We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage 201ϕ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication.

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically bacteriophage were assumed not to require a cytoskeleton. Here we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with a novel, extended C-terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in vitro formed filaments, indicating how interactions between the C-terminus and the following monomer drive polymerization. Finally, we show that PhuZ assembles a spindle-like array required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. This is the first example of a prokaryotic tubulin array that functions analogously to the microtubule-based spindles of eukaryotes.

Summary We recently demonstrated that the large Pseudomonas chlororaphis bacteriophage 201φ2-1 assembles a nucleus-like structure that encloses phage DNA and segregates proteins according to function, with DNA processing proteins inside and metabolic enzymes and ribosomes outside the nucleus. Here we investigate the replication pathway of the Pseudomonas aeruginosa bacteriophages φKZ and φPA3. Bacteriophages φKZ and φPA3 encode a proteinaceous shell that assembles a nucleus-like structure that compartmentalizes proteins and DNA during viral infection. We show that the tubulin-like protein PhuZ encoded by each phage assembles a bipolar spindle that displays dynamic instability and positions the nucleus at midcell. Our results suggest that the phage spindle and nucleus play the same functional role in all three phages, 201φ2-1, φKZ and φPA3, demonstrating that these key structures are conserved among large Pseudomonas phages.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. Methods We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of coronavirus disease (COVID-19) patients. We studied plasma from 64 COVID-19 positive patients, 17 COVID-19 negative patients, and 34 pre-pandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 COVID-19 patients. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. Results SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95%CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within one day) was statistically significant. Conclusions The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of COVID-19 positive patients to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.