Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity

BMC Genomics

Fuquan Hu²  Guangtao Huang²  Lin Zhang²  Xia Zhao²  Xinyue Yao²  Qiwen Hu²  Xiaolin Jin²  Jing Wang²  Shu Li²  Qingshan Ni²  Lingyun Zou²  Junmin Zhu²  Xiancai Rao²  Haimei Chen¹  Jianjun Huang²  Kebin Zhang²  Chang Liu¹  Ming Li²  Yinling Tan²  Shuai Le²  Shuguang Lu² 

[1] IMPLAD/PacBio joint laboratory for advanced genomic analysis, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, P. R. China;Department of Microbiology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038, P. R. China

Others  :  1139558
DOI  :  10.1186/1471-2164-15-803

 received in 2014-01-26, accepted in 2014-09-16,  发布年份 2014

【 摘 要 】

Background

Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome.

Results

After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi^- mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library.

Conclusions

This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.

【 授权许可】

   
2014 Lu et al.; licensee BioMed Central Ltd.

【 图 表 】

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