Project description:We used microarray analysis to investigate whole genome transcriptome dynamics of the marine cyanobacterium Prochlorococcus sp. strain MED4 and the T7-like podovirus P-SSP7 over a time course during the 8 hour latent period of lytic infection prior to cell lysis. Manuscript Summary: Interactions between bacterial hosts and their viruses (phages) lead to reciprocal genome evolution through a dynamic co-evolutionary process1-5. Phage-mediated transfer of host genes – often located in genome islands – has had a major impact on microbial evolution1, 4, 6. Furthermore, phage genomes have clearly been shaped by the acquisition of genes from their hosts2, 3, 5. Here we investigate whole-genome expression of a host and phage, the marine cyanobacterium Prochlorococcus and a T7-like cyanophage during lytic infection, to gain insight into these co-evolutionary processes. While most of the phage genome was linearly transcribed over the course of infection, 4 phage-encoded bacterial metabolism genes were part of the same expression cluster, even though they are physically separated on the genome. These genes — encoding photosystem II D1 (psbA), high-light inducible protein (hli), transaldolase (talC) and ribonucleotide reductase (nrd) — are transcribed together with phage DNA replication genes and appear to make up a functional unit involved in energy and deoxynucleotide production needed for phage replication in resource-poor oceans. Also unique to this system was the upregulation of numerous genes in the host during infection. These may be host stress response genes, and/or genes induced by the phage. Many of these host genes are located in genome islands and have homologues in cyanophage genomes. We hypothesize that phage have evolved to utilize upregulated host genes, leading to their stable incorporation into phage genomes and their subsequent transfer back to hosts in genome islands. Thus activation of host genes during infection may be directing the co-evolution of gene content in both host and phage genomes. Keywords: time course, viral infection, marine cyanobacteria, podovirus, bacteriophage, stress response
Project description:Bacteriophages (phages) are widespread in Streptococcus pneumoniae, with most strains carrying phage genomes integrated into the chromosome. RNA sequencing was utilised to explore whether phage gene expression could be detected. The pneumococcal reference strain PMEN3 (Spain9V-3), which contained two full-length phages and one partial phage, was grown in broth culture and mitomycin C was added to facilitate phage induction. PMEN3 culture samples were taken at sequential time points and RNA was extracted and sequenced.
Project description:Bacteriophage (phage) are viruses that can kill bacteria, but also mediate gene transfer for bacterial evolution. The telomere phages are a curious form using telomere-like structures to replicate their genomes as linear extrachromosomal elements. Here we find that telomere phages are widely distributed in bacteria, being highly prevalent in Klebsiella species. We established a model system to investigate telomere phage biology and find only a small set of phage proteins are expressed in phage-host cells, including a toxin – telocin - that kills other Klebsiella strains. We identify and validate other telocins in the genomes of other, widespread Klebsiella telomere phages. Thus, telomere phages are widespread elements encoding diverse antibacterial weapons and we discuss the prospect of using telocins for precision editing of microbial populations.
Project description:Bacteriophage (phage) therapy is a promising alternative to antibiotics, yet phage-induced immune responses can affect treatment efficacy. However, current methods for assessing phage immunogenicity are limited, hindering the development of safer, more effective therapies. Here, we introduce the Bacteriophage Risk Index (BRI), a novel metric that quantifies phage immunogenic potential based on CpG dinucleotide frequency, motif spacing, and sequence context, key factors influencing Toll-like receptor 9 (TLR9) activation. Applying the BRI to 7,011 phage genomes, we classified them into five risk tiers, revealing substantial immunogenic variability, even among phages targeting the same bacterial host. BRI scores correlated with immune responses in human lung epithelial cells, validating its predictive power. Experimental testing further confirmed this, as exposure of lung epithelial cells to two phages from distinct risk tiers showed that the high-risk phage (Category 4) induced a strong pro-inflammatory response, upregulating CXCL1, CXCL8, IRF7, and TNFAIP3, while the low-risk phage (Category 2) triggered minimal immune activation with limited cytokine expression. These findings confirm that higher BRI scores predict stronger immune responses, providing a robust tool for evaluating phage immunogenicity. By enabling the selection of phages with lower immunogenic potential, the BRI enhances the safety and efficacy of phage therapy while offering a standardized framework for regulatory agencies, clinical researchers, and biologic drug development, with applications extending beyond phage therapy to other immunogenic biologics.
Project description:Whole-genome sequencing is an important way to understand the genetic information, gene function, biological characteristics, and living mechanisms of organisms. There is no difficulty to have mega-level genomes sequenced at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. The shotgun sequencing method failed to dissect this genome. After insisting for 10 years and going over 3 generations of sequencing techniques, we successfully dissected the PaP1 genome with 91,715 bp in length. Single-molecule sequencing revealed that this genome contains lots of modified bases, including 51 N6-methyladenines (m6A) and 152 N4-methylcytosines (m4C). At the same time, further investigations revealed a novel immune mechanism of bacteria, by which the host bacteria can recognize and repel the modified bases containing inserts in large scale, and this led to the failure of the shotgun method in PaP1 genome sequencing. Strategy of resolving this problem is use of non-library dependent sequencing techniques or use of the nfi- mutant of E. coli DH5M-NM-1 as the host bacteria to construct the shotgun library. In conclusion, we unlock the mystery of phage PaP1 genome hard to be sequenced, and discover a new mechanism of bacterial immunity in present study. Methylation profiling of Pseudomonas aeruginosa phage PaP1 using kinetic data generated by single-molecule, real-time (SMRT) sequencing on the PacBio RS.
Project description:Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.
Project description:Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.
Project description:Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses1–3. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA4–10. Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae, but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.
Project description:By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to contend with suboptimal conditions that would otherwise reduce their fitness. In addition, certain types of dormancy like sporulation, can serve as a refuge from parasitic infections. Phages are unable to attach to spores, but their genomes can be entrapped in the resting structures and are able to resume infection upon host germination. Thus, dormancy has the potential to affect both the reproductive and survival components of phage fitness. Here, we characterized the distribution and diversity of sigma factors in nearly 3,500 phage genomes. Homologs of bacterial sigma factors that are responsible for directing transcription during sporulation were preferentially recovered in phages that infect spore-forming hosts. While non-essential for lytic infection, when expressed in Bacillus subtilis, we demonstrate that phage-encoded sigma factors activated sporulation gene networks and reduced spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate the expression of complex traits, like the transitions involved in bacterial dormancy.
Project description:ϕXacN1 is a novel jumbo myovirus infecting the causative agent of Asian citrus canker, Xanthomonas citri. Its linear 384,670 bp double-stranded DNA genome encodes 592 predicted protein coding genes and shows 65,875 bp direct terminal repeats (DTRs), so far the longest DTRs among sequence phage genomes. The DTRs harbor 56 tRNA genes, corresponding to all 20 amino acids. This is the highest number of tRNA genes reported in a phage genome. Codon usage analyses revealed a propensity that the phage encoded tRNAs target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes, additional seven translation-related genes as well as a chaperonin gene found in the ϕXacN1 genome suggests an increased level of independence of phage replication on host molecular machinery and a wide host range. Consistently, ϕXacN1 showed a wider host range than other X. citri phages in an infection test against a panel of X. citri strains. Phylogenetic analyses revealed a clade of phages composed of ϕXacN1 and ten other jumbo phages showing an evolutionary stability in their large genome sizes.