Institute of Industrial Genetics, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany1
Author for correspondence: Josef Altenbuchner. Tel: +49 711 685 7591. Fax: +49 711 685 6973. e-mail: joe{at}gensun.biologie.uni-stuttgart.de
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ABSTRACT |
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Keywords: DNA amplification, deletion, genetic instability, class I amplifiable elements
Abbreviations: ADS, amplified DNA sequence; AUD, amplifiable unit of DNA; HT, HickeyTresner
The GenBank accession number for the sequence reported in this paper is AF072709.
a Present address: Cardiovascular Biology, Pfizer Central Research, Pfizer Limited, Sandwich, Kent CT13 9NJ, UK.
b Present address: Klinische Chemie, Franz Josef Strauß-Allee 11, 93053 Regensburg, Germany.
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INTRODUCTION |
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Frequently, the large deletions are accompanied by DNA amplifications. Amplifications occur from specific chromosomal sequences called AUDs (amplifiable units of DNA). The AUDs amplify to several hundred copies of head-to-tail arranged units, called ADSs (amplified DNA sequences) (Fishman & Hershberger, 1983 ). Two classes of AUD elements were identified. Class II AUDs consist of two directly repeated sequences flanking an internal single-copy sequence. Independent mutants show the same amplified unit consisting of one of the two direct repeats and the internal sequence each. Class I amplifications arise in certain chromosomal regions of a strain. The size of these regions is about 100 kb. Each individual mutant amplifies a different segment of this region; some of the amplified DNAs overlap, some have no sequence in common with other amplified segments (Hütter & Eckhardt, 1988
).
Several amplifiable elements were found in S. lividans. The strain segregates chloramphenicol-sensitive (CmlS) mutants with a frequency of 0·5% of spores and these mutants again segregate arginine-auxotrophic derivatives (Arg) at about 25% of spores. The double mutants usually show amplification of AUD1, a class II element, which consists of three 1 kb and two 4·7 kb direct repeats in the order 1 kb4·7 kb1 kb4·7 kb1 kb. The amplified unit consists of a 4·7 kb and a 1 kb repeat (Altenbuchner & Cullum, 1985 ). Another class II element encoding mercury-resistance genes (AUD2) has a size of 90 kb and is flanked by two directly repeated copies of the IS element IS1372. It was amplified together with AUD1 in a Cml S Arg mutant (Eichenseer & Altenbuchner, 1994
). AUD1 is located about 800 kb away from the end of the linear chromosome (Redenbach et al., 1993
). A class I AUD region of about 70 kb was described by Rauland et al. (1995)
in 2-deoxygalactose- resistant mutants about 300 kb away from the other end of the chromosome. The 24 kb AUD3 sequence, which was found amplified together with AUD1 in a CmlS Arg mutant (Altenbuchner et al., 1988
), and a 4·3 kb sequence found by Betzler et al. (1997)
might be part of this AUD class I region.
In this paper we describe the characterization and mapping of a further 8·2 kb amplifiable element from S. lividans , which was found after transformation of a CmlS Arg - mutant with an SCP2 plasmid derivative.
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METHODS |
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Plasmid constructions.
The plasmid pJOE907 was constructed from the SCP2- and pJOE810- derived shuttle vector pJOE850 (Altenbuchner et al., 1988 ) by insertion of a 15 kb EcoRI fragment from
MT686 (Altenbuchner & Cullum, 1985
), containing the complete AUD1 region of S. lividans TK64, into the Eco RI site of the vector. The plasmid pJOE803 is a derivative of pIC20H (Marsh et al., 1984
) containing the 1·05 kb BclI fragment with the thiostrepton- resistance gene from pIJ702 (Katz et al., 1983
) integrated in the NruI site (Altenbuchner & Eichenseer, 1991
). The plasmids pEI53 and pJOE920-1 were constructed by inserting ADS4 DNA, obtained from total DNA of a S. lividans AJ100/pJOE907 transformant, as an 8·2 kb BglII fragment, into the BglII site of pJOE803 and pIC19H (Marsh et al., 1984
), respectively. The plasmids pEI52, pSC30 and pSC22 contained an incomplete copy of ADS4, which was a 5·9 kb BamHI fragment (pEI52), a 3·8 kb XhoISacI fragment (pSC30) or a 2·63 kb SacIMluI fragment (pSC22) inserted into pJOE803. The plasmids pSC23, pSC28 and pSC27 were derived from pEI53 by deleting a 5·9 kb KpnI fragment (pSC23), a 4·5 kb PstIXho I fragment (pSC28) or a 1 kb SacI fragment (pSC27). The plasmids pEI574 and pEI584-2 contain a 6 kb BamHI fragment from
EI33 or a 2·5 kb MluI fragment from
EI32 inserted into the BamHI or Eco RI site of pJOE867, respectively (Altenbuchner, 1993
). The plasmids pEI584-3 and pEI573 contain a 2·7 kb Mlu I fragment from
EI32 and a 2·7 kb Bam HI fragment from
EI33, respectively, inserted into pIC19H. The plasmid pEI586 is a SmaI-deletion derivative of pEI573, and pEI585 contains a 290 bp SmaINcoI fragment from pEI584-3 inserted into pIC19H.
Genomic library of S. lividans 1326 in RESI.
Total DNA of S. lividans 1326 was partially digested with Sau3AI, separated through a low-melting agarose gel, fragments in the range of 812 kb were purified by phenol extraction and ligated to the arms of the RESI vector (Altenbuchner, 1993
) which had been digested with BamHI and purified in the same way. The packaging was done as described by Sambrook et al. (1989)
. After plaque hybridization, plasmids were generated from the inserts in the phages by infection of E. coli HB101 F' lac::Tn1739 tnpR (Altenbuchner, 1993
).
Southern and plaque hybridization.
The blotting of DNA from agarose gels onto nitrocellulose filters was done according to Smith & Summers (1980) .
DNA was transferred from plaques to nitrocellulose filters as described by Sambrook et al. (1989
). DNA fragments (about 100 ng) were labelled with [
-32P]dCTP using the random-primed DNA labelling kit from Boehringer Mannheim. Filters were hybridized in buffer containing 50% formamide and washed under the conditions described by Hopwood et al. (1985)
.
PFGE.
DNA from the various Streptomyces strains was prepared from cultures grown for 2 d in YEME liquid medium at 30 °C, as described by Leblond et al. (1993) , and the agarose blocks digested with AseI for 12 h at 37 °C. The DNA was separated on a 0·8% agarose gel in 0·5xTBE buffer with 1·6 mM thiourea using a Chef Mapper from Bio-Rad. The running conditions were: initial switch time 1·65 s, final switch time 2·3 min, 6 V cm-1 for 20 h. Size standards were Saccharomyces cerevisiae chromosomes and
concatemeric DNA from Bio-Rad.
DNA sequence analysis.
DNA sequencing of the AUD4 fragment was carried out by the chain- termination method with double-stranded plasmid DNA. Various restriction fragments of AUD4 and ADS4 were inserted into pIC20H and sequenced using Cy5-labelled M13 universal and reverse primers with the ALFexpress AutoRead sequencing kit (Amersham Pharmacia Biotech). In addition, primer walking was performed using oligonucleotides from MWG Biotech and the Cy5-dATP labelling mix in combination with the ALFexpress AutoRead sequencing kit. The DNA was separated on a 5·5% Hydrolink Long Ranger gel matrix in an ALFexpress DNA sequencer for 12 h at 55 °C, 800 V and 0·5xTBE buffer. The nucleotide sequence was analysed with the GCG program package (Devereux et al., 1984 ). Codon usage was analysed with a codon-usage table based on the analysis of 8 Streptomyces genes as described by Sedlmeier & Altenbuchner (1992)
. Database searches were performed with the BLASTP and BLASTX programs (Altschul et al., 1990
) using the electronic mail server of the National Center for Biotechnology Information, Bethesda, MD, USA.
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RESULTS |
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The DNA of one of the earlier AJ100 transformants that had AUD4 highly amplified was digested with BglII, the DNA separated through an agarose gel and the 8·2 kb fragment isolated and inserted into the E. coli vector pIC19H. The resulting plasmid, pJOE920-1, was used to screen a genomic library of S. lividans 1326 for the corresponding AUD4 sequence. The library was constructed by insertion of S. lividans 1326 DNA, partially digested with Sau3AI, between the BamHI sites of the replacement vector
RESI. Three different phages hybridizing with pJOE920-1 were isolated. The phages were converted into plasmids as described by Altenbuchner (1993)
and the insertions mapped by restriction analysis. The location of AUD4 in the phage inserts was determined by comparing the restriction maps from pJOE920-1 and the corresponding wild-type sequences in the phages
EI32,
EI33 and
EI35 (Fig. 1
) as well as by Southern hybridization (not shown).
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Nucleotide sequence of AUD4
The nucleotide sequence of the 8·2 kb BglII fragment, comprising a complete copy of the ADS4 element, in pJOE907 was determined on both strands. To identify the ends of AUD4 and the flanking chromosomal sequences, a 2·7 kb BamHI fragment from EI33 (pEI573) and a 2·7 kb Mlu I fragment from
EI32 (pEI584-3) were inserted into pIC19H (Fig. 1
), mapped in detail and compared to the corresponding amplified ADS4 junction fragment. Finally, a 290 bp SmaINcoI fragment from pEI584-3 and a 310 bp BamHISmaI fragment from pJOE573 were sequenced. Together with the ADS4 sequence, the AUD4 sequence could be reconstructed. AUD4 was 8202 bp long and with the flanking chromosomal regions, a continuous sequence of 8366 bp was obtained (GenBank accession no. AF072709). The nucleotide sequence of the ends of AUD4 and the ADS4 junction is shown in Fig. 4
. The crossover point leading to the amplification of AUD4 must have been between two cytosine residues present on both ends of AUD4. A 11 bp sequence was found 16 nt from one end of AUD4 which is inversely repeated on the other side, here 22 nt away from the two cytosines. A comparison of the right and left AUD4 flanking region revealed long imperfect direct repeats with 25 out of 39 nt identical (Fig. 4b
). The ADS4 sequence of three additional transformants was cloned in E. coli and the junction analysed by DNA sequencing. They all showed exactly the same sequence. All the amplified strains tested came from a single transformation of AJ100 with pJOE907. They should be independent transformants, since immediately after the addition of DNA, the protoplasts were embedded in soft agar and spread on R2 agar plates.
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Several transformants of TK64 or ZX7 were also tested to see if they had still intact chromosomal ends. The total DNA of six transformants with high amplifications of AUD4 and/or the integrated plasmids (pSC27, pSC28, pSC30), of three with low amplifications or just recognizable amplification (pEI52) was digested with KpnI and hybridized with a 1·2 kb KpnI fragment from pLUS449, a plasmid containing DNA from the terminal inverted repeats of the S. lividans chromosome, just a few base pairs away from the chromosomal ends (Lin et al., 1993 ). From the six mutants with high amplifications, only one showed no hybridization, the others gave faint hybridizing bands in comparison to S. lividans TK64 wild-type. The bands of the other mutants with low amplification showed reduced intensities or had about the same intensity as the wild-type (Fig. 9
). This indicates i, a correlation of the amplification of AUD4 and the deletions of the chromosomal ends, and ii, that each single mutant contains chromosomes at different stages of deletions.
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DISCUSSION |
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DNA amplifications have been observed in many bacteria. They can be interpreted as an adaptive and reversible response to the demand of higher resistance to toxic metals or antibiotics, to conditions of nutrient limitations or to processes of pathogenic and symbiotic interactions (reviewed by Romero & Palacios, 1997 ). The number of amplified copies is low and often there are only duplications. Unequal crossover or circle excision and reinsertions at large tandem repeats were proposed as mechanisms of amplification. The low amplifications of plasmids observed in some S. lividans strains after transformation and integration of the ADS4 containing pJOE803 derivatives into the chromosome might have been selected by insufficient thiostrepton resistance mediated by just one copy of the gene. To our knowledge, a precise relationship between thiostrepton resistance and copy number of the resistance gene in a cell has not formally been tested.
The spontaneous high DNA amplifications in streptomycetes differ from amplifications in other bacteria in many respects. Large deletions removing one or both ends of the chromosome precede the amplification events. DNA sequence analysis of amplified DNA has revealed the presence of putative genes and in a few cases gene expression was demonstrated (Aigle et al., 1996 ; Betzler et al., 1997
; this work) but there is no indication that the amplification of these genes is of any selective advantage to the cells. Even in the case of AUD2, which encodes the mercury- resistance genes, amplification was spontaneous and not selected by adding high mercury concentrations to the medium (Sedlmeier & Altenbuchner, 1992
). Furthermore, the class I amplifiable regions are quite large but in each of the mutants only small and different parts of these regions are amplified. Therefore, it seems that amplification of DNA in a certain region itself is selected but the amplification of specific genes is not.
According to a hypothesis discussed by Volff & Altenbuchner (1998) , which is based on observations made with artificially circularized S. lividans chromosomes, the amplification of DNA in streptomycetes occurs in mutants with circular chromosomes or inverted fused chromosomes. Due to the lack of replication terminators, the DNA is overreplicated in the chromosomal region where the replication forks meet. The overreplicated DNA is ordered into tandem repeats by illegitimate recombination. These amplifications are unstable and replaced by more stable ones like the AUD1 amplification. This needs deletions in the vicinity of the amplifiable region which direct the meeting point of replication forks to the new positions. The frequency of amplification of AUD1 is very high due to the presence of long tandem repeats. In contrast, class I sequences are amplified rarely. The rate-limiting step seems to be a duplication of a sufficiently large single-copy sequence by illegitimate recombination events. This might also be the case for AUD4. Only the duplication of large parts of AUD4 through integration of the plasmids pEI52, pEI53, pSC27, pSC28 and pSC30 leads to an efficient amplification of AUD4 sequences regarding the copy number of the amplified DNA and number of transformants showing amplifications.
It seems unlikely that the AUD1 sequence on pJOE907 induced amplification of AUD4 in a direct way, since no significant identity was found between the two sequences using the GCG BESTFIT program for comparison. The high frequency of amplification of AUD4 in AJ100 in one transformation experiment with pJOE907 and the failure to detect it in a second experiment, or in TK64, as well as in many other transformations of AJ100, ZX7 or TK64 using derivatives similar to pJOE907 may be explained in the following way. Presumably, a large part of the AJ100 cells used for protoplasting had already spontaneously duplicated the AUD4 sequence and the protoplasting and regeneration evoked the high amplification. Other stocks of AJ100 (as well as of TK64 or ZX7) had no such duplication and therefore showed no amplification. Unfortunately, the original stock of AJ100 used in the first experiment could not be tested by Southern blot hybridization and another stock of AJ100 which gave no amplification of AUD4 when transformed with pJOE907 contained no duplication of AUD4, as expected [this was shown by digesting AJ100 DNA with BglII and hybridization with labelled ADS4 sequence (data not presented)].
S. lividans strain AJ100 has a deletion in the amplifiable AUD1 element and a circular chromosome (data not shown) that according to the hypothesis of Volff & Altenbuchner (1998) should lead to amplification of other DNA sequences like the class I element AUD4. Strains TK64 and ZX7 have linear chromosomes. The integration of plasmids like pSC27 generates long duplications which may favour amplification of AUD4 instead of AUD1 in these strains. In the mutants tested, the high amplification of AUD4 correlated with a high frequency of deletions of the chromosomal ends as shown by Southern hybridization using DNA from the chromosomal ends. Again, it seems that the high amplification of AUD4 needed the deletion of the chromosomal ends. One can conclude from this experiment that each mutant was heterogeneous and the mycelium represented different stages of chromosomal deletions. It is tempting to speculate that also the degree of amplification in each of the chromosomes of a specific mutant is different with no or low amplification in intact chromosomes and high amplification in chromosomes where the ends are deleted and the chromosomes circularized. The DNA rearrangements in such cells could be occurring in various orders. The integration of the ADS4 DNA might stimulate first low copy amplifications, followed by deletion formation and high amplification or stimulate first deletions which then induce the high copy amplification. All of the plasmid-induced amplifications can be explained by homologous recombination at long direct repeats generated by the integration. For the amplification of AUD4 in AJ100, transformed by pJOE907, there were no such long direct repeats. The first duplication must have occurred between two cytosine residues, which are the only direct repeated base pairs at the crossover site. There are also two imperfect directly repeated sequences flanking AUD4 but the crossover point is at the end of one and the beginning of the other repeat and not at the ends or within these direct repeats, as one would expect. The two inverted repeats found inside AUD4 have different distances to the crossover point. It is therefore unclear which DNA sequences directed the recombining enzymes to these crossover points.
It is also unclear what defines a chromosomal region as an AUD region. Is it the border between regions of nonessential and essential genes where deletions in an unstable strain come to a halt since any further deletions would be lethal? Are there other properties that favour the amplification of a specific DNA sequence in a AUD class I region? For the class II element AUD1 it was found that, besides the long direct repeats, the binding of the regulatory protein encoded by the 1 kb repeats to the binding sites up- and downstream of the right 1 kb repeat is necessary for efficient amplification (Volff et al., 1996 ). It is possible that the gene product of one of the ORFs in AUD4 also favours the amplification of the element.
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Received 5 May 1999;
revised 2 August 1999;
accepted 27 August 1999.