MutS and MutL are dispensable for maintenance of the genomic mutation rate in the halophilic archaeon Halobacterium salinarum NRC-1

Abstract

Background: The genome of the halophilic archaeon Halobacterium salinarum NRC-1 encodes for homologs of MutS and MutL, which are key proteins of a DNA mismatch repair pathway conserved in Bacteria and Eukarya. Mismatch repair is essential for retaining the fidelity of genetic information and defects in this pathway result in the deleterious accumulation of mutations and in hereditary diseases in humans.

Methodology/principal findings: We calculated the spontaneous genomic mutation rate of H. salinarum NRC-1 using fluctuation tests targeting genes of the uracil monophosphate biosynthesis pathway. We found that H. salinarum NRC-1 has a low incidence of mutation suggesting the presence of active mechanisms to control spontaneous mutations during replication. The spectrum of mutational changes found in H. salinarum NRC-1, and in other archaea, appears to be unique to this domain of life and might be a consequence of their adaption to extreme environmental conditions. In-frame targeted gene deletions of H. salinarum NRC-1 mismatch repair genes and phenotypic characterization of the mutants demonstrated that the mutS and mutL genes are not required for maintenance of the observed mutation rate.

Conclusions/significance: We established that H. salinarum NRC-1 mutS and mutL genes are redundant to an alternative system that limits spontaneous mutation in this organism. This finding leads to the puzzling question of what mechanism is responsible for maintenance of the low genomic mutation rates observed in the Archaea, which for the most part do not have MutS and MutL homologs.

Figure 1. Distribution of mutations in 5-FOA-resistant mutants.

Insertions, deletions and base pair substitutions (BPS) in the pyrF and pyrE2 genes were obtained by sequencing 5-FOA-resistant uracil auxotrophs of H. salinarum NRC-1.

Figure 2. Location of mutations in the pyrE2 and pyrF genes.

Mutations were identified by sequencing 5-FOA-resistant mutants. One-letter code for amino acid is under the gene nucleotide sequence; start and stop codons are boxed with solid lines; putative TATA box is boxed with a dotted line; ▿ indicates insertion of the base(s) specified in parenthesis next to the symbol; ▵ indicates deletion of bases directly located below the symbol; BPS changes are indicated above the sequence in bold; highlighted in green and yellow in pyrE2 are the two 9-nt direct repeats. (A) pyrE2 gene and (B) pyrF gene.

Figure 3. Analysis of deletions in the mutL, mutS1(mutS1A), mutS2 (mutS1B), double mutS, and uvrD genes.

Probes for Southern blot analysis were designed to hybridize to regions 500 nt downstream of the target genes coding region. PCR analysis primers were located 500 nt upstream of the start codon of the targeted gene and 1000 nt downstream of the stop codon. (A) Southern hybridizations. (B) PCR analysis: positive lanes template was wildtype H. salinarum NRC-1 DNA, negative lanes had no template DNA.

Figure 4. Survival of H. salinarum NRC-1 background and mutant strains to MNNG.

H. salinarum NRC-1 background strain Δura3 (AK07) and mutant strains ΔmutL (CB074), ΔmutS1A (CB071), ΔmutS1B (CB072), ΔmutS1AΔmutS1B (CB073), and ΔuvrD (CB081) were exposed to 50, 100, and 600mg/L of MNNG. Survival was calculated as the average ratio (N/No) of surviving CFU from treated cultures (N) and untreated (No) cultures. Data are the average of a least three independent experiments, with standard errors shown.