Mucin induces CRISPR-Cas defence in an opportunistic pathogen

Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we exposed the bacterial fish pathogen Flavobacterium columnare to its virulent phage V156 in the presence of a eukaryotic host signal (mucin). All tested conditions led to some level of innate immunity, but the presence of mucin led to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies had obtained at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the concentration of phages on metazoan surfaces may select for the diversification of bacterial immune systems.

Prophage-encoded hotspots of bacterial immune systems

The arms race between bacteria and phages led to the emergence of a variety of genetic systems used by bacteria to defend against viral infection, some of which were repurposed as powerful biotechnological tools. While numerous defense systems have been identified in genomic regions termed defense islands, it is believed that many more remain to be discovered. Here, we show that P2- like prophages and their P4-like satellites have genomic hotspots that represent a significant source of novel anti-phage systems. We validate the defense activity of 14 systems spanning various protein domains and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Immunity hotspots are present across prophages of distant bacterial species, highlighting their biological importance in the competition between bacteria and phages.

Intracellular Organization by Jumbo Bacteriophages

ABSTRACTSince their discovery more than 100 years ago, the viruses that infect bacteria (bacteriophages) have been widely studied as model systems. Largely overlooked, however, have been “jumbo phages,” with genome sizes ranging from 200 to 500 kbp. Jumbo phages generally have large virions with complex structures and a broad host spectrum. While the majority of jumbo phage genes are poorly functionally characterized, recent work has discovered many unique biological features, including a conserved tubulin homolog that coordinates a proteinaceous nucleus-like compartment that houses and segregates phage DNA. The tubulin spindle displays dynamic instability and centers the phage nucleus within the bacterial host during phage infection for optimal reproduction. The shell provides robust physical protection for the enclosed phage genomes against attack from DNA-targeting bacterial immune systems, thereby endowing jumbo phages with broad resistance. In this review, we focus on the current knowledge of the cytoskeletal elements and the specialized nuclear compartment derived from jumbo phages, and we highlight their importance in facilitating spatial and temporal organization over the viral life cycle. Additionally, we discuss the evolutionary relationships between jumbo phages and eukaryotic viruses, as well as the therapeutic potential and drawbacks of jumbo phages as antimicrobial agents in phage therapy.

Characterization of Primed Adaptation in the Escherichia coli type I-E CRISPR-Cas System

ABSTRACTCRISPR-Cas systems are bacterial immune systems that target invading nucleic acid. The hallmark of CRISPR-Cas systems is the CRISPR array, a genetic locus that includes short sequences known as “spacers”, that are derived from invading nucleic acid. Upon exposure to an invading nucleic acid molecule, bacteria/archaea with functional CRISPR-Cas systems can add new spacers to their CRISPR arrays in a process known as “adaptation”. In type I CRISPR-Cas systems, which represent the majority of CRISPR-Cas systems found in nature, adaptation can occur by two mechanisms: naïve and primed. Here, we show that, for the archetypal type I-E CRISPR-Cas system from Escherichia coli, primed adaptation occurs at least 1,000 times more efficiently than naïve adaptation. By initiating primed adaptation on the E. coli chromosome, we show that spacers can be acquired across distances of >100 kb from the initially targeted site, and we identify multiple factors that influence the efficiency with which sequences are acquired as new spacers. Thus, our data provide insight into the mechanism of primed adaptation.[This paper has been peer reviewed, with Ailong Ke (Cornell University) serving as the editor. Reviews and point-by-point response, and a marked-up version of the edited manuscript are provided as supplementary files.]

Rational design of a split-Cas9 enzyme complex

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.