Discovery and Functional Characterization of Genomic Islands for Non-model Bacterial Systems
Authors:
Catherine M. Mageeney1* ([email protected]), Benjamin P. Diaz1, Jessica Trinh1, James D. Jaryenneh1, Rohan Krishna1, Tessa House1, Brady Cress2,3, Joseph Schoeniger1
Institutions:
1Sandia National Laboratories; 2Innovative Genomics Institution, 3University of California–Berkeley
Abstract
The intrinsic control for genome and transcriptome editing in communities (InCoGenTEC) Science Focus Area aims to develop strategies for biocontainment, enable safe transformation of non-model prokaryotes using phage vectors, and understand gene mobility in microbial communities. This project’s overall goals are to mechanistically understand gene mobility events through comprehensive computational mapping of integrase and transposon driven mobility, perform functional genomics studies to identify genes and pathways responsible for mobility, identify novel genes for use in biocontainment mechanisms, and utilize the prophages from the genomic island (GI) database to transform non-model microbes towards the goal of safe microbial community transformation.
Development of phage vectors requires closing two major knowledge gaps: (1) idenifying viable phages that infect non-model bacterial species and (2) functionally understanding these phages.
Researchers have three strategies to obtain viable phages for non-model bacteria: environmental isolation, prophage induction, and synthetic phage rebooting. The team used environmental isolation in low- or high-throughput to isolate phages from environmental reservoirs. Two new environmental phages, each with unique features, were isolated from soil using traditional low-throughput methods. Pseudomonas pudita phage MiCath contains an entire queuosine biosynthesis cassette to produce modified nucleotides protecting the phage genome from nuclease activity, and Rhodococcus phage Perlina has three tRNA genes and a split lysin gene.
To speed up the discovery of novel phages, researchers have developed a high-throughput phage isolation method (HtPIP) that enables a single-soil sample to be used for screening of up to 96 strains. Researchers are currently using HtPIP to isolate a number of phages for a diverse range of bacterial isolates from soil microbial communities.
Experimental phage hunting (even in high throughput) is slower than discovery of prophages from bacterial genomic sequence data. The team has developed a genomic island database, which includes precisely defined prophage genomes that can be “mined” for any bacterial species of interest (Mageeney et al. 2020). This database contains ~20x more phages that currently found in public phage sequence repositories. Practically, however, many of the strains that harbor these prophages are inaccessible. Researchers have developed a system to synthetically rebuild and reboot these prophages from sequence alone using DNA synthesis, yeast assisted assembly, and cell-based phage production.
To understand the phage biology and engineering constraints, and to harvest useful gene products for delivery and transformation of synthetic genetic elements, researcher must understand the functions of genes contained within the phages and genomic islands. The team plans to use CRISPRi, CRISPRi-ART, and DART technologies to enable functional genomics for the non-model bacteria and their mobile genetic elements. Using these CRISPR tools, researchers will perform whole-MGE loss-of-function screens to identify candidates genes for removal or reuse. Overall, this work provides a foundation for understanding genomic islands, allows informed design of phages for vectors, and greatly increases the ability to mine prophages.
References
eney, C. M., et al. 2020. “New Candidates for Regulated Gene Integrity Revealed Through Precise Mapping of Integrative Genetic Elements,” Nucleic Acids Research 48(8), 4052–65.
Funding Information
Funding was provided by the DOE, Office of Science, through the Genomic Science Program, BER program, under the Secure Biosystems Design Initiative project Intrinsic Control for Genome and Transcriptome Editing in Communities (InCoGenTEC); Sandia National Laboratories is managed and operated by NTESS under DOE NNSA contract DE-NA0003525.