Functional Analysis of Bacteriophage Immunity through a Type I-E CRISPR-Cas System in Vibrio cholerae and Its Application in Bacteriophage Genome Engineering.

UNLABELLED: The classical and El Tor biotypes of Vibrio cholerae serogroup O1, the etiological agent of cholera, are responsible for the sixth and seventh (current) pandemics, respectively. A genomic island (GI), GI-24, previously identified in a classical biotype strain of V. cholerae, is predicted to encode clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins); however, experimental evidence in support of CRISPR activity in V. cholerae has not been documented. Here, we show that CRISPR-Cas is ubiquitous in strains of the classical biotype but excluded from strains of the El Tor biotype. We also provide in silico evidence to suggest that CRISPR-Cas actively contributes to phage resistance in classical strains. We demonstrate that transfer of GI-24 to V. cholerae El Tor via natural transformation enables CRISPR-Cas-mediated resistance to bacteriophage CP-T1 under laboratory conditions. To elucidate the sequence requirements of this type I-E CRISPR-Cas system, we engineered a plasmid-based system allowing the directed targeting of a region of interest. Through screening for phage mutants that escape CRISPR-Cas-mediated resistance, we show that CRISPR targets must be accompanied by a 3' TT protospacer-adjacent motif (PAM) for efficient interference. Finally, we demonstrate that efficient editing of V. cholerae lytic phage genomes can be performed by simultaneously introducing an editing template that allows homologous recombination and escape from CRISPR-Cas targeting. IMPORTANCE: Cholera, caused by the facultative pathogen Vibrio cholerae, remains a serious public health threat. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) provide prokaryotes with sequence-specific protection from invading nucleic acids, including bacteriophages. In this work, we show that one genomic feature differentiating sixth pandemic (classical biotype) strains from seventh pandemic (El Tor biotype) strains is the presence of a CRISPR-Cas system in the classical biotype. We demonstrate that the CRISPR-Cas system from a classical biotype strain can be transferred to a V. cholerae El Tor biotype strain and that it is functional in providing resistance to phage infection. Finally, we show that this CRISPR-Cas system can be used as an efficient tool for the editing of V. cholerae lytic phage genomes.

Post-eclosion growth in the Drosophila Ejaculatory Duct is driven by Juvenile Hormone signaling and is essential for male fertility.

The Drosophila Ejaculatory duct (ED) is a secretory tissue of the somatic male reproductive system. The ED is involved in the secretion of seminal fluid components and ED-specific antimicrobial peptides that aid in fertility and the female post-mating response. The ED is composed of secretory epithelial cells surrounded by a layer of innervated contractile muscle. The ED grows in young adult males during the first 24h post-eclosion, but the cell cycle status of the ED secretory cells and the role of post-eclosion ED growth have been unexplored. Here, we show that secretory cells of the adult Drosophila ED undergo variant cell cycles lacking mitosis called the endocycle, that lead to an increase in the cell and organ size of the ED post eclosion. The cells largely exit the endocycle by day 3 of adulthood, when the growth of the ED ceases, resulting in a tissue containing cells of ploidies ranging from 8C-32C. The size of the ED directly correlates with the ploidy of the secretory cells, with additional ectopic endocycles increasing organ size. When endoreplication is compromised in ED secretory cells, it leads to reduced organ size, reduced protein synthesis and compromised fertility. We provide evidence that the growth and endocycling in the young adult male ED is dependent on Juvenile hormone (JH) signaling and we suggest that hormone-induced early adult endocycling is required for optimal fertility and function of the ED tissue. We propose to use the ED as a post-mitotic tissue model to study the role of polyploidy in regulating secretory tissue growth and function.

Cell cycle variants during Drosophila male accessory gland development.

The Drosophila melanogaster male accessory gland (AG) is a functional analog of the mammalian prostate and seminal vesicles containing two secretory epithelial cell types, termed main and secondary cells. This tissue is responsible for making and secreting seminal fluid proteins and other molecules that contribute to successful reproduction. The cells of this tissue are binucleate and polyploid, due to variant cell cycles that include endomitosis and endocycling during metamorphosis. Here, we provide evidence of additional cell cycle variants in this tissue. We show that main cells of the gland are connected by ring canals that form after the penultimate mitosis, and we describe an additional post-eclosion endocycle required for gland maturation that is dependent on juvenile hormone signaling. We present evidence that the main cells of the D. melanogaster AG undergo a unique cell cycle reprogramming throughout organ development that results in step-wise cell cycle truncations culminating in cells containing two octoploid nuclei with under-replicated heterochromatin in the mature gland. We propose this tissue as a model to study developmental and hormonal temporal control of cell cycle variants in terminally differentiating tissues.

Racing against the clock: How flies regenerate just in time.

In this issue of Developmental Cell, Cohen et al. show that the Drosophila hindgut is a genetically tractable model for studying tissue regeneration. This tissue exhibits different regeneration strategies at different developmental times, demonstrating that the hindgut developmental clock, not tissue type, dictates the mode and capacity for regeneration.