An archaeal Cas3 protein facilitates rapid recovery from DNA damage

Miezner, Guy; Turgeman-Grott, Israela; Zatopek, Kelly M; Gardner, Andrew F.; Reshef, Lea; Choudhary, Deepak K; Altstetter, Martina; Allers, Thorsten; Marchfelder, Anita; Gophna, Uri

doi:10.1093/femsml/uqad007

Cited by 3 publications

(1 citation statement)

References 43 publications

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“…Unexpectedly however, survival under stress generated through UV-light, hydrogen peroxide and heat was significantly lower in strains carrying the cas9 gene (Figure 2 and Figure 3). Similar to our observations, a recently published study could show that deletion of the archaeal Cas3 in Haloferax volacanii led to a higher resistance towards UV exposure (49).…”

Section: Discussionsupporting

confidence: 93%

The stress of carrying CRISPR-Cas

Haider,

Bauer,

Grempels

et al. 2024

Preprint

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Streptococcus anginosus(S. anginosus) is a commensal that can cause severe invasive bacterial infections. A considerable percentage ofS. anginosusstrains harbor CRISPR-Cas systems, which apart from being a bacterial immunity system can play an important role regarding the adaptation to environmental stress. The functionality ofS. anginosusCRISPR-Cas systems has previously not been investigated. To address this, we created a set of deletion mutants in the CRISPR-Cas type II-A system of theS. anginosusSK52 type strain, targeting the nuclease Cas9 and the CRISPR array. Testing these strains in a plasmid clearance assay, we were able to confirm CRISPR-Cas activity. Furthermore, the role of theS. anginosusCRISPR-Cas system was investigated under various stress conditions such as UV light, hydrogen peroxide exposure, and high-temperatures in wildtypeS. anginosusand CRISPR-Cas mutant strains. Under these conditions, survival was significantly lower in strains carryingcas9.Bacterial growth and metabolic activity in Alamar blue assays was also negatively affected by the presence ofcas9inS. anginosus. In summary we found that the presence of a functional CRISPR-Cas system inS. anginosusleads to measurable metabolic and fitness costs for the wildtype strain. Carryingcas9was associated with an impaired stress response in our experiments and may thus explain, why many strains of this species lack CRISPR-Cas.Author SummaryThe bacterial immunity system CRIPRS-Cas provides protection against invading foreign genetic material. Despite this obvious advantage only about 50% of bacteria carry CRISPR-Cas. To investigate the CRISPR system ofStreptococcus anginosus, which can cause serious bacterial infections and has recently been linked to gastric cancer, we created a set of mutants in different loci of the CRISPR system. Exposing these mutants to stress through UV-light, hydrogen peroxide and high temperatures, we could show that carrying the CRISPR nuclease gene Cas9 is associated with impaired survival under harsh conditions. Strains lacking the nuclease gene had a better growth and higher metabolic activity than the wildtype strain. In summary we found that the presence of a functional CRISPR-Cas system inS. anginosusleads to considerable metabolic and fitness costs. Carryingcas9was associated with an impaired stress response in our experiments and may thus explain, why many strains of this species lack CRISPR-Cas.

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Section: Discussionsupporting

confidence: 93%

The stress of carrying CRISPR-Cas

Haider,

Bauer,

Grempels

et al. 2024

Preprint

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Internal in-frame translation generates Cas11b, which is important for effective interference in an archaeal CRISPR-Cas system

Sailer,

Brendel,

Chernev

et al. 2024

Preprint

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CRISPR-Cas is a sophisticated defence system used by bacteria and archaea to fend off invaders. CRISPR-Cas systems vary in their Cas protein composition and have therefore been divided into different classes and types. Type I systems of bacteria have been shown to contain the small Cas11 protein as part of the interference complex. Here we show for the first time that an archaeal CRISPR-Cas type I system also contains a Cas11 protein. In addition, we show for the first time an internal in-frame translation of an archaeal protein. The Cas11b protein from the Haloferax volcanii type I-B system is encoded in the cas8b gene. Translation initiation at an internal methionine of the cas8b open reading frame results in synthesis of Cas11b. Cas11b is required for an effective interference reaction and without Cas11b fewer Cascade complexes form. Comparison of transcriptomes from wild type and a Cas11b less strain show that the depletion of Cas11b results in differential regulation of many genes. Taken together Cas11b is important for the defence reaction of the type I-B CRISPR-Cas system and seems to play an additional cellular role.

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Transcriptional landscape of the archaeal cell cycle is broadly conserved in eukaryotes

Gomez-Raya-Vilanova,

Teulière,

Medvedeva

et al. 2024

Preprint

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The cell cycle is a series of events that occur from the moment of cell birth to cell division. In eukaryotes, cell growth, genome replication, genome segregation, and cytokinesis are strictly coordinated, defining discrete cell cycle phases. In contrast, these key processes may occur concurrently in bacteria. Thermoacidophilic archaea in the genus Saccharolobus follow a defined cell cycle program, with the first pre-replicative growth (G1) phase, followed by the chromosome replication (S) phase, the second growth (G2) phase, and rapid genome segregation (M) and cytokinesis (D) phases. However, whether other processes, such as metabolism, catabolism, protein translation, and antiviral defense also occur at specific cell cycle phases, as in eukaryotes, or are active throughout the cell cycle, as in bacteria, remains unclear. To address this question, we synchronized cultures of S. islandicus and performed an in-depth transcriptomic analysis of samples enriched in cells undergoing the M-G1, S, and G2 phases. Differential gene expression and consensus gene co-expression network analyses provided a holistic view of the S. islandicus cell cycle. In addition to the core transcriptome network, which is expressed throughout the cell cycle, we show that diverse metabolic pathways, protein synthesis, cell motility and even antiviral defense systems, are expressed in a cell cycle dependent fashion. Our data also refines understanding of the processes previously known to be linked to the cell cycle, such as DNA replication. We show that most DNA replication genes are expressed prior to the S phase, during the M-G1, whereas expression of the major chromatin genes, and accordingly, chromatinization are concomitant with replication. A statistical model was used to define sets of signature genes characteristic of each of the analyzed cell cycle phases, emphasizing transcriptional stratification of the phases. Signature genes are more conserved across Thermoproteota than non-signature genes and their peak expression, especially for the M-G1 and G2 specific genes, matches that of homologs in yeast. Collectively, our data elucidate the complexity of the S. islandicus cell cycle and suggest that it more closely resembles the cell cycle of eukaryotes than previously appreciated.

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An archaeal Cas3 protein facilitates rapid recovery from DNA damage

Cited by 3 publications

References 43 publications

The stress of carrying CRISPR-Cas

The stress of carrying CRISPR-Cas

Internal in-frame translation generates Cas11b, which is important for effective interference in an archaeal CRISPR-Cas system

Transcriptional landscape of the archaeal cell cycle is broadly conserved in eukaryotes

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