Department of Genetics, University of Stockholm, Stockholm, Sweden
Correspondence: E-mail: anders.nilsson{at}genetics.su.se.
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Abstract |
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Key Words: bacteriophage P2 enterobacteria site-specific recombination lysogenic conversion virulence factors
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Introduction |
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Until recently, Z/Fun did not show similarity to any other protein in the databases, but genome sequencing projects have revealed that this protein can be found in Neisseria meningitidis (similarly called FunZ, 531 amino acids) with over 58% identity (Tettelin et al. 2000), as well as in the Bacillus anthracis plasmid pXO2 (pXO2-71, 517 amino acids) with about 20% identity (Okinaka et al. 1999) (table 1). These organisms are not closely related to E. coli, the standard P2 host, so the distribution of this gene is a fine illustration of the view that bacteria, plasmids, and phages are all part of the same coevolving system (Hendrix et al. 2000). The high similarity of Z/Fun especially to FunZ in N. meningitidis and the fact that funZ is not located in a prophage implies additional and more general functions of this protein.
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In this paper, we report the distribution and characterization of P2-like prophages among the 72 strains in the E. coli reference collection, ECOR (Ochman and Selander 1984), and describe the large genetic variation at the position analogous to the phage P2 Z/fun locus in these phages. We also report that this variation, and corresponding variation in pathogenic bacteria and plasmids, is mediated via the same mechanism, which implies that P2-like phages can serve as a gene supply, and vector between strains, for genes affecting bacterial virulence.
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Materials and Methods |
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Bacterial Strains and DNA Extraction
All bacterial strains were grown overnight in Luria-Bertani (LB) media at 37°C. Chromosomal DNA from the ECOR strains and from the positive control, E. coli P2 lysogen strain C-117 (Bertani 1968), and the nonlysogenic E. coli negative control, strain C-1757 (Sunshine et al. 1971), was extracted with Qiagen Blood and Cell Culture DNA extraction kit. ECOR strains that had been shown to be contaminated or mixed up in some collections (Johnson et al. 2001) were acquired from an additional source.
DNA-DNA Dot-Blot Hybridization
Two milligrams of spectrophotometrically quantified DNA from each ECOR strain and from the positive and negative control strains was microfiltrated onto each of four Zeta-ProbeGT membranes with a Bio-Dot microfiltration apparatus (BioRad). The hybridization probes, specified above, were amplified directly from P2 DNA using Ready-to-Go PCR beads (Amersham Biosciences) and 18 to 26 nt oligonucleotide primers (DNA Technology, Aarhus, Denmark) designed from the nucleotide sequence of P2 (table 2). The PCR products were purified with Qiaquick PCR purification kit (Qiagen) and checked by gel electrophoresis on a 1% agarose gel, followed by staining with ethidium bromide and inspection under a UV lamp. Rediprime II random prime labeling system was used to label the probes with [-32P]dCTP (Amersham Biosciences). The hybridizations were carried out according to the protocol supplied by the manufacturer and followed by autoradiography.
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DNA Sequencing of the Z-Region, Corresponding to the P2 Z/fun locus
Sequencing of all different inserts between G and FI was initially done on PCR products, purified as described above, that were cloned into the BamHI site of pUC18. Almost all of the sequences were too long to be finished by sequencing from both ends with plasmid forward and reverse primers. Most of the remaining sequencing was done by primer walking and with PCR products as templates. Automated DNA sequencing was carried out with an ABI Prism 377 (PerkinElmer) or on an ALFexpressII (Amersham Biosciences). Additional sequencing; primer walking of inserts ECOR 30, 31, 46, 48, and 58; and contig assembly of these inserts were done by MWG-Biotech, Ebersberg, Germany. Other nucleotide contigs were assembled with ALFwin Sequence Analyzer, the GCG Fragment Assembly System (Genetics Computer Group 1999) or manually. The sequence data have been submitted to the EMBL databases under accession numbers AJ512675 to AJ512685.
Sequence Analysis
Most of the sequence analysis was accomplished with computer programs in the GCG package. Putative open reading frames (ORFs) in the inserts were found with FRAMES, translating all DNA sequences in all six frames with the bacterial translation table. Only proteins longer than 50 amino acids were considered during the search for ORFs. Promoters and ribosomal binding sites of ORFs were sought for with FINDPATTERNS. The program TESTCODE was also used to check the presence of coding genes. The program PEPTIDESORT was used for estimation of protein molecular weights and the program COMPOSITION was used for estimation of frequencies of single, di-, and trinucleotides. The expression and size of the presumed proteins were confirmed in coupled in vitro transcription/translation assays. To avoid read-trough from unwanted promoters, these were not carried out with sequences cloned into plasmids, but done with linear PCR fragments as templates. The assays were based on an E. coli S30 extract kit and performed and analyzed according to the protocol supplied by the manufacturer (Promega). MEME was used to search for conserved patterns in the proteins, but database searches for sequence motifs in the putative proteins were also done with MOTIFS, which compared the protein patterns with patterns defined in the PROSITE Dictionary of Protein Sites and Patterns.
Secondary structures in the upstream and downstream inverted repeat regions were assessed with MFOLD, REPEAT, and STEMLOOP. The insertion sequence (IS) database at http://www-is.biotoul.fr/IS.html was used to search for IS-specific patterns in inverted repeat regions, as well as within inserts. Database similarity searches of coding regions were mainly done at the European Bioinformatics Institute Web site http://www.ebi.ac.uk/fasta33/index.html with FASTX3 (Pearson et al. 1997; Pearson 2000) against the SWALL (Swissprot and translated EMBL) databases. The program translates the nucleotide sequence in all six frames before performing the search and allows frameshifts, caused by sequencing errors, between codons. Searching a database of a certain size, the resulting expect score (E score) can be interpreted as the number of times an unrelated sequence, of the same length as the query and the hit sequence, would show a higher identity just by chance. A significant match indicating a true relationship has an E score at least below 0.05, which corresponds to an expectation of finding five unrelated sequences in a 100 searches. We also report similarities in the grey zone, 0.05 to 1.0, which may contain not only weakly similar proteins but also false positives. The Blast and tBlastX programs at http://www.ncbi.nlm.nih.gov/BLAST/ were used for complementary database searches.
Phylogenetic Analysis
Prophage, bacteria, and plasmid sequences on both sides of the different inserts (i.e., both inverted repeat regions) were concatenated to 150-nt sequences and aligned with ClustalX (Thompson, Higgins, and Gibson 1994). Since there was only small variation in most parts, only a few gaps in the sequences downstream of the boxed part of IRR (fig. 1) had to be introduced. The phylogenetic relationship was analyzed with PAUP* (Swofford 1999) under maximum-parsimony criteria and with the heuristic search option. The stepwise addition of taxa was randomized 10 times per run, keeping the two shortest trees each run. All other program parameters were set to default values (e.g., no character weighting or exclusion). The degree of confidence of the resulting shortest tree for each set was tested in bootstrap analyses, each with 100 replicates. The concatenated sequences matrix was analyzed both in four divisionsthe unboxed part of IRL, the boxed part of IRL, the boxed part of IRR, and the remainderand in all the 150 nucleotide characters together.
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Results |
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Sequence Variation in the Region Analogous to the P2 Z/fun Locus, the Z-Region
PCR amplification of the region between P2 genes G and FI was only possible from 11 prophages of the 20 strains that contained P2-like phages. It was of little use to change primers; different combinations of six forward and four reverse primers were tried without success for the remaining nine prophages (table 2). The amplification of the ECOR42 prophage resulted in a fragment, but it was hard to get enough PCR products for cloning and sequencing. Only two of the 11 amplified fragments were of the same length, and no fragment was of the same length as the original P2 Z/fun locus (table 1). The subsequent DNA sequencing of the regions revealed that the large middle part of all sequences were different, with the exception of ECOR5 and 64 where the sequence variation was around 1%. The large difference between sequences made it obvious that this was no locus in the conventional meaning, but a multivariable site, which in the following will be called the Z-region.
The heterogeneous middle part of the sequences was in all cases flanked by highly similar sequences at both sides with rather sharp boundaries to the dissimilar part of the sequences (fig. 3). Brought together, the flanking sequences can form a complex single-stranded DNA structure dominated by a long, imperfect inverted repeat sequence (fig. 4). There was also two more inverted repeats within this long inverted repeat and many short, direct repeats spread all over the structure. At the 5' end there was a stemloop containing the stop codon of gene G, and at the 3' end were several inverted repeats forming three stemloops including the promoter regions and the ribosomal binding site of the FI gene (fig. 4).
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All putative proteins in table 1 were subjected to searches for known amino acid patterns that could suggest a function, but there was no clear indication of any such motif or pattern in any sequence.
The noncoding sequence of the insert in phage P2 included the left end part of an insertion sequence, IS630, starting 53 nt from the Z/fun stop codon and ending 11 nt from the left inverted repeat (IRL). The partial IS was also found in many other enterobacteria, the most similar sequence was the enterohaemorrhagic E. coli (EHEC) O157:H7 Sakai strain (Hayashi et al. 2001), which showed 64% identity in a 255 nt overlap. The P2 insert also contained the start of a transposase gene, with the first 12 amino acids being 75% identical to the Z4330 gene in O-island #122 of the other sequenced EHEC, O157:H7 EDL933 (Perna et al. 2001). However, in the P2 insert, the gene is terminated by a single base pair deletion frameshift after 20 amino acids. There was also an 87-nt fragment of a gene similar to the E. coli TnpA transposase gene between ORFs in the ECOR46 prophage insert, but with a translational frameshift after the first 11 amino acids.
Analysis of Inverted Repeats
The inverted repeats on each side of the inserts (IRL and IRR) were quite conserved in all prophages (over 80% identity), but the sequence variation increased slightly at the border to the inserts. Three prophages had a 12-bp deletion in the stemloop in IRL, and the ECOR45 prophage lacked the 5' part of IRR (fig. 3). The two longest inserts, in ECOR 46 and 48, had additional imperfect IRs within noncoding parts of the inserts. ECOR46 had an extra IRL in between orf 8 and orf 9, and ECOR48 in between orf 11 and orf 12, in both cases dividing ORFs on different coding strands (table 1). Searches, with the prophage consensus IRs as query sequences, in databases did not indicate that any part of the IR sequences were part of IS-elements or carried integron signatures. The IRs of the Z-region was however found in bacteria, and extended searches revealed that it was confined to E. coli and Salmonella chromosomes, and to enterobacterial plasmids (fig. 1). The inverted repeats in bacteria were in most cases more similar to the 33-nt ends (boxed in figure 1) of the prophage IRs than to the beginning. Most of these bacterial IRs were located between genes with unknown function and obscure origin, but several of them were located either in pathogenicity-associated islands (PAIs) or other regions containing genes encoding virulence factor genes.
We found different inserts surrounded by the same IRs not only in the uropathogenic E. coli CFT073 PAI II (Rasko et al. 2001), the O-island #172 of E. coli O157:H7 EDL933, and the multiple-drug-resistant Salmonella Typhi CT18 but also in E. coli K-12 MG1655 (Blattner et al. 1997) and Salmonella enterica serovar Typhimurium LT2 (McClelland et al. 2001) (table 1). The sequences outside the IRs were different in most cases. One of the two Z-regions in Salmonella Typhi CT18 (in AL627272) and the Z-region in Salmonella Typhimurium LT2 were clearly located in the same sequence context, but CT18 had an extra gene immediately upstream of the region. The largest insert of all (>8 kb) was found within similar IRs in the Proteus vulgaris conjugative plasmid Rts1 (Murata et al. 2002) (table 1). Half of the plasmid genome consists of a large duplicated segment, M1a and M1b. The insert was present only in M1a, which means that it most likely became inserted after the duplication took place.
The search for IRs resulted for some genomes in the discovery of only one of the IRs. There was a 70% identical 66-nt IRL in a 450-nt sequenced fragment of the Klebsiella pneumoniae plasmid SL038 (accession number AJ276856) and a 65% identical IRL, of the same size, in Shigella flexneri virulence plasmid WR100 (accession number AL391753 [Buchrieser et al. 2000]). The left IR was also found in EHEC O157:H7, the Sakai strain, in a region containing the same genes as EDL933. The start of the gene following the IRL, coding for the first 31 amino acids, was missing in the Sakai strain as compared with EDL933, which implied that the beginning of the gene and a right IR may have been deleted at the same time.
Particular attention was paid to examining the sequences surrounding the funZ gene in Neisseria meningitidis to find the same conserved inverted repeat motifs, but here the 33-bp part of IRs (boxed in figure 1) closest to the insert were missing, and instead there was 68% identity to a 32-bp sequence immediately adjacent to the boxed part of both IRs. However, there was also a 92% identical 155-bp end of the ISNme1, a variant of IS1106 in the IS5 family, only 159 bp upstream of the end of funZ. Prophages Fels2 and SopE both contain genes at a position corresponding to the P2 Z/fun gene, but these genes appear to have been inserted by some other mechanism since we did not find IRs surrounding any of them.
The phylogenetic analysis of the relationship between all concatenated IR sequences showed that prophages are more related to each other than to bacteria, even if the bootstrap analysis resulted in poor support for many nodes (fig. 6). The trees generated in separate analyses of parts of the alignment had even lower bootstrap values but did not indicate a different evolutionary history for any part. The number of homoplasious characters (e.g., nucleotide characters that spoke for an alternate tree) was about the same in all parts of the alignment. The homoplasy was also comparable to what has been shown in a phylogenetic analysis of the late genes in different P2-like phages, where it was concluded to be caused by homologous recombination (Nilsson and Haggård-Ljungquist 2001).
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Discussion |
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There are numerous ways to incorporate new DNA into a recipient chromosome, and many systems are based on identity between donor and recipient DNA. The appearance of the Z-region in the P2-like prophages, as can be seen in the alignment (fig. 3), is that extended and highly similar sequences abruptly change into completely unlike sequences with different AT-content. Although homologous recombination is known to occur between bacterial chromosomes, including prophage sequences, it is more likely that the observed insertions are the result of site-specific recombination events. At least two observations support this notion. The IRs are more conserved closer to the inserts (the boxed part in figure 5), and in a hypothetical empty site, there would be an approximately 200-nt sequence suitable for homologous recombination between two IRs, but the recombination leading to the insertion of foreign genes in the Z-region has always happened at a specific point. There are many systems able to perform site-specific recombination. It can be carried out by recombinases supplied either by transposable elements, plasmids, phages, or bacteria.
The Z-regions found in bacteria either contain, or are frequently located in the vicinity of, sequences encoding transposase genes. This may indicate that this class of recombinases causes the movement of the inserts and that the observed IRs and inserts constitute transposons. However, we believe that this association is circumstantial and depends on the nature of the target sequences. There are also many other arguments against this hypothesis. Most transposases act in cis and must accordingly have been deleted from the majority of inserts. IS elements and transposons form classes characterized by similar IRs at the ends. They generally show a preference for an insertion target sequence and generate flanking direct repeats (DRs) when they integrate. Although some transposable elements insert randomly, we cannot find any class that fit our observations. The recognized classes are generally also spread over many species, or even genera, but we have not found any site outside the enterobacterial domain. There are over 200 flanking nucleotides that are part of IRs in the Z-region of the prophages. These flanking sequences are well conserved in the P2-like prophages as well as in enterobacteria, so it is difficult to assume that they are selectively neutral. In addition, the phage head can only package a limited amount of DNA, so junk DNA would consequently be deleted in the long run in favor of more useful genes. The strongest argument against a transposase-mediated transfer is maybe the degree of vertical inheritance or clonality of the IRs of the Z-region as revealed by the phylogenetic analysis. Clonality is not expected if the region is part of a transposable element. IRs are part of the transposable element and get inserted along with it. Vertical inheritance of the region implies that the IRs of the Z-region got inserted only once and that subsequent copies of the region have acquired mutations and followed their hosts as they have branched off into separate clones. If the IRs are part of a transposable element, it becomes hard to explain the totally different sequences within the inserts. There is no relationship between them, and they cannot possibly have originated from a common ancestor. It is more likely that they would have inserted into different clones at different times in such a case, and the distribution of the mutations in the IRs would suggest lateral transfer. It would also be likely to find some similar sequences within the inserts if the IRs are part of a transposable element.
In the phylogenetic analysis, the bootstrap value for the branch leading to prophages is somewhat low (67%), but the analysis indicates that the IRs of the Z-region are of monophyletic origin and have not been recently transferred between prophages and bacteria. The placing of the region may well have happened a long time ago, and together with the assumption that unnecessary sequences are trimmed away, it implies that the entire flanking regions are needed, possibly as recognition sites in still another site-specific recombination system. The site contains enough similar neighboring sequences, and the sequences downstream of the insertion point have additional direct and inverted repeats, to meet the general prerequisite for systems used by many site-specific recombinases other than transposases. The recombination occurs between two identical, or nearly identical, attachment sites, att, and it is common that several molecules of a recombinase need to bind to these sites, and in more complex systems, also to upstream and downstream arm regions in the donor DNA. In many cases, a complete recombination reaction cannot come about without additional factors, which also bind more or less close to the att site of the donor DNA. We believe that the most conserved part of the two observed inverted repeats, the boxed part of IRL and IRR in figure 5, corresponds to the attL and attR formed as a result of a site-specific recombination event between the cores of two att sites, often designated attP and attB. Many observations at the Z-region are consistent with this hypothesis. There are additional IRs within the att site, which is a common characteristic of the core region of phage integrase attP sites (Campbell 1992) (fig. 4). There are also extra att sites between ORFs in ECOR46 and 48 that are easily explained by a secondary integration using either attL or attR as integration site. Recombination between two flanking attL and attR sites can also generate an inversion of the inserted region. Although the most conserved 33-bp part of IRL and IRR should be similar to the core att site, it is impossible to determine the exact position. In most cases, the presumed att core site of IRs of prophages consists of an 8-nt inverted repeat on each side of an 8-nt overlap region (fig. 4), but the IR of the core site of ECOR58 is extended to 13 nt (fig. 1). Apparently, there is some sequence variation, and the size and position of the site may have to be adjusted when more data on this system becomes available. Finding an att site without insert would help and may point at a specific class of recombinases.
There is no lack of candidate recombinases. There is for instance probably one, a recombinase similar to XerC, present in the E.coli CFT078 insert (table 1). Also, there are presumably many capable recombinase systems in enterobacterial hosts and probably over a dozen distinctly different temperate phage families, each with numerous phage variants, carrying integrases that could do the job. Most of them have unknown att sites and integration mechanisms. These two examples, either a stationary recombinase supplied by the host or an invading extrachromosomal element, may represent different ideas about the recombination system.
Due to the nature and complexity of bacterial evolution, it is difficult to assess the importance of this mechanism. The evolutionary time-space is full of possible vectors and horizontal transfer mechanisms, and the mechanism presented here is maybe just another one. Phages play an important part in the evolution of bacteria and can contribute with virulence factors (Miao and Miller 1999). Similar lysogenic conversion genes have also been found in prophages of unrelated commensal and pathogenic bacteria (Brüssow and Hendrix 2002). The mechanism, presented here, used for the addition of lysogenic conversion genes, appears to be a more controlled and specific mechanism than the sometimes random insertion of transposable elements or homologous recombination of horizontally transferred genes. The similarity between a Z-region in a P2-like phage and a Z-region in a bacterium means that P2-like phages can act as vectors for virulence factors. The region is not a strict lysogenic conversion locus since a bacterium can pick up a gene through site-specific recombination even without having to integrate a whole phage genome. A homologous recombination event between IRs of two Z-regions located in phages, plasmids, and/or bacteria is probably also possible with regular systems, for example, the Red system, which only requires short homologous sequences (Poteete 2001).
The importance of this site-specific recombination system is also dependent of how widespread the Z-region is. There is a sequence bias for pathogens in the large bacterial genome projects, and it is therefore possible that this site-specific recombination system is more of a general lysogenic conversion system rather than aimed at pathogenic enterobacteria. The IRs of the Z-region is also present in nonpathogens, such as the slightly different IRs in E. coli K-12, but always present in genetically unstable regions. This question and questions about the age and evolution of the site, the effectiveness and frequency of insertion, the enzymes necessary for recombination and other factors needed, and the function of the inserted genes, will have to be addressed in future investigations.
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Acknowledgements |
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Footnotes |
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