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. IMPORTANCECholera, 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. Vibrio cholerae is a Gram-negative facultative pathogen that causes the acute diarrheal disease cholera. The current cholera pandemic, the seventh in recorded history, began in 1961 and is caused by V. cholerae O1 of the El Tor biotype (1). This pandemic has affected much of the developing world, including many countries in Asia and Africa and most recently in the Caribbean (2). The sixth cholera pandemic, however, was caused by V. cholerae O1 of the classical biotype. Classical biotype strains declined following the emergence of El Tor strains and are now believed to be extinct, after last being seen in 1990 in Bangladesh (3). The mechanisms underpinn...

The Drosophila melanogaster accessory gland is a functional analog of the mammalian prostate made up of 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. Here, we show that similar to the mammalian prostate, this tissue grows with age. We find that the adult accessory gland grows in part via endocycles to increase DNA content and cell size. The differentiated, binucleated main cells remain poised to endocycle in the adult gland, and tissue damage or upregulation of signals that promote growth are sufficient to trigger dramatic endocycling leading to increases in cell size and ploidy, independent of mating status. Our data establishes that the adult accessory gland is not quiescent, but instead uses endocycles to maintain accessory gland function throughout the fruit fly's lifespan.

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