Biology subjects

Kirigo, J.

Publications and source records attributed to Kirigo, J..

4 recordsLinked to original sources

bioRxiv2025

Serine Recombinase PinR Inverts Cryptic Prophage DNA to Block Adsorption of Phages

Recombinases catalyze site-specific integration, excision, and inversion of DNA and are found in myriad defense islands; however, their function in phage-defense is unknown as they are frequently dismissed as markers of prophages. Here, we characterize the physiological role of the previously-uncharacterized serine recombinase PinR of Escherichia coli cryptic prophage rac and discover that it inhibits T2 phage infection by inverting a 1,797 bp segment in a different cryptic prophage e14 to inhibit T2 infection; this inversion leads to the formation of a novel protein from two spliced genes, StfE2, that we find blocks phage adsorption. Modeling shows StfE2 inhibits T2 phage adsorption by preventing Gp38 binding to its primary receptors porins FadL and OmpF. Corroborating the receptor-blocking hypothesis, T2 escape mutants evolve resistance to PinR phage defense by mutating gp38 to remove 16 aa in the hyper variable region 3. Therefore, we discovered the first recombinase-activated phage inhibition system.

molecular biology↗

bioRxiv2023

Phages Produce Persisters

Bacteria primarily encounter stress, and, arguably, their greatest threats are phages. It is often assumed that those bacteria that escape phage attack have mutated; however, another possibility is that a subpopulation forms the dormant persister state, in a manner similar to that demonstrated for bacterial cells undergoing nutritive, oxidative, and antibiotic stress. Persister cells do not undergo mutation and survive lethal conditions by ceasing growth transiently. Slower growth and dormancy play a key physiological role as they allow host phage defense systems more time to clear the phage infection. Here we investigated how bacteria survive lytic phage infection by isolating surviving cells from the plaques of T2, T4, and lambda (cI mutant) virulent phages. We found that bacteria in plaques can escape phage attack both by mutation (i.e., become resistant) and without mutation (i.e., become persistent). Specifically, whereas T4-resistant and lambda-resistant bacteria with over a 100,000-fold less sensitivity were isolated from plaques with obvious genetic mutations (e.g., causing mucoidy), cells were also found after T2 infection that undergo no significant mutation, retain wild-type phage sensitivity, and survive lethal doses of antibiotics. Corroborating this, adding T2 phage to persister cells resulted in 137,000-fold more survival compared to that of addition to exponentially-growing cells. Phage treatments with Klebsiella pneumonia and Pseudomonas aeruginosa also generated persister cells. Hence, along with resistant strains, bacteria also form persister cells during phage infection.

microbiology↗

bioRxiv2023

Single-Cell Analysis Reveals Cryptic Prophage Protease LfgB Protects Escherichia coli During Oxidative Stress by Cleaving Antitoxin MqsA

Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting this gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology, since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly-characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB.

molecular biology↗

bioRxiv2023

Toxin/Antitoxin Systems Induce Persistence and Work in Concert with Restriction/Modification Systems to Inhibit Phage

Myriad bacterial anti-phage systems have been described and often the mechanism of programmed cell death is invoked for phage inhibition. However, there is little evidence of suicide under physiological conditions for these systems. Instead of death to stop phage propagation, we show here that persister cells, i.e., transiently-tolerant, dormant, antibiotic-insensitive cells, are formed and survive using the Escherichia coli C496_10 tripartite toxin/antitoxin system MqsR/MqsA/MqsC to inhibit T2 phage. Specifically, MqsR/MqsA/MqsC inhibited T2 phage by one million-fold and reduced T2 titers by 500-fold. During T2 phage attack, in the presence of MqsR/MqsA/MqsC, evidence of persistence include the single-cell physiological change of reduced metabolism (via flow cytometry), increased spherical morphology (via transmission electron microscopy), and heterogeneous resuscitation. Critically, we found restriction-modification systems (primarily EcoK McrBC) work in concert with the toxin/antitoxin system to inactivate phage, likely while the cells are in the persister state. Phage attack also induces persistence in Klebsiella and Pseudomonas spp. Hence, phage attack invokes a stress response similar to antibiotics, starvation, and oxidation, which leads to persistence, and this dormant state likely allows restriction/modification systems to clear phage DNA.

molecular biology↗