4/16/2021 0 Comments Lindsay Adler Rapidshare Library
All three technologies can be assayed across many conditions at low cost, as RB-TnSeq and Dub-seq use randomized DNA barcodes to assay strain abundance (BarSeq 67 ), whereas quantification of the pooled CRISPRi strains only requires deep sequencing of the guide sequences.Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms.
However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Sequencing data have been uploaded to the Sequence Read Archive under BioProject accession number PRJNA645443. Supplementary Tables with complete CRISPRi data are deposited here. In addition, the complete data from RB-TnSeq experiments are deposited here. The underlying data for all figures are provided in supporting information file S1 Data. Funding: This project was funded by the Microbiology Program of the Innovative Genomics Institute, Berkeley (to VKM, AMD, and APA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journals policy and the authors of this manuscript have the following competing interests: VKM, AMD, and APA consult for and hold equity in Felix Biotechnology, Inc. Lindsay Adler Rapidshare Library Drivers Of MicrobialPhages represent the most abundant biological entities on earth and are key ecological drivers of microbial community dynamics, activity, and adaptation, thereby impacting environmental nutrient cycles, agricultural output, and human and animal health 1 8. Despite nearly a century of pioneering molecular work, the mechanistic insights into phage specificity for a given host, infection pathways, and the breadth of bacterial responses to different phages have largely focused on a handful of individual bacterium-phage systems 9 13. Bacterial sensitivityresistance to phages is typically characterized using phenotypic methods such as cross-infection patterns against a panel of phages 14 27 or by whole-genome sequencing of phage-resistant mutants 28 33. As such, our understanding of bacterial resistance mechanisms against phages remains limited, and the field is therefore in need of improved methods to characterize phage-host interactions, determine the generality and diversity of phage resistance mechanisms in nature, and identify the degree of specificity for each bacterial resistance mechanism across diverse phage types 13, 25, 26, 34 48. Such genome-scale studies applied to different phage-host combinations have the unique potential to identify commonalities or differences in phage resistance patterns and mechanisms 18, 25, 28, 55 57. There have been few attempts to use genetic approaches for studying genome-wide host factors essential in phage infection. These loss-of-function (LOF) genetic screens broadly use bacterial saturation mutagenesis 49, 54, 58 61 or an arrayed library of single-gene deletion strains for studying phage-host interactions 50, 51, 53, 62, 63. Consequently, these studies have generally involved laborious experiments on relatively few phages and their hosts, and scaling the approach to characterize hundreds of phages is challenging. Random barcode transposon site sequencing (RB-TnSeq) allows genome-wide insertion mutagenesis leading to LOF mutations 64; a pooled CRISPR interference (CRISPRi) approach, which allows partial inhibition of gene function via transcriptional inhibition 65; and dual-barcoded shotgun expression library sequencing (Dub-seq) 66, which queries the effects of gene overexpression.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |