1 Institut für Medizinische Mikrobiologie und Hygiene der TU Dresden, Germany
2 Institut für Hygiene und Mikrobiologie der Bayerischen Julius Maximilians Universität Würzburg, Germany
3 Lehrstuhl für Lebensmittelchemie der Bayerischen Julius Maximilians Universität Würzburg, Germany
Correspondence
Herbert Schmidt
hschmidt{at}uni-hohenheim.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AJ304858 (phage CP-1639 and chromosomal integration site) and AJ831374 (integrative element of E. coli O103 : H2 strain 2905/96).
Present address: Department of Food Microbiology, Institute of Food Technology, University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany.
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INTRODUCTION |
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In this study, we investigated the genetic structure and chromosomal integration site of the Stx1-converting prophage CP-1639. PCR analysis was performed to study the distribution of this integration site among E. coli strains of serogroups O26, O103, O111, O128, O145 and O157.
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METHODS |
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PCR.
Amplification was carried out in a total volume of 50 µl containing 200 µM of each deoxynucleoside triphosphate, 30 pmol of each primer, 5 µl of 10-fold-concentrated polymerase synthesis buffer, 1·5 M MgCl2 and 2·5 U AmpliTaq DNA polymerase (Applied Biosystems). Oligonucleotides were designed with OLIGO 4.0 (National Biosciences), and purchased from Sigma-ARK and TibMolBiol. The first step of the PCR consisted of 5 min at 94 °C for denaturation of DNA, followed by 30 cycles each with denaturing (94 °C; 30 s), annealing (for temperature see Table 1; 60 s) and extension (72 °C; for time-course see Table 1
) steps. A final extension step was carried out for 5 min at 72 °C. For a detailed description of primer sequences and PCR conditions see Table 1
.
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DNA sequencing and sequence analysis.
Nucleotide sequencing was performed on both strands with an automated DNA sequencer (model 377; Applied Biosystems) initially by using universal-forward (5'-ACG ACG TTG TAA AAC GAC GGC CAG-3') and universal-reverse primers (5'-TTC ACA CAG GAA ACA GCT ATG ACC-3') for pK18, and super fwd (5'-GCA TTT ATC AGG GTT ATT GTC-3') and super rev primers (5'-GGA AGT CAA CAA AAA GCA GAG-3') for SuperCosI and then subsequently using customized primers. Each nucleotide was determined at least twice. Contigs were aligned with PREGAP 4, GAP 4 (Staden & MacLachlan, 1982) and BIOEDIT (Hall, 2004
). ORF analysis was performed with EDITSEQ and MAPDRAW (DNASTAR, V.08) using ATG and GTG as start codons and the GENEMARK.HMM software (Lukashin & Borodovsky, 1998
). Results of both programs were compared with BLAST analyses (National Center for Biotechnology Information, NCBI, Bethesda, USA), and ORFs with concordance in at least two of the applications applied are listed in the database entries AJ304858 and AJ831374.
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RESULTS |
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Although a bacteriophage could be induced and isolated from E. coli strain 1639/77, this was not a Stx-converting phage, indicating that the suspected Stx1 phage is defective. For characterization of the genetic background of the stx1 gene, we sequenced its flanking regions. To achieve this, a genomic library was created from E. coli O111 : H strain 1639/77 comprising 1200 cosmid clones. From eight of these clones we found PCR products after amplification with stxA1- and stxB1-specific primers, and four of these were processed further. These clones were digested with EcoRI and HindIII, and restriction fragments were subcloned in pK18. Using these subclones and the original recombinant cosmids as templates for sequencing reactions, we could determine a sequence of 50 625 bp of continuous chromosomal DNA of E. coli 1639/77 DNA, which flanks stx1 on both sides. Sequence analysis of this DNA region with MAPDRAW, GENEMARK.HMM and NCBI BLAST revealed 70 ORFs (Fig. 1, GenBank accession no. AJ304858). Further ORF analysis demonstrated that stx1 is obviously located in a defective prophage genome, which has been designated CP-1639. The genetic organization of the sequenced region is depicted in Fig. 1
.
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ORF 9 is related to ORFB of IS3 and is followed by an intact IS629 element covering base pair positions 78329141 (Fig. 1). The sequence is identical to a published IS629 element (Matsutani & Ohtsubo, 1990
). Subsequently, we found six phage-related ORFs (1217), four of which appeared to be related to the corresponding region of phage P22-related phages Sf6 and HK022 (Casjens et al., 2004
). ORFs 18 and 19 represent genes of a defective IS629 element, which is truncated by 423 bp at its 3'-end.
The area starting with cII (ORF 20) and ending with a ninH-like gene (ORF 32) is similar to the corresponding region of Stx2-encoding phage BP-933W. Interestingly, the stop codon of ninG and the start codon of ninH overlap as described for other Stx and lambdoid phages (Karch et al., 1999). Analysis of ORFs 30 and 31, both with homology to different regions of the roi gene, indicates that two truncated roi-like ORFs are present in CP-1639.
The next region to consider is the Q-stx-lysis region. This region includes the antiterminator gene Q and a lysis-associated gene rZ, and is shorter than comparable regions in other Stx phages (Fig. 1). We did not find S and R homologues in this region. The Stx1A and B subunit genes of CP-1639 are identical to published stx1 genes.
ORFs 4062 are related to DNA packaging and head and tail morphology. These genes are highly homologous to the corresponding genes of other Stx phages. However, we could not identify a gene specifying a capsid protein. The order of tail fibre genes from gene Z to J is also found in the same direction in the cryptic phage CP-933U of E. coli O157 : H7 strain EDL933 (Perna et al., 2001) and phage
. The expected tail fibre gene U was not present in an intact form. It appears to be disrupted by a complete IS629 element (Fig. 1
). This region contains also a virulence-related ORF, encoding a lom-like outer-membrane protein precursor.
The last gene of the prophage genome was ORF 64, which also occurs in a similar form in Stx1 phage CP-933V (Perna et al., 2001). This gene marks the junction of phage and chromosome, the latter of which starts with the ssrA gene. ORFs 6570 are homologues to chromosomal genes which are present in E. coli K-12 (Blattner et al., 1997
).
Characterization of the chromosomal integration site of CP-1639 and identification of an integrative element
The particular structure of the left end of the prophage genome (Fig. 1) and the presence of genes of unknown origin (ORFs 5, 6 and 7) between phage CP-1639 and the chromosome impeded the determination of the phage attachment site and thereby the length of the phage. In our initial investigations, we hypothesized that the intA gene (ORF 8) belonged to CP-1639. However, the presence of an int gene at the corresponding site in E. coli K-12, the homology to CP4-57 integrases and the orientation of int transcription suggest that the genes of unknown origin (ORF 5, 6 and 7) and the truncated intA belong together and form a separate genetic unit, hereafter designated an integrative element.
If this hypothesis is true, we should be able to find E. coli strains harbouring such an integrative element consisting of ORFs 5, 6, 7 and intA. Moreover, it should be located close to ssrA without being interrupted by a prophage genome. We examined this hypothesis by PCR using primers for the detection of CP-1639 and this particular genetic element as depicted in Fig. 2. As templates for PCR we chose a number of STEC of different serotypes as well as Stx-negative E. coli (Table 2
). The presence of the integrative element was assumed when Int 2, cuo, Int 3, Int 4 and Int 6 PCR reactions were positive, and Int 1 was negative (Fig. 2
).
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Nineteen strains of serotypes O103, O26 and O128 showed an insertion of foreign DNA in this region (Int 1-PCR negative). In addition, all these strains were Int 5 negative, and 17 out of 19 strains were Int 6 positive (Table 2). This gene order indicates the presence of an integrative element which is not further associated with a phage-like structure. Here, the intA gene appeared to be close to ssrA. Two O103 strains were Int 5 and Int 6 negative. This could mean that the integrative element is associated with a further prophage which did not contain an IS629 element at the corresponding site.
Some strains showed varying PCR results, with one or more PCR reactions, which did not fit the general pattern. Either there were sequence variations resulting in a lack of primer-binding sites, fragments of the suggested genes were missing or the presence of IS-elements caused enlarged PCR products. Int 1 PCR gave no evidence for the presence of foreign DNA between b2657 and ygaR in eight E. coli O157 : H7 and O145 : H strains, suggesting the same genetic structure as in E. coli K-12-strain MG1655 in this region. In three E. coli O157 : H strains, Int 1 and Int 4 were positive, indicating the presence of a short piece of foreign DNA related to ORF 7 and intA.
To investigate the sequence of the integrative element, we analysed the region between b2657 and ssrA in E. coli O103 : H2 strain 2905/96 (GenBank AJ831374). This showed that ORFs 5, 6 and 7 are present with sequence similarities to E. coli strain 1639/77 of >99 %, and a truncated intA identical to the E. coli 1639/77 intA gene was also identified (Fig. 2c). A short DNA region of 814 bp containing gene fragments with homology to an IS3-related gene and a Salmonella integrase was found between intA and ssrA. This suggests that this region was subject to intensive recombination. This region ends up with the 3'-end of ssrA. No intact phage sequences were found.
At the right side, the prophage sequence ends with ORF 64. ORF 65 represents the ssrA gene, encoding a small, stable tmRNA. The tmRNA is well known as an integration site for phages and other integrative elements, and serves also as an integration site for CP-1639. Williams (2003) analysed a number of enterobacterial integrative elements which have been inserted at the 3'-end of the tmRNA gene ssrA. We compared the rightward CP-1639 sequence including its junction to the bacterial chromosome with the corresponding sequences of E. coli O157 : H7 strain EDL933, E. coli K-12 strain MG1655, E. coli O103 : H2 strain 2905/96 and Salmonella enterica serovar Typhimurium strain LT2, according to an alignment published by Williams (2003)
. Seven-basepair crossover segments found in attachment sites were identified at the 3'-end of ssrA, which are present in all E. coli sequences but not in S. enterica serovar Typhimurium strain LT2. Downstream of the attachment sites, factor-independent terminators were characterized by Williams (2003)
. These sequences could also be identified in E. coli strains 1639/77, EDL933, MG1655, 2905/96 and S. enterica serovar Typhimurium LT2. From this alignment it can be concluded that the rightward attachment site of E. coli 1639/77 is identical to that of E. coli O157 : H7 strain EDL933 and is located at basepair positions 46 68646 870.
Comparison of the integration region in different E. coli strains
To get more information on the site of CP-1639 integration, we compared this site in E. coli strain 1639/77 with published sequences of the corresponding regions of E. coli K-12 strain MG1655 (NC_000913) and E. coli O157 : H7 strain EDL933 (NC_002655). The prophage CP-1639 is inserted between the E. coli gene b2657 and the 3'-end of ssrA as described above. In E. coli K-12 strain MG1655, b2657 is located at position 2 785 000 and ssrA at 2 752 773. There is a region of about 32 kb which is not present in E. coli 1639/77. The ssrA gene is associated with a CP4-57 integrase, followed by a number of CP4-57-related genes. A number of genes designated yfi fills the space between the remnant of CP4-57 and ypiA. These genes are neither present in strain 1639/77 nor in strain EDL933. The function and origin of the yfi genes is not known. In E. coli O157 : H7 strain EDL933, O-island #108 (CP-933Y) is close to ssrA, encompassing a region of approximately 21·5 kb, containing phage-related genes. At the other side, ypiA is the first chromosomal gene, similar to E. coli K-12 but different from E. coli O111 : H 1639/77, where b2657 is the first chromosomal gene.
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DISCUSSION |
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STEC phages belong to the group of lambdoid phages, which displays a modular structure and a large extent of mosaicism (Campbell, 1994; Koch et al., 2003
; Makino et al., 1999
; Muniesa et al., 2000
; Neely & Friedman, 1998
; Plunkett et al., 1999
; Sato et al., 2003a
, b
). Although a number of Stx phages have genomes different from E. coli O157 : H7 phages BP-933W and H19J, the mode of stx expression seems to be uniquely phage growth-cycle-dependent (Neely & Friedman, 1998
; Unkmeir & Schmidt, 2000
; Wagner & Waldor, 2002
).
It has been speculated that stx in some strains is expressed from a chromosomal locus, since Stx phages could not be isolated (O'Brien & Holmes, 1987). The current point of view is that it is more likely that stx is expressed from defective prophages in these cases. This seems also to be true for CP-1639, which can not be induced with standard methods. On a closer look on the molecular structure of CP-1639, it seems to be immobilized due to the lack of important phage genes.
The determination of the length of the CP-1639 prophage genome is difficult since the left prophage end could not be identified with certainty. Due to rearrangements and deletions in the recombination region, we can only speculate about the extent of CP-1639. However, the DNA region between the putative CP4-57 integrase remnant and the chromosomal ssrA represents most conclusively the cryptic prophage and encompasses 39 445 bp.
The presence of two IS629 elements at the left side and a number of genes which seemed to be merged together from other phages let us suggest that this is a region of intensive recombination, which probably has caused a deletion of integrase and excisionase genes. The similarity of ORFs 1217 to Salmonella phage P22 genes and the presence of ant indicate a connection between CP-1639 and Salmonella phages. Although single genes which are similar to the recombination and immunity region of Salmonella phage P22 are present, it seems that important recombination and immunity genes are missing. By inspection of the region 3' of stx, only an Rz gene is present, which putatively encodes an endopeptidase. Moreover, no holin genes could be identified, and a gene encoding a putative head protein is absent also. The tail fibre components Z to J follow the gene order observed in phage and are most similar to the corresponding genes of E. coli O157 : H7 phage CP-933U (Perna et al., 2001
). All in all, three IS629 elements have been found in the CP-1639. Since at least two of these elements appeared functional, downstream effects can be envisaged that alter host transcription. The stx gene of S. dysenteriae type 1 is also encoded in a phage remnant. Here, the stx operon is flanked by a number of insertion elements and only a small number of phage genes remained (McDonough et al., 1999
) so that the prophage character of this element is archetypal. On the basis of sequence analysis, CP-1639 is considered to be a victim of a single or multiple recombination events, that left behind a handicapped mosaic phage genome.
The ssrA gene has been shown to be an integration site for a number of integrative elements. ssrA encodes a tmRNA, a molecule that possess tRNA and mRNA character. With its tRNA function it can transfer an attached amino acid residue to a growing protein in the ribosome, although this is not anticodon-mediated. With its mRNA properties it has a role in rescuing ribosomes from malfunction (Williams, 2003). In a recent review, Williams (2003)
demonstrated that the ssrA site is occupied in different enterobacteriaceae with a number of different integrative elements which contain ssrA-specific integrases. One group is the P4 integrase family, and the truncated intA gene identified in E. coli 1639/77 belongs to this family. Since we have found an integrative element without associated Stx phages in a number of strains, this may be considered as an ancient integrative-element, which has served as integration site for CP-1639. The mechanism of insertion and deletion of important functions of the prophage can not be deduced clearly. However, arrays of such integrative elements have been reported to occur at ssrA. Particular integrase subfamilies can trigger integration at ssrA and may leave a reconstituted ssrA site, which can serve as a convenient integration site for a further integrative element. If this occurred in E. coli 1639/77, the tandem array was probably due to recombination of these arrayed elements and subsequent deletion events may have taken place. This could be the reason for that particular gene arrangement found in CP-1639.
The results of this study clearly show that the E. coli genome contains highly variable capturing sites that may be used by mobile genetic elements for stable entry in the chromosome of pathogens. Insertion, mutation and deletion events obviously led to a defective Stx-prophage. These occurrences can be considered as pathoadaptive mutations. It is not known what benefit the cell derives from immobilization of stx. It is not likely that plasmids, transposons or temperate phages are purely parasitic and can be maintained in the chromosome without paying for their dinner. The current view is that elements that are not purely parasitic bear genes that may be beneficial for the host cells, at least in certain cases (Levin & Bergstrom, 2000). Further work on the ecology and physiology of Stx phages and their hosts is needed to gain a better understanding of the mechanisms and evolutionary forces that extend the genetic spectrum of E. coli and other bacterial pathogens.
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ACKNOWLEDGEMENTS |
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Received 17 September 2004;
revised 13 December 2004;
accepted 16 December 2004.
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