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Surveillance and Defense Mechanisms in Microbes

  • Author(s): Osuna, Beatriz Adriana
  • Advisor(s): Bondy-Denomy, Joseph
  • et al.
Abstract

To survive the harsh conditions of their environment, microbes have evolved protection mechanisms that counteract the most lethal of threats: damage to essential biosynthetic machinery and infiltration by foreign invaders. Surveillance mechanisms monitor fundamental biosynthetic processes—including DNA replication, mRNA transcription, and protein translation—to ensure their accuracy and limit the accumulation of incorrectly synthesized macromolecules in the cell. Immunity mechanisms, on the other hand, prevent cellular infiltration by external invaders such as viruses. In the first half of this study, I investigated a surveillance pathway that detects ribosomes stalled during protein translation and marks the incomplete nascent proteins for degradation. In yeast, this pathway was known to involve Carboxy-terminal Alanine and Threonine (CAT) tailing: a non-canonical, mRNA-independent peptide synthesis mechanism. However, the mechanism of CAT tailing and its role in maintaining protein homeostasis was unknown. Here, I show that CAT tailing requires the catalytic activity of fragmented 60S ribosomes and charged tRNAs, but is otherwise distinct from canonical protein synthesis. Moreover, CAT tailing of the incomplete nascent protein exposes a lysine residue that is modified with ubiquitin, a tag for protein destruction. In the second half of this study, I investigated the bacterial CRISPR-Cas9 immune system in Listeria monocytogenes, which detects and destroys invading phage (virus) DNA. The phage protein, AcrIIA1, was known to inactivate CRISPR-Cas9 but its mechanism was uncharacterized. Here, I show that AcrIIA1 binds to Cas9 with high affinity via the catalytic HNH domain to induce Cas9 degradation, thereby protecting the Listeria genome during lysogeny but not during lytic growth. AcrIIA1 also directly represses transcription of the anti-CRISPR locus and is therefore required for optimal phage replication. Finally, bacterial hosts have co-opted AcrIIA1 homologs that potentially act as “anti-anti-CRISPRs” by blocking the deployment of phage anti-CRISPRs.

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