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:The global transcriptional profile of novel T7-like Pseudomonas aeruginosa phage LUZ100 was obtained using the long read RNA sequencing technique ONT-cappable-seq. Using this approach we obtained a comprehensive genome-wide map of viral transcription start sites, terminators and transcription units and gained new insights in the molecular mechanisms of transcriptional regulation of T7-like temperate phages.
Project description:Differential RNA-seq (dRNA-seq) was performed on Pseudomonas aeruginosa alone or shortly after iinfection with the jumbo phage phiKZ
Project description:Quorum sensing (QS) is the cell density-dependent virulence factor regulator in Pseudomonas aeruginosa. Here, we elucidate PIT2, a phage-encoded inhibitor of the QS regulator LasR, derived from the lytic Pseudomonas phage LMA2. PIT2 inhibits the effectors PrpL and LasA of the type 2 secretion system of P. aeruginosa and attenuates bacterial virulence towards HeLa cells and in Galleria mellonella. Using RNAseq-based differential gene expression analysis, the effect of PIT2 on the LasR regulatory network was revealed. Moreover, the specific interaction between LasR and PIT2 was determined. These data expand our knowledge on phage-encoded modulators of the bacterial metabolism, as this examples an anti-virulence protein derived from a lytic phage. From an applied perspective, this phage protein reveals and exploits an interesting anti-virulence target in P. aeruginosa. As such, it lays the foundation for a new phage-inspired anti-virulence strategy to combat multidrug resistant pathogens and opens the door for SynBio applications.
Project description:Phage therapy is a promising adjunct therapeutic approach against bacterial multidrug-resistant infections, including Pseudomonas aeruginosa-derived infections. Nevertheless, the current knowledge about the phage-bacteria interaction within a human environment is limited. In this work, we performed a transcriptome analysis of phage-infected P. aeruginosa adhered to a human epithelium (Nuli-1 ATCC® CRL-4011™). To this end, we performed RNA-sequencing from a complex mixture comprising phage–bacteria–human cells at early, middle, and late infection and compared it to uninfected adhered bacteria. Overall, we demonstrated that phage genome transcription is unaltered by bacterial growth and phage employs a core strategy of predation through upregulation of prophage-associated genes, a shutdown of bacterial surface receptors, and motility inhibition. In addition, specific responses were captured under lung-simulating conditions, with the expression of genes related to spermidine syntheses, sulfate acquisition, spermidine syntheses, biofilm formation (both alginate and polysaccharide syntheses), lipopolysaccharide (LPS) modification, pyochelin expression, and downregulation of virulence regulators. These responses should be carefully studied in detail to better discern phage-induced changes from bacterial responses against phage. Our results establish the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, being obvious the phage versatility on bacterial cell invasion.
Project description:Purpose: The purpose of this study was to investigate the effect of quorum sensing on phage infection. Methods: We constructed the lasR gene knockout strain of Pseudomonas aeruginosa PAO1 and performed transcriptome sequencing.
