AUD4, a new amplifiable element from Streptomyces lividans

Esther Schmida,1, Christa Büchler b,1 and Josef Altenbuchner1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
After transformation of the Streptomyces lividans chloramphenicol-sensitive, arginine-auxotrophic mutant strain AJ100 with a derivative of plasmid SCP2, some of the regenerated protoplasts contained an 8·2 kb DNA sequence amplified to several hundred copies per chromosome. The corresponding non-amplified sequence, called AUD4, was isolated from a {lambda} phage genomic library of S. lividans 1326. Two cytosine residues were the only directly repeated nucleotides at the ends of the element, indicating that AUD4 is a class I amplifiable sequence. The element mapped in the AseI-D fragment of the S. lividans chromosome, where other class I amplifications have been described. The complete element was sequenced and 10 ORFs were identified. Some of the deduced proteins are highly conserved in other organisms but a putative function could be attributed to only a few of them. Duplication of AUD4 by integration of an Escherichia coli plasmid carrying various parts of AUD4 and a thiostrepton-resistance gene in S. lividans AJ100, ZX7 or TK64 induced amplification of the integrated plasmid, AUD4 or both at high frequency.

Keywords: DNA amplification, deletion, genetic instability, class I amplifiable elements

Abbreviations: ADS, amplified DNA sequence; AUD, amplifiable unit of DNA; HT, Hickey–Tresner

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Genetic instability is a widespread phenomenon in the genus Streptomyces: mutants affected in various phenotypic properties like morphological differentiation, secondary metabolism or antibiotic resistance arise at frequencies between 0·1% and 1% of colony- forming spores (for reviews see Leblond & Decaris, 1994 ; Dharmalingam & Cullum, 1996 ; Volff & Altenbuchner, 1998 ). Most of the spontaneous mutations are caused by large chromosomal deletions removing the ends of the linear chromosome. The chromosome becomes circular and is then even more unstable than the linear chromosome (Fischer et al., 1997 ; Lin & Chen, 1997 ; Volff et al., 1997 ). This explains the hypervariability and progressive degeneration of such strains, in particular in S. lividans and S. ambofaciens where circularized chromosomes have been studied.

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 kb–4·7 kb–1 kb–4·7 kb–1 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, media and culture conditions.
The bacterial strains used in this work are listed in Table 1 . Escherichia coli JM109 (Yanisch- Perron et al., 1985 ) was used as the host for construction of plasmids and DNA sequencing. It was grown in dYT liquid medium or on dYT agar plates (Sambrook et al., 1989 ) at 37 °C. For transformation, the protocol of Chung et al. (1989) was used. Transformants were selected by adding 100 µg ampicillin ml-1 to the growth medium. For infection with {lambda} phages, the E. coli strain Q358 (Karn et al., 1980 ) was grown in L broth supplemented with 10 mM MgCl2 and 0·4% maltose (Sambrook et al., 1989 ). Plaques were obtained in the following way: 0·1 ml of an overnight culture of Q358 was incubated with 0·1 ml phage dilutions for 10 min, transferred to 3 ml prewarmed (45 °C) liquid L broth soft agar (L broth containing 0·7% agar, maltose and MgCl2 ), poured onto L agar plates and incubated at 37 °C overnight. Streptomycetes were grown at 30 °C in YEME liquid medium supplemented with 27% w/v sucrose, 0·5% glycine and 5 mM MgCl2 (Hopwood et al., 1985 ). To prepare spore suspensions, the strains were grown on Hickey–Tresner (HT) agar plates (Pridham et al., 1957 ) and the spores filtered through cotton wool. S. lividans protoplasts were transformed according to Hopwood et al. (1985) and regenerated on R2YE plates. Transformants were selected by overlaying the agar after 12 h with 3 ml 0·7% soft agar containing 1·25 mg thiostrepton (kindly provided by Hoechst AG). Otherwise, thiostrepton was used at 50 µg ml-1 in YEME medium or in HT and R2YE plates.


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Table 1. Bacterial strains used in this study

 
DNA manipulation.
Restriction enzymes and DNA modifying enzymes were purchased from Boehringer Mannheim. For restriction-enzyme analysis and cloning experiments, standard procedures were used as described by Sambrook et al. (1989) . Plasmid DNA was isolated using the QIAwell 8 plasmid purification kit (Qiagen). Total DNA of Streptomyces strains was extracted from cultures grown for 2 d in 15 ml YEME liquid medium by CsCl/EtBr density-gradient centrifugation as described by Sedlmeier & Altenbuchner, 1992 ).

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 {lambda}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 XhoI–SacI fragment (pSC30) or a 2·63 kb SacI–MluI 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 PstI–Xho I fragment (pSC28) or a 1 kb SacI fragment (pSC27). The plasmids pEI574 and pEI584-2 contain a 6 kb BamHI fragment from {lambda}EI33 or a 2·5 kb MluI fragment from {lambda}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 {lambda}EI32 and a 2·7 kb Bam HI fragment from {lambda}EI33, respectively, inserted into pIC19H. The plasmid pEI586 is a SmaI-deletion derivative of pEI573, and pEI585 contains a 290 bp SmaI–NcoI fragment from pEI584-3 inserted into pIC19H.

Genomic library of S. lividans 1326 in {lambda}RESI.
Total DNA of S. lividans 1326 was partially digested with Sau3AI, separated through a low-melting agarose gel, fragments in the range of 8–12 kb were purified by phenol extraction and ligated to the arms of the {lambda}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) . {lambda} DNA was transferred from plaques to nitrocellulose filters as described by Sambrook et al. (1989 ). DNA fragments (about 100 ng) were labelled with [{alpha}-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 {lambda} 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification and cloning of the AUD4 element
Strain AJ100 has been extensively used to study amplification of AUD1 (Altenbuchner & Eichenseer, 1991 ). It is a spontaneous CmlS Arg mutant of S. lividans TK64 in which the amplifiable element AUD1 was deleted. Plasmid pJOE907 consists of a 15 kb BclI fragment containing the complete AUD1 region, which was isolated from {lambda}MT686 (Altenbuchner & Cullum, 1985 ) as an EcoRI fragment and inserted into the SCP2-derived vector pJOE850. Transformants of AJ100 containing this plasmid showed no amplification of AUD1. Instead, an 8·2 kb BglII fragment was amplified to several hundred copies per chromosome in five out of nine transformants tested. The amplified element was called ADS4, and the corresponding non-amplified sequence, AUD4. A repetition of the transformation of independently prepared AJ100 protoplasts with pJOE907 failed to evoke amplification of AUD4. Furthermore, transformation of S. lividans TK64 with pJOE907 and analysis of twelve transformants by restriction-enzyme digestion and agarose gel electrophoresis failed to reveal any amplification of AUD4. Over several years, at least 50 transformations of AJ100, TK64 and ZX7 with SCP2 derivatives containing various parts of AUD1 or just AUD1 sequences on E. coli vectors have been carried out, as described by Altenbuchner & Eichenseer (1991) or Volff et al. (1996) , and several hundred transformants analysed for DNA amplifications. In all these transformations, the amplification of AUD4 was only seen once more. It occurred in AJ100, the transformants had AUD4 amplified with a frequency similar to that described above and again AJ100 was transformed with an SCP2 derivative containing parts of AUD1 as inserts.

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 {lambda} replacement vector {lambda}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 {lambda}EI32, {lambda}EI33 and {lambda}EI35 (Fig. 1 ) as well as by Southern hybridization (not shown).



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Fig. 1. Restriction map of the chromosomal region containing AUD4. The location of AUD4 is indicated by a solid arrow. The region was isolated in three overlapping fragments in the {lambda} phages {lambda}EI32, {lambda}EI33 and {lambda}EI35. The position of restriction fragments isolated from the phages and inserted into the pIC plasmids or pJOE867 are indicated. The deletion seen in AJ100 strains with AUD4 amplified is marked by a dotted arrow.

 
Deletions in strains with ADS4 and mapping of AUD4 by PFGE
Spontaneous CmlS Arg S. lividans mutants with amplifications of AUD1 usually have one or both chromosomal ends deleted (Redenbach et al., 1993 ; Rauland et al., 1995 ; Volff et al., 1996 ). One side of the deletion ends near or within the amplified DNA. To see if the amplification of AUD4 is accompanied by a deletion, a 2·5 kb MluI fragment from {lambda}EI32 flanking AUD4 on the left side and a 6 kb BamHI fragment from {lambda}EI33 flanking AUD4 on the right side (Fig. 1) were inserted into the vector pJOE867 (to give pEI584-2 and pEI574, respectively). The plasmid DNA was labelled with [{alpha}-32 P]dCTP and hybridized to total DNA of TK64, three strains of AJ100 transformed with pJOE907 containing amplified AUD4, and AJ100. The same 6 kb BamHI fragment hybridized in all strains, regardless of whether they contained an amplification. The 2·5 kb MluI fragment was present only in strains without an amplification (Fig. 2). This indicates that the amplification of AUD4 is accompanied by deletions on one side of the element, as observed with other amplifications.



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Fig. 2. Agarose gel electrophoresis of total DNA from S. lividans TK64 (lane 1), three transformants of AJ100 with pJOE907 (lanes 2–4) and AJ100 (lane 5), digested with Bam HI (a) or MluI (b), and Southern blot hybridization with [{alpha}-32P]dCTP-labelled pEI574 (a) and pEI584-2 (b). The size standard is {lambda} DNA digested with HindIII (lane 0).

 
To localize the AUD4 sequence on the S. lividans chromosome, cells were embedded in agarose plugs, the DNA purified for PFGE and digested with the rare-cutting enzyme AseI. Since there are considerable differences between S. lividans strains, DNA was prepared from S. lividans strains 1326, TK19, TK20, TK21, TK24, TK64 (Hopwood et al., 1983 ) and ZX7 (Tsai & Chen, 1987 ; Zhou et al., 1988 ). S. lividans 1326 was cured of the linear 50 kb plasmid SLP2 so this band is missing in TK19 (Fig. 3 ). In a second step, TK19 was cured of SLP3, a plasmid identified only by the lethal zygosis phenotype; after the curing, a 380 kb AseI band (AseI-F*) was missing (TK21). Strains TK24 and TK64 are derived from TK21 and showed no differences to their progenitors in the AseI pattern. ZX7 is derived from TK64 by NTG mutagenesis and has a 900 kb and a 80 kb fragment (AseI-B* and AseI-J*, respectively) deleted. The AseI and DraI map of S. lividans was made from ZX7 and so the positions of bands B*, F* and J* are not known. TK20 is a derivative of 1326, independently cured of the plasmid SLP3. In this strain, both the AseI-F* and AseI-D fragments are missing. A new band of 670 kb is presumably the result of a deletion fusing the AseI-D and AseI-F* fragments.



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Fig. 3. PFGE of AseI-digested DNA of S. lividans TK19 (lane 1), TK20 (lane 2), TK21 (lane 3), TK24 (lane 4), TK64 (lane 5), ZX7 (lane 6) and 1326 (lane 7), and Southern-blot hybridization with labelled pJOE920-1 containing ADS4. The numbering of the AseI fragments is according to Leblond et al. (1993) . Fragments not present in the S. lividans ZX7 map are marked by asterisks. Saccharomyces cerevisiae chromosomes (Bio-Rad) were used as size standards (lane 0).

 
In all strains, the AseI-D fragment hybridized with the ADS4 sequence in plasmid pJOE920-1, except in TK20 where no hybridization was found (Fig. 3). The AseI-D fragment was mapped near the chromosomal end on the opposite side to AUD1. This region is also affected by genetic instability. Class I amplifications have been described in this region by Redenbach et al. (1993) and Rauland et al. (1995) . The class I AUD region was cloned by Rauland et al. (1995) in the cosmids A6, A7 and U31, altogether a region of about 90 kb. Hybridization of ADS4 DNA to these cosmids gave no signal (data not shown).

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 {lambda}EI33 (pEI573) and a 2·7 kb Mlu I fragment from {lambda}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 SmaI–NcoI fragment from pEI584-3 and a 310 bp BamHI–SmaI 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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Fig. 4. (a) Comparison of the nucleotide sequences of the ends of AUD4 and the junction fragment in ADS4. Inverted repeats are marked by arrows and a rectangle is drawn around the two cytosine residues common to both ends. (b) Comparison of the left and right sequence flanking AUD4. Nucleotides repeated in direct orientation are marked by rectangles. The two cytosine residues at the borders of AUD4 are given in lower-case letters.

 
The complete AUD4 sequence was analysed for codon usage with the CodonPreference program using a Streptomyces codon usage table as described by Sedlmeier & Altenbuchner (1992) . The upstream regions of each ORF were analysed for sequences matching Streptomyces ribosome-binding sites (Strohl, 1992 ). Ten putative genes were identified (Fig. 5 ). The gene products were compared with entries in various protein databases using the BLAST programs. The results are listed in Table 2. A possible function can be attributed to ORFs 3 and 4, which are highly similar in sequence to cytochrome P-450 oxidoreductase and ferredoxin, the corresponding electron carrier. ORF9 shares a significant identity with quinone oxidoreductases and ORF2 might be a regulator protein. The genes encoding ORF1 and ORF5 seem to be highly conserved in some bacteria but the function is unknown. Between 25% and 44% amino acid sequence identities with hypothetical proteins, mainly identified in genome sequencing projects, were found for the remaining ORFs.



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Fig. 5. Restriction map of the 8366 bp sequence containing AUD4 (black arrow) and the position of the ten ORFs (white arrows) identified in the sequence.

 

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Table 2. ORFs and deduced proteins in the AUD4 sequence

 
Induction of AUD4 amplification
When an E. coli plasmid with a DNA fragment homologous to the incomplete AUD1 region in strain AJ100 was integrated into the AJ100 chromosome by homologous recombination, the integrated plasmid was amplified to high copy numbers in most transformants. The only requirement for frequent, high-level amplification was a minimum length of 2 kb for the direct repeats generated by the integrating plasmid (Altenbuchner & Eichenseer, 1991 ). To see if amplification of AUD4 in AJ100 could be evoked by similar experiments, various fragments of ADS4 were inserted into the polylinker sequence of pJOE803, a pIC20H derivative containing a thiostrepton-resistance gene. Representative examples of the plasmids constructed are given in Fig. 6 . Since the DNA for the plasmid construction was taken from the ADS4 sequence, there were, in principle, two different sorts of plasmids. Some of them contained the junction from the left and right ends of AUD4, i.e. pEI53, pEI52, pSC27 and pSC30 and others, i.e. pSC22, pSC23 and pSC28, that contained fragments from the internal region of AUD4. After integration of the former group of plasmids into the chromosomal AUD4 sequence there will be two copies of AUD4 in the chromosome, one AUD4 wild-type copy and a second copy with the AUD4 ends but modified internally by the integrated plasmid sequence. With the latter group of plasmids there will be just one AUD4 unit with an internal duplication flanking the E. coli plasmid (Fig. 7 ). Strain AJ100 as well as the wild- type strains TK64 and ZX7 were transformed with these plasmids. Thiostrepton-resistant transformants were streaked out on HT agar plates containing thiostrepton and from there transferred to 10 ml YEME liquid medium supplemented with thiostrepton. After growth for about 3 d, total DNA was extracted and characterized by restriction analysis for DNA amplifications. With pSC22 a weak amplification of the integrated plasmid was seen in one in five AJ100 transformants and one in six ZX7 transformants tested (Fig. 8 ). Similar results were obtained with pSC23: one in five AJ100 and one in six ZX7 transformants showed weak amplification of the integrated plasmid. With pSC28, which had the longest ADS4 insert in this group, there was no amplification seen in AJ100 (five transformants tested), weak amplification in one out of five TK64 transformants and a strong amplification of the plasmid in three out of six ZX7 transformants. None of the transformants had the complete AUD4 element amplified together with the integrated plasmid. With pSC27, pSC30, pEI52 and pEI53, some transformed colonies showed amplification of just the integrated plasmid. Others showed amplification of the integrated plasmid and, separately, the amplification of AUD4 at the same or higher copy number. For example, with pSC30 one out of three colonies of AJ100 had the plasmid weakly amplified, whereas from seven colonies of ZX7 tested, two had the plasmid highly amplified and two other colonies had a low plasmid amplification but in addition a high amplification of AUD4. Similar frequencies were obtained with pSC27, pEI52 and pEI53. Some examples are shown in Fig. 8.



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Fig. 6. Plasmid constructions containing pIC20H (white bars), the thiostrepton-resistance gene (hatched bar) and various parts of ADS4 (black bars and arrows) which were used to transform S. lividans AJ100 and TK64. At the top of the figure, the restriction map of ADS4 (represented by two ADS4 copies) is shown. On the right side the results achieved with these plasmids in AJ100, ZX7 and TK64 are given: (+) weak amplification of the integrated plasmid; + strong amplification of the integrated plasmid, AUD4 or both.

 


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Fig. 7. Structure of AUD4 in S. lividans AJ100 or TK64 after integration of the plasmids pSC22 or pSC27 (representative for all plasmids shown in Fig. 6) by homologous recombination. For pSC27, the two possible sites of integration are shown. Symbols are the same as in Fig. 6; white arrows indicate the sequence duplicated by the integration. MluI* indicates the site destroyed during construction of the plasmid.

 


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Fig. 8. Examples of DNA amplifications found by restriction enzyme analysis in S. lividans AJ100 and TK64 transformed with plasmids as shown in Fig. 6. Lanes: 0, {lambda}x HindIII size standard; 1, AJ100/pSC22; 2, pSC22; 3, AJ100/pSC27; 4, pSC27; 5, TK64/pEI52 no. 1; 6, TK64/pEI52, no. 2; 7, TK64/pEI52 no. 3; 8, pEI52. The DNA in lanes 1 and 2 was digested with EcoRV and with SacI in lanes 3–8.

 
In general, the frequency of amplification was in a range between 10% and 40% of all transformants tested, and in TK64 and ZX7 it was slightly higher than in AJ100 in which ADS4 had been originally found. This is in contrast to integration of plasmids in the AUD1 region, where the frequency of amplification was high in AJ100 (50–75% of the transformants) and low in TK64 or ZX7 (in the latter strains, amplification was first seen after loss of the chloramphenicol resistance and argG at the usual frequencies). All the TK64 and ZX7 transformants showing high amplification of AUD4 were still chloramphenicol resistant and arginine prototrophs except one mutant of TK64 transformed with pEI52, which had the AUD1 element amplified in addition to AUD4 (not shown).

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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Fig. 9. Southern-blot hybridization of a labelled 1·2 kb KpnI fragment from the terminal inverted repeats of the S. lividans chromosome to chromosomal DNA of TK64 and ZX7 derivatives transformed with pSC27, pSC28, pSC30 and pEI52. The DNA on the agarose gel (a) was digested with KpnI. The autoradiogram (b) shows the hybridizing 1·2 KpnI fragments from the strains. Lanes: 0, {lambda}xHindIII size standard; 1 and 2, ZX7/pSC30; 3 and 4, TK64/pSC27; 5 and 7, ZX7/pSC28; 6, TK64; 8–10, TK64/pEI52. For each pair of lanes, different colonies from a transformation experiment were used for DNA preparations.

 
Finally, five TK64 derivatives transformed with pSC22, pSC27 or pSC28 without visible amplification were tested to determine whether the plasmids were correctly inserted via homologous recombination. The DNA was digested with PstI and hybridized to detect ADS4 DNA by Southern blotting. The plasmids chosen had no PstI site and in TK64 DNA the enzyme cuts outside the AUD4 DNA and gives a high molecular mass fragment of greater than 20 kb. No additional fragment was seen in the transformants (data not shown), which indicates that the plasmids were integrated by homologous recombination.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A class I AUD region was mapped in S. lividans about 300 kb away from one chromosomal end (Redenbach et al., 1993 ; Rauland et al., 1995 ). This corresponds to the AseI-D band in which AUD4 was mapped in this work by Southern hybridization. Hybridization of ADS4 DNA to cosmids containing the class I AUD region gave no signal. Therefore, AUD4 seems to be a different class I element. The AUD4 sequence was found in all S. lividans strains tested except in TK20 where the AseI-D and AseI-F* bands are missing. Ase I-F* is also deleted in ZX7 and so its position on the chromosome is unknown. However, it seems very likely that AseI-D and Ase I-F* are fused in the new band seen in TK20, which would mean that AseI-F* is located on one or the other side of the Ase I-D fragment.

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.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 5 May 1999; revised 2 August 1999; accepted 27 August 1999.