In depth bioinformatic CRISPR reconstruction from metagenomic data disclose phage -host coevolution in complex environments

ERIJMAN, L; ORELLANA, E.; GUERRERO, L.D.

Virtual

Congreso; Reunion conjunta SAIB | SAMIGE 2020; 2020

Sociedad Argentina de Microbiología General-SAMIGE

Bacteriophages are highly abundant and present in almost any habitat. They play a critical role shaping the microbiomes by infecting bacteria and archaea which carry out important processes to the environment. Many of these microbes have defense systems against phages. One of these system is present in most archaea and nearly 40% of bacteria and is known as CRISPR (clustered regularly interspaced short palindromic repeats). CRISPR-Cas systems are composed by Cas enzymes and an array of short DNA sequences, call spacers, separated by a repetitive sequence. Spacers are incorporated into CRISPR during unsuccessful phage attacks and it act as a immune system, protecting the cell against future infections by the same phage. At the same time, it keeps a chronological register of previous attacks. Lab-scale studies revealed that bacterial hosts respond to phage attacks by using a number of mechanisms that allow them to evade phage predation. In turn, genome rearrangements, mutations and antibacterial defense systems allow phages to overcome these barriers, leading to an evolutionary arms race. However, laboratory settings do not necessarily reflect the more complex interactions that bacteria and phages experience in natural ecosystems. Metagenomics may complement this gap in information. Unfortunately, universal phylogenetic marker, such as the 16S rRNA gene of prokaryotes, are not present in phages. Therefore, investigating the diversity of phage communities and prediction of phage-host relationships is not straightforward. We developed a bioinformatic pipeline to provide a comprehensive picture of phage-host coevolution in naturally evolving populations within a complex environment from metagenomic data. Reads containing repetitive CRISPR sequences were used to reconstruct all the detectable variants of each particular CRISPR array. This resulted in a network of all possible spacers (nodes) connected by repeats (edges), which was subsequently curated manually. Phages were matched to their specific bacterial host by searching the corresponding protospacers within the metagenome. This methodology was applied to predict phage-Gordonia associations and to assemble bacterial and phage variants in an environmental biotechnology system. By looking closely at single nucleotide variants and resolving CRISPR spacers that were present even at low abundance across a temporal series, we gained insight into the complexity of virus-host interaction at the population level in a real-world setting.