MutS and MutL Are Dispensable for Maintenance of the Genomic Mutation Rate in the Halophilic Archaeon Halobacterium salinarum NRC-1

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

Cloning, Purification, and Partial Characterization of the Halobacterium sp. NRC-1 Minichromosome Maintenance (MCM) Helicase

The MCM gene from the archaeon Halobacterium, with and without its intein, was cloned into an Escherichia coli expression vector, overexpressed and the protein was purified and antibodies were generated. The antibodies were used to demonstrate that in vivo only the processed enzyme, without the intein, could be detected

Coordination of frontline defense mechanisms under severe oxidative stress

Inference of an environmental and gene regulatory influence network (EGRINOS) by integrating transcriptional responses to H2O2 and paraquat (PQ) has revealed a multi-tiered oxidative stress (OS)-management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids and gas vesicles, metal trafficking, and various other aspects of metabolism.ChIP-chip, microarray, and survival assays have validated important architectural aspects of this network, identified novel defense mechanisms (including two evolutionarily distant peroxidase enxymes), and showed that general transcription factors of the transcription factor B family have an important function in coordinating the OS response (OSR) despite their inability to directly sense ROS.A comparison of transcriptional responses to sub-lethal doses of H2O2 and PQ with predictions of these responses made by an EGRIN model generated earlier from responses to other environmental factors has confirmed that a significant fraction of the OSR is made up of a generalized component that is also observed in response to other stressors.Analysis of active regulons within environment and gene regulatory influence network for OS (EGRINOS) across diverse environmental conditions has identified the specialized component of oxidative stress response (OSR) that is triggered by sub-lethal OS, but not by other stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of γ rays

Identification of a novel gene regulating amygdala-mediated fear extinction.

Recent years have seen advances in our understanding of the neural circuits associated with trauma-related disorders, and the development of relevant assays for these behaviors in rodents. Although inherited factors are known to influence individual differences in risk for these disorders, it has been difficult to identify specific genes that moderate circuit functions to affect trauma-related behaviors. Here, we exploited robust inbred mouse strain differences in Pavlovian fear extinction to uncover quantitative trait loci (QTL) associated with this trait. We found these strain differences to be resistant to developmental cross-fostering and associated with anatomical variation in basolateral amygdala (BLA) perineuronal nets, which are developmentally implicated in extinction. Next, by profiling extinction-driven BLA expression of QTL-linked genes, we nominated Ppid (peptidylprolyl isomerase D, a member of the tetratricopeptide repeat (TPR) protein family) as an extinction-related candidate gene. We then showed that Ppid was enriched in excitatory and inhibitory BLA neuronal populations, but at lower levels in the extinction-impaired mouse strain. Using a virus-based approach to directly regulate Ppid function, we demonstrated that downregulating BLA-Ppid impaired extinction, while upregulating BLA-Ppid facilitated extinction and altered in vivo neuronal extinction encoding. Next, we showed that Ppid colocalized with the glucocorticoid receptor (GR) in BLA neurons and found that the extinction-facilitating effects of Ppid upregulation were blocked by a GR antagonist. Collectively, our results identify Ppid as a novel gene involved in regulating extinction via functional actions in the BLA, with possible implications for understanding genetic and pathophysiological mechanisms underlying risk for trauma-related disorders

Phosphoinositide-binding interface proteins involved in shaping cell membranes

The mechanism by which cell and cell membrane shapes are created has long been a subject of great interest. Among the phosphoinositide-binding proteins, a group of proteins that can change the shape of membranes, in addition to the phosphoinositide-binding ability, has been found. These proteins, which contain membrane-deforming domains such as the BAR, EFC/F-BAR, and the IMD/I-BAR domains, led to inward-invaginated tubes or outward protrusions of the membrane, resulting in a variety of membrane shapes. Furthermore, these proteins not only bind to phosphoinositide, but also to the N-WASP/WAVE complex and the actin polymerization machinery, which generates a driving force to shape the membranes

5-FOA-resistant uracil auxotrophs of <i>H. salinarum</i> NRC-1 with mutations in multiple UMP biosynthetic genes.

(1)<p>no mutation in <i>pyrE1</i>.</p>(2)<p>mutation in both <i>pyrF</i> and <i>pyrE2</i>.</p><p>nd not determined.</p

Survival of <i>H. salinarum</i> NRC-1 background and mutant strains to MNNG.

<p><i>H. salinarum</i> NRC-1 background strain Δ<i>ura3</i> (AK07) and mutant strains Δ<i>mutL</i> (CB074), Δ<i>mutS1A</i> (CB071), Δ<i>mutS1B</i> (CB072), Δ<i>mutS1A</i>Δ<i>mutS1B</i> (CB073), and <i>ΔuvrD</i> (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.</p

Distribution of mutations in 5-FOA-resistant mutants.

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

Types and positions of spontaneous mutations in the <i>pyrF</i> and <i>pyrE2</i> genes.

(1)<p>insertion indicated was found prior to the stated base pair position.</p>(2)<p>non-synonymous base pair changes.</p