Engineering topics

Piya, Denish

Publications and source records attributed to Piya, Denish.

3 records

DOE OSTI2023

Systematic and scalable genome-wide essentiality mapping to identify nonessential genes in phages

Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage–host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Geochemical constraints on bacteriophage infectivity in terrestrial environments

Abstract Lytic phages can be potent and selective inhibitors of microbial growth and can have profound impacts on microbiome composition and function. However, there is uncertainty about the biogeochemical conditions under which phage predation modulates microbial ecosystem function, particularly in terrestrial systems. Ionic strength is critical for infection of bacteria by many phages, but quantitative data is limited on the ion thresholds for phage infection that can be compared with environmental ion concentrations. Similarly, while carbon composition varies in the environment, we do not know how this variability influences the impact of phage predation on microbiome function. Here, we measured the half-maximal effective concentrations (EC50) of 80 different inorganic ions for the infection of E. coli with two canonical dsDNA and ssRNA phages, T4 and MS2, respectively. Many alkaline earth metals and alkali metals enabled lytic infection but the ionic strength thresholds varied for different ions between phages. Additionally, using a freshwater nitrate-reducing microbiome, we found that the ability of lytic phages to influence nitrate reduction end-products depended upon the carbon source as well as ionic strength. For all phage:host pairs, the ion EC50s for phage infection exceeded the ion concentrations found in many terrestrial freshwater systems. Thus, our findings support a model where phages most influence terrestrial microbial functional ecology in hot spots and hot moments such as metazoan guts, drought influenced soils, or biofilms where ion concentration is locally or transiently elevated and nutrients are available to support the growth of specific phage hosts.

Carlson, Hans K. (ORCID:0000000215835313)↗

DOE OSTI2020

High-throughput mapping of the phage resistance landscape in E. coli

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. 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.

59 BASIC BIOLOGICAL SCIENCES↗