Project description:Retrons are bacterial genetic elements that encode a reverse transcriptase and, in combination with toxic effector proteins, can serve as antiphage defense systems. However, the mechanisms of action of most retron effectors, and how phages evade retrons, are not well understood. Here, we show that some phages can evade retrons and other defense systems by producing specific tRNAs. We find that expression of retron-Eco7 effector proteins (PtuA and PtuB) leads to degradation of tRNA-Tyr and abortive infection. The genomes of T5 phages that evade retron-Eco7 include a tRNA-rich region, including a highly expressed tRNA-Tyr gene, which confers protection against retron-Eco7. Furthermore, we show that other phages (T1, T7) can use a similar strategy, expressing a tRNA-Lys, to counteract a tRNA anticodon defense system (PrrC170).
Project description:Phages are viruses that infect prokaryotes and can shape microbial communitiesby lysis, thus offering applications in various fields. However, challengesexist in sampling, isolation and accurate prediction of the host specificity ofphages as well as in the identification of newly replicated virions in response toenvironmental challenges. A new workflow using biorthogonal non-canonicalamino acid tagging (BONCAT) and click chemistry (CC) allowed combinedanalysis of phages and their hosts, the identification of newly replicated virions,and the specific tagging of phages with biotin for affinity chromatography.Replication of phage λ in Escherichia coli was selected as a model for workflowdevelopment. Specific labeling of phage λ proteins with the non-canonicalamino acid 4-azido-L-homoalanine (AHA) during phage development in E. coliwas confirmed by LC–MS/MS. Subsequent tagging of AHA with fluorescentdyes via CC allowed the visualization of phages adsorbed to the cell surfaceby fluorescence microscopy. Flow cytometry enabled the automated detectionof these fluorescent phage-host complexes. Alternatively, AHA-labeled phageswere tagged with biotin for purification by affinity chromatography. Despitebiotinylation the tagged phages could be purified and were infectious afterpurification. Applying this approach to environmental samples would enablehost screening without cultivation. A flexible and powerful workflow for thedetection and enrichment of phages and their hosts in pure cultures has beenestablished. The developed method lays the groundwork for future workflowsthat could enable the isolation of phage-host complexes from diverse complexmicrobial communities using fluorescence-activated cell sorting or biotinpurification. The ability to expand and customize the workflow through thegrowing range of compounds for CC offers the potential to develop a versatiletoolbox in phage research. This work provides a starting point for these furtherstudies by providing a comprehensive standard operating procedure.
Project description:The efficacy of bacteriophages in treating bacterial infections largely depends on the phages’ vitality, which is impaired when they are naturally released from their hosts, as well as by culture media, manufacturing processes and other insults. Here, by wrapping phage-invaded bacteria individually with a polymeric nanoscale coating to preserve the microenvironment on phage-induced bacterial lysis, we show that, compared with naturally released phages, which have severely degraded proteins in their tail, the vitality of phages isolated from polymer-coated bacteria is maintained. Such latent phages could also be better amplified, and they more efficiently bound and lysed bacteria when clearing bacterial biofilms. In mice with bacterially induced enteritis and associated arthritis, latent phages released from orally administered bacteria coated with a polymer that dissolves at neutral pH had higher bioavailability and led to substantially better therapeutic outcomes than the administration of uncoated phages.
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:Many, if not all, bacteria use quorum sensing (QS) to control gene expression and collective behaviours, and more recently QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, in order to switch between lytic and lysogenic modes of infection. In this project, we studied the interaction of Vibrio cholerae, the causative agent of cholera disease, with the lysogenic vibriophage VP882. The lytic cycle of VP882 is induced by the QS molecule DPO (3,5-dimethylpyrazin-2-ol), however, the global regulatory consequences of DPO-mediated VP882 activation have remained unclear.
Project description:hvKP ATCC43816 and its lytic phage H5 were employed as a phage-antibiotic combination model. Based on the comprehensive characterization of phages, including cryo-electron microscopy, we evaluated the synergic effect of H5 on bacterial killing in vitro when combined with multiple antibiotics, and analyzed the advantages of phage-antibiotic combinations from an evolutionary perspective and proposes a novel PAS mechanism by using ceftazidime as an example.