Biology subjects

Malone, L. M.

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

3 recordsLinked to original sources

bioRxiv2022

Type III CRISPR-Cas provides resistance against nucleus-forming jumbo phages via abortive infection

Bacteria have diverse defences against phages. In response, jumbo phages evade multiple DNA-targeting defences by protecting their DNA inside a nucleus-like structure. We previously demonstrated that RNA-targeting type III CRISPR-Cas systems provide jumbo phage immunity by recognising viral mRNA exported from the nucleus for translation. Here, we demonstrate that recognition of phage mRNA by the type III system activates a cyclic triadenylate-dependent accessory nuclease, NucC. Although unable to access phage DNA in the nucleus, NucC degrades the bacterial chromosome, triggers cell death, and disrupts phage replication and maturation. Hence, type III-mediated jumbo phage immunity occurs via abortive infection, with suppression of the viral epidemic protecting the population. We further show that type III systems targeting jumbo phages have diverse accessory nucleases, including RNases that provide immunity. Our study demonstrates how type III CRISPR-Cas systems overcome the inaccessibility of jumbo phage DNA to provide robust immunity.

microbiology↗

bioRxiv2022

A lipopolysaccharide-dependent phage infects a pseudomonad phytopathogen and can evolve to evade phage resistance

Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in-depth examination of the genome of {Phi}Psa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of {Phi}Psa374, we revealed the structural proteome and that this phage possess a T=9 capsid triangulation, unusual for myoviruses. Furthermore, we show that {Phi}Psa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage-resistance is overcome by mutations in a tail fiber and structural protein of unknown function in {Phi}Psa374. This study provides new insight into the properties of {Phi}Psa374-like phages that informs their use as antimicrobials against Psa. Originality-Significance StatementThe rise of phytopathogen resistance to agrichemicals poses a significant threat to crop production, and requires urgent attention. The work presented here examines a phage genus, members of which utilize LPS as a receptor, and show potential as biocontrol agents of Psa. Studies in planta showed the development of Psa resistance to the phage, whilst maintaining bacterial virulence. This finding underscores the importance of examining phages in their ecological context for the informed design of phage cocktails that are effective in phytopathogen control.

microbiology↗

bioRxiv2019

A nucleus-forming jumbophage evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity

CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages1. However, DNA modification2,3, the production of anti-CRISPR proteins4,5 and potentially other strategies enable phages to evade CRISPR-Cas. Here we discovered a Serratia jumbophage that evaded type I CRISPR-Cas systems, but was sensitive to type III immunity. Jumbophage infection resulted in a nucleus-like structure enclosed by a proteinaceous phage shell - a phenomenon only reported recently for distantly related Pseudomonas phages6,7. All three native CRISPR-Cas complexes in Serratia - type I-E, I-F and III-A - were spatially excluded from the phage nucleus and phage DNA was not targeted. However, the type III-A system still arrested jumbophage infection by targeting phage RNA in the cytoplasm in a process requiring Cas7, Cas10 and an accessory nuclease. Type III, but not type I, systems frequently targeted nucleus-forming jumbophages that were identified in global viral sequence datasets. These findings explain why many bacteria harbour both RNA- and DNA-targeting CRISPR-Cas systems1,8. Together, our results indicate that jumbophage nucleus-like compartments serve as a barrier to DNA-targeting, but not RNA-targeting defences, and that this phenomenon is widespread amongst jumbophages.

microbiology↗