- Research article
- Open Access
Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity
© Lu et al.; licensee BioMed Central Ltd. 2014
- Received: 26 January 2014
- Accepted: 16 September 2014
- Published: 19 September 2014
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.
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.
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.
- Modify Base
- Phage Genome
- Host Bacterium
- Shotgun Library
- SMRT Sequencing
Whole-genome sequencing is a very important method to understand the genotype and phenotype of an organism. In 1976, the genome of phage MS2 (only 3.5 kb in length) was the first completely sequenced genome . The whole genome sequence of phage φX174 (with 5.3 kb genome) was then reported a year later . Early genome-sequencing studies mainly focused on small genomes. With the advancement of sequencing technologies, particularly shotgun sequencing method [3, 4], the sequencing of large genomes has become possible. Thus far, next- and third-generation sequencing technologies have become available [5–8]. Hence, genome sequencing has shown remarkable development.
However, small genomes, particularly bacteriophage genomes, are occasionally hard to be sequenced. We once encountered a tough work in sequencing a phage genome with a size of approximately 90 kb. In 2004, we isolated and characterised a Pseudomonas aeruginosa phage named PaP1 [9, 10]. Pulsed-field gel electrophoresis (PFGE) results showed that PaP1 contains a genome of approximately 90 kb, but 20 contigs obtained using the shotgun library sequencing method could not be assembled in an integral genome; the total length of these obtained contigs was approximately 47.7 kb, which is almost half of 90 kb. We subsequently submitted the PaP1 genomic DNA to another sequencing center, where this DNA was subjected to repeated sequencing with the shotgun method. We obtained almost the same result. We further verified this result by obtaining the PaP1 genome sequence with primer walking ; however, we failed again. Hence, this work was suspended.
Four years later, Roche/454 technique [12, 13], a second-generation sequencing method, was established. We re-sequenced the PaP1 genome by using the Roche/454 technique in 2008. We easily obtained the complete PaP1 genome sequence with a size of 91,715 bp. Thus, we aimed to determine why the PaP1 genome was successfully sequenced using the Roche/454 DNA sequencer but not using the shotgun sequencing method. Based on the differences of the principles of the two sequencing methods, our presumption was that the host bacterium of the shotgun library construction, Escherichia coli DH5α, may greatly repel the inserted phage-DNA fragments by a particular immune mechanism. In the present study, this hypothesis was confirmed by conducting several experiments, including gene knockout and single-molecule real-time (SMRT) DNA sequencing techniques (third-generation sequencing methods) [6, 14–16]; we also investigated the methylome of phage PaP1. We revealed a novel mechanism of bacterial immunity that could repel exogenous DNA and maintain their genetic stability via enzyme Endo V activity.
Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in this study
Strains or plasmids
Source or reference
Pseudomonas aeruginosa PA1
Belongs to serum typing group 9 of P. aeruginosa international antigenic typing system; host of phage PaP1
P. aeruginosa PA3
Belongs to serum typing group 6 of P. aeruginosa international antigenic typing system; host of phage PaP3
E. coli DH5α
Host for the construction of shotgun library clones
Promega, WI, USA
E. coli DH5α cat + :Δnfi
The nfi gene is replaced with cat.
E. coli DH5α Δnfi
The nfi gene is knocked out.
Template plasmid for Red system; Ampr, Cmr
Red expression plasmid; Ampr
Flp expression plasmid; Ampr, Cmr
Vector for the construction of shotgun library clones; Ampr
TaKaRa, Shiga, Japan
Phage propagation and purification
We isolated PaP1 and PaP3 phages from hospital sewage by using P. aeruginosa PA1 and PA3 (Table 1) as host bacteria, respectively, in accordance with standard lambda phage isolation protocol . PaP1 and PaP3 were propagated and purified in accordance with previously described protocols [9, 18, 19] with slight modifications. In brief, the liquid culture of the host bacteria during the log growth phase was inoculated with phages (multiplicity of infection of 1/100) and incubated at 37°C with shaking at 200 rpm. The culture showed signs of lysis after 5 h and a few drops of chloroform were added to ensure that all of the host bacteria were lysed. The culture was then centrifuged at 10,000 × g for 5 min; the supernatant (crude PaP1 suspensions) was concentrated and purified via PEG8000 (Sigma-Aldrich, St. Louis, MO) precipitation, as described previously . The PaP1 particles were concentrated using PEG8000 (these particles were placed in an ice bath for 1 h and centrifuged at 12,000 × g for 10 min; the precipitate was then collected) and further purified using a CsCl gradient ultracentrifuge in accordance with previously reported methods [21, 22].
DNA extraction and purification
EDTA (20 mM), proteinase K (50 μg mL-1) and sodium dodecyl sulfate (0.5%, w/v) were added to the purified phage stock solution (PaP1 or PaP3). The mixture was incubated at 56°C for 1 h; an equal volume of phenol-chloroform-isoamyl alcohol solution (25:24:1) was added and the resulting mixture was centrifuged at 5,000 × g for 10 min. An aqueous layer was collected and extracted with chloroform at 5,000 × g for 10 min. The collected aqueous layer was mixed with 0.6 volumes of isopropanol and stored overnight at -20°C. Afterward, the mixture was centrifuged for 10 min at 12,000 × g and 4°C; the precipitated DNA was collected and washed with 70% and 100% ethanol, respectively. The PaP1 DNA was suspended in TE buffer (pH 8.0) and stored at -20°C for subsequent use.
Endonuclease digestion assay
The following restriction endonucleases were used to digest the genomic DNA of PaP1 or PaP3 in 20 μL reaction systems according to the manufacturer’s instructions: PauI; VspI; AatII; SpeI; and EcoRI (New England Biolabs, Ipswich, MA, USA). The mixture was incubated at 37°C for 120 min and then used to perform PFGE. PFGE was conducted in 1% agarose gel with an initial switch time of 0.6 s and a final switch time of 1.6 s at 8 V/cm and an angle of 180° with a run time of 4.5 h. The restriction map was captured and analysed using Quantity One software (Bio-Rad, Hercules, CA, USA) to estimate the sizes of DNA bands on the gel. The commercial Endo V, or the products of E. coli gene nfi, was purchased from New England Biolabs, Ipswich, MA, USA. The PaP1 or PaP3 genomic DNA was digested by Endo V in 20 μL reaction systems according to the manufacturer’s instructions.
Sequencing of the PaP1 genome by using shotgun library method
In 2004, the genomic DNA of PaP1 was submitted to Chinese National Human Genome Center (CNHGC) in Shanghai, China for genome sequencing with the shotgun sequencing method  in an ABI 3730 DNA sequencer (ABI, Foster City, CA, USA). A shotgun library was constructed using E. coli DH5α as host bacterium. The PaP1 genomic DNA was digested by Sau3AI (New England Biolabs, Ipswich, MA, USA) or treated with ultrasonic waves; the DNA fragments with a length ranging from 1.6 kb to 2.0 kb were recovered to construct the shotgun library. The recovered DNA fragments were ligated into pUC18 and then electrotransformed into the host bacterium E. coli DH5α. Clones were selected randomly from the library and used for sequencing. A total of 1,653 clones were sequenced and the average sequence coverage reached approximately 15-fold of the PaP1 genome. The obtained reads were assembled using the Phred/Phrap/Consed software package . We obtained 20 contigs, but these contigs could not be assembled into an integral genome. To obviate mistakes caused by sequencing, we submitted the PaP1 genomic DNA to CNHGC in Beijing, China for repeat sequencing. Although the average sequence coverage also reached approximately 15-fold of the PaP1 genome, the obtained results were almost the same as those of the first sequencing. We also tried primer walking  to fill the gaps, but we failed to obtain the whole genome sequence of PaP1.
In 2012, we knocked out the nfi gene of E. coli DH5α (see below). To validate whether or not the nfi- mutant of E. coli DH5α can be used to construct a shotgun library and sequence the PaP1 genome, we repeated the sequencing of the PaP1 genome at Genemine Biotechnology Co., Ltd. (Chongqing, China). The procedures were exactly the same as described previously except the shotgun library clones were constructed with the nfi- mutant of E. coli DH5α as host bacterium. At this time, 1,017 clones were sequenced and the average sequence coverage reached approximately 10-fold of the PaP1 genome.
Sequencing of the PaP1 genome by using Roche/454 technique
In 2008, next-generation sequencing techniques were established. We then submitted the PaP1 genome to the CNHGC (Shanghai, China) for sequencing with a Roche/454 GS FLX titanium system . In brief, the purified genomic DNA of PaP1 was fragmented, ligated to adapters and separated into single strands; the DNA fragments were bound to beads and amplified by emulsion PCR. A solid-phase pyrophosphate sequencing reaction was performed to reveal the raw sequence data. The Roche/454 reads were assembled using a Newbler assembler  (454 Life Sciences). The PaP1 genome sequence and its annotation information were available for download at the NCBI GenBank (http://www.ncbi.nlm.nih.gov/genbank/) with an accession number of HQ832595.
Construction of the nfi- mutant of E. coliDH5α
Primers and other DNA sequences used in this study
Primers or other DNA sequencesa
Target genes or locations
Construction of the nfi mutant
Chloromycetin-resistant gene of pKD3
nfi gene of E. coli DH5α
Upstream of the nfi gene
Downstream of the nfi gene
Nfi-F (upstream of the gene nfi) and Nfi-R (downstream of the gene nfi) primers were designed to indicate the change in the nfi gene. PCR was performed using Nfi-F and Nfi-R primers with the genomic DNAs of E. coli DH5α, E. coli DH5α cat+:Δnfi and E. coli DH5α Δnfi as templates. The PCR products were used in 0.8% agarose gel electrophoresis (100 V for 40 min) to determine their sizes.
SMRT sequencing of the PaP1 genome
The PaP1 genome was subjected to SMRT sequencing at the Institute of Medicinal Plant Development (Beijing, China) by using a PacBio RS DNA sequencer (Pacific Biosciences, Menlo Park, CA, USA; http://www.pacificbiosciences.com/) [27, 28]. SMRT sequencing was performed in accordance with previously described protocols [6, 14, 15]. In brief, SMRTbell template libraries with DNA fragments of 2 kb were prepared [29, 30]. Sequencing was then performed using one SMRT cell (http://www.pacificbiosciences.com/products/consumables/SMRT-cells/); zero-mode waveguide (ZMW)  signals were obtained. SMRT reads were mapped to the reference sequence of the PaP1 genome by using the BLASR software (https://github.com/PacificBiosciences/blasr)  in accordance with standard mapping protocols. Interpulse durations (IPDs) were determined and processed as previously described [15, 29, 33] for all of the pulses aligned to each position in the PaP1 genome sequence. The modified bases were identified using SMRT Analysis Server v. 1.4.0 (Pacific Biosciences). The generated data sets are available for download at the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/)  with the accession number of GSE50100 [GEO: GSE50100].
DNAStar  was used to analyse the basic characteristics of the PaP1 genome sequence. The Internet tool tRNAscan-SE 1.21  was used to predict tRNA genes in the DNA sequence with a cove score cutoff of 20. DNAMAN software (http://www.lynnon.com/) was used to analyse the localisation of the 20 contigs in the PaP1 genome and to graphically describe the result. The PanDaTox database (http://www.weizmann.ac.il/pandatox)  was used to analyse the putative DNA motifs that were toxic to bacteria in the PaP1 genome.
The raw modification calls of the PaP1 genomic DNA, produced using the SMRTPortal Analysis Platform v. 1.3.3 (Pacific Biosciences; details are available at http://www.pacb.com/pdf/TN_Detecting_DNA_Base_Modifications.pdf), were collated as single Modifications.gff file. To predict modified motifs, we screened the Modifications.gff file by using publicly available R-scripts software (https://github.com/PacificBiosciences/motif-finding), as well as an online motif finding server (MEME, http://meme.nbcr.net/meme/cgi-bin/meme.cgi) . PaP1 ORF48 was blasted against NCBI non-redundant protein sequences (nr) (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=&LINK_LOC=blasttab&LAST_PAGE=blastn) to search probable correlations between ORF48 and methyltransferases. Protein sequences were subjected to multiple sequence alignments by using ClustalW  with default parameters and a phylogenetic tree was constructed and displayed using MEGA5  with a neighbor-joining method .
Shotgun strategy failed to obtain a complete PaP1 genome sequence
PaP1 genome sequence obtained by Roche/454 sequencer
Single-molecule sequencing revealed modified bases in the PaP1 genome
The PaP1 genome could be successfully sequenced with the Roche/454 technique but not with the shotgun method. The shotgun method depends on the construction of a DNA library; by contrast, the Roche/454 technique is a non-library-dependent technique. Therefore, we hypothesised that the shotgun method failed possibly because E. coli DH5α, the host bacterium of the shotgun library construction, greatly repelled the inserted DNA fragments by endonucleases; the PaP1 genome may contain modified bases that may be the recognised targets degraded by endonucleases.
Methylome analysis of the PaP1 phage
Comparison of PaP1 ORF48 against putative methyltransferases using BlastP
Pseudomonas phage JG004
Haliangium ochraceum DSM 14365
Lactococcus lactis subsp. lactis KLDS 4.0325
Haliangium ochraceum DSM 14365
Myxococcus xanthus DK 1622
Stigmatella aurantiaca DW4/3-1
Myxococcus fulvus HW-1
Stigmatella aurantiaca DW4/3-1
Digestion of the PaP1 genomic DNA by Endo V
Use of the nfi - mutant of E. coliDH5α as the host bacterium for shotgun library construction revealed the whole PaP1 genome sequence
To further validate the role of Endo V in the failure of the shotgun sequencing of the PaP1 genome and verify the aforementioned hypothesis, we knocked out the Endo V coding gene (nfi) of E. coli DH5α. The nfi gene of E. coli DH5α genome was initially substituted with a donor DNA (containing chloramphenicol-resistant gene, cat) by using a λ-red recombination system; the cat gene was then eliminated by FLP (a yeast-derived recombinase) recombination (Figure 8A). The PCR identification results showed that the sizes of the PCR products are correct (Figure 8B). These PCR products were sequenced and the results indicated that the nfi gene was completely knocked out. This mutant was designated as E. coli DH5α Δnfi or the nfi- mutant of E. coli DH5α.
In clone-based genome sequencing, some genomic DNA fragments cannot be cloned using E. coli; as a result, cloning gaps are retained when sequence reads are analysed. Although cloning-independent sequencing methods are available [5–7], the cause of the sequencing problem remains unclear. Previous findings indicated that some restriction enzymes  and toxic small RNA are present in a shotgun-unclonable genome region. Furthermore, some DNA fragments in shotgun-unclonable regions suppress the growth of E. coli. However, the PanDaTox database reveals that the PaP1 genome does not have any evident DNA motifs that are toxic to bacteria; in this study, a different viewpoint was proposed, in which the Endo V-mediated immunity of E. coli is responsible for the failure of the shotgun method to sequence a phage genome that contains modified bases.
This study was initiated when we found that the shotgun library method failed to sequence the genome of the PaP1 phage with a size of 90 kb in 2004. Several years later, Roche/454 sequencing method was established. We used the Roche/454 technique to sequence the PaP1 genome again in 2008. We easily obtained the complete genome sequence (91,715 bp) of the PaP1 genome. As such, we wondered why the PaP1 genome could be successfully sequenced using Roche/454 technique but could not be sequenced using the shotgun method. In contrast to the Roche/454 strategy, the shotgun strategy requires shotgun library construction. Based on the principle difference of the two sequencing methods, our presumption was that E. coli DH5α, the host bacterium of the shotgun library construction, probably repel the inserted phage-DNA fragments via a particular immune mechanism.
The shotgun strategy has been successfully applied to sequence the genomes of many organisms, including bacteria, plants and animals, as well as viruses. The host bacteria of the constructed shotgun library did not repel the inserted DNA fragments of these organisms. Therefore, the PaP1 genome, as a hard-to-sequence genome, should exhibit a unique characteristic in its genome composition. Considering previous studies, we found that some phage genomes contain modified bases. For instance, deoxycytidines in the genome of Enterobacteria phage T4 are replaced with 5-hydroxymethyldeoxycytidines (5-hmdC) [47, 48]; thymines in the genome of Bacillus subtilis phage PBS-1 are substituted by uracils (U) . Thymines in the genomes of B. subtilis phage SPO1  and Delftia acidovorans phage ΦW-14 [51, 52] are replaced with 5-hydroxymethyldeoxyuridines (5-hmdU). The phage genomes with modified bases may be commonly observed. These modified bases in a phage genome perform essential functions [53, 54], such as escaping the exclusion of host immune mechanism. During evolution, bacteria most likely develop an immune mechanism that aims directly at these modified bases in exogenous DNA.
Several known bacterial immune mechanisms, such as R-M , T-A , Abi  and CRISPR-Cas  systems exist, but any of these mechanisms does not directly aim at varied modified bases in exogenous DNA. We then focused on the enzyme Endo V because this enzyme can recognise many kinds of modified bases in DNA strands [42, 45, 59]. The mechanism of Endo V activity is different from that of general restriction endonucleases in an R-M system because these restriction endonucleases of the R-M system generally recognise and cut at unmodified base sites ; by contrast, Endo V recognises and cuts at modified base sites. Endo V also exhibits endonuclease and exonuclease activities [61, 62], which provide Endo V with a more effective DNA destruction activity than general restriction endonucleases.
Endo V was originally reported as a DNA repair enzyme [43, 44, 63] encoded by the nfi gene; most bacteria contain the nfi gene in their genome. This enzyme can recognise and cleave various modified bases and abnormal structures, such as deaminated bases, abasic (AP) sites, base mismatches, methylated bases, flap DNA, pseudo-Y structures and small insertions/deletions [42, 45, 59, 63] in DNA molecules, with a cleavage site at the second phosphodiester bond in the 3′ direction from the recognition site; as a result, a nick with 5′-phosphate and 3′-hydroxyl groups is formed and DNA strands are greatly disrupted because of the exonuclease activity of this enzyme. To determine whether or not Endo V can destroy the PaP1 genomic DNA, Endo V (a product of E. coli nfi gene) was used to digest the PaP1 genomic DNA. The result indicated that Endo V degraded the PaP1 genomic DNA into a smear band (Figure 7A).
To further validate the role of Endo V in the failure of the shotgun sequencing of the PaP1 genome, we knocked out Endo V-coding nfi gene and constructed an nfi- mutant of E. coli DH5α. This mutant was then used as the host bacterium to construct the PaP1 genomic DNA shotgun library. Consequently, the obtained sequences covered 92.3% of the PaP1 genome when the sequencing amount of the PaP1 genome reached a 10-fold coverage and the largest gap between contigs was <1.5 kb (Figure 4), which is very easy to close. This result further confirmed that the activity of Endo V is responsible for the failure of the shotgun sequencing of the PaP1 genome.The SMRT DNA sequence of the PaP1 genome showed that 7,557 bases of this genome were substituted with modified bases, including 51 m6A, 152 m4C and 7,354 other modified bases (unidentified modified types, Figures 3 and 4). The positions of each modified base in the PaP1 genome (Figure 4) indicated the presence of modified bases in this genome. We also investigated the methylome of the PaP1 phage, which may be the first phage methylome revealed by SMRT technology; this methylome may be significant in future studies on phage biology and host interaction.
This work revealed the whole PaP1 genome sequence that contains numerous modified bases, provided complete information of the epigenetic information map of the PaP1 phage with 7,557 modified bases and investigated the methylome of PaP1. We found that the shotgun sequencing method is unsuitable for genomes containing many modified bases. To resolve this problem, we may use the nfi- mutant of E. coli DH5α as the host bacterium of DNA library construction. Moreover, we revealed a new mechanism of bacterial immunity to repel exogenous DNA by Endo V activity. Considering that bacteriophage is a virus infecting bacteria and modified bases are commonly found in a phage genome, the new mechanism of bacterial immunity we first demonstrated in this study, may be particularly necessary for bacteria to evade DNA invasion and retain their genetic stability.
Availability of supporting data
The nucleotide sequence of PaP1 phage was deposited in the GenBank database with the accession number of HQ832595 (http://www.ncbi.nlm.nih.gov/nuccore/HQ832595). The data sets supporting the results of this article are available in the NCBI GEO repository  with the accession number of GSE50100 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE50100).
This work was supported by the National Natural Science Foundation of China (31070153) and the Chongqing Education Committee Foundation of China (101207). We would like to thank Professor Weiguo Cao (who works at the Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, Clemson, USA) for providing relevant information related to Endo V.
- Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D, Merregaert J, Min Jou W, Molemans F, Raeymaekers A, Van den Berghe A, Volckaert G, Ysebaert M: Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene. Nature. 1976, 260 (5551): 500-507.PubMedView ArticleGoogle Scholar
- Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA, Hutchison CA, Slocombe PM, Smith M: Nucleotide sequence of bacteriophage phi X174 DNA. Nature. 1977, 265 (5596): 687-695.PubMedView ArticleGoogle Scholar
- Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM: Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995, 269 (5223): 496-512.PubMedView ArticleGoogle Scholar
- Fuchs TM, Brandt K, Starke M, Rattei T: Shotgun sequencing of Yersinia enterocolitica strain W22703 (biotype 2, serotype O:9): genomic evidence for oscillation between invertebrates and mammals. BMC Genomics. 2011, 12: 168-PubMed CentralPubMedView ArticleGoogle Scholar
- Gupta PK: Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 2008, 26 (11): 602-611.PubMedView ArticleGoogle Scholar
- McCarthy A: Third generation DNA sequencing: pacific biosciences’ single molecule real time technology. Chem Biol. 2010, 17 (7): 675-676.PubMedView ArticleGoogle Scholar
- Shendure J, Ji H: Next-generation DNA sequencing. Nat Biotechnol. 2008, 26 (10): 1135-1145.PubMedView ArticleGoogle Scholar
- Krebes J, Morgan RD, Bunk B, Sproer C, Luong K, Parusel R, Anton BP, Konig C, Josenhans C, Overmann J, Roberts RJ, Korlach J, Suerbaum S: The complex methylome of the human gastric pathogen Helicobacter pylori. Nucleic Acids Res. 2013, 42 (4): 2415-2432.PubMed CentralPubMedView ArticleGoogle Scholar
- Lu S, Le S, Tan Y, Zhu J, Li M, Rao X, Zou L, Li S, Wang J, Jin X, Huang G, Zhang L, Zhao X, Hu F: Genomic and proteomic analyses of the terminally redundant genome of the pseudomonas aeruginosa phage PaP1: establishment of genus PaP1-like phages. PLoS One. 2013, 8 (5): e62933-PubMed CentralPubMedView ArticleGoogle Scholar
- Le S, He XS, Tan YL, Huang GT, Zhang L, Lux R, Shi WY, Hu FQ: Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of pseudomonas aeruginosa bacteriophages PaP1 and JG004. PLoS One. 2013, 8 (7): e68562-PubMed CentralPubMedView ArticleGoogle Scholar
- Benes V, Kilger C, Voss H, Paabo S, Ansorge W: Direct primer walking on P1 plasmid DNA. Biotechniques. 1997, 23 (1): 98-100.PubMedGoogle Scholar
- Zheng Z, Advani A, Melefors O, Glavas S, Nordstrom H, Ye W, Engstrand L, Andersson AF: Titration-free 454 sequencing using Y adapters. Nat Protoc. 2011, 6 (9): 1367-1376.PubMedView ArticleGoogle Scholar
- Clark MS, Thorne MA, Vieira FA, Cardoso JC, Power DM, Peck LS: Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing. BMC Genomics. 2010, 11: 362-PubMed CentralPubMedView ArticleGoogle Scholar
- Davis BM, Chao MC, Waldor MK: Entering the era of bacterial epigenomics with single molecule real time DNA sequencing. Curr Opin Microbiol. 2013, 16 (2): 192-198.PubMed CentralPubMedView ArticleGoogle Scholar
- Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC, Clark TA, Korlach J, Turner SW: Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods. 2010, 7 (6): 461-465.PubMed CentralPubMedView ArticleGoogle Scholar
- Murray IA, Clark TA, Morgan RD, Boitano M, Anton BP, Luong K, Fomenkov A, Turner SW, Korlach J, Roberts RJ: The methylomes of six bacteria. Nucleic Acids Res. 2012, 40 (22): 11450-11462.PubMed CentralPubMedView ArticleGoogle Scholar
- Sambrook J, Russell DW: The Condensed Protocols from Molecular Cloning : A Laboratory Manual. 2006, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory PressGoogle Scholar
- Sun WZ, Tan YL, Jia M, Hu XM, Rao XC, Hu FQ: Functional characterization of the endolysin gene encoded by Pseudomonas aeruginosa bacteriophage PaP1. Afr J Microbiol Res. 2010, 4 (10): 933-939.Google Scholar
- Tan Y, Zhang K, Rao X, Jin X, Huang J, Zhu J, Chen Z, Hu X, Shen X, Wang L, Hu F: Whole genome sequencing of a novel temperate bacteriophage of P. aeruginosa: evidence of tRNA gene mediating integration of the phage genome into the host bacterial chromosome. Cell Microbiol. 2007, 9 (2): 479-491.PubMedView ArticleGoogle Scholar
- Govind R, Fralick JA, Rolfe RD: Genomic organization and molecular characterization of Clostridium difficile bacteriophage PhiCD119. J Bacteriol. 2006, 188 (7): 2568-2577.PubMed CentralPubMedView ArticleGoogle Scholar
- Casas V, Rohwer F: Phage metagenomics. Methods Enzymol. 2007, 421: 259-268.PubMedView ArticleGoogle Scholar
- Vandersteegen K, Kropinski AM, Nash JH, Noben JP, Hermans K, Lavigne R: Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications. J Virol. 2013, 87 (6): 3237-3247.PubMed CentralPubMedView ArticleGoogle Scholar
- de la Bastide M, McCombie WR: Assembling genomic DNA sequences with PHRAP. Current protocols in bioinformatics / editoral board, Andreas D Baxevanis [et al]. 2007, Chapter 11: Unit11 14-Google Scholar
- Chaisson MJ, Pevzner PA: Short read fragment assembly of bacterial genomes. Genome Res. 2008, 18 (2): 324-330.PubMed CentralPubMedView ArticleGoogle Scholar
- Cherepanov PP, Wackernagel W: Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene. 1995, 158 (1): 9-14.PubMedView ArticleGoogle Scholar
- Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000, 97 (12): 6640-6645.PubMed CentralPubMedView ArticleGoogle Scholar
- Powers JG, Weigman VJ, Shu J, Pufky JM, Cox D, Hurban P: Efficient and accurate whole genome assembly and methylome profiling of E. coli. BMC Genomics. 2013, 14: 675-PubMed CentralPubMedView ArticleGoogle Scholar
- Wittmann J, Dreiseikelmann B, Rohde M, Meier-Kolthoff JP, Bunk B, Rohde C: First genome sequences of Achromobacter phages reveal new members of the N4 family. Virol J. 2014, 11: 14-PubMed CentralPubMedView ArticleGoogle Scholar
- Clark TA, Murray IA, Morgan RD, Kislyuk AO, Spittle KE, Boitano M, Fomenkov A, Roberts RJ, Korlach J: Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing. Nucleic Acids Res. 2012, 40 (4): e29-PubMed CentralPubMedView ArticleGoogle Scholar
- Travers KJ, Chin CS, Rank DR, Eid JS, Turner SW: A flexible and efficient template format for circular consensus sequencing and SNP detection. Nucleic Acids Res. 2010, 38 (15): e159-PubMed CentralPubMedView ArticleGoogle Scholar
- Levene MJ, Korlach J, Turner SW, Foquet M, Craighead HG, Webb WW: Zero-mode waveguides for single-molecule analysis at high concentrations. Science. 2003, 299 (5607): 682-686.PubMedView ArticleGoogle Scholar
- Chaisson MJ, Tesler G: Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory. BMC Bioinformatics. 2012, 13: 238-PubMed CentralPubMedView ArticleGoogle Scholar
- Lluch-Senar M, Luong K, Llorens-Rico V, Delgado J, Fang G, Spittle K, Clark TA, Schadt E, Turner SW, Korlach J, Serrano L: Comprehensive Methylome Characterization of Mycoplasma Genitalium and Mycoplasma Pneumoniae at Single-Base Resolution. PLoS Genet. 2013, 9 (1): e1003191-PubMed CentralPubMedView ArticleGoogle Scholar
- Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Holko M, Yefanov A, Lee H, Zhang N, Robertson CL, Serova N, Davis S, Soboleva A: NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Res. 2013, 41 (Database issue): D991-D995.PubMed CentralPubMedView ArticleGoogle Scholar
- Rosseel T, Scheuch M, Hoper D, De Regge N, Caij AB, Vandenbussche F, Van Borm S: DNase SISPA-next generation sequencing confirms Schmallenberg virus in Belgian field samples and identifies genetic variation in Europe. PLoS One. 2012, 7 (7): e41967-PubMed CentralPubMedView ArticleGoogle Scholar
- Schattner P, Brooks AN, Lowe TM: The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005, 33 (Web Server issue): W686-W689.PubMed CentralPubMedView ArticleGoogle Scholar
- Kimelman A, Levy A, Sberro H, Kidron S, Leavitt A, Amitai G, Yoder-Himes DR, Wurtzel O, Zhu YW, Rubin EM, Sorek R: A vast collection of microbial genes that are toxic to bacteria. Genome Res. 2012, 22 (4): 802-809.PubMed CentralPubMedView ArticleGoogle Scholar
- Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS: MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009, 37 (Web Server issue): W202-W208.PubMed CentralPubMedView ArticleGoogle Scholar
- Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD: Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 2003, 31 (13): 3497-3500.PubMed CentralPubMedView ArticleGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28 (10): 2731-2739.PubMed CentralPubMedView ArticleGoogle Scholar
- Som A, Fuellen G: The effect of heterotachy in multigene analysis using the neighbor joining method. Mol Phylogenet Evol. 2009, 52 (3): 846-851.PubMedView ArticleGoogle Scholar
- Dalhus B, Arvai AS, Rosnes I, Olsen OE, Backe PH, Alseth I, Gao H, Cao W, Tainer JA, Bjoras M: Structures of endonuclease V with DNA reveal initiation of deaminated adenine repair. Nat Struct Mol Biol. 2009, 16 (2): 138-143.PubMed CentralPubMedView ArticleGoogle Scholar
- Gates FT, Linn S: Endonuclease V of Escherichia coli. J Biol Chem. 1977, 252 (5): 1647-1653.PubMedGoogle Scholar
- Liu J, He B, Qing H, Kow YW: A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V. Mutat Res. 2000, 461 (3): 169-177.PubMedView ArticleGoogle Scholar
- Weiss B: Endonuclease V of Escherichia coli prevents mutations from nitrosative deamination during nitrate/nitrite respiration. Mutat Res. 2001, 461 (4): 301-309.PubMedView ArticleGoogle Scholar
- Zheng Y, Posfai J, Morgan RD, Vincze T, Roberts RJ: Using shotgun sequence data to find active restriction enzyme genes. Nucleic Acids Res. 2009, 37 (1): e1-PubMed CentralPubMedView ArticleGoogle Scholar
- Childs JD, Ellison MJ, Pilon R: Formation of 5-hydroxymethylcytosine-containing pyrimidine dimers in UV-irradiated bacteriophage T4 DNA. Photochem Photobiol. 1983, 37 (5): 513-519.PubMedView ArticleGoogle Scholar
- Lehman IR, Pratt EA: On the structure of the glucosylated hydroxymethylcytosine nucleotides of coliphages T2, T4, and T6. J Biol Chem. 1960, 235: 3254-3259.PubMedGoogle Scholar
- Takahashi I, Marmur J: Replacement of thymidylic acid by deoxyuridylic acid in the deoxyribonucleic acid of a transducing phage for Bacillus subtilis. Nature. 1963, 197: 794-795.PubMedView ArticleGoogle Scholar
- Kallen RG, Simon M, Marmur J: The new occurrence of a new pyrimidine base replacing thymine in a bacteriophage DNA:5-hydroxymethyl uracil. J Mol Biol. 1962, 5: 248-250.PubMedView ArticleGoogle Scholar
- Kropinski AM, Bose RJ, Warren RA: 5-(4-Aminobutylaminomethyl)uracil, an unusual pyrimidine from the deoxyribonucleic acid of bacteriophage phiW-14. Biochemistry. 1973, 12 (1): 151-157.PubMedView ArticleGoogle Scholar
- Maltman KL, Neuhard J, Warren RA: 5-[(Hydroxymethyl)-O-pyrophosphoryl]uracil, an intermediate in the biosynthesis of alpha-putrescinylthymine in deoxyribonucleic acid of bacteriophage phi W-14. Biochemistry. 1981, 20 (12): 3586-3591.PubMedView ArticleGoogle Scholar
- Gommers-Ampt JH, Borst P: Hypermodified bases in DNA. FASEB J. 1995, 9 (11): 1034-1042.PubMedGoogle Scholar
- Warren RA: Modified bases in bacteriophage DNAs. Annu Rev Microbiol. 1980, 34: 137-158.PubMedView ArticleGoogle Scholar
- Xu SY, Nugent RL, Kasamkattil J, Fomenkov A, Gupta Y, Aggarwal A, Wang X, Li Z, Zheng Y, Morgan R: Characterization of type II and III restriction-modification systems from Bacillus cereus strains ATCC 10987 and ATCC 14579. J Bacteriol. 2012, 194 (1): 49-60.PubMed CentralPubMedView ArticleGoogle Scholar
- Fineran PC, Blower TR, Foulds IJ, Humphreys DP, Lilley KS, Salmond GP: The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Proc Natl Acad Sci U S A. 2009, 106 (3): 894-899.PubMed CentralPubMedView ArticleGoogle Scholar
- Friedman DI, Mozola CC, Beeri K, Ko CC, Reynolds JL: Activation of a prophage-encoded tyrosine kinase by a heterologous infecting phage results in a self-inflicted abortive infection. Mol Microbiol. 2011, 82 (3): 567-577.PubMedView ArticleGoogle Scholar
- Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E: Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun. 2012, 3: 945-PubMedView ArticleGoogle Scholar
- Childs JD, Paterson MC, Smith BP, Gentner NE: Evidence for a near UV-induced photoproduct of 5-hydroxymethylcytosine in bacteriophage T4 that can be recognized by endonuclease V. Mol Gen Genet. 1978, 167 (1): 105-112.PubMedGoogle Scholar
- Bickle TA, Kruger DH: Biology of DNA restriction. Microbiol Rev. 1993, 57 (2): 434-450.PubMed CentralPubMedGoogle Scholar
- Huang J, Lu J, Barany F, Cao W: Multiple cleavage activities of endonuclease V from Thermotoga maritima: recognition and strand nicking mechanism. Biochemistry. 2001, 40 (30): 8738-8748.PubMedView ArticleGoogle Scholar
- Majorek KA, Bujnicki JM: Modeling of Escherichia coli Endonuclease V structure in complex with DNA. J Mol Model. 2009, 15 (2): 173-182.PubMedView ArticleGoogle Scholar
- Rosnes I, Rowe AD, Vik ES, Forstrom RJ, Alseth I, Bjoras M, Dalhus B: Structural basis of DNA loop recognition by endonuclease V. Structure. 2013, 21 (2): 257-265.PubMedView ArticleGoogle Scholar
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