Silicibacter phage DSS3-P1
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ABSTRACT:
PROVIDER: PRJNA47567 | ENA |
REPOSITORIES: ENA
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Project description:Bacteriophage P1 along with λ and T4 phages are among the best described bacterial viruses in molecular biology. For years, P1 features as well as its life cycle have been studied and its complete genome was published. Undeciphered phenomenon of improved P1vir lytic development in the absence of DksA protein in cell engaged us to more holistic experimental approach. Bacterial wild type and dksA strains were cultured to OD600 = 0.2. Next, P1vir was added, samples were withdrawn at 0, 10 and 30 minutes after P1vir infection. Total RNA was isolated and checked for quality using the Bioanalyzer 2100. The sequencing run was conducted on the Illumina NovaSeq6000 platform. 30 million pair-end reads per samples were assessed with 101 pb read length. Reference P1 phage genome sequence and annotations were downloaded from GenBank. We have discovered many changes in virus transcriptome. For instance: downregulation of phage genes encoding the main repressor of lysogeny C1 or proteins triggering cell lysis (e.g., lysozyme, holin) and upregulation of genes encoding antiholins in dksA mutant. This results support our gentle lysis hypothesis – less efficient lysis, combined with minor improvements of phage development which may lead to higher phage yield in DksA-devoid cells. We have observed upregulated expression of phage genes responsible for virion-parts production in the dksA mutant. Interestingly, expression of lysogeny-related c8 gene is upregulated in the dksA mutant. We speculate that P1vir developing in the dksA host is at the brink of lysogeny but is unable to established it and eventually enters the lytic pathway. We also found some interesting events in host cells upon infection. P1vir is taking control of the cellular protein, sugar and lipid metabolism in both, the wild type and dksA mutant hosts. However, in dksA mutant several genes involved in sulfur metabolism were uniquely upregulated. It remains unclear if this associates with obtaining new energy sources or with global reprograming via H2S signaling functions. Generally, the hosts are reacting by activating SOS response or upregulating the heat shock proteins. But we also found downregulation of proteolysis which was unique for the dksA strain. We believe that this extensive and comprehensive study not only finds reasonable explanations for the improved P1vir development in dksA strain, but also makes a great contribution to the field of P1 phage biology. Funding: This research was funded by the National Science Center, Poland (grant PRELUDIUM 2013/09/N/NZ2/01899 to G.M.C.)
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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.
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