Klinische Forschergruppe, Zentrum Biochemie und Zentrum Kinderheilkunde, OE 6711, Medizinische Hochschule Hannover,Carl-Neuberg-Str. 1, D-30623 Hannover, Germany1
Author for correspondence: Burkhard Tümmler. Tel: +49 511 5322920. Fax: +49 511 5326723. e-mail: tuemmler.burkhard{at}mh-hannover.de
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ABSTRACT |
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Keywords: cystic fibrosis, genome evolution, Pseudomonas aeruginosa, recombination, tRNA gene
Abbreviations: CF, cystic fibrosis
The GenBank accession numbers for the sequences reported in this paper are AF285416AF285426.
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INTRODUCTION |
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Current knowledge about genome diversity results mostly from comparative mapping and sequencing of selected isolates (Casjens, 1998 ) or propagation of bacterial populations in defined laboratory environments (Papadopoulos et al., 1999
). These approaches inherently provide only indirect evidence of how microbial genomes evolve in complex natural habitats. For the past 15 years we have collected P. aeruginosa airway isolates taken from patients with cystic fibrosis (CF) since the onset of colonization (Römling et al., 1994
). The P. aeruginosa infections in CF are a paradigm of how versatile environmental bacteria can conquer, adapt and persist in an atypical habitat, and successfully evade defence mechanisms and chemotherapy in a susceptible host (Tümmler & Kiewitz, 1999
). This strain collection provides a unique opportunity to monitor genome evolution. Here we report on the aetiology of genome rearrangements in sequential isolates from a patient with CF who has been and still is harbouring the most prevalent P. aeruginosa clones C and K (Römling et al., 1995
). A large plasmid was found to integrate and mobilize repetitively from an attB site located within two separate copies of a lysine tRNA gene. Both tRNA loci are located in hypervariable regions: one copy is located adjacent to phnAB (Heuer et al., 1998a
, b
) and the other is adjacent to pilA (Römling et al., 1995
).
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METHODS |
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Restriction mapping.
P. aeruginosa bacteria (5x109 P. aeruginosa cells ml-1), grown to late exponential phase were encapsulated into agarose plugs, lysed extensively with detergents and proteinase K, and the intact chromosomes cleaved with SpeI or I-CeuI as described previously (Römling et al., 1997 ). In one-dimensional PFGE, the SpeI digests were separated in a Bio-Rad CHEF-DRIII cell using three linear ramps of 560 s (17 h), 1025 s (14 h) and 15 s (6 h) (E=6 V cm-1, 13 °C, 1% agarose gel with 10 µM thiourea in 0·5x TBE buffer).
For two-dimensional macrorestriction mapping (see Schmidt et al., 1996 ) the genome was completely digested with I-CeuI and separated in the first dimension in a Bio-Rad CHEF-DRIII cell (E=1·3 V cm-1, one linear ramp of 4001000 s in 1 s increments, 96 h, 13 °C, 0·6% agarose gel with 10 µM thio-urea). The entire lane of each strain was cut out, digested with SpeI and separated perpendicular to the first run in the second dimension using three linear ramps from 1050 s (24 h), 1025 s (22 h) and 110 s (14 h) (E=6 V cm-1, 13 °C, 1·5% agarose gel with 10 µM thiourea).
For high-resolution Smith/Birnstiel mapping, SpeI complete/n partial double digestions (n: EcoRI, BglII, XhoI, NdeI) were carried out, the fragments separated by PFGE and hybridized with cloned SpeI fragment ends as described previously (Heuer et al., 1998b ).
SpeI maps of strains K, K1 and K2 covering the toxAhemA region were reconstructed by partial SpeI digestions of agarose-embedded chromosomes (1x1010 P. aeruginosa cells ml-1) using 0·025 U, 0·01 U or 0·005 U SpeI (New England Biolabs) for 1 h. The partial SpeI digests were separated in a Bio-Rad CHEF-DRIII cell with three linear ramps of 5100 s (30 h), 1040 s (10 h) and 70100 s (10 h) (E=6 V cm-1, 13 °C, 1·5% agarose gel with 10 µM thiourea) and transferred onto nylon membranes by capillary blotting. Southern hybridizations were performed with PCR-amplified PAO sequences (http://www.pathogenesis.com/), cloned genes (Schmidt et al., 1996 ), cloned SpeI fragment ends (Heuer et al., 1998a
) and SpeI linking clones (Römling & Tümmler, 1993
). Probes were labelled with digoxigenin-dUTP and hybridized with the pulsed-field blot as described previously (Schmidt et al., 1996
). Hybridized fragments were detected by chemiluminescence using an alkaline phosphatase-conjugated anti-digoxigenin antibody and CDP-Star (Tropix) as substrate.
Isolation and analysis of P. aeruginosa plasmids pKLC102 and pKLK106.
High-molecular-mass plasmids were prepared on a large scale by modified alkaline lysis. Plasmid size was determined by adding up the sizes of restriction fragments after conventional or PFGE gel separation of BamHI or SpeI digests, respectively. A library of partially BamHI-restricted pKLC102 was maintained as pLAFR3-derived cosmids (Staskawicz et al., 1987 ) in Escherichia coli DH5
. For cosmid ordering, about 60 clones were digested with BamHI to completion and manually assembled by restriction fingerprint analysis. The fragment assembly was confirmed by Southern hybridization where selected cosmids, single BamHI fragments (e.g. BmQ) or the small SpeI fragment SpAQ served as probes.
PCR amplification and sequencing.
Chromosomal DNA was prepared using a rapid method for Gram-negative bacteria (Chen & Kuo, 1993 ). PCR was performed from purified DNA as described previously (Spangenberg et al., 1995
). Primer sequences for PCR were designed to anneal to the PAO genome sequence between phnAB and oprL, the PAO sequence upstream of the pilA locus and the BamHI fragment sequence BmQ of pKLC102. The sequences are shown in Table 1
. Selected PCR products were sequenced in both directions.
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Sequence data.
Novel nucleotide sequences were deposited in GenBank. The accession numbers are AF285416AF285426.
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RESULTS |
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SpeI fragment patterns of patient 11s clone K sequential isolates changed within the first 3 years of colonization (Fig. 1). The basis for these shifts of fragment size was not that these were nucleotide substitutions in SpeI recognition sites, but gross genome rearrangements, as became evident from two-dimensional PFGE of complete I-CeuI/SpeI digestions of clone K, K1 and K2 chromosomes (Fig. 2
). The intron-encoded endonuclease I-CeuI cleaves within the four ribosomal operons of P. aeruginosa (Liu et al., 1993
). The similarity of the SpeI fragment patterns reflects the conservation of most fragment sizes in the four I-CeuI fragments CeA, CeB, CeC and CeD, but a few SpeI fragments on CeA and CeD were unique for strains K, K1 and K2, and a 29 kb fragment was either located in CeA (K) or in CeD (K1 and K2) (Fig. 2
). Two of these unique fragments (strain K: 46 kb; strains K1 and K2: 169 kb; Fig. 2
) were found to hybridize with strain PAO fragment SpU. We knew from previous Southern hybridizations that the phnAB hypervariable region, which maps to SpU in strain PAO, was affected by the complex genome rearrangements in the sequential clone K isolates (Heuer et al., 1998a
, b
). The 1·5 Mb region flanking this hypervariable region was mapped by Southern hybridization of SpeI complete and partial digests of strain K, K1 and K2 chromosomes with PAO-derived gene probes (Fig. 3
). The global gene contig turned out to be conserved between PAO and the clone K strains as had been expected from the comparative two-dimensional analysis (see Fig. 2
). The SpeI fragment contig and sizes were identical within experimental error for the adjacent four fragments towards the terminus of replication, but were divergent towards the origin of replication (Fig. 3
).
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The recombination site of pKLC102 was mapped with cosmid-derived probes to fragment BmQ (Fig. 5b). To identify the plasmidchromosome junction and putative targeting signals, the BmQ fragment was sequenced and searched for homologies in the completed PAO genomic sequence. Perfect matches of BmQ with PAO sequence were found in the hypervariable pilA (SpE) and phnABoprL regions (SpU/SpF). A 45 bp sequence of BmQ was identical to the 3' region of a tRNALys gene of which single copies are encoded on SpE and SpF. The 3' ends of tRNA genes are typical targets for the integration of phages (att sites) (Campbell, 1996
) and, correspondingly, we have designated the 45 terminal nucleotides of the tRNA gene plus an additional T as the attB site. The perfect match of chromosomal attB and plasmid attP sequences is shown in Fig. 6(a)
. The attB site in the BmQ fragment is followed by 295 bp non-coding sequence and a 1356 bp large ORF that encodes an integrase (int; GenBank accession no. AF285416) showing highest homology to the integrase of Actinobacillus actinomycetemcomitans (AF006830). An attspacerint contig is typical for phage attachment sites (Campbell, 1996
), but unusual for a plasmid sequence.
Reversible chromosomal integration of plasmid at tRNALys loci
Combinatorial PCR was applied to test the hypothesis that the 3' sequence of the tRNALys gene had been utilized by the clone K and C strains to incorporate their respective plasmids into the chromosome. Several primers were designed downstream and upstream of the attP site in BmQ and the two tRNALys genes in the PAO genome. One plasmid-derived and one chromosome-derived primer were combined to amplify chromosomal DNA from strains C, K, K1 and K2 by PCR. Single PCR products were obtained and sequenced. In all cases, the 3' segment of the tRNALys gene was identified at the junctions between species-specific chromosomal and clone-specific plasmid sequence. In the pilA region the sequence contig in strains C, K1 and K2 reads hemAchromosome5' tRNALys(BmQplasmidBmQ)att 3'chromosomepilA (Fig. 3). Sequence alignments of the recombination sites are shown in Fig. 6
(b
, c
). In strain K the plasmid is integrated into the phnABoprL region at the tRNALys gene (Figs 3
and 4
), and the sequence contig correspondingly reads oprLchromosome5' tRNALys(BmQplasmidBmQ)att 3'chromosomephnAB(Fig. 6b
, c
).
Interestingly, the same genomic organization also applies to the 8·9 kb block of PAO-specific DNA at the SpU/SpF border (Fig. 4). The left junction consists of the tRNALys gene and the right junction of a 21 bp segment of the 3' end of the tRNALys gene (att*). At the left junction, the PAO chromosome and the plasmid-derived DNA of strain K exhibit an extended sequence homology of about 800 bp beyond the 3' end of the tRNALys gene (Fig. 6b
). In other words, the strong sequence similarity between PAO and clone K strains ends about 800 bp downstream of the 3' end of the tRNALys gene and starts again at the att* element (Fig. 4
).
Our conclusion that the two tRNALys genes are hotspots for the integration and excision of DNA was supported by the subculturing of single colonies of clone K isolates in vitro. Numerous descendants of a K1 ancestor carried pKLK106 DNA in the phnAB instead of the pilA region (for example strain K1', Fig. 1), implying that the plasmid sequence was rapidly mobilized from one att site to the other. In summary, the genome rearrangements observed in sequential clone K isolates were based on the reversible integration of pKLK106 in either one or the other chromosomal tRNALys gene. One site has also been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements, the other site by clone C to target its plasmid pKLC102 into the chromosome.
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DISCUSSION |
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An 8·9 kb island was integrated into the PAO chromosome at the same tRNA recognition site in the phnABoprL region as pKLK106, albeit the discernible att 3' sequence was truncated (att*). The annotation of the 8·9 kb insertion provides evidence that the DNA block was acquired by horizontal gene transfer (Fig. 4). Two different IS elements are located close to the borders of the element and encode transposases. One large ORF encodes a bactericidal S-type pyocin. Otherwise, no function could be ascribed to the ORFs of the insertion. Twelve out of 17 identified ORFs upstream of and within the DNA block are orphan genes with no significant homology to any entry in the public sequence databases (Fig. 4
), supporting the notion that a hypervariable region should be devoid of (essential) housekeeping genes.
The 8·9 kb insertion is stably maintained in strain PAO and hence behaves like a pathogenicity island (Hacker et al., 1997 ; Hou, 1999
). tRNAs play a role in the horizontal transfer of virulence gene clusters between different pathogens. An example is the tRNASel locus of E. coli that has served as the site of integration of two distinct pathogenicity islands that are responsible for converting benign strains into uro- and enteropathogens (Blanc-Potard & Groisman, 1997
).
tRNAs play versatile roles in prokaryotes and eukaryotes. They are central components of the translational machinery and are essential for replication of retroviruses because the tRNAs bind to viral genomes through their 3'-end sequences and act as primers for initiation of viral replication (Hou, 1999 ). Many temperate bacteriophages integrate into the bacterial chromosome via site-specific integration at an attB site that is typically within, or overlaps with, the 3' end of a tRNA gene (Campbell, 1996
). In P. aeruginosa, the cytotoxin-converting temperate phage
CTX was found to integrate at a chromosomal serine tRNA gene (Hayashi et al., 1993
; Nakayama et al., 1999
).
Whereas the integration of phage genomes into their host chromosome at the 3' end of tRNA genes has been demonstrated for numerous taxospecies (Campbell, 1996 ), reports that plasmids can recombine with chromosomal tRNA genes are rare. Examples are the elements pSAM2 from Streptomyces ambofaciens (Raynal et al., 1998
) and pSE101 from Saccharopolyspora erythraea (Brown et al., 1994
), which integrate into the chromosome at the 3' ends of the tRNAPro (pSAM2) and tRNAThr (pSE101) genes. Interestingly, pSAM2, pSE101 and our P. aeruginosa plasmids pKLC102 and pKLK106 all encode an integrase gene (int) in close vicinity to the attP end.
We compared the att sequences, their localization within the tRNA genes and the organization of the attspacerint sequence contig of pKLK106 with that of conjugative mobile genetic elements pSAM2 (Raynal et al., 1998 ), pSE101 (Brown et al., 1994
) and that of the temperate phages mv4 (Auvray et al., 1997
; 1999
), Sfi21 (Bruttin et al., 1997
) and VWB (Van Mellaert et al., 1998
) and the classical paradigm phage
(Campbell, 1996
). Amongst this set of selected examples, pKLK106 and pKLC102 are conspicuous by containing the largest spacer sequence of 295 bp between att and int. The attint sequence contigs of the plasmid-like mobile genetic elements are no more closely related to each other than they are to those of the phages. No general rules were seen with respect to the overlap of tRNA and att gene sequences, primary sequences and length of the att site, GC-contents and length of the spacer, and the coding sequence of int and its orientation with respect to att. All investigated examples share the localization of att at a tRNA gene locus and an adjacent int gene, but otherwise each analysed phage or conjugative genetic element is characterized by individual features of its primary sequence at the att locus.
In conclusion, by the analysis of sequential clone K isolates from CF airways, we have detected the reversible integration of a 106 kb plasmid at two identical att sites within the P. aeruginosa chromosome. The utilization of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms that allows P. aeruginosa to generate the mosaic genome structure of blocks of species-, clone- and strain-specific DNA. Our example demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes. The comparative genomics of strains that are collected from natural habitats over an extended period of time under well-documented sampling conditions provides robust primary data about genomic evolution in natural bacterial populations that are not biased by any heuristic assumptions or models and hence should withstand the test of time.
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ACKNOWLEDGEMENTS |
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Received 30 March 2000;
revised 17 July 2000;
accepted 21 July 2000.