Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye

Egido, Julia E; Toner-Bartelds, Catherine; Costa, Ana Rita; Brouns, Stan J. J.; Rooijakkers, Suzan H. M.; Bardoel, Bart W.; Haas, Pieter-Jan

doi:10.1038/s41598-023-27734-w

Cited by 12 publications

(14 citation statements)

References 61 publications

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“…PhiKZ PicA T367P mutant phage have a delay in importing PhiKZ proteins and a delay in phage nucleus growth. To determine whether these delays affect the timing of the PhiKZ lytic cycle, we performed a single-step lysis curve in which we monitored cell lysis with SYTOX Green, a cell impermeable fluorescent DNA dye, as a reporter for the completion of the phage lytic cycle ( 43 ). Cells infected with wild-type PhiKZ lysed at an average of 62 mpi (n = 12) as determined by the time at which 50% maximal fluorescence was reached, whereas lysis of cells infected with PhiKZ PicA T367P was delayed until an average of 75 mpi (n = 12) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Morgan,

Enustun,

Armbruster

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

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Section: Resultsmentioning

confidence: 99%

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Morgan,

Enustun,

Armbruster

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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“…Experiment was performed essentially as described in Egido et Al. (38). In short, P. aeruginosa K2733 was grown at 37 °C to OD 600 ∼0.25 in LB.…”

Section: Methodsmentioning

confidence: 99%

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Morgan,

Enustun,

Armbruster

et al. 2024

Preprint

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Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

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“…To achieve this, we quantified the production of free DNA during a bacterial growth curve using Sytox DNA stain. This method has previously been used to look at the lysis rate of cells by both bacteriocins and bacteriophages (Harhala et al, 2021, Egido et al, 2023). Here we use it as a proxy to understand if lysogens are dying significantly faster than either other lysogenised cells or non-lysogens.…”

Section: Methodsmentioning

confidence: 99%

What makes a temperate phage an effective bacterial weapon?

Thomas,

Brockhurst,

Coyte

2023

Preprint

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Temperate bacteriophages (phages) are common features of bacterial genomes and can act as self-amplifying biological weapons, killing susceptible competitors and thus increasing the fitness of their bacterial hosts (lysogens). Despite their prevalence, however, the key characteristics of an effective temperate phage weapon remain unclear. Here we use systematic mathematical analyses coupled with experimental tests to understand what makes an effective temperate phage weapon. We find that effectiveness is controlled by phage life history traits – in particular, the probability of lysis, and induction rate – but that the optimal combination of traits varies with the initial frequency of a lysogen within a population. As a consequence, certain phage weapons can be detrimental when their hosts are rare, yet beneficial when their hosts are common, while subtle changes in individual life history traits can completely reverse the impact of an individual phage weapon on lysogen fitness. We confirm key predictions of our model experimentally, using temperate phages isolated from the clinically relevant Liverpool Epidemic Strain ofPseudomonas aeruginosa. Through these experiments, we further demonstrate that nutrient availability can also play a critical role in driving frequency-dependent patterns in phage-mediated competition. Together, these findings highlight the complex and context-dependent nature of temperate phage weapons, and highlight the importance of both ecological and evolutionary processes in shaping microbial community dynamics more broadly.ImportanceTemperate bacteriophage – viruses that integrate within bacterial DNA – are incredibly common within bacterial genomes. These phages are thought to act as powerful self-amplifying weapons, allowing their bacterial hosts to kill nearby competitors and thus gain a fitness advantage within a given niche. But what makes an effective phage weapon? Here we first use a simple mathematical model to explore the factors determining phage weapon utility. Our models suggest that phage weapons are nuanced and context-dependent: an individual phage may be beneficial or costly depending upon tiny changes to how it behaves, or to the bacterial community in which it resides. We then confirm these mathematical predictions experimentally, using phage isolated from Cystic Fibrosis patients. But, in doing so, we also find that another factor – nutrient availability – plays a key role in shaping phage-mediated competition. Together our results provide new insights into how temperate phage modulate bacterial communities.

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Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye

Cited by 12 publications

References 61 publications

An essential and highly selective protein import pathway encoded by nucleus-forming phage

An essential and highly selective protein import pathway encoded by nucleus-forming phage

An essential and highly selective protein import pathway encoded by nucleus-forming phage

What makes a temperate phage an effective bacterial weapon?

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