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DNA Research Advance Access originally published online on February 22, 2006
DNA Research 2005 12(6):389-401; doi:10.1093/dnares/dsi021
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© The Author 2006. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Genome Comparison In Silico in Neisseria Suggests Integration of Filamentous Bacteriophages by their Own Transposase

Mikihiko Kawai1,2,3, Ikuo Uchiyama4 and Ichizo Kobayashi1,2,5,*

1Department of Medical Genome Sciences, Graduate School of Frontier Science, The University of Tokyo Japan
2Institute of Medical Science, The University of Tokyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
3Division of Pathology, Immunology and Microbiology, Graduate School of Medicine, The University of Tokyo Japan
4Research Center for Computational Science, National Institutes of Natural Sciences Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan
5Graduate Program in Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo Japan

Received 24 August 2005; revised 24 November 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Supplementary Data
 Acknowledgements
 References
 
We have identified filamentous prophages, Nf (Neisserial filamentous phages), during an in silico genome comparison in Neisseria. Comparison of three genomes of Neisseria meningitidis and one of Neisseria gonorrhoeae revealed four subtypes of Nf. Eleven intact copies are located at different loci in the four genomes. Each intact copy of Nf is flanked by duplication of 5'-CT and, at its right end, carries a transposase homologue (pivNM/irg) of RNaseH/Retroviral integrase superfamily. The phylogeny of these putative transposases and that of phage-related proteins on Nfs are congruent. Following circularization of Nfs, a promoter-like sequence forms. The sequence at the junction of these predicted circular forms (5'-atCTtatat) was found in a related plasmid (pMU1) at a corresponding locus. Several structural variants of Nfs—partially inverted, internally deleted and truncated—were also identified. The partial inversion seems to be a product of site-specific recombination between two 5'-CTtat sequences that are in inverse orientation, one at its end and the other upstream of pivNM/irg. Formation of internally deleted variants probably proceeded through replicative transposition that also involved two 5'-CTtat sequences. We concluded that the PivNM/Irg transposase on Nfs integrated their circular forms into the chromosomal 5'-CT-containing sequences and probably mediated the above rearrangements.

Key words: transposase; filamentous bacteriophage; integration; prophage; genome comparison


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and Methods
 3. Results and Discussion
 Supplementary Data
 Acknowledgements
 References
 
Genome comparison involving closely related organisms can be useful for understanding the mechanisms of genome rearrangements and for identifying elements that participate in them.1Go,2Go During examination of a large chromosomal rearrangement that was seen when a comparison was made between two genomes of Neisseria meningitidis,3Go we encountered filamentous prophages.

The filamentous bacteriophages have a single-stranded circular DNA genome.4Go Some of them, such as M13, propagate in a double-stranded circular form in their host bacteria,4Go whereas others, such as CTX{varphi} of Vibrio cholerae, integrate themselves into the host chromosome.5Go So far, their integration is known to be mediated by one of two types of tyrosine recombinases: integration into a dif-like site by the host-encoded XerC/D recombinase6Go and integration into a tRNA gene by the phage-encoded recombinase.7Go Quite different from these tyrosine recombinases in sequence and molecular mechanism are the pivNM/irg genes3Go,8Go–10Go that belong to the Piv subfamily of the IS110/IS492 transposase family of RNaseH/Retrovial Integrase superfamily.9Go,11Go The irg genes in Neisseria gonorrhoeae were regarded as the transposases of small insertion sequence (IS) elements,10Go which were found linked with two homologues of filamentous phage proteins and were thought to be integrated into the host chromosome as part of the bacteriophage genome.10Go

In the present work, we identified prophages of this family in four Neisseria genomes and named this phage family Nf (Neisserial filamentous phages). We found that each intact copy of Nf carries a pivNM/irg homologue at their right end. Our further analysis strongly indicates that this phage family is integrated by a novel mechanism using its own PivNM/Irg transposase. This transposase is probably responsible for variously rearranged prophage genomes. Recently, Bille et al.12Go published an experimental work that was in agreement with ours (see Section 3.6).


   2. Materials and Methods
Top
 Abstract
 1. Introduction
 2. Materials and Methods
3. Results and Discussion
 Supplementary Data
 Acknowledgements
 References
 
2.1. Accession numbers and gene names
Accession numbers of the complete genomes are as follows: N. meningitidis serogroup A strain Z2491 (NmeA) (NC_003116 [GenBank] .1),8Go N. meningitidis serogroup B strain MC58 (NmeB) (NC_003112 [GenBank] .1),3Go N. gonorrhoeae strain FA1090 (Ngo) (NC_002946 [GenBank] .2) (http://www.genome.ou.edu/gono.html; http://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=635). The genome sequence of N. meningitidis serogroup C strain FAM18 (NmeC) was obtained from The Sanger Institute (ftp://ftp.sanger.ac.uk/pub/pathogens/nm/). Accession numbers for the sequences of plasmids, filamentous phages and IS are as follows: pJS-B (NC_004758 [GenBank] .1),13Go pJTPS1 (NC_001399 [GenBank] .1),14Go pMU1 (NC_007093 [GenBank] .1),15Go M13 (NC_003287 [GenBank] .2),16Go Pf1 (NC_001331 [GenBank] .1),17Go Pf3 (NC_001418 [GenBank] .1),18Go VGJ{varphi} (NC_004736 [GenBank] .1),19Go B5 (NC_003460 [GenBank] .1),20Go IS621(AB097054 [GenBank] -AB097056).9Go

We used the entries of the complete genome sequences of NmeA, NmeB and Ngo in the NCBI RefSeq (ftp://ftp.ncbi.nlm.nih.gov./genomes/Bacteria/) for gene names, amino acid sequences and coordinates. Genes of Nfs in NmeC are predicted by GeneMark.hmm for Prokaryotes21Go (Version 2.4; http://opal.biology.gatech.edu/GeneMark/gmhmm2_prok.cgi), with the nucleotide sequence of Nfs (from the left position to the right position, as shown in Table 1) as input sequences and N. meningitidis as species.


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  		Table 1. Nfs found in four Neisseria genomes

 
2.2. Bioinformatic analysis
The searches for macroscopic genome rearrangements and conserved gene clusters were performed by CGAT22Go and MBGD23Go (http://mbgd.genome.ad.jp/), respectively. CGAT was used to detect nucleotide sequence homology of a locus with the other parts of the genome. MBGD was used to detect homologous open reading frames (ORFs) and their neighbouring genes.

A homology search was performed by BLAST24Go and fasta2 package.25Go Multiple sequence alignments were constructed by ClustalW.26Go Default parameters were used unless otherwise specified. A phylogenetic tree was constructed using ClustalW, NJplot27Go and TreeViewPPC (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).


   3. Results and Discussion
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
Supplementary Data
 Acknowledgements
 References
 
3.1. Filamentous prophages in the genomes of Neisseria
3.1.1. Nomenclature and terminology of Nf prophages
In Table 1, we have listed all Nf prophages that were found in the four Neisseria genomes.

First, we describe the nomenclature of Nf prophages. Nf4-G1(del) may be used as an example of our nomenclature of Nf prophages. Here (i) ‘Nf’ stands for Neisseria filamentous phage, (ii) ‘4’ stands for subtype (subtype 1–4, as classified in Section 3.1.3), (iii) ‘G’ stands for strain (A for NmeA, B for NmeB, C for NmeC and G for Ngo), (iv) ‘1’ stands for ordinal number within a subtype that is found within the same genome (a smaller number was given to the copy with smaller genome coordinates; for exceptions, see footnotes f–h of Table 1), and (v) ‘(del)’ stands for internal deletion. Partially inverted, truncated and internally deleted copies are designated as ‘(inv)’, ‘(tr)’ and ‘(del)’, respectively. The putative intact copies are not given such a description.

We followed the following terminology. ‘A copy of Nf’ means a stretch of DNA sequence that encodes at least one gene that is homologous to the genes of ‘consensus, intact’ Nfs. By ‘the left/right of Nfs’, we mean the left/right of Nfs, as shown in Fig. 1A. ‘Intact’ copies are those that encode a potentially full set of homologues of phage genes that are oriented in the same direction and carry the expected left and right junction sequences (see Section 3.2 for a determination of the junctions). An only ‘partially inverted’ copy [Nf4-G2(inv)] resembles the intact copies, except for partial inversion of its irg gene (irg2) region. ‘Truncated’ copies of Nfs are the copies with truncation of the right end or the left end. Three of the irg copies (irg1, 4 and 8) are not neighboured by the homologues of the above conserved genes.10Go However, three regions containing these irg genes have the expected left and right junctions (see Section 3.3.2), so we regarded them as ‘internally deleted’ copies of Nfs and named them Nf4-G1(del), Nf4-G4(del) and Nf4-G8(del) (Fig. 1A).


Figure 1
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  		Figure 1. Nf filamentous prophages on Neisseria chromosome, other filamentous bacteriophages and related plasmids. In the box are shown keys to gene families (the corresponding gene of M13 in parentheses) and DNA features (upper case letters, CT, indicate direct repeats flanking Nfs). Gene names are written above the arrows for colored genes. The alternative names for pivNM/irg3Go,9Go,10Go are shown in parentheses. Note that members listed in (B) and (C) have been shown to be present in circular DNA form. (A) Nf prophages located at different loci in Neisseria genomes. The left half brackets indicate subtypes. Shown are Nfs located at different loci (Table 1) on four strains—NmeA Z2491, NmeB MC58, NmeC FAM18 and Ngo FA1090. Genes of Nf1-C1, C2, C3 and C4 are annotated by GeneMark.hmm for Prokaryotes.21Go Nf2-B1(tr) and Nf2-C1(tr) are not shown because they are located at the same locus and are quite similar to Nf2-A1(tr), as are Nf2-B2(tr) and Nf2-C2(tr) with Nf2-A2(tr) (Table 1). Nomenclature of Nf is described in Section 3.1.1 and the footnote for Table 1. Supplementary data is available at www.dnaresearch.oxfordjournals.org for details about colored genes and about the cg stem–loop region, of which the consensus sequence is 5'-cccccctnnnctaayaggggggg. (B) Filamentous bacteriophages. M134 is shown with functional modules, along with four other representative filamentous phages.17Go–20Go Grouping of genes by color is based on previous literature18Go–20Go,37Go–41Go and/or on our following bioinformatic analysis: Pf1p05 of Pf1 and p06 of VGJ{varphi} as gIII and Pf3_4 of Pf3 and p07 of VGJ{varphi} as gVI (see Supplementary data are available at www.dnaresearch.oxfordjournals.org), Pf3_7 as gII (homologous with replication protein of pVT736-1(AAC37125 [GenBank] )). Phages listed in (B) are aligned with the first base of the replication initiator gene as the left end. (C) Plasmids that have genes homologous to those of Nf. pJS-B13Go from Neisseria meningitidis ET37, pMU115Go from Eikenella corrodens and pJTPS114Go from Ralstonia solanacearum. pJS-B is aligned with the 1501st bp of the original sequence as the left end. pMU1 is aligned with tatat in the presumed junction atCTtatat as the left end. pJTPS1 is aligned with the first base pair of the replication gene as the left end.

 
We used Courier font for nucleotide and amino acid sequences for clarity.

3.1.2. Nf prophages in four Neisseria genomes
A large chromosomal rearrangement is seen as a comparison between two genomes of N. meningitidis, NmeA and NmeB.3Go During examination of this rearrangement, we encountered homologues of filamentous phages. We also found several homologous prophages on the other loci. We named these prophages Nfs (for Neisserial filamentous phages).

By investigating four sequenced Neisseria genomes (NmeA, NmeB, NmeC and Ngo), we identified 23 copies of Nf, including 11 intact, 1 partially inverted, 8 truncated and 3 internally deleted copies (Table 1). Two right-truncated homologues were located at the corresponding loci in three N. meningitidis genomes but not in the N. gonorrhoeae genome—that is, Nf2-A1(tr), Nf2-B1(tr) and Nf2-C1(tr) are at one corresponding locus and Nf2-A2(tr), Nf2-B2(tr) and Nf2-C2(tr) are at the other locus. So, there are 23 copies of Nf at 19 different loci in the four genomes. Notably, all the 11 intact and the 1 partially inverted copies are located at 12 different loci.

All the 11 intact copies and the 1 partially inverted copy of Nfs are ~8 kb in length (Table 1), with gene organization being similar to that of filamentous phages (Fig. 1A versus B), except for the presence of a pivNM/irg transposase gene homologue at the right end (Fig. 1A).

As well as previously noticed homologues of the rstA protein of filamentous phage CTX{varphi} (for phage DNA replication; corresponding to gII of M13) (NMA1792 and other brown arrows in Fig. 1A) and gI protein (for assembly) (NMA1799 and other light-blue arrows in Fig. 1A),10Go we found other Nf genes corresponding to filamentous phage genes (gV, gVIII, gIII and gVI) by in silico research (Fig. 1; Supplementary Figure S1 is available at www.dnaresearch.oxfordjournals.org). In Fig. 1, these functionally corresponding, but not necessarily homologous, genes are shown with the same color.

Genes that are conserved among Nfs are oriented in the same direction (Fig. 1A), except for NGO1137 (irg2) of Nf4-G2(inv) (as discussed in Section 3.3.1). We assumed that their sense strand represents their single-strand (plus strand) within the presumed virion.

Plasmids that have genes that are homologous to those of Nf are also shown (Fig. 1C). pJS-B13Go from N. meningitidis ET37, and pJTPS114Go from Ralstonia solanacearum, do not carry a pivNM/irg homologue, whereas pMU115Go from Eikenella corrodens does.

3.1.3. Classification of Nf into four subtypes
We classified the Nfs into four subtypes, Nf1–Nf4, based on similarity of their three long ORFs: phage DNA replication protein homologues (corresponding to pII; homologous with rstA of CTX{varphi}; brown arrows in Fig. 1A), adsorption protein homologues (corresponding to pIII; previously annotated as TspB) (yellow arrows in Fig. 1A) and pI homologues (light-blue arrows in Fig. 1A) [Fig. 2A, (1)–(3)], and by overall nucleotide sequence similarity (data not shown).


Figure 2
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  		Figure 2. Comparison of phylogenetic trees of three Nf proteins and transposase on Nf. (A) (1) pII analogues. (2) pIII analogues. (3) pI homologues. (B) PivNM/Irg transposase. Proteins without a gene name are not annotated in the entry in NCBI's RefSeq. A bar indicates substitution of 0.1 amino acid. Bootstrap values based on 1000 computer-generated trees are indicated for the nodes separating the four subtypes. In (A)(1) and (2), regions corresponding to the shortest gene [NMA1167 for (A)(1) and a gene on Nf1-C1 for (A)(2)] are used.

 
The only one exception to a clear classification of sequence similarity was seen for the DNA replication protein homologue of Nf4-G2(inv). The branch of Nf4-G2(inv), which was located differently from those of the other Nf4 copies, can be explained by the assumption that Nf4-G2(inv) might have resulted from recombination between an Nf3-like copy and an Nf4 copy. In fact, the right 5942 bp part of Nf4-G2(inv) is similar to the corresponding part, if any, of the other Nf4 copies (data not shown), whereas its left part contains three regions: the left junction (see Section 3.2.2), the phage DNA replication protein (NGO1146) gene [Fig. 2A, (1)] and the cg stem–loop (Supplementary Figure S1E is available at www.dnaresearch.oxfordjournals.org), all of which are highly similar to those of Nf3-A but not to those of other Nf4s.

3.2. Junctions of integrated Nf prophages and the host genome
Except for the right junction of Nf2 and Nf3, all the junctions were determined by alignment of copies of the same subtype, as detailed below (Sections 3.2.1–3.2.5). To determine the right junctions of Nf2 and Nf3, we took advantage of the formation of a promoter-like sequence by joining the right and the left ends of Nfs, as described below (Section 3.2.6).

3.2.1. Sequences flanking Nfs
The dRS3 repeats constitute one family of repeated sequences in Neisseria and have a consensus sequence of attcccnnnnnnnngggaat.8Go Alignment of Nf1 copies (as shown in Fig. 3A) can be interpreted as Nf1s being able to target 5'-CT in specific members of the dRS3 repeat family, attccc(g/a)cCT(g/a)cgcgg(g/a)aa(t/g), and were integrated forming flanking direct repeats of 5'-CT. The consensus sequence that was targeted by Nf1 (5'-attcccgcCTgcgcgggaat) is the most abundant sequence of the dRS3 repeats (291 out of 672 copies in NmeA).


Figure 3
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  		Figure 3. DNA features of Nf prophages. In (D) and (E), as in Fig. 1, a triangle, a filled circle and an inverted triangle indicate three conserved motifs of Nf, the left junction, the right junction and a 5 bp sequence (ataAG) that is complementary to the left junction. A black arrow indicates a pivNM/irg homologue. (A) Left and right junctions of Nf1. The dRS3 sequences constitute one family of repeated sequences in Neisseria.8Go Two base pair direct repeats of ‘CT’ are boxed with solid line. The two dotted boxes along with two boxed 2 bp direct repeats indicate a dRS3 sequence split as a result of insertion of Nf1. (B) Left junction of intact, truncated or partially inverted Nfs. (1) Nf2 and Nf4. (2) Nf3-A and Nf4-G2(inv). (3) Sequence common to all the Nf copies. The common left junction sequence is boxed. (C) Right junction. (1) Nf4. (2) Sequence common to Nf1 and Nf4. The common right junction sequence is boxed. (D) Diagrams of Nf and pMU1. (1) Nf present on the chromosome. (2) The predicted circular form of Nf. (3) pMU1. (E) Promoter-like sequence predicted to form by circularization of Nf1 and Nf4. The numbers indicate the length between the stop codon of pivNM/irg genes and ‘a’ of atCT. Both the junctions of Nf1, Nf4 and IS621, and the homologous locus of pMU1 are shown. The ends of IS621 are shown in the left. Boxes in thick line and boxes in dotted line indicate sequences resembling the –35 and –10 sequences, respectively, of typical eubacterial promoters. The lengths between the –35-like sequence and the –10-like sequence of the predicted circular form of Nfs are indicated below the alignment. In the last line, a typical eubacterial promoter sequence recognized by the primary {sigma} factors28Go,29Go is illustrated. (F) Predicted right junction of Nf2-B3 and Nf3-A. Candidate sequences for flanking atCT of Nf2-B3 and Nf3-A are aligned and compared with the –35 sequence.

 
For the other three subtypes, we could not find any obvious similarity or pattern in the flanking sequences, although some sequences were observed several times—that is, aaac flanks the left of two Nf2 copies and two Nf4 copies and cgtca flanks the right of four Nf4 copies (Fig. 3B and C).

3.2.2. Left junction
From a comparison of the left junction of three groups—Nf1 (Fig. 3A), Nf2 and Nf4 [Fig. 3B, (1)], and Nf3-A and Nf4-G2(inv) [Fig. 3B, (2)]—we concluded that the sequence 5'-CTtatat represents the left junction sequence for all of the Nfs examined [Fig. 3B, (3)].

3.2.3. Right junction of Nf1 and Nf4
From a comparison of the right junction sequences of Nf1 (Fig. 3A) and Nf4 [Fig. 3C, (1)], we found that the right junction sequence that is common to Nf1 and Nf4 is 5'-atCT [Fig. 3C, (2)]. The ‘T’ of the atCT of Nf4 is the residue that is reported to be at the 3' end of aligned downstream regions of irg genes.10Go

3.2.4. Predicted circular form of Nf1 and Nf4
As filamentous phages have a circular phage genome,4Go it is reasonable to assume that Nf1 and Nf4 would have a circular phage genome before integration. From the comparison of the common left and right junctions of Nf1 and Nf4 [Fig. 3D, (1)], the sequence at the junction of the hypothetical circular form is predicted to be 5'-atCTtatat [Fig. 3D, (2)].

Notably, we found that pMU1, which has the homologous gene set with Nf (see Section 3.1.2 and Fig. 1C), has this predicted junction sequence (5'-atCTtatat) at the corresponding locus—that is, between the pivNM/irg homologue and the replication protein homologue [at position 3757–3749 bp; Fig. 3D, (3)].

3.2.5. Assembly of a promoter-like sequence by circularization of Nf1 and Nf4
The sequence that is shared between the right end of Nf1 and Nf4 (5'-tgttgncn) partially matched with 5'-ttgaca, a typical –35 sequence of the –35 element of eubacterial promoters that is recognized by primary {sigma} factors28Go,29Go (boxed in solid, thick line in Fig. 3E). Because the predicted junction of the hypothetical circular form of Nf, atCTtatatnnt, contains a –10 promoter-like sequence (boxed with dotted line in Fig. 3E), we assumed that the junction region forms a promoter in the circular form.

The related plasmid pMU1 (see Fig. 1C) also carries this promoter-like sequence (Fig. 3E). The pivNM/irg homologue of pMU1 is most similar to the pivNM/irg of Nfs so far.

The transposase gene of IS621 is another close homologue of pivNM/irg.9Go The left end of IS621 is CTtgtat and the right end is atCT (Fig. 3E), the former being similar to the left end of Nfs and the latter being identical to their right end. IS621 would also form a promoter-like sequence after assumed circularization (Fig. 3E).

The promoter-like sequence of the predicted circular form of Nfs and pMU1 could regulate the expression of phage replication gene, although some Nfs code another short ORF between the promoter-like sequence and this gene [e.g. Nf1-B1, Nf1-B2, Nf3-A and Nf4-G2(inv); Fig. 1A].

3.2.6. Predicted right junction of Nf2 and Nf3
We cannot determine the right junction of Nf2 and Nf3 by alignment, as with Nf1 and Nf4, because Nf2-B3 and Nf3-A are the single examples of the Nf2 and Nf3 subtypes, respectively, that have an intact right junction (Fig. 1A).

Instead, we assumed that both Nf2-B3 and Nf3-A would have an atCT sequence at the right junction, as Nf1 and Nf4 copies. Because the right junction sequence atCT of Nf1 and Nf4 is located at 142 and 129 bp, respectively, downstream of the stop codon of pivNM/irg (Fig. 3E), we searched the range of 250 bp downstream of the stop codon of pivNM in Nf2-B3 and Nf3-A and found atCT at only one site in Nf2-B3 and at two sites in Nf3-A (Fig. 3F). Next, we looked for a potential –35 element upstream of these three candidate sequences and found a –35-like ttgann sequence in two of them (boxed with solid line in Fig. 3F). From these, we concluded that the right junction of Nf2-B3 and Nf3-A is atCT that is located 162 and 145 bp, respectively, downstream of the pivNM gene.

3.2.7. Junction sequences common to four Nf subtypes
Altogether, we concluded that Nfs are flanked with CT at both ends. The common sequence at the left end of Nf is CTtatat, and that at the right end is atCT, as seen in Fig. 3D, (1).

3.3. Rearranged Nfs
3.3.1. A partially inverted copy of Nf [Nf4-G2(inv)]
The irg2 gene of Nf4-G2(inv), a transposase homologue, is inverted relative to the other three genes10Go and the remaining Nf4 homology (Fig. 1A). From a comparison with the intact copies of Nf4, the recombination sites of this partial inversion are identified as the 5 bp sequence (boxed in alignment of Fig. 4A). One recombination site, CTtat, is a part of the right junction CTtatat (an open triangle and underlined in Fig. 4A), which is composed of the right direct repeat (CT) and the flanking 5 bp host sequence (tatat; shown in italics). The other recombination site, ataAG (complement is CTtat), is located between irg2 and the other genes of Nf4-G2(inv) (Fig. 4A). This sequence is complementary to the 5' bp sequence of the left common junction sequence of Nf, CTtat, and is located 10, 8 and 9 bp upstream of the start codon of pivNM/irg in Nf1, Nf2 and Nf4, respectively. Nf3 has a similar sequence (gtaAG) at 10 bp upstream. The ataAG sequence partially overlaps with the ribosome binding site aaggr, according to Skaar et al.10Go


Figure 4
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  		Figure 4. Rearranged Nfs. Sequence alignments are shown for the relevant regions. A filled triangle, a filled circle and an inverted filled triangle indicate three conserved motifs of Nf, the left junction, the right junction, and a 5 bp sequence that is complementary to the left junction as in Figs 1 and 3. A black arrow indicates a pivNM/irg homologue. (A) Nf with a partial inversion [Nf4-G2(inv)]. A box in thick line indicates the 5 bp junction sequence of the inversion. The right junction of Nf4-G2(inv), CTtatat (underlined and marked with an open triangle) is composed of the right direct repeat (CT) that is shared by all Nfs and the flanking host sequence (tatat; shown in italics). The dotted underline of the hypothetical parental form indicates the sequence that is identical to the predicted junction sequence of hypothetical circular Nf. (B) Nfs with an internal deletion. (1) Nf4-G4(del). Comparison with the left junction and the ataAG region of Nf4-G3 and Nf4-G5. Inverted arrows indicate inverted repeats. Dotted arrows indicate the homologous regions between the left end of intact Nf4 and the complement of Nf4-G4(del). (2) Nf4-G1(del) and Nf4-G8(del). Boxes in dashed line (ccct) and boxes in dotted line (tgcgt) indicate short repeats that are probably responsible for deletions that generated Nf4-G8(del) and Nf4-G1(del), respectively. (3) Possible steps of formation of Nf4-G4(del). An open circle with a dotted line indicates a hypothetical site that the transposase recognized. The thick arrow indicates a sequence that is involved in the replicative transposition event to form long inverted repeats. Boxes in dashed line with dots indicate short repeats that are probably responsible for deletion.

 
This inversion structure is perfectly consistent with conservative site-specific recombination involving the 5 bp sequences. The inversion was probably mediated by PivNM/Irg itself on an integrated form of a presumed parental Nf4-G2 (the second line of Fig. 4A) because the right junction (atCT) of the parental Nf4-G2 sequence, as well as the flanking host sequence (tatat) is the same as the predicted junction of the hypothetical circular form of Nfs, atCTtatat (underlined with a dotted line in Fig. 4A). Alternatively, the inversion could have occurred on the circular form of an Nf4 member. If so, the partially inverted circular form may have been integrated into the chromosome by Irg2-mediated recombination between the junction sequence ...ccCTtatatat... [Fig. 4A and 3B, (2)] and a chromosomal sequence ...acCTtatatgc... [Fig. 3B, (2) and 4A].

3.3.2. Internally deleted copies of Nf
The irg1, 4 and 8 genes are the only exceptions of the pivNM/irg homologues that are not neighboured by the homologues of phage genes at the left (Fig. 1A). The region downstream of these irg genes to atCT, the right junction of Nfs, is highly homologous to that in the other Nf4 copies [Fig. 3C, (1)]. At a variable distance upstream from the irg genes, we identified the left junction sequence of Nfs, CTtatat [Fig. 4B, (1) and (2)]. We named these regions bound by these junction sequences: Nf4-G1(del), Nf4-G4(del) and Nf4-G8(del).

Indeed, the left 33 bp sequence of Nf4-G4(del) (solid, rightward arrow) aligned well with the left end of the intact Nf4 copies (Nf4-G3 and Nf4-G5) [Fig. 4B, (1)]. Notably, the complementary strand of the sequence just upstream of irg4 aligned longer with the left end of intact Nf4 [dotted arrows in Fig. 4B, (1)]. In Nf4-G4(del), the left end of the 33 bp sequence (CTtatat...) and the 34 bp sequence upstream of irg [...atataAG; solid, leftward arrow in Nf4-G4(del) of Fig. 4B, (1)] form inverted repeats (33/34 bp match). Note that the right junction (ataAG; boxed and indicated by an inverted triangle in Fig. 4B, (1)] is the same site as the recombination sequence for partial inversion (see Section 3.3.1 and Fig. 4A).

Another putative deletion derivative, Nf4-G8(del), aligned very well with Nf4-G4(del) with a deletion [Fig. 4B, (2)]. Nf4-G1(del) aligned well with these two deletion derivatives, [Nf4-G4(del) and Nf4-G8(del)], and with the intact Nf4s (Nf4-G3 and Nf4-G5) with a deletion [Fig. 4B, (2)].

Formation of Nf4-G8(del) is readily explained by illegitimate recombination involving short direct repeats, ccct (boxed in dashed line), in an Nf4-G4(del)-type parent [Fig. 4B, (2)]. Deletions stimulated by palindromic sequences were shown to occur between short direct repeats in the laboratory.30Go,31Go

Similarly, Nf4-G1(del) would be formed through illegitimate recombination involving short direct repeats, tgcgt (boxed in dotted line), from these types of deletion derivatives [Nf4-G4(del) and Nf4-G8(del)] or from the intact Nf4 copies [Fig. 4B, (2)]. As the parent, we prefer the structure of Nf4-G4(del), or similar, with long inverted repeats, as the inverted repeats will stimulate and select this type of deletion.31Go

Formation of Nf4-G4(del) from intact Nf4 copies can be explained by the following replicative transposition model. (i) A copy of a region of the left terminus (longer than 33 bp) is inserted into the left of the ataAG sequence upstream of the transposase gene. This is a reaction that is similar to replicative transposition. The transposase may have recognized a secondary site within Nf as in one-ended transposition.32Go (ii) The resulting long inverted repeats make the entire region unstable and cause or select an illegitimate recombination event involving a short sequence identity (aaggg). This mechanism is the same as we proposed for the other two deletion derivatives. This model can provide a uniform explanation to the formation of the three deletion derivatives.

Although these three copies are probably deletion derivatives of intact Nfs, the possibility remains that they are integrated into these sites after the deletion event. Unfortunately, we do not know how much of the Nf sequence is necessary in cis for integration or whether these three copies can express a functional transposase. The deletion in Nf4-G1(del) apparently removed the first residue of the start codon of the irg gene [Fig. 4B, (2)] and resulted in the start codon of NGO0772 (irg1) to be 33 bp upstream from the left end of Nf4-G1(del).

3.4. Simple insertion revealed by comparison with another genome without Nf
Comparison of Nf1-A integrated into NmeA with the corresponding unoccupied locus of Ngo (Fig. 5A) made it clear that the target sequence of Nf1-A integration is CT. Unfortunately, with the other intact copies of Nf1, such an obvious assignment was not possible because of the variability of the dRS3 repeats.


Figure 5
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  		Figure 5. Alignment with an unoccupied integration site. A box indicates a target CT sequence. (A) Nf1-A. Nf1-A is integrated at the loop of a dRS3 repeat. The consensus of dRS3 is shown below with its inverted repeats indicated by arrows. (B) Nf4-G1(del).

 
Comparison of one of the internally deleted copies, Nf4-G1(del), in Ngo with the corresponding unoccupied locus in NmeA strongly indicated that it also targeted CT (Fig. 5B). According to the comparison between Ngo and NmeA, the target sequence of Nf4-G8(del) also seems to be CT, although the two base pairs that are 3' to CT are different from those in the corresponding unoccupied locus. For the other copies, comparison is not straight forward because of the complex structures that flank them.

3.5. Alternative models for Nf integration
In some of the above arguments (see Section 3.3), we implicitly assumed that PivNM/Irg transposase that is present on the Nf phage genome is likely to have mediated integration of the phage genome into the chromosome. In this section, we will list the alternative models and provide evidence against them.

3.5.1. Four possible models
The first model we have been pursuing (A) is that the Nf phage was integrated by its own pivNM/irg transposase (transposase-as-integrase model). Three other models are as follows: (B) Nf without pivNM/irg was integrated by some unspecified mechanism and later the pivNM/irg was inserted as IS (phage-then-IS model); (C) pivNM/irg was inserted into the Nf phage genome and later the phage genome with pivNM/irg was integrated by some unspecified mechanism unrelated to pivNM/irg (phage-with-IS model) and (D) the pivNM/irg was inserted into the chromosome as IS and later, by some unspecified mechanism, Nf without pivNM/irg was integrated between the left junction of the IS and the start codon of pivNM/irg (IS-then-phage model). An idea similar to the phage-with-IS model was presented earlier.10Go

3.5.2. Evidences for or/and against the models
Below, the transposase-as-integrase model (model A) is discussed in comparison with the other three models (models B–D).

  1. Models B–D provide no explanation as to how the phage was integrated into the chromosome. On the other hand, model A is straightforward by explaining that pivNM/irg transposase acts as the integrase for integration of Nf.
  2. Tight linkage of phage-related genes of the Nf and pivNM/irg gene is a straightforward consequence of models A and C, but is not a straightforward consequence of models B and D. The apparently solitary irg genes [Nf4-G1(del), Nf4-G4(del) and Nf4-G8(del)] can be explained by deletion from an intact copy of Nf as detailed above (see Section 3.3.2). This supports model A. These deletion events may have happened after the integration of Nf, although the possibility that the deletion derivatives were integrated as simple IS cannot be excluded (see Section 3.3.2). Thus, we cannot exclude model D from this argument, but it is difficult to explain, by model D, why Nfs without pivNM/irg specifically target the fixed point between the left junction of ISpivNM/irg and the start codon of pivNM/irg and why they are not integrated to the other loci of Neisseria genomes.
  3. Similarity of the junction sequences of Nf and IS621, the second closest homologue of pivNM/irg of Nf (see Section 3.2.5), supports the idea that both junctions of Nf were derived from the activity of PivNM/Irg and supports models A and D but not the other two models.
  4. The closest homologue of pivNM/irg is the one that is coded on pMU1. pMU1 has the pivNM/irg homologue, along with other homologues of Nf phage genes (see Section 3.1.2 above and Fig. 1C). pMU1 encodes pivNM/irg and has an atCTtatat sequence, which is exactly the predicted junction sequence for the hypothetical circular form of Nf at the corresponding locus (see Section 3.2.4 above and Fig. 3E). This form resembles an intermediate form of transposition of several IS families, such as the IS3 family.33Go,34Go Nf and pMU1, along with IS621, have the assembled promoter-like sequence (see Section 3.2.5 and Fig. 3). IS492 of the same IS110/IS492 family as pivNM/irg takes a circular form with the junction consisting of a single copy of the direct repeats and with a promoter-like sequence.35Go These points argue for the possibility that Nfs are likely to have a circular form together with pivNM/irg and that the predicted junction sequence ‘CT’ is similar to that of pMU1 and support models A and D but not the other two models.
  5. Each of the subtypes, Nf1–Nf4, carries a distinct type of pivNM/irg gene (pivNM1/irg7 for Nf1, pivNM2 for Nf2, pivNM3 for Nf3 and irg1-6, 8 for Nf4), as revealed by phylogenetic analyses (Fig. 2A compared with 2B). This means that the copies of one subtype of Nf are linked with one specific subtype of pivNM/irg and vice versa; this is compatible with models A and C. On the other hand, in models B and D, there is no obvious reason to assume that copies of the same subtype of Nf carry pivNM/irg genes of the same type.
  6. Amino acid sequences of phage-related genes of Nf2 and Nf4 are similar [Fig. 2A, (1)–(3)], but those of their transposases, pivNM2 and irg, are not similar to each other (Fig. 2B). Nucleotide sequences of the central regions of Nf2 and Nf4 are also similar but those of pivNM2 and irg are diverged (data not shown). This might support models B and D, which indicate that there are different origins for phages and pivNM/irg. However, if we assume that the pivNM/irg of each subtype was somehow inserted independently into Nf (Nf without pivNM/irg), models A and C would be supported. pJS-B and pJTPS1 have similar gene organizations to that of Nf, but do not encode the pivNM/irg homologue nor have an atCTtatat junction sequence (Fig. 1C). Independent insertion of pivNM/irg into the circular form of Nf without pivNM/irg (like pJS-B) might have occurred during evolution. The mechanism of this insertion might be different from that of Nf integration into bacterial chromosomes because Nf integration is marked by flanking CT repeats, whereas we could not find such duplication for pivNM/irg insertion into hypothetical Nf without pivNM/irg, although we cannot exclude the possibility that loss of the CT sequence was selected.

From the above observations and considerations, we concluded that the most likely mechanism is the transposase-as-integrase model (model A). The circular form of Nf before integration into (and after excision, if any, from) the chromosome would have an atCTtatat sequence at the junction and recombine with the target CT of the chromosome by the activity of the PivNM/Irg transposase. Although this reaction mechanism is similar to that of site-specific recombinase, Piv family proteins do not have amino acid motifs that are conserved among the tyrosine- or serine-recombinase families,11Go but are similar to DDE transposases.36Go

This mechanism is distinct from the two currently known mechanisms for filamentous phage integration, integration into a dif-like site mediated by XerC/D site-specific tyrosine-recombinase6Go and integration into tRNA probably mediated by an integrase of the tyrosine-recombinase family.7Go

3.6. Comparison with the work by Bille et al.12Go
After this work was completed, Bille et al.12Go published an experimental work of which some results are in agreement with those that we described above. Their results related to ours are listed below [(i)–(vi)], whereas the findings that are specific to our work are also listed [(a)–(e)].

(i) Discovery of filamentous phages (corresponding to seven intact copies of Nf1, according to our naming) in the genomes of Neisseria based on observations of their low G+C content, homology of the first ORF to bacteriophage replication proteins and the similarity to the order and size of ORFs that are encoded by other filamentous phages. (ii) Those phages (Nf1) are inserted into dRS3. (iii) Extrachromosomal circular single-stranded DNA of one prophage (Nf1-A, according to our naming) was detected by polymerase chain reaction (PCR). (iv) Junction of the circular form was sequenced. (v) The region containing the pivNM gene was contained in the circular form, as judged from the length of PCR products (supporting our result that pivNM/irg genes are integral part of Nfs). (vi) The inactivation of four genes—NMA1792, 1797 and 1799 on Nf1-A and pilQ on a locus of the host chromosome—had predicted effects on extracellular and cytoplasmic circular DNA forms.

Our findings that are not overlapping with theirs are as follows: (a) we found and characterized not only the Nf1 subtype, but also three other subtypes (Nf2, Nf3 and Nf4). (b) The left and right sequences common to all the four Nf subtypes are CTtatat and atCT, respectively. (c) Our analyses strongly indicate that the integration of Nf is mediated by its own PivNM/Irg transposase (transposase-as-integrase model). (d) We identified and characterized structural variants of Nfs. We were able to explain their origin in terms of aberrant activity of this transposase. (e) We also added further bioinformatic evidence that ORFs of Nfs correspond to proteins of filamentous phages.

Note that their work12Go further supports the transposase-as-integrase model. Our results proved the power of bioinformatic approach through genome comparison for detailed and thorough analysis of mobile elements and genome rearrangements.


   Supplementary Data
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Supplementary Data
Acknowledgements
 References
 
Supplementary data are available online at www.dnaresearch.oxfordjournals.org.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Supplementary Data
 Acknowledgements
References
 
We thank Noriko Takahashi and Takeshi Tsuru for helpful suggestions on the manuscript. This work was supported by grants from MEXT of the Japanese government to I.K. (Genome Biology, Genome Homeostasis, DNA Repair, Kiban-evolution, Kiban-genome, 21COE: genome language).


   Footnotes
 
*To whom correspondence should be addressed. Tel. +81-3-5449-5326, Fax. +81-3-5449-5422, E-mail: ikobaya{at}ims.u-tokyo.ac.jp

Communicated by Kenta Nakai


   References
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Supplementary Data
 Acknowledgements
 References
 

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C. Schoen, J. Blom, H. Claus, A. Schramm-Gluck, P. Brandt, T. Muller, A. Goesmann, B. Joseph, S. Konietzny, O. Kurzai, et al.
Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis
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