an arginine-ornithine antiporter; no non-acid adapted strains had arcD mutations. Mutations also affected the arcR regulator of arginine catabolism, which protects bacteria from acid stress by release of ammonia. Two acid-adapted strains shared a common mutation in bop, which encodes bacterio-opsin, apoprotein for the bacteriorhodopsin light-driven proton pump. Thus, in the haloarchaeon NRC-1, as in bacteria, pH adaptation was associated with genes involved in arginine catabolism and proton transport. Our study is among the first to report experimental evolution with multiple resequenced genomes of an archaeon. Haloarchaea are polyextremophiles capable of growth under environmental conditions such as concentrated NaCl and desiccation, but little is known about pH stress. Interesting parallels appear between the molecular basis of pH adaptation in NRC-1 and in bacteria, particularly the acid-responsive arginine-ornithine system found in oral streptococci.

Genome comparison reveals that Halobacterium salinarum 63‐R2 is the origin of the twin laboratory strains NRC‐1 and R1

“…2.5 | Validation that a plasmid corresponding to pHS4 from strain R1 is absent from strain NRC-1 This is based on an analysis of Illumina sequence reads obtained upon resequencing of strain NRC-1 (Kunka et al, 2020) (SRA:SRR9025102) and is described in Supplementary Methods: https://doi.org/10. 5281/zenodo.7780801.…”

Genome comparison reveals that Halobacterium salinarum 63-R2 is the origin of the twin laboratory strains NRC-1 and R1

“…This is based on an analysis of Illumina sequence reads obtained upon resequencing of strain NRC-1 (Kunka et al, 2020) (SRA:SRR9025102) and is described in Supplementary Methods: https://doi.org/10.5281/zenodo.7780801.…”

A Genome-Scale Atlas Reveals Complex Interplay of Transcription and Translation in an Archaeon

“…Here, we have investigated the scale of the interplay between transcriptional and post-transcriptional mechanisms in regulating protein levels in the halophilic archaeon H. salinarum NRC-1, which has served as a model to investigate the traits of organisms in the domain Archaea . In particular, H. salinarum NRC-1 has been widely used as a model organism to dissect hallmark traits of halophilic archaea, including niche adaptation via expanded families of general transcription factors ( 38 ), large-scale genome organization by genomic repeats and insertion sequences (ISs) ( 39 , 40 ), flotation by gas vesicle biogenesis ( 41 ), phototransduction by bacteriorhodopsin ( 42 ), and how the modularity of translational complexes enables rapid acclimation to environmental changes ( 43 ). Previous work characterized many aspects of the global transcriptional regulatory network of H. salinarum NRC-1 at a systems level and in mechanistic detail ( 2 , 3 ), with extensive validations through genetic perturbation studies and physical mapping of genome-wide protein-DNA interactions of multiple transcription factors ( 4 , 5 ).…”