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.
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.
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