Identification and characterization of two CRISPR-Cas systems associated with the mosquito microbiome

Abstract

The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host-microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two CRISPR-Cas systems in a novel bacterium, Serratia Sp. Ag1, that was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR-Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a Type I-E CRISPR-Cas system and has a single array, CRISPR1. CRISPR2 is a Type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequence of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp, including S. marcescens and S. fonticola, whereas CRISPR1 was not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9% of CRISPR1 (Type I-E) targets are in phage sequences and 91% are in plasmid sequences. Conversely, ~66% of CRISPR2 (Type I-F), targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time.