Biology subjects

Littlehale, M. L.

Publications and source records attributed to Littlehale, M. L..

4 recordsLinked to original sources

bioRxiv2022

Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome

The ancient, ongoing coevolutionary battle between bacteria and their viral predators, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Dozens of additional anti-phage systems have been identified based on their co-location within so-called defense islands, but whether these computational screens are exhaustive is unclear. Here, we developed an experimental selection scheme agnostic to genomic context to identify defense systems encoded in 71 diverse E. coli strains. Our results unveil 21 new and conserved defense systems, none of which were previously detected as enriched in defense islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defense systems in E. coli, with defense systems typically carried in specific locations, or hotspots. These hotspots in homologous prophages and mobile genetic elements encode dozens of additional, as-yet uncharacterized defense system candidates. Collectively, our findings reveal an extended landscape of antiviral immunity in E. coli and provide a generalizable approach for mapping defense systems in other species.

microbiology↗

bioRxiv2021

The DarTG toxin-antitoxin system provides phage defense by ADP-ribosylating viral DNA

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify TA systems that protect E. coli MG1655 against phage, we searched for those frequently encoded near known phage defense genes in bacterial genomes. Two of the systems tested provide strong protection against phage infection and are homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. We demonstrate that phage infection triggers the release of DarT toxin, a DNA ADP-ribosyltransferase, to modify viral DNA and prevent replication, thereby blocking the production of mature virions. Phages can evolve to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

microbiology↗

bioRxiv2021

High-resolution, genome-wide mapping of positive supercoiling in chromosomes

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the "twin-domain" model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

molecular biology↗

bioRxiv2020

Stress induces the transcription of toxin-antitoxin systems but does not activate toxin

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking 10 TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA-sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems likely do not strongly promote survival following stress.

microbiology↗